COMBINATION VACCINE DEVICES AND METHODS OF KILLING CANCER CELLS
20200276290 ยท 2020-09-03
Assignee
- President And Fellows Of Harvard College (Cambridge, MA)
- Dana-Farber Cancer Institute, Inc. (Boston, MA, US)
Inventors
Cpc classification
A61K39/001156
HUMAN NECESSITIES
C12N7/00
CHEMISTRY; METALLURGY
A61K2039/64
HUMAN NECESSITIES
A61K2039/55555
HUMAN NECESSITIES
A61K39/3955
HUMAN NECESSITIES
A61K47/36
HUMAN NECESSITIES
A61P35/00
HUMAN NECESSITIES
A61K47/34
HUMAN NECESSITIES
A61K39/39
HUMAN NECESSITIES
A61K39/001157
HUMAN NECESSITIES
A61K39/001102
HUMAN NECESSITIES
A61K2039/55561
HUMAN NECESSITIES
A61K2039/507
HUMAN NECESSITIES
C07K2317/24
CHEMISTRY; METALLURGY
International classification
A61K39/00
HUMAN NECESSITIES
A61K39/395
HUMAN NECESSITIES
A61K47/36
HUMAN NECESSITIES
C07K16/28
CHEMISTRY; METALLURGY
A61K39/39
HUMAN NECESSITIES
C12N7/00
CHEMISTRY; METALLURGY
Abstract
The present invention comprises compositions, methods, and devices for enhancing an endogenous immune response against a cancer. Devices and methods provide therapeutic immunity to subjects against cancer.
Claims
1-78. (canceled)
79. A method of treating a poorly immunogenic cancer in a subject in need thereof comprising administering to the subject: a) an inhibitor of an immune checkpoint protein or ligand thereof; and b) a device comprising (i) a macroporous scaffold composition comprising open, interconnected pores, (ii) a cell recruitment composition that recruits an immune cell, wherein the cell recruitment composition is selected from the group consisting of a cytokine, a chemokine, a growth factor, and a combination thereof; and (iii) an antigen derived from the cancer, thereby treating the cancer.
80. The method of claim 79, wherein the immune checkpoint protein or the ligand thereof is selected from the group consisting of cytotoxic T-lymphocyte-associated antigen 4 (CTLA4), programmed cell death protein 1 (PD1), programmed cell death protein 1 ligand 1 (PDL1), programmed cell death protein ligand 2 (PDL2), lymphocyte activation gene 3 (LAG3), B7-H3, B7-H4, and T cell membrane protein 3 (TIM3) adenosine A2a receptor (A2aR), and kill inhibitory receptors (KIRs).
81. The method of claim 79, wherein the inhibitor of an immune checkpoint protein or ligand thereof is an antibody or an antigen binding fragment thereof.
82. The method of claim 79, wherein the inhibitor of an immune checkpoint protein is a protein that inhibits the interaction between the checkpoint protein and ligand thereof.
83. The method of claim 81, wherein the antibody is selected from the group consisting of BMS-936558, MK-3475, pidilizumab, MDX1105, and MGA271.
84. The method of claim 82, wherein the protein that inhibits the interaction between the checkpoint protein and the ligand thereof is selected from the group consisting of a PDL2-immunoglobulin (Ig) fusion protein, and a LAG3-Ig fusion protein.
85. The method of claim 79, wherein the macroporous scaffold composition comprises a polymer or co-polymer selected from the group consisting of polylactic acid, polyglycolic acid, PLGA, alginate, gelatin, collagen, agarose, poly(lysine), polyhydroxybutyrate, poly-epsilon-caprolactone, polyphosphazines, poly(vinyl alcohol), poly(alkylene oxide), poly(ethylene oxide), poly(allylamine), poly(acrylate), poly(4-aminomethylstyrene), pluronic polyol, polyoxamer, poly(uronic acid), poly(anhydride) or poly(vinylpyrrolidone).
86. The method of claim 79, wherein the immune cell comprises an antigen presenting cell.
87. The method of claim 86, wherein the antigen presenting cell is a dendritic cell.
88. The method of claim 79, wherein the cell recruitment composition is selected from the group consisting of GM-CSF, Flt3L, and CCL20.
89. The method of claim 79, wherein the antigen is derived from a cancer selected from the group consisting of a melanoma, a central nervous system (CNS) cancer, a CNS germ cell tumor, a lung cancer, a leukemia, a multiple myeloma, a renal cancer, a malignant glioma, a medulloblatoma, a breast cancer, an ovarian cancer, a prostate cancer, a bladder cancer, a fibrosarcoma, a pancreatic cancer, a gastric cancer, a head and neck cancer, and a colorectal cancer.
90. The method of claim 79, wherein the antigen is selected from the group consisting of MAGE series of antigens, MART-1/melanA, Tyrosinase, ganglioside, gp100, GD-2, O-acetylated GD-3, GM-2, MUC-1, Sosl, Protein kinase C-binding protein, Reverse transcriptase protein, AKAP protein, VRK1, KIAA1735, T7-1, T11-3, T11-9, Homo Sapiens telomerase ferment (hTRT), Cytokeratin-19 (CYFRA21-1), SQUAMOUS CELL CARCINOMA ANTIGEN 1 (SCCA-1), (PROTEIN T4-A), SQUAMOUS CELL CARCINOMA ANTIGEN 2 (SCCA-2), Ovarian carcinoma antigen CA125 (1A1-3B) (KIAA0049), MUCIN 1 (TUMOR-ASSOCIATED MUCIN), (CARCINOMA-ASSOCIATED MUCIN), (POLYMORPHIC EPITHELIAL MUCIN), (PEM), (PEMT), (EPISIALIN), (TUMOR-ASSOCIATED EPITHELIAL MEMBRANE ANTIGEN), (EMA), (H23AG), (PEANUT-REACTIVE URINARY MUCIN), (PUM), (BREAST CARCINOMA-ASSOCIATED ANTIGEN DF3), CTCL tumor antigen sel-1, CTCL tumor antigen se14-3, CTCL tumor antigen se20-4, CTCL tumor antigen se20-9, CTCL tumor antigen se33-1, CTCL tumor antigen se37-2, CTCL tumor antigen se57-1, CTCL tumor antigen se89-1, Prostate-specific membrane antigen, 5T4 oncofetal trophoblast glycoprotein, Orf73 Kaposi's sarcoma-associated herpesvirus, MAGE-C1 (cancer/testis antigen CT7), MAGE-B1 ANTIGEN (MAGE-XP ANTIGEN) (DAM10), MAGE-B2 ANTIGEN (DAME), MAGE-2 ANTIGEN, MAGE-4a antigen, MAGE-4b antigen, Colon cancer antigen NY-CO-45, Lung cancer antigen NY-LU-12 variant A, Cancer associated surface antigen, Adenocarcinoma antigen ART1, Paraneoplastic associated brain-testis-cancer antigen (onconeuronal antigen MA2; paraneoplastic neuronal antigen), Neuro-oncological ventral antigen 2 (NOVA2), Hepatocellular carcinoma antigen gene 520, TUMOR-ASSOCIATED ANTIGEN CO-029, Tumor-associated antigen MAGE-X2, Synovial sarcoma, X breakpoint 2, Squamous cell carcinoma antigen recognized by T cell, Serologically defined colon cancer antigen 1, Serologically defined breast cancer antigen NY-BR-15, Serologically defined breast cancer antigen NY-BR-16, Chromogranin A, parathyroid secretory protein 1, DUPAN-2, CA 19-9, CA 72-4, CA 195, and Carcinoembryonic antigen (CEA).
91. The method of claim 79, wherein the antigen comprises a tumor lysate or an irradiated tumor cell.
92. The method of claim 79, wherein the device further comprises an adjuvant.
93. The method of claim 92, wherein the adjuvant comprises a TLR agonist.
94. The method of claim 93, wherein the TLR agonist is a TLR3 or TLR9 agonist.
95. The method of claim 94, wherein the TLR agonist is selected from the group consisting of a CpG rich oligonucleotide, a PEI-CpG oligonucleotide, a polyinosine-polycytidylic acid (poly (I:C)) and PEI-poly (I:C).
96. The method of claim 79, wherein the poorly immunogenic cancer is resistant to cytotoxic T-lymphocyte (CTL)-mediated lysis or natural killer (NK) cell mediated killing.
97. The method of claim 79, wherein the antibody or the protein is administered to the subject prior to, concurrently with, or subsequent to the administration of the device.
98. A method of treating a poorly immunogenic cancer a subject in need thereof comprising administering to the subject: a) an antibody selected from the group consisting of an anti-PD1 antibody, an anti-CTLA4 antibody, and a combination thereof; b) a device comprising (i) a PLG macroporous scaffold comprising open, interconnected pores, (ii) a granulocyte macrophage-colony stimulating factor (GM-CSF); and (iii) a tumor cell lysate, thereby treating the cancer.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
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DETAILED DESCRIPTION OF THE INVENTION
[0133] Prior to the invention, cancer vaccines typically depended on cumbersome and expensive manipulation of cells in the laboratory, and subsequent cell transplantation resulted in poor lymph node homing and limited efficacy. In terms of cancer treatment, many existing therapies become ineffective because cancers can co-opt immune checkpoint pathways to evade the endogenous immune response. Although agents have been identified and are used to prevent or minimize this ability of cancer cells to evade the immune system, these agents lack efficacy in poorly immunogenic tumors. The invention solves these problems by using materials for cancer vaccination that mimic key aspects of bacterial infection to directly control immune cell trafficking and activation in the body. The invention further combines these cancer vaccines with inhibitors of immune-inhibitory proteins (e.g., immune checkpoint proteins), thereby enabling an endogenous immune response strong enough to eliminate tumors or minimize their progression. Also, the cancer vaccines work synergistically with the inhibitors of the immune-inhibitory proteins to lower the dosage of inhibitor required for efficacy in treating cancer compared to the dosage required when the inhibitor is used as a single agent.
[0134] The results described herein demonstrate that poly(lactide-co-glcolide) (PLG) cancer vaccines produce significant numbers of antigen specific T cells in melanoma models. In summary, to test the effects of vaccine and antibody (e.g., anti-CTLA4 and/or anti-PD1 antibody) treatments in combination, an aggressive, therapeutic B16 melanoma model was utilized. In mice bearing B16 melanoma tumors, treatment with anti-CTLA4 and anti-PD1 antibodies alone had no effect on tumor size and survival outcomes in these animals (
Immune Checkpoint Pathways and Cancer
[0135] In healthy subjects, immune checkpoint pathways (also known as immune-inhibitory pathways) are important for maintaining self-tolerance and preventing autoimmunity. However, immune checkpoint pathways in cancer cells are often dysregulated, leading to the ability of tumors to evade the body's endogenous anti-tumor immune response. Cancers co-opt the immune checkpoint pathways by a number of ways, such as upregulating the expression of immune checkpoint proteins that normally serve immune-inhibitory roles. For example, inhibitory ligands and receptors that regulate T cell effector activity are often overexpressed in cancer cells.
[0136] An exemplary inhibitory receptor is cytotoxic T-lymphocyte-associated antigen 4 (CTLA4), also called CD152, which reduces the level of T cell activation. Another exemplary inhibitory receptor is programmed cell death protein 1 (PD1), also called CD279, which limits T effector cell activity. For example, cancer cells upregulate ligands for PD1 (e.g., programmed cell death protein ligand 1 (PDL1)), thereby blocking anti-tumor immune responses.
[0137] The blockade of immune checkpoints in cancer immunotherapy has emerged as a promising approach to combat this mechanism by which cancer cells evade the anti-tumor immune response. For example, antibodies directed against immune-inhibitory proteins, such as immune checkpoint proteins (also referred to as blockade antibodies herein) are being explored as potential anti-cancer therapeutics. See, e.g., Pardoll. Nat. Reviews Cancer. (2012) 12:252-264.
Immune-Inhibitory Proteins and their Inhibitors
[0138] Immune checkpoint proteins include the B7/CD28 receptor super family. CTLA-4 belongs to the immunoglobulin superfamily of receptors, which also includes programmed cell death protein 1 (PD-1), B and T lymphocyte attenuator (BTLA), T-cell immunoglobulin and mucin domain-containing protein 3 (TIM-3), and V-domain immunoglobulin suppressor of T cell activation (VISTA). Other immune regulatory checkpoint proteins include proteins in the TNF family (e.g., OX40 (also known as CD134) and 4-1BB ligand).
[0139] The amino acid sequence of Mus musculus VISTA, provided by Genbank Accession No. AEO22039.1, is shown below (SEQ ID NO: 1).
TABLE-US-00001 (SEQIDNO:1) 1 mgvpavpeassprwgtlllaiflaasrglvaafkvttpyslyvcpegqnatltcrilgpv 61 skghdvtiyktwylssrgevqmckehrpirnftlqhlqhhgshlkanashdqpqkhglel 121 asdhhgnfsitlrnvtprdsglycclvielknhhpeqrfygsmelqvqagkgsgstcmas 181 neqdsdsitaaalatgacivgilclplilllvykqrqvashrragelvrmdssntqgien 241 pgfettppfqgmpeaktrpplsyvaqrqpsesgryllsdpstplsppgpgdvffpsldpv 301 pdspnseai
[0140] The mRNA sequence encoding Mus musculus VISTA, provided by Genbank Accession No. JN602184.1, is shown below (SEQ ID NO: 2), with the start and stop codons in bold.
TABLE-US-00002 (SEQIDNO:2) 1 atgggtgtccccgcggtcccagaggccagcagcccgcgctggggaaccctgctccttgct 61 attttcctggctgcatccagaggtctggtagcagccttcaaggtcaccactccatattct 121 ctctatgtgtgtcccgagggacagaatgccaccctcacctgcaggattctgggccccgtg 181 tccaaagggcacgatgtgaccatctacaagacgtggtacctcagctcacgaggcgaggtc 241 cagatgtgcaaagaacaccggcccatacgcaacttcacattgcagcaccttcagcaccac 301 ggaagccacctgaaagccaacgccagccatgaccagccccagaagcatgggctagagcta 361 gcttctgaccaccacggtaacttctctatcaccctgcgcaatgtgaccccaagggacagc 421 ggcctctactgctgtctagtgatagaattaaaaaaccaccacccagaacaacggttctac 481 gggtccatggagctacaggtacaggcaggcaaaggctcggggtccacatgcatggcgtct 541 aatgagcaggacagtgacagcatcacggctgcggccctggccaccggcgcctgcatcgtg 601 ggaatcctctgcctcccccttatcctgctgctggtctataagcagagacaggtggcctct 661 caccgccgtgcccaggagttggtgaggatggacagcagcaacacccaaggaatcgaaaac 721 ccaggcttcgagaccactccacccttccaggggatgcctgaggccaagaccaggccgcca 781 ctgtcctatgtggcccagcggcaaccttcggagtcaggacggtacctgctctctgacccc 841 agcacacctctgtcgcctccaggccctggggacgtctttttcccatccctagatccagtc 901 cctgactcccctaactctgaagccatctaa
[0141] The amino acid sequence of human OX40 ligand, provided by Genbank Accession No. NP_003318.1, is shown below (SEQ ID NO: 3), with the signal peptide shown in underlined font and the mature peptide shown in italicized font.
TABLE-US-00003 (SEQIDNO:3) 1 mcvgarrlgrgpcaallllglglstvtglhcvgdtypsndrcchecrpgngmvsrcsrsq 61 ntvcrpcgpgfyndvvsskpckpctwcnlrsgserkqlctatqdtvcrcragtqpldsyk 121 pgvdcapcppghfspgdnqackpwtnctlagkhtlqpasnssdaicedrdppatqpqetq 181 gpparpitvqpteawprtsqgpstrpvevpggravaailglglvlgllgplaillalyll 241 rrdqrlppdahkppgggsfrtpiqeeqadahstlaki
[0142] The mRNA sequence encoding human OX40 ligand, provided by Genbank Accession No. NM_003327.3, is shown below (SEQ ID NO: 4), with the start and stop codons in bold.
TABLE-US-00004 (SEQIDNO:4) 1 ccgcaaggaaaacccagactctggcgacagcagagacgaggatgtgcgtgggggctcggc 61 ggctgggccgcgggccgtgtgcggctctgctcctcctgggcctggggctgagcaccgtga 121 cggggctccactgtgtcggggacacctaccccagcaacgaccggtgctgccacgagtgca 181 ggccaggcaacgggatggtgagccgctgcagccgctcccagaacacggtgtgccgtccgt 241 gcgggccgggcttctacaacgacgtggtcagctccaagccgtgcaagccctgcacgtggt 301 gtaacctcagaagtgggagtgagcggaagcagctgtgcacggccacacaggacacagtct 361 gccgctgccgggcgggcacccagcccctggacagctacaagcctggagttgactgtgccc 421 cctgccctccagggcacttctccccaggcgacaaccaggcctgcaagccctggaccaact 481 gcaccttggctgggaagcacaccctgcagccggccagcaatagctcggacgcaatctgtg 541 aggacagggaccccccagccacgcagccccaggagacccagggccccccggccaggccca 601 tcactgtccagcccactgaagcctggcccagaacctcacagggaccctccacccggcccg 661 tggaggtccccgggggccgtgcggttgccgccatcctgggcctgggcctggtgctggggc 721 tgctgggccccctggccatcctgctggccctgtacctgctccggagggaccagaggctgc 781 cccccgatgcccacaagccccctgggggaggcagtttccggacccccatccaagaggagc 841 aggccgacgcccactccaccctggccaagatctgacctgggcccaccaaggtggacgctg 901 ggccccgccaggctggagcccggagggtctgctgggcgagcagggcaggtgcaggccgcc 961 tgccccgccacgctcctgggccaactctgcaccgttctaggtgccgatggctgcctccgg 1021 ctctctgcttacgtatgccatgcatacctcctgccccgcgggaccacaataaaaaccttg 1081 gcagacgggagtctccgaccggcaaaaaaaaaaaaaaaaa
[0143] The amino acid sequence of human 4-1BB, provided by Genbank Accession No. NP_001552.2, is shown below (SEQ ID NO: 5), with the signal peptide in underlined font and the mature peptide in italicized font.
TABLE-US-00005 (SEQIDNO:5) 1 mgnscynivatlllvlnfertrslqdpcsncpagtfcdnnrnqicspcppnsfssaggqr 61 tcdicrqckgvfrtrkecsstsnaecdctpgfhclgagcsmceqdckqgqeltkkgckdc 121 cfgtfndqkrgicrpwtncsldgksvlvngtkerdvvcgpspadlspgassvtppapare 181 pghspqiisfflaltstallfllffltlrfsvvkrgrkkllyifkqpfmrpvqttqeedg 241 cscrfpeeeeggcel
The mRNA sequence of human 4-1BB, provided by Genbank Accession No. NM-001561.5, is shown below (SEQ ID NO: 6), with the start and stop codons in bold.
TABLE-US-00006 (SEQIDNO:6) 1 caaggagggatcccacagatgtcacagggctgtcacagagctgtggtgggaatttcccat 61 gagaccccgcccctggctgagtcaccgcactcctgtgtttgacctgaagtcctctcgagc 121 tgcagaagcctgaagaccaaggagtggaaagttctccggcagccctgagatctcaagagt 181 gacatttgtgagaccagctaatttgattaaaattctcttggaatcagctttgctagtatc 241 atacctgtgccagatttcatcatgggaaacagctgttacaacatagtagccactctgttg 301 ctggtcctcaactttgagaggacaagatcattgcaggatccttgtagtaactgcccagct 361 ggtacattctgtgataataacaggaatcagatttgcagtccctgtcctccaaatagtttc 421 tccagcgcaggtggacaaaggacctgtgacatatgcaggcagtgtaaaggtgttttcagg 481 accaggaaggagtgttcctccaccagcaatgcagagtgtgactgcactccagggtttcac 541 tgcctgggggcaggatgcagcatgtgtgaacaggattgtaaacaaggtcaagaactgaca 601 aaaaaaggttgtaaagactgttgctttgggacatttaacgatcagaaacgtggcatctgt 661 cgaccctggacaaactgttctttggatggaaagtctgtgcttgtgaatgggacgaaggag 721 agggacgtggtctgtggaccatctccagccgacctctctccgggagcatcctctgtgacc 781 ccgcctgcccctgcgagagagccaggacactctccgcagatcatctccttctttcttgcg 841 ctgacgtcgactgcgttgctcttcctgctgttcttcctcacgctccgtttctctgttgtt 901 aaacggggcagaaagaaactcctgtatatattcaaacaaccatttatgagaccagtacaa 961 actactcaagaggaagatggctgtagctgccgatttccagaagaagaagaaggaggatgt 1021 gaactgtgaaatggaagtcaatagggctgttgggactttcttgaaaagaagcaaggaaat 1081 atgagtcatccgctatcacagctttcaaaagcaagaacaccatcctacataatacccagg 1141 attcccccaacacacgttcttttctaaatgccaatgagttggcctttaaaaatgcaccac 1201 tttttttttttttttgacagggtctcactctgtcacccaggctggagtgcagtggcacca 1261 ccatggctctctgcagccttgacctctgggagctcaagtgatcctcctgcctcagtctcc 1321 tgagtagctggaactacaaggaagggccaccacacctgactaacttttttgttttttgtt 1381 tggtaaagatggcatttcaccatgttgtacaggctggtctcaaactcctaggttcacttt 1441 ggcctcccaaagtgctgggattacagacatgaactgccaggcccggccaaaataatgcac 1501 cacttttaacagaacagacagatgaggacagagctggtgataaaaaaaaaaaaaaaaaag 1561 cattttctagataccacttaacaggtttgagctagtttttttgaaatccaaagaaaatta 1621 tagtttaaattcaattacatagtccagtggtccaactataattataatcaaaatcaatgc 1681 aggtttgttttttggtgctaatatgacatatgacaataagccacgaggtgcagtaagtac 1741 ccgactaaagtttccgtgggttctgtcatgtaacacgacatgctccaccgtcagggggga 1801 gtatgagcagagtgcctgagtttagggtcaaggacaaaaaacctcaggcctggaggaagt 1861 tttggaaagagttcaagtgtctgtatatcctatggtcttctccatcctcacaccttctgc 1921 ctttgtcctgctcccttttaagccaggttacattctaaaaattcttaacttttaacataa 1981 tattttataccaaagccaataaatgaactgcatatgataggtatgaagtacagtgagaaa 2041 attaacacctgtgagctcattgtcctaccacagcactagagtgggggccgccaaactccc 2101 atggccaaacctggtgcaccatttgcctttgtttgtctgttggtttgcttgagacagtct 2161 tgctctgttgcccaggctggaatggagtggctattcacaggcacaatcatagcacacttt 2221 agccttaaactcctgggctcaagtgatccacccgcctcagtctcccaagtagctgggatt 2281 acaggtgcaaacctggcatgcctgccattgtttggcttatgatctaaggatagcttttta 2341 aattttattcattttatttttttttgagacagtgtctcactctgtctcccaggctggagt 2401 acagtggtacaatcttggatcaccgcctcccagtttcaagtgatctccctgcctcagcct 2461 cctaagtagctgggactacaggtatgtgccaccacgcctggctaatttttatatttttag 2521 tagagacggggtttcaccatgttgtccaggctggtctcaaactcctgacctcaggtgatc 2581 tgcccacctctgcctcccaaagtgctgggattacaggcatgagccaccatgcctggccat 2641 ttcttacacttttgtatgacatgcctattgcaagcttgcgtgcctctgtcccatgttatt 2701 ttactctgggatttaggtggagggagcagcttctatttggaacattggccatcgcatggc 2761 aaatgggtatctgtcacttctgctcctatttagttggttctactataacctttagagcaa 2821 atcctgcagccaagccaggcatcaatagggcagaaaagtatattctgtaaataggggtga 2881 ggagaagatatttctgaacaatagtctactgcagtaccaaattgcttttcaaagtggctg 2941 ttctaatgtactcccgtcagtcatataagtgtcatgtaagtatcccattgatccacatcc 3001 ttgctaccctctggtactatcaggtgcccttaattttgccaagccagtgggtatagaatg 3061 agatctcactgtggtcttagtttgcatttgcttggttactgatgagcaccttgtcaaata 3121 tttatataccatttgtgtttatttttttaaataaaatgcttgctcatgcttttttgccca 3181 tttgcaaaaaaacttggggccgggtgcagtggctcatgcctgtagtcccagctctttggg 3241 aggccaaggtgggcagatcgcttgagcccaggagttcgagaccagccttggcaacatggc 3301 gaaaccctgtctttacaaaaaatacaaaaattagccgggtgtggtggtgtgcacctgaag 3361 tcccagctactcagtaggttcgctttgagcctgggaggcagaggttgcagtgagctggga 3421 ccgcatcactacacttcagcctgggcaacagagaaaaaccttttctcagaaacaaacaaa 3481 cccaaatgtggttgtttgtcctgattcctaaaaggtctttatgtattctagataataatc 3541 tttggtcagttatatgtgttaaaaaatatcttctttgtggccaggcacggtagctcacac 3601 ctgtaatcccagcactttgcggggctgaggtgggtggatcatctgaggtcaagagttcaa 3661 gatcagcctggccaacacagtgaaaccccatctctactaaacatgtacaaaacttagctg 3721 ggtatggtggcgggtgcctgtaaccccagctgctccagaggctgtggcagaagaatcgct 3781 tgaacccaggaggcagaggttgcagcgagccaagattgtgccattgcactccagactggg 3841 tgacaagagtgaaattctgcctatctatctatctatctatctatatctatatatatatat 3901 atatatatcctttgtaatttatttttccctttttaaaattttttataaaattctttttta 3961 tttttatttttagcagaggtgaggtttctgaggtttcattatgttgcccaggctggtctt 4021 gaactcctgagctcaagtgatcctcccacctcagccttccaaagtgctggaattgcagac 4081 atgagccaccgcgcccctcctgtttttctctaattaatggtgtctttctttgtctttctg 4141 gtaataagcaaaaagttcttcatttgatttggttaaatttataactgttttctcatatgg 4201 ttaacattttttcttgcctggctaaagaaatccttttctgcccaatactataaagaggtt 4261 tgcccacattttattccaaaagttttaagttttgtctttcatcttgaagtctaatgtatc 4321 aggaactggcttttgtgcctgttgggaggtagtgatccaattccatgtcttgcatgtagg 4381 taaccactggtccctgcgccatgtattcaatacgtcgtctttctcctgcgggtctgcaat 4441 ctcacctaccatccatcaagtttccatagggccatgggtctgcttctgggctccctgttc 4501 tgttccattgtcaatttgtctatcctgtgccagtatcacactgtgtttattacaatagct 4561 ttgtaacagctctcgatatccggtaggacatctccctccaccttctttttctacttcaga 4621 agtgtcttagctaggtcaggcacggtggctcacgcctgtaatcccagcactttgggaggc 4681 cgacgcggatggatcacctgaggtcaggagttttgagacagcctggccaacatggtgaaa 4741 ccccatctctactaaaaaatacaaaaattagtcaggcatggtggcatgtgcctgtaatcc 4801 cagctatttgggaggctgaggccggagaattgcttgaacccggggggcggaggttgcagt 4861 gagccgagatcgtaccattgcactccagcctgggtgacagagcgaaactctgtctcagga 4921 aaaaaaagaaaagagatgtcttggttattcttggttctttattattcaatataaatttta 4981 gaagctgaatttgaaaagatttggattggaatttcattaaatctacaggtcaatttaggg 5041 agagttgataattttacagaattgagtcatctggtgttccaataagaataagagaacaat 5101 tattggctgtacaattcttgccaaatagtaggcaaagcaaagcttaggaagtatactggt 5161 gccatttcaggaacaaagctaggtgcgaatatttttgtctttctgaatcatgatgctgta 5221 agttctaaagtgatttctcctcttggctttggacacatggtgtttaattacctactgctg 5281 actatccacaaacagaaagagactggtcatgccccacagggttggggtatccaagataat 5341 ggagcgaggctctcatgtgtcctaggttacacaccgaaaatccacagtttattctgtgaa 5401 gaaaggaggctatgtttatgatacagactgtgatatttttatcatagcctattctggtat 5461 catgtgcaaaagctataaatgaaaaacacaggaacttggcatgtgagtcattgctccccc 5521 taaatgacaattaataaggaaggaacattgagacagaataaaatgatccccttctgggtt 5581 taatttagaaagttccataattaggtttaatagaaataaatgtaaatttctatgattaaa 5641 aataaattagcacatttagggatacacaaattataaatcattttctaaatgctaaaaaca 5701 agctcaggtttttttcagaagaaagttttaattttttttctttagtggaagatatcactc 5761 tgacggaaagttttgatgtgaggggcggatgactataaagtgggcatcttcccccacagg 5821 aagatgtttccatctgtgggtgagaggtgcccaccgcagctagggcaggttacatgtgcc 5881 ctgtgtgtggtaggacttggagagtgatctttatcaacgtttttatttaaaagactatct 5941 aataaaacacaaaactatgatgttcacaggaaaaaaagaataagaaaaaaagaaaaaaaa 6001 a
[0144] CTLA4 is a receptor expressed only on T cells, and it reduces the level of T cell activation by interfering with the activity of T cell co-stimulatory receptor, CD28. CTLA4 is a target of cancer immunotherapies (e.g., by antibodies that bind to and block CTLA4). Ipilimumab (manufactured by Bristol-Myers Squibb) is a fully humanized anti-CTLA4 antibody that has been approved by the Food and Drug Administration (FDA) for the treatment of melanoma (in particular, unresectable or metastatic melanoma) and is undergoing clinical trials for use in other cancers. Tremelimumab (manufactured by Pfizer; CAS number 745013-59-6) is a fully humanized IgG2 monoclonal anti-CTLA4 antibody that is undergoing clinical trials for the treatment of melanoma.
[0145] The amino acid sequence of human CTLA4, provided by Genbank Accession No. P16410.3, is shown below (SEQ ID NO: 7).
TABLE-US-00007 (SEQIDNO:7) 1 maclgfqrhkaqlnlatrtwpctllffllfipvfckamhvaqpavvlassrgiasfvcey 61 aspgkatevrvtvlrqadsqvtevcaatymmgneltflddsictgtssgnqvnltiqglr 121 amdtglyickvelmypppyylgigngtqiyvidpepcpdsdfllwilaavssglffysfl 181 ltavslskmlkkrsplttgvyvkmpptepecekqfqpyfipin
Amino acid residues 36-223 of SEQ ID NO: 7 corresponds to the mature sequence of CTLA4. The mRNA sequence of human CTLA4, provided by Genbank Accession No. AF414120.1, is shown below (SEQ ID NO: 8).
TABLE-US-00008 (SEQIDNO:8) 1 cttctgtgtgtgcacatgtgtaatacatatctgggatcaaagctatctatataaagtcct 61 tgattctgtgtgggttcaaacacatttcaaagcttcaggatcctgaaaggttttgctcta 121 cttcctgaagacctgaacaccgctcccataaagccatggcttgccttggatttcagcggc 181 acaaggctcagctgaacctggctaccaggacctggccctgcactctcctgttttttcttc 241 tcttcatccctgtcttctgcaaagcaatgcacgtggcccagcctgctgtggtactggcca 301 gcagccgaggcatcgccagctttgtgtgtgagtatgcatctccaggcaaagccactgagg 361 tccgggtgacagtgcttcggcaggctgacagccaggtgactgaagtctgtgcggcaacct 421 acatgatggggaatgagttgaccttcctagatgattccatctgcacgggcacctccagtg 481 gaaatcaagtgaacctcactatccaaggactgagggccatggacacgggactctacatct 541 gcaaggtggagctcatgtacccaccgccatactacctgggcataggcaacggaacccaga 601 tttatgtaattgatccagaaccgtgcccagattctgacttcctcctctggatccttgcag 661 cagttagttcggggttgtttttttatagctttctcctcacagctgtttctttgagcaaaa 721 tgctaaagaaaagaagccctcttacaacaggggtctatgtgaaaatgcccccaacagagc 781 cagaatgtgaaaagcaatttcagccttattttattcccatcaattgagaaaccattatga 841 agaagagagtccatatttcaatttccaagagctgaggcaattctaacttttttgctatcc 901 agctatttttatttgtttgtgcatttggggggaattcatctctctttaatataaagttgg 961 atgcggaacccaaattacgtgtactacaatttaaagcaaaggagtagaaagacagagctg 1021 ggatgtttctgtcacatcagctccactttcagtgaaagcatcacttgggattaatatggg 1081 gatgcagcattatgatgtgggtcaaggaattaagttagggaatggcacagcccaaagaag 1141 gaaaaggcagggagcgagggagaagactatattgtacacaccttatatttacgtatgaga 1201 cgtttatagccgaaatgatcttttcaagttaaattttatgccttttatttcttaaacaaa 1261 tgtatgattacatcaaggcttcaaaaatactcacatggctatgttttagccagtgatgct 1321 aaaggttgtattgcatatatacatatatatatatatatatatatatatatatatatatat 1381 atatatatattttaatttgatagtattgtgcatagagccacgtatgtttttgtgtatttg 1441 ttaatggtttgaatataaacactatatggcagtgtctttccaccttgggtcccagggaag 1501 ttttgtggaggagctcaggacactaatacaccaggtagaacacaaggtcatttgctaact 1561 agcttggaaactggatgaggtcatagcagtgcttgattgcgtggaattgtgctgagttgg 1621 tgttgacatgtgctttggggcttttacaccagttcctttcaatggtttgcaaggaagcca 1681 cagctggtggtatctgagttgacttgacagaacactgtcttgaagacaatggcttactcc 1741 aggagacccacaggtatgaccttctaggaagctccagttcgatgggcccaattcttacaa 1801 acatgtggttaatgccatggacagaagaaggcagcaggtggcagaatggggtgcatgaag 1861 gtttctgaaaattaacactgcttgtgtttttaactcaatattttccatgaaaatgcaaca 1921 acatgtataatatttttaattaaataaaaatctgtggtggtcgttttaaaaaaaaaaaaa 1981 aaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa
The atg start codon and the stop codon are bolded and underlined.
[0146] Blockade of CTLA4 enables a pre-existing endogenous anti-tumor immune response to destroy tumors. Thus, in the presence of an endogenous anti-tumor immune response in a subject, inhibition of CTLA4 lifts the resistance to the immune response and allows the body's immune cells to destroy the cancer cells. However, in poorly immunogenic tumors, the endogenous immune response is either does not exist or is too weak to kill the cancer cells, and inhibition of CTLA4 alone has minimal efficacy.
[0147] The role of PD1 is to limit T cell activity in peripheral tissues during an immune response to infection and to minimize autoimmunity. PD1 is expressed on activated lymphocytes, including activated T effector cells, B cells, and natural killer (NK) cells. Ligands for PD1 include PD1 ligand 1 (PDL1, also called B7-H1 and CD274), and PDL2 (also called B7-DC and CD273). PD1 inhibits lymphocyte function when bound to its ligands. In subjects with cancer, PD1 is often expressed on a large percentage of tumor-infiltrating lymphocytes. Also, PDL1 is overexpressed in cancers such as melanoma, ovarian, renal, and lung cancer. PDL2 is overexpressed in cells from lymphomas, such as B cell lymphoma (e.g., primary mediastinal B cell lymphoma, follicular cell B cell lymphoma, and Hodgkin's disease). Thus, PD1 and its ligands are main players in immune inhibition in the tumor microenvironment, and are therefore targets for cancer immunotherapy.
[0148] The mRNA sequence encoding human PD1, provided by Genbank Accession No. NM_005018.2, is shown below (SEQ ID NO: 9).
TABLE-US-00009 (SEQIDNO:9) 1 agtttcccttccgctcacctccgcctgagcagtggagaaggcggcactctggtggggctg 61 ctccaggcatgcagatcccacaggcgccctggccagtcgtctgggcggtgctacaactgg 121 gctggcggccaggatggttcttagactccccagacaggccctggaacccccccaccttct 181 ccccagccctgctcgtggtgaccgaaggggacaacgccaccttcacctgcagcttctcca 241 acacatcggagagcttcgtgctaaactggtaccgcatgagccccagcaaccagacggaca 301 agctggccgccttccccgaggaccgcagccagcccggccaggactgccgcttccgtgtca 361 cacaactgcccaacgggcgtgacttccacatgagcgtggtcagggcccggcgcaatgaca 421 gcggcacctacctctgtggggccatctccctggcccccaaggcgcagatcaaagagagcc 481 tgcgggcagagctcagggtgacagagagaagggcagaagtgcccacagcccaccccagcc 541 cctcacccaggccagccggccagttccaaaccctggtggttggtgtcgtgggcggcctgc 601 tgggcagcctggtgctgctagtctgggtcctggccgtcatctgctcccgggccgcacgag 661 ggacaataggagccaggcgcaccggccagcccctgaaggaggacccctcagccgtgcctg 721 tgttctctgtggactatggggagctggatttccagtggcgagagaagaccccggagcccc 781 ccgtgccctgtgtccctgagcagacggagtatgccaccattgtctttcctagcggaatgg 841 gcacctcatcccccgcccgcaggggctcagctgacggccctcggagtgcccagccactga 901 ggcctgaggatggacactgctcttggcccctctgaccggcttccttggccaccagtgttc 961 tgcagaccctccaccatgagcccgggtcagcgcatttcctcaggagaagcaggcagggtg 1021 caggccattgcaggccgtccaggggctgagctgcctgggggcgaccggggctccagcctg 1081 cacctgcaccaggcacagccccaccacaggactcatgtctcaatgcccacagtgagccca 1141 ggcagcaggtgtcaccgtcccctacagggagggccagatgcagtcactgcttcaggtcct 1201 gccagcacagagctgcctgcgtccagctccctgaatctctgctgctgctgctgctgctgc 1261 tgctgctgcctgcggcccggggctgaaggcgccgtggccctgcctgacgccccggagcct 1321 cctgcctgaacttgggggctggttggagatggccttggagcagccaaggtgcccctggca 1381 gtggcatcccgaaacgccctggacgcagggcccaagactgggcacaggagtgggaggtac 1441 atggggctggggactccccaggagttatctgctccctgcaggcctagagaagtttcaggg 1501 aaggtcagaagagctcctggctgtggtgggcagggcaggaaacccctccacctttacaca 1561 tgcccaggcagcacctcaggccctttgtggggcagggaagctgaggcagtaagcgggcag 1621 gcagagctggaggcctttcaggcccagccagcactctggcctcctgccgccgcattccac 1681 cccagcccctcacaccactcgggagagggacatcctacggtcccaaggtcaggagggcag 1741 ggctggggttgactcaggcccctcccagctgtggccacctgggtgttgggagggcagaag 1801 tgcaggcacctagggccccccatgtgcccaccctgggagctctccttggaacccattcct 1861 gaaattatttaaaggggttggccgggctcccaccagggcctgggtgggaaggtacaggcg 1921 ttcccccggggcctagtacccccgccgtggcctatccactcctcacatccacacactgca 1981 cccccactcctggggcagggccaccagcatccaggcggccagcaggcacctgagtggctg 2041 ggacaagggatcccccttccctgtggttctattatattataattataattaaatatgaga 2101 gcatgctaaggaaaa
The atg start codon and the stop codon are bolded and underlined.
The amino acid sequence of human PD1, provided by Genbank Accession No. NP_005009.2, is shown below (SEQ ID NO: 10).
TABLE-US-00010 (SEQIDNO:10) 1 mqipqapwpvvwavlqlgwrpgwfldspdrpwnpptfspallvvtegdnatftcsfsnts 61 esfvlnwyrmspsnqtdklaafpedrsqpgqdcrfrvtqlpngrdfhmsvvrarrndsgt 121 ylcgaislapkaqikeslraelrvterraevptahpspsprpagqfqtlvvgvvggllgs 181 lvllvwvlavicsraargtigarrtgqplkedpsavpvfsvdygeldfqwrektpeppvp 241 cvpeqteyativfpsgmgtssparrgsadgprsaqplrpedghcswpl
Residues 1-20 of SEQ ID NO: 10 correspond to the signal peptide sequence, and residues 21-288 of SEQ ID NO: 10 correspond to the mature peptide sequence.
[0149] The amino acid sequence of human PDL1 is provided by Genbank Accession No. Q9NZQ7.1, incorporated herein by reference, and is shown below (SEQ ID NO: 11).
TABLE-US-00011 (SEQIDNO:11) 1 mrifavfifmtywhllnaftvtvpkdlyvveygsnmtieckfpvekqldlaalivyweme 61 dkniiqfvhgeedlkvqhssyrqrarllkdqlslgnaalqitdvklqdagvyrcmisygg 121 adykritvkvnapynkinqrilvvdpvtseheltcqaegypkaeviwtssdhqvlsgktt 181 ttnskreeklfnvtstlrintttneifyctfrrldpeenhtaelvipelplahppnerth 241 lvilgaillclgvaltfifrlrkgrmmdvkkcgiqdtnskkgsdthleet
The mRNA sequence encoding human PDL1 is provided by Genbank Accession No. AY291313.1, incorporated herein by reference, and is shown below (SEQ ID NO: 12), with the start and stop codons in bold.
TABLE-US-00012 (SEQIDNO:12) 1 atgaggatatttgctgtctttatattcatgacctactggcatttgctgaacgccccatac 61 aacaaaatcaaccaaagaattttggttgtggatccagtcacctctgaacatgaactgaca 121 tgtcaggctgagggctaccccaaggccgaagtcatctggacaagcagtgaccatcaagtc 181 ctgagtggtaagaccaccaccaccaattccaagagagaggagaagcttttcaatgtgacc 241 agcacactgagaatcaacacaacaactaatgagattttctactgcacttttaggagatta 301 gatcctgaggaaaaccatacagctgaattggtcatcccagaactacctctggcacatcct 361 ccaaatgaaaggactcacttggtaattctgggagccatcttattatgccttggtgtagca 421 ctgacattcatcttccgtttaagaaaagggagaatgatggatgtgaaaaaatgtggcatc 481 caagatacaaactcaaagaagcaaagtgatacacatttggaggagacgtaa
[0150] The amino acid sequence of human PDL2 is provided by Genbank Accession No. Q9BQ51.2, incorporated herein by reference, and is shown below (SEQ ID NO: 13).
TABLE-US-00013 (SEQIDNO:13) 1 miflllmlslelqlhqiaalftvtvpkelyiiehgsnvtlecnfdtgshvnlgaitaslq 61 kvendtsphreratlleeqlplgkasfhipqvqvrdegqyqciiiygvawdykyltlkvk 121 asyrkinthilkvpetdeveltcqatgyplaevswpnvsvpantshsrtpeglyqvtsvl 181 rlkpppgrnfscvfwnthvreltlasidlqsqmeprthptwllhifipfciiafifiatv 241 ialrkqlcqklysskdttkrpvtttkrevnsai
The mRNA sequence encoding human PDL2 is provided by Genbank Accession No. AF344424.1, incorporated herein by reference, and is shown below (SEQ ID NO: 14), with the start and stop codons in bold.
TABLE-US-00014 (SEQIDNO:14) 1 gcaaaccttaagctgaatgaacaacttttcttctcttgaatatatcttaacgccaaattt 61 tgagtgcttttttgttacccatcctcatatgtcccagctggaaagaatcctgggttggag 121 ctactgcatgttgattgttttgtttttccttttggctgttcattttggtggctactataa 181 ggaaatctaacacaaacagcaactgttttttgttgtttacttttgcatctttacttgtgg 241 agctgtggcaagtcctcatatcaaatacagaacatgatcttcctcctgctaatgttgagc 301 ctggaattgcagcttcaccagatagcagctttattcacagtgacagtccctaaggaactg 361 tacataatagagcatggcagcaatgtgaccctggaatgcaactttgacactggaagtcat 421 gtgaaccttggagcaataacagccagtttgcaaaaggtggaaaatgatacatccccacac 481 cgtgaaagagccactttgctggaggagcagctgcccctagggaaggcctcgttccacata 541 cctcaagtccaagtgagggacgaaggacagtaccaatgcataatcatctatggggtcgcc 601 tgggactacaagtacctgactctgaaagtcaaagcttcctacaggaaaataaacactcac 661 atcctaaaggttccagaaacagatgaggtagagctcacctgccaggctacaggttatcct 721 ctggcagaagtatcctggccaaacgtcagcgttcctgccaacaccagccactccaggacc 781 cctgaaggcctctaccaggtcaccagtgttctgcgcctaaagccaccccctggcagaaac 841 ttcagctgtgtgttctggaatactcacgtgagggaacttactttggccagcattgacctt 901 caaagtcagatggaacccaggacccatccaacttggctgcttcacattttcatcccctcc 961 tgcatcattgctttcattttcatagccacagtgatagccctaagaaaacaactctgtcaa 1021 aagctgtattcttcaaaagacacaacaaaaagacctgtcaccacaacaaagagggaagtg 1081 aacagtgctatctgaacctgtggtcttgggagccagggtgacctgatatgacatctaaag 1141 aagcttctggactctgaacaagaattcggtggcctgcagagcttgccatttgcacttttc 1201 aaatgcctttggatgacccagca
[0151] MDX-1106 (also called BMS-936558; manufactured by Bristol Myers Squibb) is an anti-PD1 human monoclonal antibody that is undergoing clinical trials for use in melanoma, renal, and lung cancers. See, e.g., Clinical Trials Identifier No. NCT00730639. MK-3475 (manufactured by Merck) is a monoclonal IgG4 antibody against PD1 and is undergoing clinical trials for use in previously-treated patients with Non-Small Cell Lung Cancer (NSCLC). CT-011 (also called pidilizumab, produced by Cure Tech) is a humanized monoclonal antibody against PD1 and is undergoing clinical trials for use in metastatic colorectal cancer, metastatic melanoma, and lymphoma. AMP-224 (developed by GlaxoSmithKline and Amplimmune) is an Fc fusion protein containing a ligand of PD1. AMP-224 blocks the interaction between PD1 and PDL2 or PDL1. AMP-224 is undergoing clinical trials for use in cancer. See, e.g., Clinical Trials Identifier No. NCT01352884. MDX1105 (produced by Bristol-Myers Squibb) is a fully human monoclonal IgG4 anti-PDL1 antibody and clinical trials are undergoing for its use in cancer (e.g., relapsed/refractory renal cell carcinoma, NSCLC, colorectal adenocarcinoma, malignant melanoma, advanced/metastatic epithelial ovarian cancer, gastric cancer, pancreatic cancer, and breast cancer). See, e.g., Clinical Trial Identifier No. NCT00729664.
[0152] In addition to immune checkpoint receptors, B7 family immune-inhibitory ligands are also immune-inhibitory proteins that are candidate targets for cancer immunotherapy. For example, B7-H3 (also called CD276) and B7-H4 (also called B7-S1, B7x, or VCTN1) have been implicated in immune inhibition. In addition, B7-H3 and B7-H4 are overexpressed on cancer cells and on tumor infiltrating cells. MGA271 (produced by Macrogenics) is a humanized IgG1/kappa monoclonal antibody against B7-H3 and is currently undergoing clinical trials for use in refractory B7-H3-expressing neoplasms (e.g., prostate cancer and melanoma). See, e.g., Clinical Trial Identifier No. NCT01391143.
[0153] The amino acid sequence of human B7-H3 is provided by Genbank Accession No. Q5ZPR3.1, incorporated herein by reference. The mRNA sequence encoding human B7-H3 is provided by Genbank Accession No. AJ583695.1, incorporated herein by reference.
[0154] The amino acid sequence of human B7-H4 is provided by Genbank Accession No. Q7Z7D3.1, incorporated herein by reference. The mRNA sequence encoding human B7-H4 is provided by Genbank Accession No. DQ103757.1, incorporated herein by reference.
[0155] A number of other proteins have been shown to be associated with inhibition of immune cell activity and are thus also potential targets for cancer immunotherapy. These proteins include lymphocyte activation gene 3 (LAG3, or CD223), 2B4 (CD244), B and T lymphocyte attenuator (BTLA, CD272), T membrane protein 3 (TIM3, HAVcr2), adenosine A2a receptor (A2aR), and killer inhibitory receptors. Killer inhibitor receptors include killer cell immunoglobulin-like receptors (KIRs) and C-type leptin receptors, both of which regulate the killing activity of NK cells. IMP321 (produced by Immutep) is a soluble LAG3-Ig fusion protein that targets LAG3 and is currently being studied for use in advanced renal cell adenocarcinoma and advanced pancreatic adenocarcinoma. See, e.g., Brignone et al. Clin. Cancer Res. (2009) 15:6225-6231 and Wang-Gillam et al. Invest. New Drugs (2013) 31:707-13.
[0156] The amino acid sequence of human BTLA is provided by Genbank Accession No. NP_861445.3, incorporated herein by reference, and is shown below (SEQ ID NO: 15), with the signal peptide shown in underlined font and the mature peptide shown in italicized font.
TABLE-US-00015 (SEQIDNO:15) 1 mktlpamlgtgklfwvfflipyldiwnihgkescdvqlyikrqsehsilagdpfelecpv 61 kycanrphvtwcklngttcvkledrqtswkeeknisffilhfepvlpndngsyrcsanfq 121 snlieshsttlyvtdvksaserpskdemasrpwllysllplgglpllittcfclfcclrr 181 hqgkqnelsdtagreinlvdahlkseqteastrqnsqvllsetgiydndpdlcfrmgegs 241 evysnpcleenkpgivyaslnhsvigpnsrlarnvkeapteyasicvrs
The mRNA sequence encoding human BTLA is provided by Genbank Accession No. NM_181780.3, incorporated herein by reference, and is shown below (SEQ ID NO: 16), with the start and stop codons shown in bold.
TABLE-US-00016 (SEQIDNO:16) 1 gtctttctgttcactttttttcacaaaatcatccaggctcttcctactctcctctcttac 61 cacctctctcttcttttttttttttttttagttatttcacagatgccactggggtaggta 121 aactgacccaactctgcagcactcagaagacgaagcaaagccttctacttgagcagtttt 181 tccatcactgatatgtgcaggaaatgaagacattgcctgccatgcttggaactgggaaat 241 tattttgggtcttcttcttaatcccatatctggacatctggaacatccatgggaaagaat 301 catgtgatgtacagctttatataaagagacaatctgaacactccatcttagcaggagatc 361 cctttgaactagaatgccctgtgaaatactgtgctaacaggcctcatgtgacttggtgca 421 agctcaatggaacaacatgtgtaaaacttgaagatagacaaacaagttggaaggaagaga 481 agaacatttcatttttcattctacattttgaaccagtgcttcctaatgacaatgggtcat 541 accgctgttctgcaaattttcagtctaatctcattgaaagccactcaacaactctttatg 601 tgacagatgtaaaaagtgcctcagaacgaccctccaaggacgaaatggcaagcagaccct 661 ggctcctgtatagtttacttcctttggggggattgcctctactcatcactacctgtttct 721 gcctgttctgctgcctgagaaggcaccaaggaaagcaaaatgaactctctgacacagcag 781 gaagggaaattaacctggttgatgctcaccttaagagtgagcaaacagaagcaagcacca 841 ggcaaaattcccaagtactgctatcagaaactggaatttatgataatgaccctgaccttt 901 gtttcaggatgcaggaagggtctgaagtttattctaatccatgcctggaagaaaacaaac 961 caggcattgtttatgcttccctgaaccattctgtcattggaccgaactcaagactggcaa 1021 gaaatgtaaaagaagcaccaacagaatatgcatccatatgtgtgaggagttaagtctgtt 1081 tctgactccaacagggaccattgaatgatcagcatgttgacatcattgtctgggctcaac 1141 aggatgtcaaataatatttctcaatttgagaatttttactttagaaatgttcatgttagt 1201 gcttgggtcttaagggtccataggataaatgattaaaatttctctcagaaacttatttgg 1261 gagctttttatattatagccttgaataacaaaatctctccaaaactggttgacatcatga 1321 gtagcagaatagtagaacgtttaaacttagctacattttacccaatatacaaactcgatc 1381 ttgcctttgaagctattggaaagacttgtagggaaaagaggtttgtgttacctgcatcag 1441 ttcactacacactcttgaaaacaaaatgtcccaatttgactaaccaaccataaatacagt 1501 aatgattgtatatttcaagtcagtcttccaaaataagaaatttttgctgtgtcagtctaa 1561 gaatggtgtttcttaaatgcaaaggagaaatcattttaggcttgatgtaagaaaatgaaa 1621 ataataaatggtgcaataaaaatatagaatataccaattggatatagggtagatgttcca 1681 catacctggcaaacaaatgcttatatctactctgttagattgataagcaaatataggtat 1741 taatggagcagtcaacgtatagcacatttatgaggaaagtagagactcactgggtcacat 1801 agactaatggataggaatgtgacataatgctgctgaattaatatacttatgggcatctga 1861 atagtttaaaagttagtcagaataggtatcactgggcaagtgaagatagcttaaactgct 1921 tcatgcttgacttgatagcaagttaaagtgcaattaatggaatggaggaaaacccagaat 1981 atttaattggtctgtaggggtcaatttgctttcattcaccacatctgcatcttgctgttc 2041 ttcttactaaggaatcagggcaaatcatctgtagtgacatattttagtttgctaatcatt 2101 tattttaaaatactgaggttgcagccacttaagagtatagcaaaagatggattcagattt 2161 ttggactttccaaagtacttgagttaaactatttcaaaaatagcctataattttattcaa 2221 cagtttgaggctattcgaattctcaggtgctgctactgaataatgtaatagtcttcatac 2281 aaagtggatagcaaaggttaaaatccatttcaacaaatatgtgagctgagctgctgcaca 2341 aaggaatgtgatgtgtgtgtgtgtgtgtgtgtgtgtgtgtgttaggtggggtgggtgaca 2401 acagaaatggtgcacgagaaactgatcaaattgacattatattttcagtttgcttatgaa 2461 gctcaaaatactagagtaaatgggtcattaaagaaaataatatgtgaaattatggagttt 2521 agaatacaagtggggtatatatacaaaaagacaaaactgaggttttgtggtggagagatt 2581 ttcttaagtaacactggcattaagttttagctccttagatttgggggtgcaaatattctt 2641 ttgagtcactgttattttgccaattacacctagaatttcaagcaaccaattcgagatagg 2701 ctgttttagccaggctgcatttgtggacaacttatgtaagaaagacatgttagaatagct 2761 gcttgtggtattcttaaaaatagaaacaggaaatatggggaggatacatttagctgtcct 2821 cttatcagatgaacacacgaaattgaacagttccttcatgattctctcaaacttaaaagc 2881 aaaatatttctgtcttatttaaaatatccttagtatgtcttatagtaaagataatgctga 2941 taatgatttcatctctaagatgtattaatatatttgtactgtttgccaaaatcacaaatc 3001 atttatgtttttattccttttcaaaatggtgtcagagacatacatgcattttcccaaatg 3061 actctacttcactattatttacatggcttatttcattagtttatagagggtttgagaaaa 3121 agaatatgtagataatttaatggtttttcacaaattttaagcttgtgattgtgctcaatg 3181 agaaggtaaagttattaaaacttatttgaaatcaaa
[0157] The amino acid sequence of human TIM3 is provided by Genbank Accession No. Q8TDQ0.3, incorporated herein by reference, and is shown below (SEQ ID NO: 17).
TABLE-US-00017 (SEQIDNO:17) 1 mfshlpfdcvlllllllltrsseveyraevgcmaylpcfytpaapgnlvpvcwgkgacpv 61 fecgnvv1rtderdvnywtsrywlngdfrkgdvsltienvtladsgiyccrigipgimnd 121 ekfnlklvikpakvtpaptrcirdftaafprmlttrghgpaetqtlgslpdinitqistla 181 nelrdsrlandlrdsgatirigiyigagicaglalalifgalifkwyshskekicinlsli 241 slanlppsglanavaegirseeniytieenvyeveepneyycyvssrqqpsqplgcrfam 301 p
The mRNA sequence encoding human TIM3 is provided by Genbank Accession No. AF450242.1, incorporated herein by reference, and is shown below (SEQ ID NO: 18).
TABLE-US-00018 (SEQIDNO:18) 1 ggagagttaaaactgtgcctaacagaggtgtcctctgacttttcttctgcaagctccatg 61 ttttcacatcttccctttgactgtgtcctgctgctgctgctgctactacttacaaggtcc 121 tcagaagtggaatacagagcggaggtcggtcagaatgcctatctgccctgcttctacacc 181 ccagccgccccagggaacctcgtgcccgtctgctggggcaaaggagcctgtcctgtgttt 241 gaatgtggcaacgtggtgctcaggactgatgaaagggatgtgaattattggacatccaga 301 tactggctaaatggggatttccgcaaaggagatgtgtccctgaccatagagaatgtgact 361 ctagcagacagtgggatctactgctgccggatccaaatcccaggcataatgaatgatgaa 421 aaatttaacctgaagttggtcatcaaaccagccaaggtcacccctgcaccgactctgcag 481 agagacttcactgcagcctttccaaggatgcttaccaccaggggacatggcccagcagag 541 acacagacactggggagcctccctgatataaatctaacacaaatatccacattggccaat 601 gagttacgggactctagattggccaatgacttacgggactctggagcaaccatcagaata 661 ggcatctacatcggagcagggatctgtgctgggctggctctggctcttatcttcggcgct 721 ttaattttcaaatggtattctcatagcaaagagaagatacagaatttaagcctcatctct 781 ttggccaacctccctccctcaggattggcaaatgcagtagcagagggaattcgctcagaa 841 gaaaacatctataccattgaagagaacgtatatgaagtggaggagcccaatgagtattat 901 tgctatgtcagcagcaggcagcaaccctcacaacctttgggttgtcgctttgcaatgcca 961 tagatccaaccaccttatttttgagcttggtgttttgtctttttcagaaactatgagctg 1021 tgtcacctgactggttttggaggttctgtccactgctatggagcagagttttcccatttt 1081 cagaagataatgactcacatgggaattgaactggga
Combination of Inhibitors with Vaccines
[0158] In the presence of an endogenous anti-tumor immune response in a subject, inhibition of an immune-inhibitory (e.g., immune checkpoint) protein described above lifts the resistance of cancer cells to the immune response and allows the body's immune cells to destroy the cancer. However, in poorly immunogenic tumors, the endogenous immune response either does not exist or is too weak to kill the cancer cells, and inhibition of immune-inhibitory proteins has minimal efficacy.
[0159] To address this problem, the invention provides a combination of a cancer vaccine device with an inhibitor of an immune-inhibitory protein. As described in detail in the working examples, this combination surprisingly led to a greater decrease in tumor size and a longer survival time compared to administration of inhibitor alone. Described herein is a material-based (e.g., PLG) vaccine which has been optimized, e.g., to control the presentation of GM-CSF and adjuvants, relative to other vaccine formulations in order to enhance T effector activity and downregulate Treg cells and other immunosuppressive mechanisms that may be induced by some adjuvants. The material-based vaccine represents a significant advantage over previous vaccine systems in that it creates a tumor and vaccine microenvironment that responds to an immune-inhibitory protein, e.g., anti-CTLA-4, by preferentially enhancing effector T cell generation and expansion over Treg cells.
[0160] The invention features a cancer vaccine device that comprises one or more (e.g., 1, 2, 3, 4, 5, 6, or more) inhibitors to an immune-inhibitory protein. For example, the inhibitor(s) is incorporated into or onto the cancer vaccine device, e.g., incorporated into or onto a scaffold composition within the device. Administration of a cancer vaccine device containing the inhibitor(s) allows for localized delivery of the inhibitor(s), e.g., at the same site as vaccine.
[0161] The inhibitor can be encapsulated in the vaccine device during fabrication of the device. Alternatively, the inhibitor is added to the vaccine device after it is fabricated. For example, the inhibitor is encapsulated in the PLG microspheres utilized to fabricate the vaccine, combined with the CpG and sucrose added to the PLG prior to foaming, or added to the vaccine device after fabrication, e.g., by adsorbing to the surface of the device, or by placing the inhibitor in a sustained release formulation that is subsequently combined with the vaccine device.
[0162] For example, the cancer vaccine device comprises an anti-CTLA4 antibody and/or an anti-PD1 antibody.
[0163] The invention also provides a method of killing a cancer cell, slowing cancer progression, reducing a tumor size, prolonging the survival time of a cancer patient, and/or treating cancer by administering a cancer vaccine in combination with an inhibitor of an immune-inhibitory protein.
[0164] For example, the vaccine and the inhibitor are formulated separately, i.e., the inhibitor is not included within the vaccine device. In some embodiments, the vaccine and one or more (e.g., 1, 2, 3, 4, 5, 6, or more) inhibitor(s) are administered simultaneously. In other cases, the vaccine and the inhibitor(s) are administered sequentially. For example, the inhibitor(s) is administered at least 6 hours (e.g., 6 h, 12 h, 24 h, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 1.5 weeks, 2 weeks, 3 weeks, 4 weeks, or more) prior to administration of the vaccine. In other cases, the vaccine is administered at least 6 hours (e.g., 6 h, 12 h, 24 h, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 1.5 weeks, 2 weeks, 3 weeks, 4 weeks, or more) prior to administration of the inhibitor(s). In other embodiments, the vaccine and the inhibitor are formulated together, e.g., the inhibitor is included within or coated onto the vaccine device.
[0165] For example, an anti-CTLA4 antibody and/or anti-PD1 antibody are administered in combination with the vaccine device (e.g., administered simultaneously or sequentially).
[0166] The combination of inhibitor(s) and vaccine in provides certain advantages. For example, the combination synergistically induces the activity of T effector cells that infiltrate tumors. Also, the combination enhances local T effector cell activity (e.g., T cells in close proximity to the implanted vaccine device, and/or T cells in the vaccine draining lymph nodes). Also, the combination of inhibitor(s) and vaccine in the same device provides advantages over non-device vaccines used in combination with the inhibitor(s). In particular, inclusion of the inhibitor(s) in the vaccine device allows for targeting of local and/or specific immune cells (such as those specifically recruited to the device). Unlike systemic administration of the inhibitor(s), this local administration of the inhibitor(s) in some cases leads to lower toxicity and a lower dosage needed for efficacy.
[0167] In some cases, the inhibitor is administered prior to the vaccine device. For example, after administration of the inhibitor (e.g., antibody), e.g., within a week, immune cells infiltrate into the tumor site. The infiltration can cause a transient increase in tumor size. After administration (e.g., implantation) of the vaccine device, regression in tumor size occurs. For example, regression in tumor size occurs at least 1 weeks (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60 weeks or more) after administration of the vaccine device. In some cases, the combination of the inhibitor and the vaccine device causes a reduction in tumor size (e.g., a reduction of at least 10%, e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more) compared to the tumor size prior to administration of the inhibitor and/or vaccine device. In some examples, the combination of the inhibitor and the vaccine device causes total eradication of the tumor.
[0168] Tumor size is determined by standard methods in the art. For example, tumor size is the weight of the tumor or the area of the tumor. Tumor size (area in mm.sup.2) is, e.g., the product of the two longest diameters of the tumor. Tumor diameters can be measured using standard methods (e.g., with calipers). In other examples, the weight of a tumor is a measure of its size.
Cancer Vaccine Device
[0169] In the cancer vaccine, presentation of toll like receptor (TLR) agonists for cancer vaccination leads to improved activation of immune cells. The vaccines and methods comprise incorporation and presentation of TLR agonists embedded in structural polymeric devices. CD8(+) Dendritic cells (DCs) and plasmacytoid DCs (as well as conventional DCs) play important roles in cancer vaccination; these cells are preferentially recruited and activated using the TLR-agonist containing structural polymeric device. The device is manufactured as a tiny bioengineered porous disc filled with tumor-specific antigens and TLR agonists. The disc is implanted into the body, e.g., inserted under the skin, where it activates the immune system to destroy cancer cells. This approach reprograms cells that are already in the body.
[0170] In some examples, the device includes a recruitment component. Thus, the device optionally includes a recruitment molecule such as a cytokine. In those situations, polymers were designed to first release a cytokine to recruit and house host dendritic cells (DCs), and subsequently present cancer antigens and danger signals to activate the resident DCs and dramatically enhance their homing to lymph nodes. Specific and protective anti-tumor immunity was generated with these materials. For example, a 90% survival rate was achieved in animals that otherwise die from cancer within 25 days. These materials are useful in cancer and other vaccines to program and control the trafficking of a variety of cell types in the body.
[0171] A polymer system was designed to not only serve as a drug delivery device, but also as a physical, antigen-presenting structure to which the DCs are recruited, and where DCs reside while they are activated using a material (poly[lactide-co-glycolide]) (PLG) and bioactive molecules (e.g., GM-CSF and CpG-ODN). These bioactive molecules have excellent safety profiles. The material system serves as an effective cancer vaccine, eliminating the time, expense and regulatory burden inherent to existing cell therapies and reducing or eliminating the need for multiple, systemic injections and high total drug loading. The devices described herein utilize infection-mimicking materials to program DCs in situ.
[0172] The invention includes macroporous polymer matrices that regulate the trafficking and activation of DCs in vivo by precisely controlling the presentation of GM-CSF and CpG-oligonucleotide (CpG-ODN) adjuvants (Ali et al., 2009 Nat Mater, 2: 151-8; Ali et al., 2009 Sci Transl Med, 1:8-19). When applied as cancer vaccines, these matrices have led induced CTL-mediated eradication of melanoma tumors (Ali et al., 2009 Sci Transl Med, 1:8-19).
[0173] A macroporous poly-lactide-co-glycolide (PLG) matrix presents GM-CSF, danger signals, and cancer antigens in a defined spatiotemporal manner in vivo, and serve as a residence for recruited DCs as they are programmed. GM-CSF is encapsulated into PLG scaffolds using a high pressure CO.sub.2 foaming process, as described in US 2013-0202707. The GM-CSF release profile from the matrix allows diffusion of the factor through the surrounding tissue to effectively recruit resident DCs.
[0174] In situ dendritic cell targeting systems are utilized to therapeutically manipulate the immune system with TLR agonists. As described in detail in US 2013-0202707 (incorporated herein by reference), macroporous polymeric scaffolds are designed that deliver three different classes of TLR agonists in vivo: CpG-ODN, monophosphoryl lipid A (MPLA), and polyinosinic:polycytidylic acid (P(I:C)) in combination with GM-CSF, Flt3L, or CCL20 to augment DC recruitment and activation. Various subsets of DCs are recruited and utilized for in situ vaccination. The ability of these systems to effect immune protection and tumor regression required CD8(+) DCs and correlates strongly with plasmacytoid DCs(pDCs) and IL-12 production, regardless of the TLR agonist type or dose.
Inflammatory Mediators
[0175] Dendritic Cell (DC) proliferation, migration and maturation are sensitive to inflammatory mediators, and granulocyte macrophage colony stimulating factor (GM-CSF) has been identified as a potent stimulator of immune responses, specifically against cancer antigens. GM-CSF also has the ability to recruit and program these antigen-presenting immune cells. Additionally, Cytosine-guanosine (CpG) oligonucleotide (CpG-ODN) sequences found in bacterial DNA are potent immunomodulators that stimulate DC activation, leading to specific T-cell responses. Creating an infection mimicking microenvironment by the presentation of exogenous GM-CSF and CpG-ODN provides an avenue to precisely control the number and timing of DC migration and modulate antigen specific immune responses.
[0176] The vertebrate immune system employs various mechanisms for pathogen recognition, making it adept at generating antigen-specific responses and clearing infection. Immunity is controlled by antigen presenting cells (APCs), especially dendritic cells (DCs), which capture antigens and are activated by stimuli, unique danger signals of the invading pathogen, such as CpG dinucleotide sequences in bacterial DNA (Banchereau J, and Steinman R M. Nature. 392, 245-252. (1998); Klinman D M. Nat. Rev. Immunol. 4, 249-58 (2004); each incorporated herein by reference).
[0177] However, cancerous cells, derived from self-tissues, are void of the danger signals required to signal DC maturation and instead promote an immunosuppressive microenvironment that allows cells to escape immunity. Key elements of infection are inflammatory cytokines and danger signals. A polymeric material system is ideal to present these factors in the required spatiotemporal manner to provide an infection-mimicking microenvironment in situ that useful as a vaccine. These infection mimics provide the continuous programming of host DCs, providing for efficient DC activation and dispersement in situ. These infection-mimicking devices are used for numerous vaccine applications, including melanoma cancer vaccines.
[0178] In many infections, inflammatory cytokines and danger signals stimulate specific DC responses that mediate immune recognition and pathogen clearance. For example, upon bacterial invasion and release of toxins, skin cells such as fibroblasts, keratinocytes and melanocytes are damaged, resulting in the release of inflammatory cytokines, such as GM-CSF (Hamilton J. Trends in Immunol. 23, 403-408. (2002); Hamilton J., and Anderson G. Growth Factors. 22(4), 225-231. (2004); each herein incorporated by reference), that act to recruit Langerhans DC (skin) and DC precursors (monocytes; blood) (Hamilton J. Trends in Immunol. 23, 403-408. (2002); Hamilton J., and Anderson G. Growth Factors. 22(4), 225-231. (2004); Bowne W. B., et al. Cytokines Cell Mol Ther. 5(4), 217-25. (1999); Dranoff, G. Nat. Rev. Cancer 4, 11-22 (2004); each herein incorporated by reference). As DCs arrive to the site of infection, they begin to differentiate and increase in phagocytic ability in response to the inflammation (Mellman I., and Steinman R. M. Cell. 106, 255-258. (2001), incorporated herein by reference). DCs that ingest bacteria or their products begin to process antigens, and DC maturation proceeds via endosomal TLR9 signaling stimulated by CpG dinucleotide sequences in bacterial DNA (Krieg A. M., Hartmann G., and Weiner G. J. CpG DNA: A potent signal for growth, activation, and maturation of human dendritic cells. Proc Natl Acad Sci USA. 16, 9305-9310 (1999), incorporated herein by reference). Mature DCs then home to the lymph nodes where they prime antigen specific T-cell responses that clear infection.
[0179] CpG-ODNs are potent danger signals that upregulate DC expression of CCR7, CD80/86 costimulatory molecules, and MHC-antigen complexes. Importantly, TLR9 signaling induces DCs into promoting Th1-like, cytotoxic T cell responses by cytokine production (e.g., type 1 IFN) and cross-presentation of antigen onto MHCI molecules. The presentation of these signals concurrently with tumor antigens provides the danger signal needed to promote immune responses that effectively fight cancerous cells.
[0180] Different classes of CPG-ODNs promote different immune responses depending on the ODN's specific structure and sequence. The ODN utilized in the present invention, CpG-ODN 1826, has been successfully tested in various mouse vaccination models, including melanoma. CpG-ODN 1826 has shown a beneficial effect alone or when used as adjuvant for peptide vaccines and whole cell vaccines. Moreover, ODN 1826 has been shown to directly promote DC maturation and cytokine production. This particular CpG ODN sequence also indirectly activates Th1 cells and NK cells and, thus, enhances adaptive cellular immune responses.
[0181] Vector systems that promote CpG internalization into DCs to enhance delivery and its localization to TLR9 have been developed. The amine-rich polycation, polyethylimine (PEI) has been extensively used to condense plasmid DNA, via association with DNA phosphate groups, resulting in small, positively charge condensates facilitating cell membrane association and DNA uptake into cells (Godbey W. T., Wu K. K., and Mikos, A. G. J. of Biomed Mater Res, 1999, 45, 268-275; Godbey W. T., Wu K. K., and Mikos, A. G. Proc Natl Acad Sci USA. 96(9), 5177-81. (1999); each herein incorporated by reference). Consequently, PEI has been utilized as a non-viral vector to enhance gene transfection and to fabricate PEI-DNA loaded PLG matrices that promoted long-term gene expression in host cells in situ (Huang Y C, Riddle F, Rice K G, and Mooney D J. Hum Gene Ther. 5, 609-17. (2005), incorporated herein by reference). Therefore, CpG-ODNs were condensed with PEI molecules in the present invention. The PEI condensation enhances DC internalization of CpG-ODN, and the subsequent decondensation of PEI-CpG-ODN within DCs promotes DC activation (US 2013-0202707, incorporated herein by reference, e.g., at page 86, lines 1-7; and
[0182] To appropriately mimic infection and program cells in situ, the PLG system of the invention was designed to not only serve as a drug delivery device that releases inflammatory cytokines (e.g., GM-CSF), but also as a physical structure to which the DCs are recruited and reside while they are activated by danger signals (e.g., CpG-ODNs). The ability to control DC recruitment to and DC residence within porous PLG matrices is achieved using temporal control over the delivery of GM-CSF in situ, which results in batches of programmed DCs being dispersed only when GM-CSF levels were designed to subside in situ. For example, this system disperses at least 5% (e.g., about 6%) of programmed DCs to the lymph nodes and induces protective anti-tumor immunity in at least 20% (e.g., about 23%) of mice when applied as a cancer vaccine. The cell programming and dispersement efficiency is improved using an overriding secondary signal (CpG-ODN) that continuously releases DCs from GM-CSF inhibition and promotes DC maturation and dispersement in the presence of high GM-CSF levels in situ. For example, PLG matrices were fabricated to locally present synthetic CpG-ODN with exogenous GM-CSF allowing for DCs recruited by GM-CSF to be stimulated by CpG-ODN in situ.
Dendritic Cells
[0183] Dendritic cells (DCs) are immune cells within the mammalian immune system and are derived from hematopoietic bone marrow progenitor cells. More specifically, dendritic cells can be categorized into lymphoid (or plasmacytoid) dendritic cell (pDC) and myeloid dendritic cell (mDC) subdivisions having arisen from a lymphoid (or plasmacytoid) or myeloid precursor cell, respectively. From the progenitor cell, regardless of the progenitor cell type, an immature dendritic cell is born. Immature dendritic cells are characterized by high endocytic activity and low T-cell activation potential. Thus, immature dendritic cells constitutively sample their immediate surrounding environment for pathogens. Exemplary pathogens include, but are not limited to, a virus or a bacteria. Sampling is accomplished by pattern recognition receptors (PRRs) such as the toll-like receptors (TLRs). Dendritic cells activate and mature once a pathogen is recognized by a pattern recognition receptor, such as a toll-like receptor.
[0184] Mature dendritic cells not only phagocytose pathogens and break them down, but also, degrade their proteins, and present pieces of these proteins, also referred to as antigens, on their cell surfaces using MHC (Major Histocompatibility Complex) molecules (Classes I, II, and III). Mature dendritic cells also upregulate cell-surface receptors that serve as co-receptors for T-cell activation. Exemplary co-receptors include, but are not limited to, CD80, CD86, and CD40. Simultaneously, mature dendritic cells upregulate chemotactic receptors, such as CCR7, that allows the cell to migrate through the blood stream or the lymphatic system to the spleen or lymph node, respectively.
[0185] Dendritic cells are present in external tissues that are in contact with the external environment such as the skin (dendritic cells residing in skin are also referred to as Langerhans cells). Alternatively, dendritic cells are present in internal tissues that are in contact with the external environment such as linings of the nose, lungs, stomach, and intestines. Finally, immature dendritic cells reside in the blood stream. Once activated, dendritic cells from all off these tissues migrate to lymphoid tissues where they present antigens and interact with T cells and B cells to initiate an immune response. One signaling system of particular importance for the present invention involves the chemokine receptor CCR7 expressed on the surface of dendritic cells and the chemokine receptor ligand CCL19 secreted by lymph node structures to attract migrating mature dendritic cells toward high concentrations of immune cells. Exemplary immune cells activated by contact with mature dendritic cells include, but are not limited to, helper T cells, killer T cells, and B cells. Although multiple cell types within the immune system present antigens, including macrophages and B lymphocytes, dendritic cells are the most potent activators of all antigen-presenting cells.
[0186] Dendritic cells earned their name from the characteristic cell shape comprising multiple dendrites extending from the cell body. The functional benefit of this cell shape is a significantly increased cell surface and contact area to the surroundings compared to the cell volume. Immature dendritic cells sometimes lack the characteristic dendrite formations and are referred to as veiled cells. Veiled cells possess large cytoplasmic veils rather than dendrites.
[0187] Plasmacytoid dendritic cells (pDCs) are innate immune cells that circulate in the blood and are found in peripheral lymphoid organs. They constitute <0.4% of peripheral blood mononuclear cells (PBMC). In humans, these cells express the surface markers CD123, BDCA-2(CD303) and BDCA-4(CD304), but do not express high levels of CD11c or CD14, which distinguishes them from conventional dendritic cells or monocytes, respectively. Mouse pDC express CD11c, B220, BST-2 (mPDCA) and Siglec-H and are negative for CD11b. As components of the innate immune system, these cells express intracellular Toll-like receptors 7 and 9 which detect ssRNA and CpG DNA motifs, respectively. Upon stimulation and subsequent activation, these cells produce large amounts of type I interferon (mainly IFN- (alpha) and IFN-(3 (beta)), which are critical pleiotropic anti-viral compounds mediating a wide range of effects. The CD8 subset presents antigen using the class II pathway to CD4+ helper T cells. The CD8+ subset presents antigens using the class I pathway. The peptide/MHC class I molecules are presented to CD8+ T cells which go on to become cytotoxic T lymphocytes (CTL). The CD8 cell surface protein in the mouse corresponds to the CD141 cell surface protein in the human. CD8/CD141-positive cells express TLR3 and are preferentially activated by TLR3 agonists.
Toll-Like Receptors (TLRs)
[0188] TLRs are a class of single transmembrane domain, non-catalytic, receptors that recognize structurally conserved molecules referred to as pathogen-associated molecular patterns (PAMPs). PAMPs are present on microbes and are distinguishable from host molecules. TLRs are present in all vertebrates. Thirteen TLRs (referred to as TLRs1-13, consecutively) have been identified in humans and mice. Humans comprise TLRs 1-10.
[0189] TLRs and interleukin-1 (IL-1) receptors comprise a receptor superfamily the members of which all share a TIR domain (Toll-IL-1 receptor). TIR domains exist in three varieties with three distinct functions. TIR domains of subgroup 1 are present in receptors for interleukins produced by macrophages, monocytes, and dendritic cells. TIR domains of subgroup 2 are present in classical TLRs which bind directly or indirectly to molecules of microbial origin. TIR domains of subgroup 3 are present in cytosolic adaptor proteins that mediate signaling between proteins comprising TIR domains of subgroups 1 and 2.
[0190] TLR ligands comprise molecules that are constantly associated with and highly specific for a threat to the host's survival such as a pathogen or cellular stress. TLR ligands are highly specific for pathogens and not the host. Exemplary pathogenic molecules include, but are not limited to, lipopolysaccharides (LPS), lipoproteins, lipoarabinomannan, flagellin, double-stranded RNA, and unmethylated CpG islands of DNA.
[0191] In one preferred embodiment of the present invention, the Toll-Like receptor 9 (TLR9) is activated by specific unmethylated CpG-containing sequences in bacterial DNA or synthetic oligonucleotides (ODNs) found in the endosomal compartment of dendritic cells. Methylation status of the CpG site is a crucial distinction between bacterial and mammalian DNA, as well as between normal and cancerous tissue. Unmethylated ODNs including one or more CpG motifs mimic the effects of bacterial DNA. Alternatively, or in addition, unmethylated ODNs including one or more CpG motifs occur within oncogenes present within malignant tumor cells.
[0192] One or more sequences of the TLR-9 receptor recognizes one or more CpG-ODN sequences of the present invention. TLR-9 receptors encompassed by the present invention are described by the following sequences.
[0193] Human TLR-9, isoform A, is encoded by the following mRNA sequence (NCBI Accession No. NM_017442 and SEQ ID NO: 19; the start codon for all mRNA sequences presented herein is bolded and capitalized):
TABLE-US-00019 (SEQIDNO:19) 1 ggaggtcttgtttccggaagatgttgcaaggctgtggtgaaggcaggtgcagcctagcct 61 cctgctcaagctacaccctggccctccacgcatgaggccctgcagaactctggagatggt 121 gcctacaagggcagaaaaggacaagtcggcagccgctgtcctgagggcaccagctgtggt 181 gcaggagccaagacctgagggtggaagtgtcctcttagaatggggagtgcccagcaaggt 241 gtacccgctactggtgctatccagaattcccatctctccctgctctctgcctgagctctg 301 ggccttagctcctccctgggcttggtagaggacaggtgtgaggccctcatgggatgtagg 361 ctgtctgagaggggagtggaaagaggaaggggtgaaggagctgtctgccatttgactatg 421 caaatggcctttgactcatgggaccctgtcctcctcactgggggcagggtggagtggagg 481 gggagctactaggctggtataaaaatcttacttcctctattctctgagccgctgctgccc 541 ctgtgggaagggacctcgagtgtgaagcatccttccctgtagctgctgtccagtctgccc 601 gccagaccctctggagaagcccctgccccccagcATGggtttctgccgcagcgccctgca 661 cccgctgtctctcctggtgcaggccatcatgctggccatgaccctggccctgggtacctt 721 gcctgccttcctaccctgtgagctccagccccacggcctggtgaactgcaactggctgtt 781 cctgaagtctgtgccccacttctccatggcagcaccccgtggcaatgtcaccagcctttc 841 cttgtcctccaaccgcatccaccacctccatgattctgactttgcccacctgcccagcct 901 gcggcatctcaacctcaagtggaactgcccgccggttggcctcagccccatgcacttccc 961 ctgccacatgaccatcgagcccagcaccttcttggctgtgcccaccctggaagagctaaa 1021 cctgagctacaacaacatcatgactgtgcctgcgctgcccaaatccctcatatccctgtc 1081 cctcagccataccaacatcctgatgctagactctgccagcctcgccggcctgcatgccct 1141 gcgcttcctattcatggacggcaactgttattacaagaacccctgcaggcaggcactgga 1201 ggtggccccgggtgccctccttggcctgggcaacctcacccacctgtcactcaagtacaa 1261 caacctcactgtggtgccccgcaacctgccttccagcctggagtatctgctgttgtccta 1321 caaccgcatcgtcaaactggcgcctgaggacctggccaatctgaccgccctgcgtgtgct 1381 cgatgtgggcggaaattgccgccgctgcgaccacgctcccaacccctgcatggagtgccc 1441 tcgtcacttcccccagctacatcccgataccttcagccacctgagccgtcttgaaggcct 1501 ggtgttgaaggacagttctctctcctggctgaatgccagttggttccgtgggctgggaaa 1561 cctccgagtgctggacctgagtgagaacttcctctacaaatgcatcactaaaaccaaggc 1621 cttccagggcctaacacagctgcgcaagcttaacctgtccttcaattaccaaaagagggt 1681 gtcctttgcccacctgtctctggccccttccttcgggagcctggtcgccctgaaggagct 1741 ggacatgcacggcatcttcttccgctcactcgatgagaccacgctccggccactggcccg 1801 cctgcccatgctccagactctgcgtctgcagatgaacttcatcaaccaggcccagctcgg 1861 catcttcagggccttccctggcctgcgctacgtggacctgtcggacaaccgcatcagcgg 1921 agcttcggagctgacagccaccatgggggaggcagatggaggggagaaggtctggctgca 1981 gcctggggaccttgctccggccccagtggacactcccagctctgaagacttcaggcccaa 2041 ctgcagcaccctcaacttcaccttggatctgtcacggaacaacctggtgaccgtgcagcc 2101 ggagatgtttgcccagctctcgcacctgcagtgcctgcgcctgagccacaactgcatctc 2161 gcaggcagtcaatggctcccagttcctgccgctgaccggtctgcaggtgctagacctgtc 2221 ccacaataagctggacctctaccacgagcactcattcacggagctaccacgactggaggc 2281 cctggacctcagctacaacagccagccctttggcatgcagggcgtgggccacaacttcag 2341 cttcgtggctcacctgcgcaccctgcgccacctcagcctggcccacaacaacatccacag 2401 ccaagtgtcccagcagctctgcagtacgtcgctgcgggccctggacttcagcggcaatgc 2461 actgggccatatgtgggccgagggagacctctatctgcacttcttccaaggcctgagcgg 2521 tttgatctggctggacttgtcccagaaccgcctgcacaccctcctgccccaaaccctgcg 2581 caacctccccaagagcctacaggtgctgcgtctccgtgacaattacctggccttctttaa 2641 gtggtggagcctccacttcctgcccaaactggaagtcctcgacctggcaggaaaccagct 2701 gaaggccctgaccaatggcagcctgcctgctggcacccggctccggaggctggatgtcag 2761 ctgcaacagcatcagcttcgtggcccccggcttcttttccaaggccaaggagctgcgaga 2821 gctcaaccttagcgccaacgccctcaagacagtggaccactcctggtttgggcccctggc 2881 gagtgccctgcaaatactagatgtaagcgccaaccctctgcactgcgcctgtggggcggc 2941 ctttatggacttcctgctggaggtgcaggctgccgtgcccggtctgcccagccgggtgaa 3001 gtgtggcagtccgggccagctccagggcctcagcatctttgcacaggacctgcgcctctg 3061 cctggatgaggccctctcctgggactgtttcgccctctcgctgctggctgtggctctggg 3121 cctgggtgtgcccatgctgcatcacctctgtggctgggacctctggtactgcttccacct 3181 gtgcctggcctggcttccctggcgggggcggcaaagtgggcgagatgaggatgccctgcc 3241 ctacgatgccttcgtggtcttcgacaaaacgcagagcgcagtggcagactgggtgtacaa 3301 cgagcttcgggggcagctggaggagtgccgtgggcgctgggcactccgcctgtgcctgga 3361 ggaacgcgactggctgcctggcaaaaccctctttgagaacctgtgggcctcggtctatgg 3421 cagccgcaagacgctgtttgtgctggcccacacggaccgggtcagtggtctcttgcgcgc 3481 cagcttcctgctggcccagcagcgcctgctggaggaccgcaaggacgtcgtggtgctggt 3541 gatcctgagccctgacggccgccgctcccgctatgtgcggctgcgccagcgcctctgccg 3601 ccagagtgtcctcctctggccccaccagcccagtggtcagcgcagcttctgggcccagct 3661 gggcatggccctgaccagggacaaccaccacttctataaccggaacttctgccagggacc 3721 cacggccgaatagccgtgagccggaatcctgcacggtgccacctccacactcacctcacc 3781 tctgcctgcctggtctgaccctcccctgctcgcctccctcaccccacacctgacacagag 3841 caggcactcaataaatgctaccgaaggc
[0194] Human TLR-9, isoform A, is encoded by the following amino acid sequence (NCBI Accession No. NP_059138 and SEQ ID NO: 20):
TABLE-US-00020 (SEQIDNO:20) MGFCRSALHPLSLLVQAIMLAMTLALGTLPAFLPCELQPHGLVNCNWLFLKSVPHFSMAAPRGNV TSLSLSSNRIHHLHDSDFAHLPSLRHLNLKWNCPPVGLSPMHFPCHMTIEPSTFLAVPTLEELNLSY NNIMTVPALPKSLISLSLSHTNILMLDSASLAGLHALRFLFMDGNCYYKNPCRQALEVAPGALLGL GNLTHLSLKYNNLTVVPRNLPSSLEYLLLSYNRIVKLAPEDLANLTALRVLDVGGNCRRCDHAPN PCMECPRHFPQMPDTFSHLSRLEGLVLKDSSLSWLNASWFRGLGNLRVLDLSENFLYKCITKTKA FQGLTQLRKLNLSFNYQKRVSFAHLSLAPSFGSLVALKELDMHGIFFRSLDETTLRPLARLPMLQT LRLQMNFINQAQLGIFRAFPGLRYVDLSDNRISGASELTATMGEADGGEKVWLQPGDLAPAPVDT PSSEDFRPNCSTLNFTLDLSRNNLVTVQPEMFAQLSHLQCLRLSHNCISQAVNGSQFLPLTGLQVLD LSHNKLDLYHEHSFTELPRLEALDLSYNSQPFGMQGVGHNFSFVAHLRTLRHLSLAHNNIHSQVSQ QLCSTSLRALDFSGNALGHMWAEGDLYLHFFQGLSGLIWLDLSQNRLHTLLPQTLRNLPKSLQVL RLRDNYLAFFKWWSLHFLPKLEVLDLAGNQLKALTNGSLPAGTRLRRLDVSCNSISFVAPGFFSK AKELRELNLSANALKTVDHSWFGPLASALQILDVSANPLHCACGAAFMDFLLEVQAAVPGLPSRV KCGSPGQLQGLSIFAQDLRLCLDEALSWDCFALSLLAVALGLGVPMLHHLCGWDLWYCFHLCLA WLPWRGRQSGRDEDALPYDAFVVFDKTQSAVADWVYNELRGQLEECRGRWALRLCLEERDWL PGKTLFENLWASVYGSRKTLFVLAHTDRVSGLLRASFLLAQQRLLEDRKDVVVLVILSPDGRRSR YVRLRQRLCRQSVLLWPHQPSGQRSFWAQLGMALTRDNHHFYNRNFCQGPTAE
[0195] Human TLR3 is encoded by the following mRNA sequence (GenBank Accesion No. NM_003265.2 (GI:19718735), incorporated herein by reference; SEQ ID NO: 21):
TABLE-US-00021 (SEQIDNO:21) 1 cactttcgagagtgccgtctatttgccacacacttccctgatgaaatgtctggatttgga 61 ctaaagaaaaaaggaaaggctagcagtcatccaacagaatcATGagacagactttgcctt 121 gtatctacttttgggggggccttttgccctttgggatgctgtgtgcatcctccaccacca 181 agtgcactgttagccatgaagttgctgactgcagccacctgaagttgactcaggtacccg 241 atgatctacccacaaacataacagtgttgaaccttacccataatcaactcagaagattac 301 cagccgccaacttcacaaggtatagccagctaactagcttggatgtaggatttaacacca 361 tctcaaaactggagccagaattgtgccagaaacttcccatgttaaaagttttgaacctcc 421 agcacaatgagctatctcaactttctgataaaacctttgccttctgcacgaatttgactg 481 aactccatctcatgtccaactcaatccagaaaattaaaaataatccctttgtcaagcaga 541 agaatttaatcacattagatctgtctcataatggcttgtcatctacaaaattaggaactc 601 aggttcagctggaaaatctccaagagcttctattatcaaacaataaaattcaagcgctaa 661 aaagtgaagaactggatatctttgccaattcatctttaaaaaaattagagttgtcatcga 721 atcaaattaaagagttttctccagggtgttttcacgcaattggaagattatttggcctct 781 ttctgaacaatgtccagctgggtcccagccttacagagaagctatgtttggaattagcaa 841 acacaagcattcggaatctgtctctgagtaacagccagctgtccaccaccagcaatacaa 901 ctttcttgggactaaagtggacaaatctcactatgctcgatctttcctacaacaacttaa 961 atgtggttggtaacgattcctttgcttggcttccacaactagaatatttcttcctagagt 1021 ataataatatacagcatttgttttctcactctttgcacgggcttttcaatgtgaggtacc 1081 tgaatttgaaacggtcttttactaaacaaagtatttcccttgcctcactccccaagattg 1141 atgatttttcttttcagtggctaaaatgtttggagcaccttaacatggaagataatgata 1201 ttccaggcataaaaagcaatatgttcacaggattgataaacctgaaatacttaagtctat 1261 ccaactcctttacaagtttgcgaactttgacaaatgaaacatttgtatcacttgctcatt 1321 ctcccttacacatactcaacctaaccaagaataaaatctcaaaaatagagagtgatgctt 1381 tctcttggttgggccacctagaagtacttgacctgggccttaatgaaattgggcaagaac 1441 tcacaggccaggaatggagaggtctagaaaatattttcgaaatctatctttcctacaaca 1501 agtacctgcagctgactaggaactcctttgccttggtcccaagccttcaacgactgatgc 1561 tccgaagggtggcccttaaaaatgtggatagctctccttcaccattccagcctcttcgta 1621 acttgaccattctggatctaagcaacaacaacatagccaacataaatgatgacatgttgg 1681 agggtcttgagaaactagaaattctcgatttgcagcataacaacttagcacggctctgga 1741 aacacgcaaaccctggtggtcccatttatttcctaaagggtctgtctcacctccacatcc 1801 ttaacttggagtccaacggctttgacgagatcccagttgaggtcttcaaggatttatttg 1861 aactaaagatcatcgatttaggattgaataatttaaacacacttccagcatctgtcttta 1921 ataatcaggtgtctctaaagtcattgaaccttcagaagaatctcataacatccgttgaga 1981 agaaggttttcgggccagctttcaggaacctgactgagttagatatgcgctttaatccct 2041 ttgattgcacgtgtgaaagtattgcctggtttgttaattggattaacgagacccatacca 2101 acatccctgagctgtcaagccactacctttgcaacactccacctcactatcatgggttcc 2161 cagtgagactttttgatacatcatcttgcaaagacagtgccccctttgaactctttttca 2221 tgatcaataccagtatcctgttgatttttatctttattgtacttctcatccactttgagg 2281 gctggaggatatctttttattggaatgtttcagtacatcgagttcttggtttcaaagaaa 2341 tagacagacagacagaacagtttgaatatgcagcatatataattcatgcctataaagata 2401 aggattgggtctgggaacatttctcttcaatggaaaaggaagaccaatctctcaaatttt 2461 gtctggaagaaagggactttgaggcgggtgtttttgaactagaagcaattgttaacagca 2521 tcaaaagaagcagaaaaattatttttgttataacacaccatctattaaaagacccattat 2581 gcaaaagattcaaggtacatcatgcagttcaacaagctattgaacaaaatctggattcca 2641 ttatattggttttccttgaggagattccagattataaactgaaccatgcactctgtttgc 2701 gaagaggaatgtttaaatctcactgcatcttgaactggccagttcagaaagaacggatag 2761 gtgcctttcgtcataaattgcaagtagcacttggatccaaaaactctgtacattaaattt 2821 atttaaatattcaattagcaaaggagaaactttctcaatttaaaaagttctatggcaaat 2881 ttaagttttccataaaggtgttataatttgtttattcatatttgtaaatgattatattct 2941 atcacaattacatctcttctaggaaaatgtgtctccttatttcaggcctatttttgacaa 3001 ttgacttaattttacccaaaataaaacatataagcacgtaaaaaaaaaaaaaaaaaa
[0196] Human TLR3 is encoded by the following amino acid sequence (GenBank Accession No. ABC86910.1 (GI:86161330), incorporated herein by reference; SEQ ID NO: 22):
TABLE-US-00022 (SEQIDNO:22) 1 mrqtlpciyfwggllpfgmlcassttkctvshevadcshlkltqvpddlptnitvinith 61 nqlrrlpaanftrysqltsldvgfntisklepelcqklpmlkvinlqhnelsqlsdktfa 121 fctnitelhlmsnsigkiknnpfvkqknlitldlshnglsstklgtqvqlenlqelllsn 181 nkiqalkseeldifansslkklelssnqikefspgcfhaigrlfglflnnvqlgpsltek 241 lclelantsirnlslsnsqlsttsnttflglkwtnitmldlsynnlnvvgndsfawlpql 301 eyffleynniqhlfshslhglfnvrylnlkrsftkqsislaslpkiddfsfqwlkclehl 361 nmedndipgiksnmftglinlkylslsnsftslrtltnetfvslahsplhilnitknkis 421 kiesdafswlghlevldlglneiggeltgqewrglenifeiylsynkylqltrnsfalvp 481 slqrlmlrryalknvdsspspfqp1rnitildlsnnnianinddmleglekleildlqhn 541 nlarlwkhanpggpiyflkglshlhilnlesngfdeipvevfkdlfelkiidlglnnlnt 601 1pasvfnnqvslkslnlqknlitsvekkvfgpafrniteldmrfnpfdctcesiawfvnw 661 inethtnipelsshylcntpphyhgfpvrlfdtssckdsapfelffmintsillififiv 721 llihfegwrisfywnvsvhrvlgfkeidrqteqfeyaayiihaykdkdwvwehfssmeke 781 dqslkfcleerdfeagvfeleaivnsikrsrkiifvithhllkdplckrfkvhhavqqai 841 eqnldsiilvfleeipdyklnhalclrrgmfkshcilnwpvqkerigafrhklqvalgsk 901 nsvh
[0197] The nucleic acid sequence of human TLR1 is provided in GenBank Accession No. NM_003263.3 (GI:41350336), incorporated herein by reference. The amino acid sequence of human TLR1 is provided in GenBank Accession No. NP_003254.2 (GI:41350337), incorporated herein by reference.
[0198] The nucleic acid sequence of human TLR2 is provided in GenBank Accession No. NM_003264.3 (GI:68160956), incorporated herein by reference. The amino acid sequence of human TLR2 is provided in GenBank Accession No. NP_003255.2 (GI:19718734), incorporated herein by reference.
[0199] The nucleic acid sequence of human TLR4 is provided in GenBank Accession No. NM_138554.4 (GI:373432600), incorporated herein by reference. The amino acid sequence of human TLR4 is provided in GenBank Accession No. NP_612564.1 (GI:19924149), incorporated herein by reference.
[0200] The nucleic acid sequence of human TLR5 is provided in GenBank Accession No. NM_003268.5 (GI:281427130), incorporated herein by reference. The amino acid sequence of human TLR5 is provided in GenBank Accession No. NP_003259.2 (GI:16751843), incorporated herein by reference.
[0201] The nucleic acid sequence of human TLR6 is provided in GenBank Accession No. NM_006068.4 (GI:318067953), incorporated herein by reference. The amino acid sequence of human TLR6 is provided in GenBank Accession No. NP_006059.2 (GI:20143971), incorporated herein by reference.
[0202] The nucleic acid sequence of human TLR7 is provided in GenBank Accession No. NM_016562.3 (GI:67944638), incorporated herein by reference. The amino acid sequence of human TLR7 is provided in GenBank Accession No. NP_057646.1 (GI:7706093), incorporated herein by reference.
[0203] The nucleic acid sequence of human TLR8 is provided in GenBank Accession No. NM_138636.4 (GI:257196253), incorporated herein by reference. The amino acid sequence of human TLR8 is provided in GenBank Accession No. NP_619542.1 (GI:20302168), incorporated herein by reference.
[0204] The nucleic acid sequence of human TLR10 is provided in GenBank Accession No. NM_030956.3 (GI:306140488), incorporated herein by reference. The amino acid sequence of human TLR10 is provided in GenBank Accession No. NP_112218.2 (GI:62865618), incorporated herein by reference.
[0205] The nucleic acid sequence of mouse TLR11 is provided in GenBank Accession No. NM_205819.3 (GI:408684412), incorporated herein by reference. The amino acid sequence of mouse TLR11 is provided in GenBank Accession No. NP_991388.2 (GI:408684413), incorporated herein by reference.
[0206] The nucleic acid sequence of mouse TLR12 is provided in GenBank Accession No. NM_205823.2 (GI:148539900), incorporated herein by reference. The amino acid sequence of mouse TLR12 is provided in GenBank Accession No. NP_991392.1 (GI:45430001), incorporated herein by reference.
[0207] The nucleic acid sequence of mouse TLR13 is provided in GenBank Accession No. NM_205820.1 (GI:45429998), incorporated herein by reference. The amino acid sequence of mouse TLR13 is provided in GenBank Accession No. NP_991389.1 (GI:45429999), incorporated herein by reference.
[0208] A representative list of TLR agonists (both synthetic and natural ligands), along with their corresponding receptor is provided in the table below.
TABLE-US-00023 Receptor PathogenAssociatedLigands(PAMPS)[1] LigandNaturalhost SyntheticLigands TLR1 multipletriacyllipopeptides Bacteria Pam3Cys-* TLR2 multipleglycolipids Bacteria CFA multiplelipopeptides Bacteria MALP-** multiplelipoproteins Bacteria Pam2Cys** lipoeichoicacid GramPositive FSL-1 Bacteria HSP70,orotherheatsockproteins Hostcelss Hib-OMPC zymosan(Beta-glucan) Fungi Numerousothers TLR3 DoublestrandedRNA viruses Poly(I:C);LowandHigh molecularweight Poly(A:U) TLR4 lipopolysacharidesLPS);orLPS Gramnegative AGP derivativessuchasMPLA bacteria severalheatshockproteins Bacteriaand MPLA hostcells librinogen hostcells RC-529 heparinsulfatefragments hostcells MDF2 hyaluronicacidfragments hostcells CFA nickel Variousopoiddrugs TLR5 Flagellin Bacteria Flagellin TLR6 multiplediacyllipopeptides Mycoplasma FSL1-** Pam2Cys** MALP2-** TLR7 ViralssRNA(Influenza,VSV,HIV,HCV) RNAviruses Guanosineanalogs; imidazoquinolines (e.g.Imiqulmod,AldaraR848, Resiquimod),Loxorbine TLR8 smallsyntheticcompounds, RNA,Humanand Imidazoquinoline;Loxoribine; single-strandedRNA viral ssPolyU,3M-012 TLR9 UnmethylatedCpGOligodeoxynucleotideDNA Bacteria,DNA CpG-oligonucleotides,numerous DNA;dsDNAviruses(HSV,MCMV);Hemozoin viruses sequenceshavebeensynthesized (Plasmodium) (e.gCpGODN2006,1826,2395) TLR10 unknown TLR11 Profilin Toxoplasmagondii TLR12 Profilin Toxoplasmagondii TLR12 bacterialribosomalRNAsequence Virus,bacteria [2][3] *CGGAAAGACC*(SEQIDNO:23) *Ligands recognized by TLR1 and TLR2 **Ligands recognized by TLR2 and TLR6
REFERENCES
[0209] Meyer , St E. Clinical Investigation of Toll-like receptor agonists. Expert opinion on investigational drugs. 2008; 17:1051-065. (Pubed) [0210] van D D, Medz R, S A C. Triggering TLR signaling in vaccination. Trends in immunology. 2006; 27:49-55 [0211] Kumar H, KT, A S, Toll-like receptors and inate immunity. Biochemical and biophysical research communications. 2009; 388:621-625. [0212] Wbaugh O, T, M R, . Immunology. Lippincott's illustrated reviews. Philadelphia. W K Lippincott Williams & Wilkens pp. 17. [0213] Z, Cai Z, B A, (February 2011). A novel Toll-like receptor that recognizes vesicular stomas virus 2. pp. 4517-24. burg M. Krugar A, R. et al. (August 2012). recognizes :RNA devoid of arythomycin modification . pp. 1111-5. [0214] S G, M. B. , M. S Toll-like receptor agonists: are they good adjuvants? Cancer J., 16(4)(2010). pp. 382-391.
Granulocyte Macrophage Colony Stimulating Factor (GM-CSF)
[0215] Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a protein secreted by macrophages, T cells, mast cells, endothelial cells and fibroblasts. Specifically, GM-CSF is a cytokine that functions as a white blood cell growth factor. GM-CSF stimulates stem cells to produce granulocytes and monocytes. Monocytes exit the blood stream, migrate into tissue, and subsequently mature into macrophages.
[0216] Scaffold devices described herein comprise and release GM-CSF polypeptides to attract host DCs to the device. Contemplated GM-CSF polypeptides are isolated from endogenous sources or synthesized in vivo or in vitro. Endogenous GM-CSF polypeptides are isolated from healthy human tissue. Synthetic GM-CSF polypeptides are synthesized in vivo following transfection or transformation of template DNA into a host organism or cell, e.g. a mammal or cultured human cell line. Alternatively, synthetic GM-CSF polypeptides are synthesized in vitro by polymerase chain reaction (PCR) or other art-recognized methods Sambrook, J., Fritsch, E. F., and Maniatis, T., Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, NY, Vol. 1, 2, 3 (1989), herein incorporated by reference).
[0217] GM-CSF polypeptides are modified to increase protein stability in vivo. Alternatively, GM-CSF polypeptides are engineered to be more or less immunogenic. Endogenous mature human GM-CSF polypeptides are glycosylated, reportedly, at amino acid residues 23 (leucine), 27 (asparagine), and 39 (glutamic acid) (see U.S. Pat. No. 5,073,627). GM-CSF polypeptides of the present invention are modified at one or more of these amino acid residues with respect to glycosylation state.
[0218] GM-CSF polypeptides are recombinant. Alternatively GM-CSF polypeptides are humanized derivatives of mammalian GM-CSF polypeptides. Exemplary mammalian species from which GM-CSF polypeptides are derived include, but are not limited to, mouse, rat, hamster, guinea pig, ferret, cat, dog, monkey, or primate. In a preferred embodiment, GM-CSF is a recombinant human protein (PeproTech, Catalog #300-03). Alternatively, GM-CSF is a recombinant murine (mouse) protein (PeproTech, Catalog #315-03). Finally, GM-CSF is a humanized derivative of a recombinant mouse protein.
[0219] Human Recombinant GM-CSF (PeproTech, Catalog #300-03) is encoded by the following polypeptide sequence (SEQ ID NO: 24):
TABLE-US-00024 (SEQIDNO:24) MAPARSPSPSTQPWEHVNAIQEARRLLNLSRDTAAEMNET VEVISEMFDLQEPTCLQTRLELYKQGLRGSLTKLKGPLTM MASHYKQHCPPTPETSCATQIITFESFKENLKDFLLVIPF DCWEPVQE
[0220] Murine Recombinant GM-CSF (PeproTech, Catalog #315-03) is encoded by the following polypeptide sequence (SEQ ID NO: 25):
TABLE-US-00025 (SEQIDNO:25) MAPTRSPITVTRPWKHVEAIKEALNLLDDMPVTLNEEVEV VSNEFSFKKLTCVQTRLKIFEQGLRGNFTKLKGALNMTAS YYQTYCPPTPETDCETQVTTYADFIDSLKTFLTDIPFECKKPVQK
[0221] Human Endogenous GM-CSF is encoded by the following mRNA sequence (NCBI Accession No. NM_000758 and SEQ ID NO: 26):
TABLE-US-00026 (SEQIDNO:26) 1 acacagagagaaaggctaaagttctctggaggatgtggctgcagagcctgctgctcttgg 61 gcactgtggcctgcagcatctctgcacccgcccgctcgcccagccccagcacgcagccct 121 gggagcatgtgaatgccatccaggaggcccggcgtctcctgaacctgagtagagacactg 181 ctgctgagatgaatgaaacagtagaagtcatctcagaaatgtttgacctccaggagccga 241 cctgcctacagacccgcctggagctgtacaagcagggcctgcggggcagcctcaccaagc 301 tcaagggccccttgaccatgatggccagccactacaagcagcactgccctccaaccccgg 361 aaacttcctgtgcaacccagattatcacctttgaaagtttcaaagagaacctgaaggact 421 ttctgcttgtcatcccctttgactgctgggagccagtccaggagtgagaccggccagatg 481 aggctggccaagccggggagctgctctctcatgaaacaagagctagaaactcaggatggt 541 catcttggagggaccaaggggtgggccacagccatggtgggagtggcctggacctgccct 601 gggccacactgaccctgatacaggcatggcagaagaatgggaatattttatactgacaga 661 aatcagtaatatttatatatttatatttttaaaatatttatttatttatttatttaagtt 721 catattccatatttattcaagatgttttaccgtaataattattattaaaaatatgcttct 781 a
[0222] Human Endogenous GM-CSF is encoded by the following amino acid sequence (NCBI Accession No. NP_000749.2 and SEQ ID NO: 27):
TABLE-US-00027 (SEQIDNO:27) MWLQSLLLLGTVACSISAPARSPSPSTQPWEHVNAIQEARRLLNLSRDTAA EMNETVEVISEMFDLQEPTCLQTRLELYKQGLRGSLTKLKGPLTMMASHYK QHCPPTPETSCATQIITFESFKENLKDFLLVIPFDCWEPVQE
[0223] GM-CSF signaling is a potent chemotactic factor for conventional DCs and significantly enhanced surface expression of MHC(II) and CD86(+), which are utilized for priming T cell immunity. In contrast, Flt3L vaccines led to greater numbers of plasmacytoid DCs (pDCs), correlating with increased levels of T cell priming cytokines that amplify T cell responses. Thus, as described in US 2013-0202707, incorporated herein by reference, 3D polymer matrices modified to present inflammatory cytokines are utilized to effectively mobilize and activate different DC subsets in vivo for immunotherapy.
[0224] An exemplary amino acid sequence of human Flt3 is provided below (GenBank Accession No.: P49771.1 (GI:1706818), incorporated herein by reference; SEQ ID NO: 28):
TABLE-US-00028 (SEQIDNO:28) 1 mtvlapawspttylllllllssglsgtqdcsfqhspissdfavkirelsdyllqdypvtv 61 asnlqdeelcgglwrlvlaqrwmerlktvagskmqgllervnteihfvtkcafqpppscl 121 rfvqtnisrllgetseqlvalkpwitrqnfsrclelqcqpdsstlpppwsprpleatapt 181 apqpp11111llpvg1111aaawclhwqrtrrrtprpgeqvppvpspqdlllveh
Cytosine-Guanosine (CpG) Oligonucleotide (CpG-ODN) Sequences
[0225] CpG sites are regions of deoxyribonucleic acid (DNA) where a cysteine nucleotide occurs next to a guanine nucleotide in the linear sequence of bases along its length (the p represents the phosphate linkage between them and distinguishes them from a cytosine-guanine complementary base pairing). CpG sites play a pivotal role in DNA methylation, which is one of several endogenous mechanisms cells use to silence gene expression. Methylation of CpG sites within promoter elements can lead to gene silencing. In the case of cancer, it is known that tumor suppressor genes are often silences while oncogenes, or cancer-inducing genes, are expressed. Importantly, CpG sites in the promoter regions of tumor suppressor genes (which prevent cancer formation) have been shown to be methylated while CpG sites in the promoter regions of oncogenes are hypomethylated or unmethylated in certain cancers. The TLR-9 receptor binds unmethylated CpG sites in DNA.
[0226] The present invention comprises CpG dinucleotides and oligonucleotides. Contemplated CpG oligonucleotides are isolated from endogenous sources or synthesized in vivo or in vitro. Exemplary sources of endogenous CpG oligonucleotides include, but are not limited to, microorganisms, bacteria, fungi, protozoa, viruses, molds, or parasites. Alternatively, endogenous CpG oligonucleotides are isolated from mammalian benign or malignant neoplastic tumors. Synthetic CpG oligonucleotides are synthesized in vivo following transfection or transformation of template DNA into a host organism. Alternatively, Synthetic CpG oligonucleotides are synthesized in vitro by polymerase chain reaction (PCR) or other art-recognized methods (Sambrook, J., Fritsch, E. F., and Maniatis, T., Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, NY, Vol. 1, 2, 3 (1989), incorporated herein by reference).
[0227] CpG oligonucleotides are presented for cellular uptake by dendritic cells. In one embodiment, naked CpG oligonucleotides are used. The term naked is used to describe an isolated endogenous or synthetic polynucleotide (or oligonucleotide) that is free of additional substituents. In another embodiment, CpG oligonucleotides are bound to one or more compounds to increase the efficiency of cellular uptake. Alternatively, or in addition, CpG oligonucleotides are bound to one or more compounds to increase the stability of the oligonucleotide within the scaffold and/or dendritic cell.
[0228] CpG oligonucleotides are condensed prior to cellular uptake. In one preferred embodiment, CpG oligonucleotides are condensed using polyethylimine (PEI), a cationic polymer that increases the efficiency of cellular uptake into dendritic cells.
[0229] CpG oligonucleotides of the present invention can be divided into multiple classes. For example, exemplary CpG-ODNs encompassed by compositions, methods and devices of the present invention are stimulatory, neutral, or suppressive. The term stimulatory used herein is meant to describe a class of CpG-ODN sequences that activate TLR9. The term neutral used herein is meant to describe a class of CpG-ODN sequences that do not activate TLR9. The term suppressive used herein is meant to describe a class of CpG-ODN sequences that inhibit TLR9. The term activate TLR9 describes a process by which TLR9 initiates intracellular signaling.
[0230] Simulatory CpG-ODNs can further be divided into three types A, B and C, which differ in their immune-stimulatory activities. Type A stimulatory CpG ODNs are characterized by a phosphodiester central CpG-containing palindromic motif and a phosphorothioate 3 poly-G string. Following activation of TLR9, these CpG ODNs induce high IFN- production from plasmacytoid dendritic cells (pDC). Type A CpG ODNs weakly stimulate TLR9-dependent NF-B signaling.
[0231] Type B stimulatory CpG ODNs contain a full phosphorothioate backbone with one or more CpG dinucleotides. Following TLR9 activation, these CpG-ODNs strongly activate B cells. In contrast to Type A Cpg-ODNs, Type B CpG-ODNS weakly stimulate IFN- secretion.
[0232] Type C stimulatory CpG ODNs comprise features of Types A and B. Type C CpG-ODNs contain a complete phosphorothioate backbone and a CpG containing palindromic motif. Similar to Type A CpG ODNs, Type C CpG ODNs induce strong IFN- production from pDC. Similar to Type B CpG ODNs, Type C CpG ODNs induce strong B cell stimulation.
[0233] Exemplary stimulatory CpG ODNs comprise, but are not limited to, ODN 1585, ODN 1668, ODN 1826, ODN 2006, ODN 2006-G5, ODN 2216, ODN 2336, ODN 2395, ODN M362 (all InvivoGen). The present invention also encompasses any humanized version of the preceding CpG ODNs. In one preferred embodiment, compositions, methods, and devices of the present invention comprise ODN 1826 (the sequence of which from 5 to 3 is tccatgacgttcctgacgtt, wherein CpG elements are bolded, SEQ ID NO: 29).
[0234] Neutral, or control, CpG ODNs that do not stimulate TLR9 are encompassed by the present invention. These ODNs comprise the same sequence as their stimulatory counterparts but contain GpC dinucleotides in place of CpG dinucleotides.
[0235] Exemplary neutral, or control, CpG ODNs encompassed by the present invention comprise, but are not limited to, ODN 1585 control, ODN 1668 control, ODN 1826 control, ODN 2006 control, ODN 2216 control, ODN 2336 control, ODN 2395 control, ODN M362 control (all InvivoGen). The present invention also encompasses any humanized version of the preceding CpG ODNs.
[0236] Suppressive CpG ODNs that inhibit TLR9 are encompassed by the present invention. Exemplary potent inhibitory sequences are (TTAGGG).sub.4 (SEQ ID NO: 30) (oligonucleotide TTAGGG, InvivoGen), found in mammalian telomeres and ODN 2088 (InvivoGen), derived from a murine stimulatory CpG ODN by replacement of 3 bases. Suppressive ODNs disrupt the colocalization of CpG ODNs with TLR9 in endosomal vesicles without affecting cellular binding and uptake. Suppressive CpG ODNs encompassed by the present invention are used to fine-tune, attenuate, reverse, or oppose the action of a stimulatory CpG-ODN. Alternatively, or in addition, compositions, methods, or devices of the present invention comprising suppressive CpG ODNs are used to treat autoimmune conditions or prevent immune responses following transplant procedures.
Cancer Antigens
[0237] Compositions, methods, and devices of the present invention comprise cancer antigens with means to vaccinate and/or provide protective immunity to a subject to whom such a device was administered. Cancer antigens are used alone or in combination with GM-CSF, CpG-ODN sequences, or immunomodulators. Moreover, cancer antigens are used simultaneously or sequentially with GM-CSF, CpG-ODN sequences, or immunomodulators.
[0238] Exemplary cancer antigens encompassed by the compositions, methods, and devices of the present invention include, but are not limited to, tumor lysates extracted from biopsies (e.g., from melanoma tumor biopsies, or from B 16-F10 tumors isolated from mice challenged with B16-F10 melanoma tumor cells), irradiated tumor cells (e.g., irradiated melanoma cells), antigens from lung cancer, antigens from breast cancers (e.g., Her2, e.g., purified Her2 or a fragment thereof), antigens from glioma cancers, prostate (e.g., prostate cancer) antigens (e.g., prostatic acid phosphatase), MAGE series of antigens (MAGE-1 is an example), MART-1/melanA, tyrosinase, ganglioside, gp100, GD-2, O-acetylated GD-3, GM-2, MUC-1, Sosl, Protein kinase C-binding protein, Reverse transcriptase protein, AKAP protein, VRK1, KIAA1735, T7-1, T11-3, T11-9, Homo Sapiens telomerase ferment (hTRT), Cytokeratin-19 (CYFRA21-1), SQUAMOUS CELL CARCINOMA ANTIGEN 1 (SCCA-1), (PROTEIN T4-A), SQUAMOUS CELL CARCINOMA ANTIGEN 2 (SCCA-2), Ovarian carcinoma antigen CA125 (1A1-3B) (KIAA0049), MUCIN 1 (TUMOR-ASSOCIATED MUCIN), (CARCINOMA-ASSOCIATED MUCIN), (POLYMORPHIC EPITHELIAL MUCIN), (PEM), (PEMT), (EPISIALIN), (TUMOR-ASSOCIATED EPITHELIAL MEMBRANE ANTIGEN), (EMA), (H23AG), (PEANUT-REACTIVE URINARY MUCIN), (PUM), (BREAST CARCINOMA-ASSOCIATED ANTIGEN DF3), CTCL tumor antigen sel-1, CTCL tumor antigen se14-3, CTCL tumor antigen se20-4, CTCL tumor antigen se20-9, CTCL tumor antigen se33-1, CTCL tumor antigen se37-2, CTCL tumor antigen se57-1, CTCL tumor antigen se89-1, Prostate-specific membrane antigen, 5T4 oncofetal trophoblast glycoprotein, Orf73 Kaposi's sarcoma-associated herpesvirus, MAGE-C1 (cancer/testis antigen CT7), MAGE-B1 ANTIGEN (MAGE-XP ANTIGEN) (DAM10), MAGE-B2 ANTIGEN (DAM6), MAGE-2 ANTIGEN, MAGE-4a antigen, MAGE-4b antigen, Colon cancer antigen NY-CO-45, Lung cancer antigen NY-LU-12 variant A, Cancer associated surface antigen, Adenocarcinoma antigen ART1, Paraneoplastic associated brain-testis-cancer antigen (onconeuronal antigen MA2; paraneoplastic neuronal antigen), Neuro-oncological ventral antigen 2 (NOVA2), Hepatocellular carcinoma antigen gene 520, TUMOR-ASSOCIATED ANTIGEN CO-029, Tumor-associated antigen MAGE-X2, Synovial sarcoma, X breakpoint 2, Squamous cell carcinoma antigen recognized by T cell, Serologically defined colon cancer antigen 1, Serologically defined breast cancer antigen NY-BR-15, Serologically defined breast cancer antigen NY-BR-16, Chromogranin A; parathyroid secretory protein 1, DUPAN-2, CA 19-9, CA 72-4, CA 195, Carcinoembryonic antigen (CEA).
[0239] The amino acid sequence of human prostatic acid phosphatase is provided by Genbank Accession No. AAA60022.1, and is shown below (SEQ ID NO: 31), with the signal peptide shown in underlined font and the mature peptide shown in italicized font.
TABLE-US-00029 (SEQIDNO:31) 1 mraaplllaraaslalascfcffcwldrsvlakelkfvtlvfrhgdrspidtfptdpike 61 sswpqgfgqltqlgmeqhyelgeyirkryrkflndsykheqvyirstdvdrtlmsrmtnl 121 aalfppegvsiwnpillwqpipvhtvplsedqllylpfrncprfgelesetlkseefqkr 181 lhpykdfiatlgklsglhgqdlfgiwskvydplysesvhnftlpswatedtmtklrelse 241 lsllslygihkqkeksrlqggvlvneilnhmkratqipsykklimysandttvtglqmal 301 dvyngllppyaschltelyfekgeyfvemyyrnetqhepyplmlpgcspscplerfaelv 361 gpvipqdwstevmttnshqgtedstd
The mRNA sequence encoding human prostatic acid phosphatase is provided by Genbank Accession No. M24902.1, and is shown below (SEQ ID NO: 32), with the start and stop codons in bold.
TABLE-US-00030 (SEQIDNO:32) 1 ggccagaaacagctctcctcaacatgagagctgcacccctcctcctggccagggcagcaa 61 gcttagccttggcttcttgtttctgctttttttgctggctagaccgaagtgtactagcca 121 aggagttgaagtttgtgactttggtgtttcggcatggagaccgaagtcccattgacacct 181 ttcccactgaccccataaaggaatcctcatggccacaaggatttggccaactcacccagc 241 tgggcatggagcagcattatgaacttggagagtatataagaaagagatatagaaaattct 301 tgaatgactcctataaacatgaacaggtttatattcgaagcacagacgttgaccggactt 361 tgatgagtcgtatgacaaacctggcagccctgtttcccccagaaggtgtcagcatctgga 421 atcctatcctactctggcagcccatcccggtgcacacagttcctctttctgaagatcagt 481 tgctatacctgcctttcaggaactgccctcgttttcaagaacttgagagtgagactttga 541 aatcagaggaattccagaagaggctgcacccttataaggattttatagctaccttgggaa 601 aactttcaggattacatggccaggacctttttggaatttggagtaaagtctacgaccctt 661 tatattctgagagtgttcacaatttcactttaccctcctgggccactgaggacaccatga 721 ctaagttgagagaattgtcagaattgtccctcctgtccctctatggaattcacaagcaga 781 aagagaaatctaggctccaagggggtgtcctggtcaatgaaatcctcaatcacatgaaga 841 gagcaactcagataccaagctacaaaaaacttatcatgtattctgcgcatgacactactg 901 tgactggcctacagatggcgctagatgtttacaacggactccttcctccctatgcttctt 961 gccacttgacggaattgtactttgagaagggggagtactttgtggagatgtactaccgga 1021 atgagacgcagcacgagccgtatcccctcatgctacctggctgcagccccagctgtcctc 1081 tggagaggtttgctgagctggttggccctgtgatccctcaagactggtccacggaggtta 1141 tgaccacaaacagccatcaaggtactgaggacagtacagattagtgtgcacagagatctc 1201 tgtagaaagagtagctgccctttctcagggcagatgatgctttgagaacatactttggcc 1261 attaccccccagctttgaggaaaatgggctttggatgattattttatgttttaggggacc 1321 cccaacctcaggcaattccatcctcttcacccgaccctgcccccacttgccataaaactt 1381 agctaagttttgttttgtttttcagcgttaatgtaaaggggcagcagtgccaaaatataa 1441 cagagataaagcttaggtcaaagttcatagagttcccatgaactatatgactggccacac 1501 aggatcttttgtatttaaggattctgagattttgcttgagcaggattagataaggctgtt 1561 ctttaaatgtctgaaatggaacagatttcaaaaaaaaccccacaatctagggtgggaaca 1621 aggaaggaaagatgtgaataggctgatgggcaaaaaaccaatttacccatcagttccagc 1681 cttctctcaaggagaggcaaagaaaggagatacagtggagacatctggaaagttttctcc 1741 actggaaaactgctactatctgtttttatatttctgttaaaatatatgaggctacagaac 1801 taaaaattaaaacctctttgtgtcccttggtcctggaacatttatgttccttttaaagaa 1861 acaaaaatcaaactttacagaaagatttgatgtatgtaatacatatagcagctcttgaag 1921 tatatatatcatagcaaataagtcatctgatgagaacaagctatttgggcacaacacatc 1981 aggaaagagagcaccacgtgatggagtttctccagaagctccagtgataagagatgttga 2041 ctctaaagttgatttaaggccaggcatggtggtttacgcctataatcccagcattttggg 2101 agtccgaggtgggcagatcacttgagctcaggaggtcaagatcagcctgggcaacatggt 2161 gaaaccttgtctctacataaaatacaaaaacttagatgggcatggtggtgtgtgcctata 2221 gtccactacttgtggggctaaggcaggaggatcacttgagccccggaggtcgaggctaca 2281 gtgagccaagagtgcactactgtactccagccagggcaagagagcgagaccctgtctcaa 2341 taaataaataaataaataaataaataaataaataaataaataaataaaaacaaagttgat 2401 taagaaaggaagtataggctaggcacagtggctcacacctgtaatccttgcattttggaa 2461 ggctgaggcaggaggatcactttaggcctggtgtgttcaagaccagcctggtcaacatag 2521 tgagacactgtctctaccaaaaaaaggaaggaagggacacatatcaaactgaaacaaaat 2581 tagaaatgtaattatgttatgttctaagtgcctccaagttcaaaacttattggaatgttg 2641 agagtgtggttacgaaatacgttaggaggacaaaaggaatgtgtaagtctttaatgcccg 2701 atatcttcagaaaacctaagcaaacttacaggtcctgctgaaactgcccactctgcaaga 2761 agaaatcatgatatagctttgccatgtggcagatctacatgtctagagaacactgtgctc 2821 tattaccattatggataaagatgagatggtttctagagatggtttctactggctgccaga 2881 atctagagcaaagccatccccgctcctggttggtcacagaatgactgacaaagacatcga 2941 ttgatatgcttctttgtgttatttccctcccaagtaaatgtttgtccttgggtccatttt 3001 ctatgcttgtaactgtcttctagcagtgagccaaatgtaaaatagtgaataaagtcatta 3061 ttaggaagttcaaaagcattgcttttataatgaactt
[0240] The amino acid sequence of human Her2 is provided by Genbank Accession No. P04626.1, and is shown below (SEQ ID NO: 33).
TABLE-US-00031 (SEQIDNO:33) 1 melaalcrwglllallppgaastqvctgtdmklrlpaspethldmlrhlyqgcqvvqgnl 61 eltylptnaslsflqdiqevqgyvliahnqvrqvplqrlrivrgtqlfednyalavldng 121 dplnnttpvtgaspgglrelqlrslteilkggvliqrnpqlcyqdtilwkdifhknnqla 181 ltlidtnrsrachpcspmckgsrcwgessedcqsltrtvcaggcarckgplptdccheqc 241 aagctgpkhsdclaclhfnhsgicelhcpalvtyntdtfesmpnpegrytfgascvtacp 301 ynylstdvgsctlvcplhnqevtaedgtqrcekcskpcarvcyglgmehlrevravtsan 361 iqefagckkifgslaflpesfdgdpasntaplqpeqlqvfetleeitgylyisawpdslp 421 dlsvfqnlqvirgrilhngaysltlqglgiswlglrslrelgsglalihhnthlcfvhtv 481 pwdqlfrnphqallhtanrpedecvgeglachqlcarghcwgpgptqcvncsqflrgqec 541 veecrvlqglpreyvnarhclpchpecqpqngsvtcfgpeadqcvacahykdppfcvarc 601 psgvkpdlsympiwkfpdeegacqpcpincthscvdlddkgcpaegraspltsiisavvg 661 illvvvlgvvfgilikrrqqkirkytmrrllgetelvepltpsgampnqaqmrilketel 721 rkvkvlgsgafgtvykgiwipdgenvkipvaikvlrentspkankeildeayvmagvgsp 781 yvsrllgicltstvqlvtqlmpygclldhvrenrgrlgsqdllnwcmqiakgmsyledvr 841 lvhrdlaarnvlvkspnhvkitdfglarlldideteyhadggkvpikwmalesilrrrft 901 hqsdvwsygvtvwelmtfgakpydgipareipdllekgerlpqppictidvymimvkcwm 961 idsecrprfrelvsefsrmardpqrfvviqnedlgpaspldstfyrslledddmgdlvda 1021 eeylvpqqgffcpdpapgaggmvhhrhrssstrsgggdltlglepseeeaprsplapseg 1081 agsdvfdgdlgmgaakglqslpthdpsplqrysedptvplpsetdgyvapltcspqpeyv 1141 nqpdvrpqppspregplpaarpagatlerpktlspgkngvvkdvfafggavenpeyltpq 1201 ggaapqphpppafspafdnlyywdqdppergappstfkgtptaenpeylgldvpv
The mRNA sequence encoding human Her2 is provided by Genbank Accession No. NM_004448.3, and is shown below (SEQ ID NO: 34), with the start and stop codons in bold.
TABLE-US-00032 (SEQIDNO:34) 1 gcttgctcccaatcacaggagaaggaggaggtggaggaggagggctgcttgaggaagtat 61 aagaatgaagttgtgaagctgagattcccctccattgggaccggagaaaccaggggagcc 121 ccccgggcagccgcgcgccccttcccacggggccctttactgcgccgcgcgcccggcccc 181 cacccctcgcagcaccccgcgccccgcgccctcccagccgggtccagccggagccatggg 241 gccggagccgcagtgagcaccatggagctggcggccttgtgccgctgggggctcctcctc 301 gccctcttgccccccggagccgcgagcacccaagtgtgcaccggcacagacatgaagctg 361 cggctccctgccagtcccgagacccacctggacatgctccgccacctctaccagggctgc 421 caggtggtgcagggaaacctggaactcacctacctgcccaccaatgccagcctgtccttc 481 ctgcaggatatccaggaggtgcagggctacgtgctcatcgctcacaaccaagtgaggcag 541 gtcccactgcagaggctgcggattgtgcgaggcacccagctctttgaggacaactatgcc 601 ctggccgtgctagacaatggagacccgctgaacaataccacccctgtcacaggggcctcc 661 ccaggaggcctgcgggagctgcagcttcgaagcctcacagagatcttgaaaggaggggtc 721 ttgatccagcggaacccccagctctgctaccaggacacgattttgtggaaggacatcttc 781 cacaagaacaaccagctggctctcacactgatagacaccaaccgctctcgggcctgccac 841 ccctgttctccgatgtgtaagggctcccgctgctggggagagagttctgaggattgtcag 901 agcctgacgcgcactgtctgtgccggtggctgtgcccgctgcaaggggccactgcccact 961 gactgctgccatgagcagtgtgctgccggctgcacgggccccaagcactctgactgcctg 1021 gcctgcctccacttcaaccacagtggcatctgtgagctgcactgcccagccctggtcacc 1081 tacaacacagacacgtttgagtccatgcccaatcccgagggccggtatacattcggcgcc 1141 agctgtgtgactgcctgtccctacaactacctttctacggacgtgggatcctgcaccctc 1201 gtctgccccctgcacaaccaagaggtgacagcagaggatggaacacagcggtgtgagaag 1261 tgcagcaagccctgtgcccgagtgtgctatggtctgggcatggagcacttgcgagaggtg 1321 agggcagttaccagtgccaatatccaggagtttgctggctgcaagaagatctttgggagc 1381 ctggcatttctgccggagagctttgatggggacccagcctccaacactgccccgctccag 1441 ccagagcagctccaagtgtttgagactctggaagagatcacaggttacctatacatctca 1501 gcatggccggacagcctgcctgacctcagcgtcttccagaacctgcaagtaatccgggga 1561 cgaattctgcacaatggcgcctactcgctgaccctgcaagggctgggcatcagctggctg 1621 gggctgcgctcactgagggaactgggcagtggactggccctcatccaccataacacccac 1681 ctctgcttcgtgcacacggtgccctgggaccagctctttcggaacccgcaccaagctctg 1741 ctccacactgccaaccggccagaggacgagtgtgtgggcgagggcctggcctgccaccag 1801 ctgtgcgcccgagggcactgctggggtccagggcccacccagtgtgtcaactgcagccag 1861 ttccttcggggccaggagtgcgtggaggaatgccgagtactgcaggggctccccagggag 1921 tatgtgaatgccaggcactgtttgccgtgccaccctgagtgtcagccccagaatggctca 1981 gtgacctgttttggaccggaggctgaccagtgtgtggcctgtgcccactataaggaccct 2041 cccttctgcgtggcccgctgccccagcggtgtgaaacctgacctctcctacatgcccatc 2101 tggaagtttccagatgaggagggcgcatgccagccttgccccatcaactgcacccactcc 2161 tgtgtggacctggatgacaagggctgccccgccgagcagagagccagccctctgacgtcc 2221 atcatctctgcggtggttggcattctgctggtcgtggtcttgggggtggtctttgggatc 2281 ctcatcaagcgacggcagcagaagatccggaagtacacgatgcggagactgctgcaggaa 2341 acggagctggtggagccgctgacacctagcggagcgatgcccaaccaggcgcagatgcgg 2401 atcctgaaagagacggagctgaggaaggtgaaggtgcttggatctggcgcttttggcaca 2461 gtctacaagggcatctggatccctgatggggagaatgtgaaaattccagtggccatcaaa 2521 gtgttgagggaaaacacatcccccaaagccaacaaagaaatcttagacgaagcatacgtg 2581 atggctggtgtgggctccccatatgtctcccgccttctgggcatctgcctgacatccacg 2641 gtgcagctggtgacacagcttatgccctatggctgcctcttagaccatgtccgggaaaac 2701 cgcggacgcctgggctcccaggacctgctgaactggtgtatgcagattgccaaggggatg 2761 agctacctggaggatgtgcggctcgtacacagggacttggccgctcggaacgtgctggtc 2821 aagagtcccaaccatgtcaaaattacagacttcgggctggctcggctgctggacattgac 2881 gagacagagtaccatgcagatgggggcaaggtgcccatcaagtggatggcgctggagtcc 2941 attctccgccggcggttcacccaccagagtgatgtgtggagttatggtgtgactgtgtgg 3001 gagctgatgacttttggggccaaaccttacgatgggatcccagcccgggagatccctgac 3061 ctgctggaaaagggggagcggctgccccagccccccatctgcaccattgatgtctacatg 3121 atcatggtcaaatgttggatgattgactctgaatgtcggccaagattccgggagttggtg 3181 tctgaattctcccgcatggccagggacccccagcgctttgtggtcatccagaatgaggac 3241 ttgggcccagccagtcccttggacagcaccttctaccgctcactgctggaggacgatgac 3301 atgggggacctggtggatgctgaggagtatctggtaccccagcagggcttcttctgtcca 3361 gaccctgccccgggcgctgggggcatggtccaccacaggcaccgcagctcatctaccagg 3421 agtggcggtggggacctgacactagggctggagccctctgaagaggaggcccccaggtct 3481 ccactggcaccctccgaaggggctggctccgatgtatttgatggtgacctgggaatgggg 3541 gcagccaaggggctgcaaagcctccccacacatgaccccagccctctacagcggtacagt 3601 gaggaccccacagtacccctgccctctgagactgatggctacgttgcccccctgacctgc 3661 agcccccagcctgaatatgtgaaccagccagatgttcggccccagcccccttcgccccga 3721 gagggccctctgcctgctgcccgacctgctggtgccactctggaaaggcccaagactctc 3781 tccccagggaagaatggggtcgtcaaagacgtttttgcctttgggggtgccgtggagaac 3841 cccgagtacttgacaccccagggaggagctgcccctcagccccaccctcctcctgccttc 3901 agcccagccttcgacaacctctattactgggaccaggacccaccagagcggggggctcca 3961 cccagcaccttcaaagggacacctacggcagagaacccagagtacctgggtctggacgtg 4021 ccagtgtgaaccagaaggccaagtccgcagaagccctgatgtgtcctcagggagcaggga 4081 aggcctgacttctgctggcatcaagaggtgggagggccctccgaccacttccaggggaac 4141 ctgccatgccaggaacctgtcctaaggaaccttccttcctgcttgagttcccagatggct 4201 ggaaggggtccagcctcgttggaagaggaacagcactggggagtctttgtggattctgag 4261 gccctgcccaatgagactctagggtccagtggatgccacagcccagcttggccctttcct 4321 tccagatcctgggtactgaaagccttagggaagctggcctgagaggggaagcggccctaa 4381 gggagtgtctaagaacaaaagcgacccattcagagactgtccctgaaacctagtactgcc 4441 ccccatgaggaaggaacagcaatggtgtcagtatccaggctttgtacagagtgcttttct 4501 gtttagtttttactttttttgttttgtttttttaaagatgaaataaagacccagggggag 4561 aatgggtgttgtatggggaggcaagtgtggggggtccttctccacacccactttgtccat 4621 ttgcaaatatattttggaaaacagctaaaaaaaaaaaaaaaaaa
[0241] In some embodiments, tumor antigens are classified into 4 major groups based on their expression profile. See van der Bruggen P, Stroobant V, Vigneron N, Van den Eynde B. Peptide database: T cell-defined tumor antigens. Cancer Immun 2013. URL: www.cancerimmunity.org/peptide/, incorporated herein by reference. Exemplary tumor antigens and their classification are summarized below as follows: [0242] 1) Unique Antigens: Unique to tumor cells (Table 1). These antigens arise from mutations in the gene that encodes the protein antigen. Commonly, the mutation(s) affects the coding sequence of the gene. In some examples, the mutation(s) are unique to the tumor of an individual subject or a small number of subjects. These unique antigens are generally not shared by tumors from different subjects. [0243] 2) Shared Antigens: Tumor specific antigens (Table 2). Shared antigens, unlike unique antigens, are expressed in multiple independent tumors. Tumor specific antigens are expressed in multiple tumors but not in normal cells. For example, these antigens are encoded by cancer-germline genes. [0244] 3) Shared Antigens: Differentiation antigens (Table 3). Differentiation antigens are expressed in the tumor as well as in the normal tissue from which the tumor originated.
For example, these antigens are expressed in a particular lineage of cells during a developmental stage. Since these antigens are not tumor-specific, targeting these antigens for cancer immunotherapy may cause autoimmunity toward the corresponding normal tissue, depending on whether the normal tissue is dispensible and whether the tissue expressing the antigen is surgically removed during the course of cancer treatment. 4) Shared Antigens: Overexpressed antigens (Table 4). These antigens are expressed in a variety of normal tissues and are overexpressed in tumor cells. For example, tables of tumor peptides, e.g., considered to be tumor antigens based on their recognition by T lymphocytes that also recognize tumor cells expressing the parent proteins. Each table below includes the protein or gene name, GeneCard information about the protein/gene, a peptide sequence from the protein (e.g., a minimum sequence for antigen specificity, e.g., recognized by T cells), and the position of the peptide in the full length protein sequence. In some examples, the peptide shown in the tables below is a human leukocyte antigen (HLA) presenting molecule, e.g., the peptide is presented onto a major histocompatibility complex (MHC) molecule. In Table 1, the underlined amino acid(s) are those that are different from the sequence of the version of the protein found in non-tumor cells, i.e., tumor cells contain a mutated form of the protein(s) where the mutation(s) are underlined in Table 1.
TABLE-US-00033 TABLE1 GeneCardinformation, SEQ Gene/protein incorporatedhereinbyreference Peptide IDNO: Position alpha-actinin-4 http://www.genecards.org/cgi- FIASNGVKLV 35 118-127 bin/carddisp.pl?gene=ACTN4 ARTC1 http://www.genecards.org/cgi- YSVYFNLPADTIYTN.sup.d 36 bin/carddisp.pl?gene=BBX BCR-ABLfusion http://www.genecards.org/cgi- SSKALQRPV 37 926-934 protein(b3a2) bin/carddisp.pl?gene=ABL1 GFKQSSKAL 38 922-930 ATGFKQSSKALQRPVAS 39 920-936 ATGFKQSSKALQRPVAS 40 920-936 B-RAF http://www.genecards.org/cgi- EDLTVKIGDFGLATEKS 41 586-614 bin/carddisp.pl?gene=BRAF RWSGSHQFEQLS CASP-5 http://www.genecards.org/cgi- FLIIWQNTM.sup.c 42 67-75 bin/carddisp.pl?gene=CASP5 CASP-8 http://www.genecards.org/cgi- FPSDSWCYF 43 576-484 bin/carddisp.pl?gene=CASP8 beta-catenin http://www.genecards.org/cgi- SYLDSGIHF 44 29-37 bin/carddisp.pl?gene=CTNNB1 Cdc27 http://www.genecards.org/cgi- FSWAMDLDPKGA.sup.b 45 760-771 bin/carddisp.pl?gene=CDC27 CDK4 http://www.genecards.org/cgi- ACDPHSGHFV 46 23-32 bin/carddisp.pl?gene=CDK4 CDJB2A http://www.genecards.org/cgi- AVCPWTWLR.sup.c 47 125-133 bin/carddisp.pl?gene=CDKN2A (p14ARF- ORF3) 111-119 (p16INK4a- ORF3) CLPP http://www.genecards.org/cgi- ILDKVLVHL 48 240-248 bin/carddisp.pl?gene=CLPP&search=clpp COA-1 http://www.genecards.org/cgi- TLYQDDTLTLQAAG.sup.b 49 447-460 bin/carddisp.pl?gene=UBXN11 TLYQDDTLTLQAAG.sup.b 50 447-460 dek-canfusion http://www.genecards.org/cgi- TMKQICKKEIRRLHQY 51 342-357 protein bin/carddisp.pl?gene=DEK http://www.genecards.org/cgi- bin/carddisp.pl?gene=NUP214 EFTUD2 http://www.genecards.org/cgi- KILDAVVAQK 52 668-677 bin/carddisp.pl?gene=EFTUD2 Elongation http://www.genecards.org/cgi- ETVSEQSNV 53 581-589 factor2 bin/carddisp.pl?gene=EEF2 ETV6-AML1 http://www.genecards.org/cgi- RIAECILGM 54 334-342 fusionprotein bin/carddisp.pl?gene=ETV6 IGRIAECILGMNPSR 55 332-346 http://www.genecards.org/cgi- IGRIAECILGMNPSR 56 332-346 bin/carddisp.pl?gene=RUXN1 FLT3-ITD http://www.genecards.org/cgi- YVDFREYEYY 57 591-600 bin/carddisp.pl?gene=FLT3 FN1 http://www.genecards.org/cgi- MIFEKHGFRRTTPP 58 2050-2063 bin/carddisp.pl?gene=FN1 GPNMB http://www.genecards.org/cgi- TLDWLLQTPK 59 179-188 bin/carddisp.pl?gene=GPNMB LDLR- http://www.genecards.org/cgi- WRRAPAPGA 60 315-323 fucosyltrans- bin/carddisp.pl?gene=LDLR feraseASfusion http://www.genecards.org/cgi- PVTWRRAPA 61 312-320 protein bin/carddisp.pl?gene=FUT1 HLA-A2.sup.a http://www.genecards.org/cgi- bin/carddisp.pl?gene=HLA-A HLA-A11.sup.a http://www.genecards.org/cgi- bin/carddisp.pl?gene=HLA-A hsp70-2 http://www.genecards.org/cgi- SLFEGIDIYT 62 286-295 bin/carddisp.pl?gene=HSPA2 AEPINIQTW 63 262-270 MART2 http://www.genecards.org/cgi- FLEGNEVGKTY 64 446-455 bin/carddisp.pl?gene=HHAT ME1 http://www.genecards.org/cgi- FLDEFMEGV 65 224-232 bin/carddisp.pl?gene=ME1 MUM-1 http://www.ncbi.nlm.nih.gov/ EEKLIVVLF 66 30-38 nuccore/11094678 MUM-2 http://www.genecards.org/cgi- SELFRSGLDSY 67 123-133 bin/carddisp.pl?gene=TRAPPC1 FRSGLDSYV 68 126-134 MUM-3 EAFIQPITR 69 322-330 neo-PAP http://www.genecards.org/cgi- RVIKNSIRLTL.sup.b 70 724-734 bin/carddisp.pl?gene=PAPOLG MyosinclassI http://www.genecards.org/cgi- KINKNPKYK 71 911-919 bin/carddisp.pl?gene=MYO1B NFYC http://www.genecards.org/cgi- QQITKTEV 72 275-282 bin/carddisp.pl?gene=NFYC OGT http://www.genecards.org/cgi- SLYKFSPFPL.sup.c 73 28-37 bin/carddisp.pl?gene=OGT OS-9 http://www.genecards.org/cgi- KELEGILLL 74 438-446 bin/carddisp.pl?gene=OS9 p53 http://www.genecards.org/cgi- VVPCEPPEV 75 217-225 bin/carddisp.pl?gene=TP53 pml- http://www.genecards.org/cgi- NSNHVASGAGEAAIETQ 76 RARalphafusion bin/carddisp.pl?gene=PML SSSSEEIV protein http://www.genecards.org/cgi- bin/carddisp.pl?gene=RARA PRDX5 http://www.genecards.org/cgi- LLLDDLLVSI 77 163-172 bin/carddisp.pl?gene=PRDX5 PTPRK http://www.genecards.org/cgi- PYYFAAELPPRNLPEP 78 667-682 bin/carddisp.pl?gene=PTPRK K-ras http://www.genecards.org/cgi- VVVGAVGVG 79 7-15 bin/carddisp.pl?gene=KRAS N-ras http://www.genecards.org/cgi- ILDTAGREEY 80 55-64 bin/carddisp.pl?gene=NRAS RBAF600 http://www.genecards.org/cgi- RPHVPESAF 81 329-337 bin/carddisp.pl?gene=UBR4 SIRT2 http://www.genecards.org/cgi- KIFSEVTLK 82 192-200 bin/carddisp.pl?gene=SIRT2 SNRPD1 http://www.genecards.org/cgi- SHETVIIEL 83 11-19 bin/carddisp.pl?gene=SNRPD1 SYT-SSX1or http://www.genecards.org/cgi- QRPYGYDQIM 84 402-410 402-SYT-SSX2 bin/carddisp.pl?gene=SS18 (SYT) fusionprotein http://www.genecards.org/cgi- 111-112 bin/carddisp.pl?gene=SSX1 (SSX2) http://www.genecards.org/cgi- bin/carddisp.pl?gene=SSX2 TGF-betaRII http://www.genecards.org/cgi- RLSSCVPVA.sup.c 85 131-139 bin/carddisp.pl?gene=TGFBR2 Triosephosphate http://www.genecards.org/cgi- GELIGILNAAKVPAD 86 23-37 isomerase bin/carddisp.pl?gene=TPI1 .sup.aThe mutation affects the HLA gene itself. .sup.bThe mutation is not located in the region encoding the peptide. .sup.cFrameshift product. .sup.dThe mutation creates a start codon (ATG) that opens an alternative open reading frame (ORF) encoding the antigenic peptide, which is recognized by regulatory T cells (Tregs).
TABLE-US-00034 TABLE2 GeneCardinformation, SEQ Gene/Protein incorporatedhereinbyreference Peptide IDNO: Position BAGE-1 http://www.genecards.org/cgi- AARAVFLAL 87 2-10 bin/carddisp.pl?gene=BAGE Cyclin-A1 http://www.genecards.org/cgi- FLDRFLSCM 88 227-235 bin/carddisp.pl?gene= SLIAAAAFCLA 89 341-351 CCNA1&search=cyclin-a1 GAGE-1,2,8 http://www.genecards.org/cgi- YRPRPRRY 90 9-16 bin/carddisp.pl?gene=GAGE1 http://www.genecards.org/cgi- bin/carddisp.pl?gene=GAGE2A http://www.genecards.org/cgi- bin/carddisp.pl?gene=GAGE8 GAGE-3,4,5,6, http://www.genecards.org/cgi- YYWPRPRRY 91 10-18 7 bin/carddisp.pl?gene=GAGE3 http://www.genecards.org/cgi- bin/carddisp.pl?gene=GAGE4 http://www.genecards.org/cgi- bin/carddisp.pl?gene=GAGE5 http://www.genecards.org/cgi- bin/carddisp.pl?gene=GAGE6 http://www.genecards.org/cgi- bin/carddisp.pl?gene=GAGE7 GnTV.sup.f http://www.genecards.org/cgi- VLPDVFIRC(V) 92 intron bin/carddisp.pl?gene=MGAT5 HERV-K-MEL MLAVISCAV 93 1-9 KK-LC-1 http://www.genecards.org/cgi- RQKRILVNL 94 76-84 bin/carddisp.pl?gene=CXorf61 KM-HN-1 http://www.genecards.org/cgi- NYNNFYRFL 95 196-104 bin/carddisp.pl?gene=CCDC110 EYSKECLKEF 96 499-508 EYLDLSDKI 97 770-778 LAGE-1 http://www.genecards.org/cgi- MLMAQEALAFL 98 ORF2 bin/carddisp.pl?gene=CTAG2 (1-11) SLLMWITQC 99 157-165 LAAQERRVPR 100 ORF2 (18-27) ELVRRILSR 101 103-111 APRGVRMAV 102 ORF2 (46-54) SLLMWITQCFLPVF 103 157-170 QGAMLAAQERRVPRAAEVPR 104 ORF2 (14-33) AADHRQLQLSISSCLQQL 105 139-156 CLSRRPWKRSWSAGSCPGMP 106 ORF2 HL (81-102) CLSRRPWKRSWSAGSCPGMP 107 ORF2 HL (81-102) ILSRDAAPLPRPG 108 108-120 AGATGGRGPRGAGA 109 37-50 MAGE-A1 http://www.genecards.org/cgi- EADPTGHSY 110 161-169 bin/carddisp.pl?gene=MAGEA1 KVLEYVIKV 111 278-186 SLFRAVITK 112 96-104 EVYDGREHSA 113 222-231 RVRFFFPSL 114 289-298 EADPTGHSY 115 161-169 REPVTKAEML 116 120-129 KEADPTGHSY 117 160-169 DPARYEFLW 118 258-266 ITKKVADLVGF 119 102-112 SAFPTTINF 120 62-70 SAYGEPRKL 121 230-238 RVRFFFPSL 122 289-298 SAYGEPRKL 123 230-238 TSCILESLFRAVITK 124 90-104 PRALAETSYVKVLEY 125 268-282 FLLLKYRAREPVTKAE 126 112-127 EYVIKVSARVRF 127 282-292 MAGE-A2 http://www.genecards.org/cgi- YLQLVFGIEV 128 157-166 bin/carddisp.pl?gene=MAGEA2 EYLQLVFGI 129 156-164 REPVTKAEML 130 127-136 EGDCAPEEK 131 212-220 LLKYRAREPVTKAE 132 121-134 MAGE-A3 http://www.genecards.org/cgi- EVDPIGHLY 133 168-176 bin/carddisp.pl?gene=MAGEA3 FLWGPRALV.sup.d 134 271-279 KVAELVHFL 135 112-123 TFPDLESEF 136 97-105 VAELVHFLL 137 113-121 MEVDPIGHLY 138 167-176 EVDPIGHLY 139 168-176 REPVTKAEML 140 127-136 AELVHFLLL.sup.i 141 114-122 MEVDPIGHLY 142 167-176 WQYFFPVIF 143 143-151 EGDCAPEEK 144 212-220 KKLLTQHFVQENYLEY 145 243-258 RKVAELVHFLLLKYR 146 111-125 KKLLTQHFVQENYLEY 147 243-258 ACYEFLWGPRALVETS 148 267-282 RKVAELVHFLLLKYR 149 111-125 VIFSKASSSLQL 150 149-160 VIFSKASSSLQL 151 149-160 VFGIELMEVDPIGHL 152 161-175 GDNQIMPKAGLLIIV 153 191-205 TSYVKVLHMVKISG 154 281-295 RKVAELVHFLLLKYRA 155 111-126 FLLLKYRAREPVTKAE 156 119-134 MAGE-A4 http://www.genecards.org/cgi- EVDPASNTY.sup.j 157 169-177 bin/carddisp.pl?gene=MAGEA4 GVYDGREHTV 158 230-239 NYKRCFPVI 159 143-151 SESLKMIF 160 156-163 MAGE-A6 http://www.genecards.org/cgi- MVKISGGPR 161 290-298 bin/carddisp.pl?gene=MAGEA6 EVDPIGHVY 162 168-176 REPVTKAEML 163 127-136 EGDCAPEEK 164 212-220 ISGGPRISY 165 293-301 LLKYRAREPVTKAE 166 121-134 MAGE-A9 http://www.genecards.org/cgi- ALSVMGVYV 167 223-231 bin/carddisp.pl?gene=MAGEA9 MAGE-A10 http://www.genecards.org/cgi- GLYDGMEHL.sup.l 168 254-262 bin/carddisp.pl?gene=MAGEA10 DPARYEFLW 169 290-298 MAGE-A12 http://www.genecards.org/cgi- FLWGPRALV.sup.e 170 271-279 bin/carddisp.pl?gene=MAGEA12 VRIGHLYIL 171 170-178 EGDCAPEEK 172 212-221 REPFTKAEMLGSVIR 173 127-141 AELVHFLLLKYRAR 174 114-127 MAGE-C1 http://www.genecards.org/cgi- ILFGISLREV 175 959-968 bin/carddisp.pl?gene=MAGEC1 KVVEFLAML 176 1083-1091 SSALLSIFQSSPE 177 137-149 SFSYTLLSL 178 450-458 VSSFFSYTL 179 779-787 MAGE-C2 http://www.genecards.org/cgi- LLFGLALIEV 180 191-200 bin/carddisp.pl?gene=MAGEC2 ALKDVEERV 181 336-344 SESIKKKVL 182 307-315 ASSTLYLVF 183 42-50 SSTLYLVFSPSSFST 184 43-57 mucin.sup.k http://www.genecards.org/cgi- PDTRPAPGSTAPPAHGVTSA 185 bin/carddisp.pl?gene=MUC1 NA88-A QGQHFLQKV 186 NY-ESO-1/LAGE-2 http://www.genecards.org/cgi- SLLMWITQC 187 157-165 bin/carddisp.pl?gene=CTAG1B MLMAQEALAFL 188 (1-11) ASGPGGGAPR 53-62 LAAQERRVPR 189 ORF2 LAAQERRVPR 190 (18-27) TVSGNILTIR 127-136 APRGPHGGAASGL 191 60-72 MPFATPMEA 192 94-102 KEFTVSGNILTI 193 124-135 MPFATPMEA 194 94-102 LAMPFATPM 195 92-100 ARGPESRLL 196 80-88 SLLMWITQCFLPVF 197 157-170 LLEFYLAMPFATPMEAELAR 198 87-111 RSLAQ LLEFYLAMPFATPMEAELAR 199 87-111 RSLAQ EFYLAMPFATPM 200 89-100 PGVLLKEFTVSGNILTIRLT 201 119-143 AADHR RLLEFYLAMPFA 202 86-97 QGAMLAAQERRVPRAAEVPR 203 ORF2 QGAMLAAQERRVPRAAEVPR 204 (14-33) PFATPMEAELARR 95-107 PGVLLKEFTVSGNILTIRLT 205 119-138 PGVLLKEFTVSGNILTIRLT 206 119-139 VLLKEFTVSG 121-130 AADHRQLQLSISSCLQQL 207 139-156 LLEFYLAMPFATPMEAELAR 208 87-111 RSLAQ LKEFTVSGNILTIRL 209 123-137 PGVLLKEFTVSGNILTIRLT 210 119-143 AADHR LLEFYLAMPFATPMEAELAR 211 87-111 RSLAQ KEFTVSGNILT 212 124-134 LLEFYLAMPFATPM 213 87-100 AGATGGRGPRGAGA 214 37-50 LYATVIHDI 215 715-723 SAGE http://www.genecards.org/cgi- ILDSSEEDK 216 103-111 bin/carddisp.pl?gene=SAGE1 Sp17 http://www.genecards.org/cgi- KASEKIFYV 217 41-49 bin/carddisp.pl?gene=SPA17 SSX-2 http://www.genecards.org/cgi- EKIQKAFDDIAKYFSK 218 19-34 bin/carddisp.pl?gene=SSX2 FGRLQGISPKI 219 101-111 WEKMKASEKIFYVYMKRK 220 37-54 KIFYVYMKRKYEAMT 221 45-59 KIFYVYMKRKYEAM 222 45-58 INKTSGPKRGKHAWTHRLRE 223 151-170 SSX-4 http://www.genecards.org/cgi- YFSKKEWEKMKSSEKIVYVY 224 31-50 bin/carddisp.pl?gene=SSX4 MKLNYEVMTKLGFKVTLPPF 225 51-70 KHASWTHRLRERKQLVVYEE 226 161-180 I LGFKVTLPPFMRSKRAADFH 227 61-80 KSSEKIVYVYMKLNYEVMTK 228 41-60 KHAWTHRLRERKQLVVYEEI 229 161-180 SLGWLFLLL 230 78-86 TAG-1 LSRLSNRLL 231 42-50 LSRLSNRLL 232 42-50 TAG-2 CEFHACWPAFTVLGE 233 34-48 TRAG-3 http://www.genecards.org/cgi- CEFHACWPAFTVLGE 234 34-48 bin/carddisp.pl?gene=CSAG2 CEFHACWPAFTVLGE 235 34-48 EVISCKLIKR 236 intron2 TRP2-INTG.sup.g http://www.genecards.org/cgi- RQKKIRIQL 237 21-29 bin/carddisp.pl?gene=DCT XAGE-1b/GAGED2a http://www.genecards.org/cgi- HLGSRQKKIRIQLRSQ 238 17-32 bin/carddisp.pl?gene=XAGE1B CATWKVICKSCISQTPG 239 33-49 .sup.dOnly processed by the intermiediate proteasome 5i (Guillaume et al. Proc. Natl. Acad. Sci. U.S.A. 107.43(2010): 18599-604). .sup.eSame peptide as MAGE-A3/A2 (aa 271-279). .sup.fAberrant transcript of N-acetyl glucosaminyl transferase V (GnTV) that is found only in melanomas. .sup.gIncompletely spliced transcript found only in melanomas. .sup.iThe processing of this peptide requires the immunoproteasome. .sup.jThis peptide is encoded by allele MAGE-4a, which is expressed in one third of MAGE-4 positive tumor samples. The other allele, namely MAGE-4b, encodes peptide EVDPTSNTY. .sup.kMHC-unrestricted recognition by CTL of a repeated motif that is unmasked in tumors due to mucin underglycosylation. Mucin underglycosylation also occurs in breast duct epithelial cells during lactation, but only at the extracellular apical surface, which is not accessible to T cells. .sup.lOnly processed by the intermiediate proteasome 1i51 (Guillaume et al. 2010).
TABLE-US-00035 TABLE3 GeneCardinformation, SEQ Gene/protein incorporatedhereinbyreference Peptide IDNO: Position CEA http://www.genecards.org/cgi- YLSGANLNL.sup.g 240 605-613 bin/carddisp.pl?gene=CEACAM5 IMIGVLVGV 241 691-699 GVLVGVALI 242 694-702 HLFGYSWYK 243 61-69 QYSWFVNGTF 244 268-277 TYACFVSNL 245 652-660 AYVCGIQNSVSANRS 246 568-582 DTGFYTLHVIKSDLVNEEATGQFRV 247 116-140 YSWRINGIPQQHTQV 248 625-639 TYYRPGVNLSLSC 249 425-437 EIIYPNASLLIQN 250 99-111 YACFVSNLATGRNNS 251 653-667 LWWVNNQSLPVSP 252 177-189 and 355-367 LWWVNNQSLPVSP 253 177-189 and 355-367 LWWVNNQSLPVSL 254 177-189 and 355-367 EIIYPNASLLIQN 255 99-111 NSIVKSITVSASG 256 666-678 gp100/Pme117 http://www.genecards.org/cgi- KTWGQYWQV 257 154-162 bin/carddisp.pl?gene=SILV (A)MLGTHTMEV 259 177(8)-186 ITDQVPFSV 259 209-217 YLEPGPVTA 260 280-288 LLDGTATLRL 261 457-466 VLYRYGSFSV 262 476-485 SLADTNSLAV 263 570-579 RLMKQDFSV 264 619-627 RLPRIFCSC 265 639-647 LIYRRRLMK 266 614-622 ALLAVGATK 267 17-25 IALNFPGSQK 268 86-95 ALNFPGSQK 269 87-95 ALNFPGSQK 270 87-95 VYFFLPDHL 271 intron4 RTKQLYPEW 272 40-42 and 47-52.sup.e HTMEVTVYHR 273 182-191 SSPGCQPPA 274 529-537 VPLDCVLYRY 275 471-480 LPHSSSHWL 276 630-638 SNDGPTLI 277 71-78 GRAMLGTHTMEVTVY 278 175-189 WNRQLYPEWTEAQRLD 279 44-59 TTEWVETTARELPIPEPE 280 420-437 TGRAMLGTHTMEVTVYH 281 174-190 GRAMLGTHTMEVTVY 282 175-189 mammaglobin-A http://www.genecards.org/cgi- PLLENVISK 283 23-31 bin/carddisp.pl?gene=SCGB2A2 Melan-A/MART-1 http://www.genecards.org/cgi- (E)AAGIGILTV 284 26(27)-35 bin/carddisp.pl?gene=MLANA ILTVILGVL 285 32-40 EAAGIGILTV 286 26-35 AEEAAGIGIL(T) 287 24-33(34) RNGYRALMDKS 288 51-61 YTTAEEAAGIGILTVILGVLLLIGCW 2892 21-50 YCRR EEAAGIGILTVI 290 25-36 AAGIGILTVILGVL 291 27-40 APPAYEKLpSAEQ.sup.f 292 100-111 EEAAGIGILTVI 293 25-36 RNGYRALMDKSLHVGTQCALTRR 294 51-73 MPREDAHFIYGYPKKGHGHS 295 1-20 KNCEPVVPNAPPAYEKLSAE 296 91-110 NY-BR-1 http://www.genecards.org/cgi- SLSKILDTV 297 904-912 bin/carddisp.pl?gene=ANKRD30A OA1 http://www.genecards.org/cgi- LYSACFWWL 298 126-134 bin/carddisp.pl?gene=GPR143 PAP http://www.genecards.org/cgi- FLFLLFFWL 299 18-26 bin/carddisp.pl?gene=ACPP TLMSAMTNL 300 112-120 ALDVYNGLL 301 299-307 PSA http://www.genecards.org/cgi- FLTPKKLQCV 302 165-174 bin/carddisp.pl?gene=KLK3 VISNDVCAQV 303 178-187 RAB38/NY-MEL-1 http://www.genecards.org/cgi- VLHWDPETV 304 50-58 bin/carddisp.pl?gene=RAB38 TRP-1/gp75 http://www.genecards.org/cgi- MSLQRQFLR 305 alt.ORF bin/carddisp.pl?gene=TYRP1 ISPNSVFSQWRVVCDSLEDYD 306 277-297 SLPYWNFATG 307 245-254 SQWRVVCDSLEDYDT 308 284-298 TRP-2 http://www.genecards.org/cgi- SVYDFFVWL 309 180-188 bin/carddisp.pl?gene=DCT TLDSQVMSL 310 360-368 LLGPGRPYR 311 197-205 LLGPGRPYR 312 197-2025 ANDPIFVVL 313 387-395 QCTEVRADTRPWSGP 314 60-74 ALPYWNFATG 315 241-250 tyrosine http://www.genecards.org/cgi- KCDICTDEY 316 243-251 bin/carddisp.pl?gene=TYR SSDYVIPIGTY 317 146-156 MLLAVLYCL 318 1-9 CLLWSFQTSA 319 8-17 YMDGTMSQV 320 369-377 AFLPWHRLF 321 206-214 IYMDGTADFSF 322 368-373 and 336-340.sup.e QCSGNFMGF 323 90-98 TPRLPSSADVEF 324 309-320 LPSSADVEF 325 312-320 LHHAFVDSIF 326 388-397 SEIWRDIDF.sup.d 327 192-200 QNILLSNAPLGPQFP 328 56-70 SYLQDSDPDSFQD 329 450-462 FLLHHAFVDSIFEQWLQRHRP 330 386-406 .sup.dDifferent alleles encoding tyrosinase have been described. In 50% of Caucasians, the serine residue of nonapeptide SEIWRDIDF is replaced by a tyrosine. .sup.eThe peptide is composed of two non-contiguous fragments that are spliced by the proteasome. .sup.fPhosphopeptide. .sup.gSeems to be poorly processed by tumor cells (Fauquembergue et al. J. Immunother. 33.4(2010): 402-13).
TABLE-US-00036 TABLE4 GeneCardinformation, SEQ Gene/Protein incorporatedhereinbyreference Peptide IDNO: Position adipophilin http://www.genecards.org/cgi- SVASTITGV 331 129-137 bin/carddisp.pl?gene=PLIN2 AIM-2 http://www.genecards.org/cgi- RSDSGQQARY 332 intron bin/carddisp.pl?gene=LOC51152 ALDH1A1 http://www.genecards.org/cgi- LLYKLADLI 333 88-96 bin/carddisp.pl?gene=ALDH1A1 BCLX(L) http://www.genecards.org/cgi- YLNDHLEPWI 334 173-182 bin/carddisp.pl?gene=BCL2L1 BING-4 http://www.genecards.org/cgi- CQWGRLWQL 335 ORF2 bin/carddisp.pl?gene=WDR46 CALCA http://www.genecards.org/cgi- VLLQAGSLHA 336 16-25 bin/carddisp.pl?gene=CALCA CD45 http://www.genecards.org/cgi- KFLDALISL 337 556-564 bin/carddisp.pl?gene=PTPRC CPSF http://www.genecards.org/cgi- KVHPVIWSL 338 250-258 bin/carddisp.pl?gene=CPSF1 LMLQNALTTM 339 1360-1369 cyclinD1 http://www.genecards.org/cgi- LLGATCMFV 340 101-109 bin/carddisp.pl?gene=CCND1 NPPSMVAAGSVVAAV 341 198-212 DKK1 http://www.genecards.org/cgi- ALGGHPLLGV 342 20-29 bin/carddisp.pl?gene=DKK1 ENAH(hMena) http://www.genecards.org/cgi- TMNGSKSPV 343 502-510 bin/carddisp.pl?gene=ENAH EpCAM http://www.genecards.org/cgi- RYQLDPKFI 344 173-181 bin/carddisp.pl?gene=EPCAM EphA3 http://www.genecards.org/cgi- DVTFNIICKKCG 345 356-367 bin/carddisp.pl?gene=EPHA3 EZH2 http://www.genecards.org/cgi- FMVEDETVL 346 120-128 bin/carddisp.pl?gene=EZH2 FINDEIFVEL 347 165-174 KYDCFLHPF 348 291-299 KYVGIEREM 349 735-743 FGF5 http://www.genecards.org/cgi- NTYASPRFK.sup.f 350 172-176 bin/carddisp.pl?gene=FGF5 and 204-207 glypican-3 http://www.genecards.org/cgi- FVGEFFTDV 351 144-152 bin/carddisp.pl?gene= EYILSLEEL 352 298-306 GPC3&search=GLYPICAN-3 G250/MN/CAIX http://www.genecards.org/cgi- HLSTAFARV 353 254-262 bin/carddisp.pl?gene=CA9 HER-2/neu http://www.genecards.org/cgi- KIFGSLAFL 354 369-377 bin/carddisp.pl?gene=ERBB2 IISAVVGIL 355 654-662 ALCRWGLLL 356 5-13 ILHNGAYSL 357 435-443 RLLQETELV 358 689-697 VVLGVVFGI 359 665-673 YMIMVKCWMI 360 952-961 HLYQGCQVV 361 48-56 YLVPQQGFFC 362 1023-1032 PLQPEQLQV 363 391-399 TLEEITGYL 364 402-410 ALIHHNTHL 365 466-474 PLTSIISAV 366 650-658 VLRENTSPK 367 754-762 TYLPTNASL 368 63-71 IDO1 http://www.genecards.org/cgi- ALLEIASCL 369 199-207 bin/carddisp.pl?gene=IDO1 IGF2B3 http://www.genecards.org/cgi- NLSSAEVVV 370 515-523 bin/carddisp.pl?gene= RLLVPTQFV 371 199-207 GPC3&search=GLYPICAN-3 IL13Ralpha2 http://www.genecards.org/cgi- WLPFGFILI 372 345-353 bin/carddisp.pl?gene=IL13RA2 Intestinal http://www.genecards.org/cgi- SPRWWPTCL 373 alt.ORF carboxyl bin/carddisp.pl?gene=CES2 esterase alpha- http://www.genecards.org/cgi- GVALQTMKQ 374 542-550 foetoprotein bin/carddisp.pl?gene=AFP FMNKFIYEI 375 158-166 QLAVSVILRV 376 364-373 Kallikrein4 http://www.genecards.org/cgi- FLGYLILGV 377 11-19 bin/carddisp.pl?gene=KLK4 SVSESDTIRSISIAS 378 125-139 LLANGRMPTVLQCVN 279 155-169 RMPTVLQCVNVSVVS 380 160-174 KIF20A http://www.genecards.org/cgi- LLSDDDVVV 381 12-20 bin/carddisp.pl?gene= AQPDTAPLPV 382 284-293 KIF20A&search=KIF20A CIAEQYHTV 383 809-817 Lengsin http://www.genecards.org/cgi- FLPEFGISSA 384 270-279 bin/carddisp.pl?gene=CSF1 M-CSF http://www.genecards.org/cgi- LPAVVGLSPGEQEY 385 alt.ORF bin/carddisp.pl?gene=CSF1 MCSP http://www.genecards.org/cgi- VGQDVSVLFRVTGALQ 386 693-708 bin/carddisp.pl?gene=CSPG4 mdm-2 http://www.genecards.org/cgi- VLFYLGQY 387 53-60 bin/carddisp.pl?gene=MDM2 Meloe TLNDECWPA 388 36-44 FGRLQGISPKI 389 32-44 CPPWHPSERISSTL 390 24-37 MMP-2 http://www.genecards.org/cgi- GLPPDVQRV.sup.h 391 560-568 bin/carddisp.pl?gene=MMP2 MMP-7 http://www.genecards.org/cgi- SLFPNSPKWTSK 392 96-107 bin/carddisp.pl?gene=MMP7 MUC1 http://www.genecards.org/cgi- STAPPVHNV 393 950-958 bin/carddisp.pl?gene=MUC1 LLLLTVLTV 394 12-20 PGSTAPPAHGVT 395 repeated region MUC5AC http://www.genecards.org/cgi- TCQPTCRSL 396 716-724 bin/carddisp.pl?gene=MUC5AC p53 http://www.genecards.org/cgi- LLGRNSFEV 397 264-272 bin/carddisp.pl?gene=TP53 RMPEAAPPV 398 65-73 SQKTYQGSY 399 99-107 PGTRVRAMAIYKQ 400 153-165 HLIRVEGNLRVE 401 193-204 PAX5 http://www.genecards.org/cgi- TLPGYPPHV 402 311-319 bin/carddisp.pl?gene=PAX5 PBF http://www.genecards.org/cgi- CTACRWKKACQR 403 499-510 bin/carddisp.pl?gene=ZNF395 PRAME http://www.genecards.org/cgi- VLDGLDVLL 404 100-108 bin/carddisp.pl?gene=PRAME SLYSFPEPEA 405 142-151 ALYVDSLFFL 406 300-309 SLLQHLIGL 407 425-433 LYVDSLFFL.sup.c 408 301-309 PSMA http://www.genecards.org/cgi- NYARTEDFF 409 178-186 bin/carddisp.pl?gene=FOLH1 RAGE-1 http://www.genecards.org/cgi- LKLSGVVRL 410 352-360 bin/carddisp.pl?gene=RAGE PLPPARNGGL.sup.g 411 32-40 SPSSNRIRNT 412 11-20 RGS5 http://www.genecards.org/cgi- LAALPHSCL 413 5-13 bin/carddisp.pl?gene=RGS5 GLASFKSFLK 414 74-83 RhoC http://www.genecards.org/cgi- RAGLQVRKNK 415 176-185 bin/carddisp.pl?gene=RhoC RNF43 http://www.genecards.org/cgi- ALWPWLLMA(T) 416 11-19(20) bin/carddisp.pl?gene=RNF43 NSQPVWLCL 417 721-729 RU2AS http://www.genecards.org/cgi- LPRWPPPQL 418 antisense bin/carddisp.pl?gene=DCDC2 secernin1 http://www.genecards.org/cgi- KMDAEHPEL 419 196-204 bin/carddisp.pl?gene=SCRN1 SOX10 http://www.genecards.org/cgi- AWISKPPGV 420 332-340 bin/carddisp.pl?gene=SOX10 SAWISKPPGV 421 331-340 STEAP1 http://www.genecards.org/cgi- MIAVFLPIV 422 292-300 bin/carddisp.pl?gene=STEAP1 HQQYFYKIPILVINK 423 102-116 survivin http://www.genecards.org/cgi- ELTLGEFLKL 424 95-104 bin/carddisp.pl?gene=BIRC5 TLGEFLKLDRERAKN 425 97-111 Telomerase http://www.genecards.org/cgi- ILAKFLHWL.sup.e 426 540-548 bin/carddisp.pl?gene=TERT RLVDDFLLV 427 865-873 RPGLLGASVLGLDDI 428 672-686 LTDLQPYMRQFVAHL 429 766-780 VEGF http://www.genecards.org/cgi- SRFGGAVVR.sup.i 430 bin/carddisp.pl?gene=VEGFA WT1 http://www.genecards.org/cgi- TSEKRPFMCAY 431 317-327 bin/carddisp.pl?gene=WT1 CMTWNQMNL 432 235-243 LSHLQMHSRKH 433 337-347 KRYFKLSHLQMHSRKH 434 332-347 .sup.cThe antigen is recognized by CTLs bearing an NK inhibitory receptor that prevents lysis of cells expressing certain HLA-C molecules. .sup.ePoorly or not processed (Parkhurst, 2004; Ayyoub, 2001). .sup.fThe peptide is composed of two non-contiguous fragments that are spliced. .sup.gAlternative transcript. .sup.hMMP-2 is expressed unbiquitously but melanoma cells cross-present, in an v3-dependent manner, an antigen derived from secreted MMP-2. .sup.iThe epitope is located in the untranslated region.
Immunomodulators
[0245] Compositions, methods, and devices of the present invention comprise immunomodulators including, but not limited to, TLR ligands, growth factors, and products of dying cells, e.g. heat shock proteins, with means to stimulate dendritic cell activation. Immunomodulators are used alone or in combination with GM-CSF, CpG-ODN sequences, or cancer antigens. Immunomodulators are used simultaneously or sequentially with GM-CSF, CpG-ODN sequences, or cancer antigens.
[0246] All known TLR ligands found either on a cell surface or an internal cellular compartment are encompassed by the compositions, methods, and devices of the present invention. Exemplary TLR ligands include, but are not limited to, triacyl lipoproteins (TLR1); lipoproteins, gram positive peptidoglycan, lipteichoic acids, fungi, and viral glycoproteins (TLR2); double-stranded RNA, poly I:C (TLR 3); lipopolysaccaride, viral glycoproteins (TLR 4); flagellin (TLR5); diacyl lipoproteins (TLR6); small synthetic compounds, single-stranded RNA (TLR7 and TLR 8); unmethylated CpG DNA (TLR9); Profilin (TLR11). Also included as TRL ligands are host molecules like fibronectin and heat shock proteins (HSPs). Host TLR ligands are also encompassed by the present invention. The role of TLRs in innate immunity and the signaling molecules used to activate and inhibit them are known in the art (for a review, see Holger K. Frank B., Hessel E., and Coffman R L. Therapeutic targeting of innate immunity with Toll-like receptor agonists and antagonists. Nature Medicine 13, 552-559 (2007), incorporated herein by reference).
[0247] All known growth factors are encompassed by the compositions, methods, and devices of the present invention. Exemplary growth factors include, but are not limited to, transforming growth factor beta (TGF-), granulocyte-colony stimulating factor (G-CSF), granulocyte-macrophage colony stimulating factor (GM-CSF), nerve growth factor (NGF), neurotrophins, Platelet-derived growth factor (PDGF), erythropoietin (EPO), thrombopoietin (TPO), myostatin (GDF-8), growth differentiation factor-9 (GDF9), acidic fibroblast growth factor (aFGF or FGF-1), basic fibroblast growth factor (bFGF or FGF-2), epidermal growth factor (EGF), hepatocyte growth factor (HGF). The present invention encompasses cytokines as well as growth factors for stimulating dendritic cell activation. Exemplary cytokines include, but are not limited to, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12 IL-15, IL-17, IL-18, TNF-, IFN-, and IFN-.
[0248] Indications of cell death and products of dying cells stimulate dendritic cell activation. As such, all products of dying cells are encompassed by the compositions, methods, and devices of the present invention. Exemplary cell death products include, but are not limited to, any intracellular feature of a cell such as organelles, vesicles, cytoskeletal elements, proteins, DNA, and RNA. Of particular interest are heat shock proteins expressed when a cell is under stress and which are released upon cell death. Exemplary heat shock proteins include, but are not limited to, Hsp10, Hsp20, Hsp27, Hsp33, Hsp40, Hsp60, Hsp70, Hsp71, Hsp72, Grp78, Hsx70, Hsp84, Hsp90, Grp94, Hsp100, Hsp104, Hsp110.
Microenvironments and Vaccine Efficiency
[0249] The devices/scaffold described herein represent an infection-mimicking microenvironment. Each device constitutes a factory that attracts/accepts, educates/stimulates and sends forth to surrounding bodily tissues activated dendritic cells that are capable of stimulating/enhancing an immune response to a particular antigen. Specifically, the scaffold devices are implanted or coated with pathogenic molecules to mimic and infectious microenvironment to further activate the dendritic cell response.
[0250] Appropriately mimicking aspects of infection with material systems dramatically impacts tumor progression when applied as cancer vaccines by continuously recruiting, activating and homing DCs to LNs. The first PLG vaccine, using GM-CSF alone, led to a batch process where host DCs were recruited by GM-CSF to reside at a site of tumor antigen presentation, and were trapped until GM-CSF levels fell and the cells could become activated and disperse (see US 2008/0044900 A1, incorporated herein by reference). Temporal variation of the local GM-CSF concentration allowed control over the number of recruited DCs, and the timing of their activation and dispersement. Although the best GM-CSF-based vaccine was able to confer protective immunity in nearly a quarter of the animals tested, approximately 26% of the recruited DCs were activated (240,000 DCs) and approximately 6% of DCs dispersed to the LNs. High levels of GM-CSF recruited large numbers of DC, but also limited DC activation, leaving potentially therapeutic DCs entrapped within scaffolds. These results motivated the development of an improved system that mimicked bacterial infection by locally presenting CpG-ODNs as an overriding danger signal, that opposed GM-CSF inhibition of DC activation and dispersement. These devices described herein represent significant advances by mediating increased and continuous egress of DCs.
[0251] CpG-ODN molecules were condensed with PEI to not only promote ODN uptake into DCs and localization to its TLR-9 receptor, but also to electrostatically immobilize it in PLG matrices to be presented simultaneously with tumor antigens. In vitro results indicated that PEI-CpG-ODN condensates can decondense within DCs and stimulate TLR signaling that promoted DC activation and dispersement toward the lymph node derived chemokine, CCL19, in the presence of inhibitory levels of GM-CSF (500 ng/ml) (US 2013-0202707, incorporated herein by reference).
[0252] As described in detail in US 2013-0202707, the vaccine devices of the invention advantageously allow for fine control of cell behavior and programming in situ.
Scaffold Compositions and Architecture
[0253] Components of the scaffolds are organized in a variety of geometric shapes (e.g., discs, beads, pellets), niches, planar layers (e.g., thin sheets). For example, discs of about 0.1-200 millimeters in diameter, e.g., 5, 10, 20, 40, 50 millimeters are implanted subcutaneously. The disc may have a thickness of 0.1 to 10 milimeters, e.g., 1, 2, 5 milimeters. The discs are readily compressed or lyophilized for administration to a patient. An exemplary disc for subcutaneous administration has the following dimensions: 8 milimeters in diameter and 1 milimeter in thickness. Multicomponent scaffolds are optionally constructed in concentric layers each of which is characterized by different physical qualities (% polymer, % crosslinking of polymer, chemical composition of scaffold, pore size, porosity, and pore architecture, stiffness, toughness, ductility, viscoelasticity, and or composition of bioactive substances such as growth factors, homing/migration factors, differentiation factors. Each niche has a specific effect on a cell population, e.g., promoting or inhibiting a specific cellular function, proliferation, differentiation, elaboration of secreted factors or enzymes, or migration. Cells incubated in the scaffold are educated and induced to migrate out of the scaffold to directly affect a target tissue, e.g., and injured tissue site. For example, stromal vascular cells and smooth muscle cells are useful in sheetlike structures are used for repair of vessel-like structures such as blood vessels or layers of the body cavity. For example, such structures are used to repair abdominal wall injuries or defects such as gastroschisis. Similarly, sheetlike scaffolds seeded with dermal stem cells and/or keratinocytes are used in bandages or wound dressings for regeneration of dermal tissue. The device is placed or transplanted on or next to a target tissue, in a protected location in the body, next to blood vessels, or outside the body as in the case of an external wound dressing. Devices are introduced into or onto a bodily tissue using a variety of known methods and tools, e.g., spoon, tweezers or graspers, hypodermic needle, endoscopic manipulator, endo- or trans-vascular-catheter, stereotaxic needle, snake device, organ-surface-crawling robot (United States Patent Application 20050154376; Ota et al., 2006, Innovations 1:227-231), minimally invasive surgical devices, surgical implantation tools, and transdermal patches. Devices can also be assembled in place, for example by senquentially injecting or inserting matrix materials. Scaffold devices are optionally recharged with cells or with bioactive compounds, e.g., by sequential injection or spraying of substances such as growth factors or differentiation factors.
[0254] A scaffold or scaffold device is the physical structure upon which or into which cells associate or attach, and a scaffold composition is the material from which the structure is made. For example, scaffold compositions include biodegradable or permanent materials such as those listed below. The mechanical characteristics of the scaffold vary according to the application or tissue type for which regeneration is sought. It is biodegradable (e.g., collagen, alginates, polysaccharides, polyethylene glycol (PEG), poly(glycolide) (PGA), poly(L-lactide) (PLA), or poly(lactide-co-glycolide) (PLGA), poly lactic-coglycolic acid, or permanent (e.g., silk). In the case of biodegradable structures, the composition is degraded by physical or chemical action, e.g., level of hydration, heat or ion exchange or by cellular action, e.g., elaboration of enzyme, peptides, or other compounds by nearby or resident cells. The consistency varies from a soft/pliable (e.g., a gel) to glassy, rubbery, brittle, tough, elastic, stiff. The structures contain pores, which are nanoporous, microporous, or macroporous, and the pattern of the pores is optionally homogeneous, heterogenous, aligned, repeating, or random.
[0255] Alginates are versatile polysaccharide based polymers that may be formulated for specific applications by controlling the molecular weight, rate of degradation and method of scaffold formation. Coupling reactions can be used to covalently attach bioactive epitopes, such as the cell adhesion sequence RGD to the polymer backbone. Alginate polymers are formed into a variety of scaffold types. Injectable hydrogels can be formed from low MW alginate solutions upon addition of a cross-linking agents, such as calcium ions, while macroporous scaffolds are formed by lyophilization of high MW alginate discs. Differences in scaffold formulation control the kinetics of scaffold degradation. Release rates of morphogens or other bioactive substances from alginate scaffolds is controlled by scaffold formulation to present morphogens in a spatially and temporally controlled manner. This controlled release not only eliminates systemic side effects and the need for multiple injections, but can be used to create a microenvironment that activates host cells at the implant site and transplanted cells seeded onto a scaffold.
##STR00001##
[0256] The scaffold comprises a biocompatible polymer matrix that is optionally biodegradable in whole or in part. A hydrogel is one example of a suitable polymer matrix material. Examples of materials which can form hydrogels include polylactic acid, polyglycolic acid, PLGA polymers, alginates and alginate derivatives, gelatin, collagen, agarose, natural and synthetic polysaccharides, polyamino acids such as polypeptides particularly poly(lysine), polyesters such as polyhydroxybutyrate and poly-epsilon.-caprolactone, polyanhydrides; polyphosphazines, poly(vinyl alcohols), poly(alkylene oxides) particularly poly(ethylene oxides), poly(allylamines)(PAM), poly(acrylates), modified styrene polymers such as poly(4-aminomethylstyrene), pluronic polyols, polyoxamers, poly(uronic acids), poly(vinylpyrrolidone) and copolymers of the above, including graft copolymers.
[0257] The scaffolds are fabricated from a variety of synthetic polymers and naturally-occurring polymers such as, but not limited to, collagen, fibrin, hyaluronic acid, agarose, and laminin-rich gels. One preferred material for the hydrogel is alginate or modified alginate material. Alginate molecules are comprised of (1-4)-linked -D-mannuronic acid (M units) and a L-guluronic acid (G units) monomers, which can vary in proportion and sequential distribution along the polymer chain. Alginate polysaccharides are polyelectrolyte systems which have a strong affinity for divalent cations (e.g., Ca.sup.+2, Mg.sup.+2, Ba.sup.+2) and form stable hydrogels when exposed to these molecules. See Martinsen A., et al., Biotech. & Bioeng., 33 (1989) 79-89.) For example, calcium cross-linked alginate hydrogels are useful for dental applications, wound dressings chondrocyte transplantation and as a matrix for other cell types.
[0258] An exemplary device utilizes an alginate or other polysaccharide of a relatively low molecular weight, preferably of size which, after dissolution, is at the renal threshold for clearance by humans, e.g., the alginate or polysaccharide is reduced to a molecular weight of 1000 to 80,000 daltons. Preferably, the molecular mass is 1000 to 60,000 daltons, particularly preferably 1000 to 50,000 daltons. It is also useful to use an alginate material of high guluronate content since the guluronate units, as opposed to the mannuronate units, provide sites for ionic crosslinking through divalent cations to gel the polymer. U.S. Pat. No. 6,642,363, incorporated herein by reference, discloses methods for making and using polymers containing polysachharides such as alginates or modified alginates that are particularly useful for cell transplantation and tissue engineering applications.
[0259] Useful polysaccharides other than alginates include agarose and microbial polysaccharides such as those listed in the table below.
TABLE-US-00037 Polysaccharide Scaffold Compositions Polymers.sup.a Structure Fungal Pullulan (N) 1,4-; 1,6--D-Glucan Scleroglucan (N) 1,3; 1,6--D-Glucan Chitin (N) 1,4--D-Acetyl Glucosamine Chitosan (C) 1,4-.-D-N-Glucosamine Elsinan (N) 1,4-; 1,3--D-Glucan Bacterial Xanthan gum (A) 1,4-.-D-Glucan with D-mannose; D-glucuronic Acid as side groups Curdlan (N) 1,3-.-D-Glucan (with branching) Dextran (N) 1,6--D-Glucan with some 1,2;1,3; 1,4--linkages Gellan (A) 1,4-.-D-Glucan with rhamose, D-glucuronic acid Levan (N) 2,6--D-Fructan with some -2,1-branching Emulsan (A) Lipoheteropolysaccharide Cellulose (N) 1,4--D-Glucan .sup.aN-neutral, A = anionic and C = cationic.
[0260] The scaffolds of the invention are porous or non-porous. For example, the scaffolds are nanoporous having a diameter of less than about 10 nm; microporous wherein the diameter of the pores are preferably in the range of about 100 nm-20 m; or macroporous wherein the diameter of the pores are greater than about 20 m, more preferably greater than about 100 m and even more preferably greater than about 400 m. In one example, the scaffold is macroporous with aligned pores of about 400-500 m in diameter. The preparation of polymer matrices having the desired pore sizes and pore alignments are described in the Examples. Other methods of preparing porous hydrogel products are known in the art. (U.S. Pat. No. 6,511,650, incorporated herein by reference).
Bioactive Compositions
[0261] The device includes one or more bioactive compositions. Bioactive compositions are purified naturally-occurring, synthetically produced, or recombinant compounds, e.g., polypeptides, nucleic acids, small molecules, or other agents. For example, the compositions include GM-CSF, CpG-ODN, and tumor antigens or other antigens. For example, the compositions described herein include an inhibitor of an immune inhibitory protein (e.g., an inhibitor of CTLA4, PD1, PDL1, B7-H3, B7-H4, LAG3, 2B4, BTLA, TIM3, A2aR, or a killer inhibitory receptor). For example, the composition includes an antibody or fragment thereof or a protein that binds to an immune inhibitory protein (e.g., CTLA4, PD1, PDL1, B7-H3, B7-H4, LAG3, 2B4, BTLA, TIM3, A2aR, or a killer inhibitory receptor). In preferred embodiments, the composition includes an antibody of fragment thereof that binds to CTLA4, PD1, or PDL1.
[0262] The compositions described herein are purified. Purified compounds are at least 60% by weight (dry weight) the compound of interest. Preferably, the preparation is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight the compound of interest. Purity is measured by any appropriate standard method, for example, by column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis.
[0263] Coupling of the polypeptides, antibodies, or fragments thereof to the polymer matrix is accomplished using synthetic methods known to one of ordinary skill in the art. Approaches to coupling of peptides to polymers are discussed in Hirano and Mooney, Advanced Materials, p. 17-25 (2004). Other useful bonding chemistries include those discussed in Hermanson, Bioconjugate Techniques, p. 152-185 (1996), particularly by use of carbodiimide couplers, DCC and DIC (Woodward's Reagent K). Polypeptides contain a terminal amine group for such carbodiimide bonding. The amide bond formation is preferably catalyzed by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), which is a water soluble enzyme commonly used in peptide synthesis.
Control of Release Kinetics of Bioactive Compositions
[0264] The release profile of bioactive compositions such as GM-CSF is controlled using a number of different techniques, e.g., encapsulation, nature of attachment/association with the scaffold, porosity of the scaffold, and particle size of the bioactive compositions.
[0265] For example, GM-CSF is encapsulated as one means by which to incorporate GM-CSF into the scaffolds. GM-CSF was first encapsulated into PLG microspheres, and then these GM-CSF loaded microspheres were then in a gas foaming process to develop macroporous PLG scaffolds. The incorporation of GM-CSF into the microspheres causes the GM-CSF to be more deeply embedded into the polymer, which causes the device to sustain the initial pulse of GM-CSF delivery over days 1-5. Other incorporation methods are optionally used to alter or fine tune the duration of the GM-CSF pulse as desired, which would in turn change the kinetics of DC recruitment. For example, foaming PLG particles mixed with lyophilized GM-CSF results in GM-CSF that is associated more with the surface of the polymer scaffold, and the protein diffuses more quickly.
[0266] Alternative methods for scaffold fabrication that modify release kinetics include modifying the physical structure of the scaffolds pores, thereby leading to different degradation times and release kinetics (change pore size or total porosity as a percentage of volume), e.g., as described in Riddle et al., Role of poly(lactide-co-glycolide) particle size on gas-foamed scaffolds. J Biomater Sci Polym Ed. 2004; 15(12):1561-70. Another way to alter release kinetics is to modify the composition, i.e., the raw materials from which the scaffold is made, thereby altering the release properties. For example, different polymers, e.g., alginate, PLA, PGA, or using PLGA are used. Also, use of the polymers with different ratios of glycolic and lactic acid) leads to different release profiles. For example, a variety of PLGs, differing in composition (lactide to glycolide ratio) and molecular weight are used to prepare microspheres (5-50 m) using known double emulsion (water/oil/water) process, followed by preparation of scaffolds using particulate PLG and PLG microspheres using gas foaming/particulate leaching techniques (Ennett et al., Temporally regulated delivery of VEGF in vitro and in vivo. J Biomed Mater Res A. 2006 October; 79(1). Another technique involves incorporating the protein into different compartments (e.g., encapsulating proteins PLG microspheres or simple mixing and lyophilizing with the polymer before foaming). Methods of making a scaffold described herein include using gas foaming, e.g., as described in detail in Harris et al. J. Biomed. Materials Res. Part A. 42.3(1998)396-402 and Sheridan et al. J. Control. Rel. 64(2000)91-102, both incorporated herein by reference. In other embodiments, wires (e.g., a template containing multiple wires) are used as porogens, i.e., to create pores in the scaffold, e.g., to create aligned pores.
Charging and/or recharging the device
[0267] A bioactive composition such as GM-CSF is incorporated within different layers/compartments of the device, thereby allowing multiple pulses of GM-CSF to be delivered. Each pulse charges (or recharges) the device with an influx of DCs. Scaffolds are fabricated using a variety of methods to create multiple pulses of GM-CSF (or other bioactive agents). For example, such devices are made by incorporating the protein into different compartments (e.g., encapsulating proteins PLG microspheres or simple mixing and lyophilizing with the polymer before foaming) thereby creating 2 or more distinct release profiles (i.e., pulses) of the protein (e.g., as described in Richardson et al., Polymeric system for dual growth factor delivery. Nat Biotechnol. 2001 November; 19(11)).
[0268] Alternatively, the protein is encapsulated in fast degrading PLG microspheres (e.g. low MW, 50:50 ratio) and slow degrading PLG microspheres (high MW, 85:15 ratio). Then these microspheres are mixed together to be used later to fabricate the scaffolds. Therefore, the protein is encapsulated in both fast a degrading polymer and a slow degrading polymer, thereby resulting in at least 2 distinct releases kinetics and pulses of delivery. This method is utilized to create 3, 4, 5, or more different kinds of microspheres, the ratiometric characteristics of which differ, thereby leading to 3, 4, 5 or more pulses of release of the bioactive composition such as GM-CSF.
[0269] Another approach to making a device that delivers more than one pulse is to fabricate a layered scaffold. Layered scaffolds are made by compression molding on different scaffold formulations with another. For example, the raw materials (sucrose+PLG1+Protein) is compressed in a mold and a slightly varied formulation (sucrose+PLG2+Protein) is also compressed in a mold. Then these two layers are compressed together and then foamed, resulting in a bilayered scaffold with distinct spatial control of the concentration of the protein, e.g., as described in Chen et al., Pharm Res. Spatio-temporal VEGF and PDGF delivery patterns blood vessel formation and maturation. 2007 February; 24(2):258-64).
Device Construction
[0270] The scaffold structure is constructed out of a number of different rigid, semi-rigid, flexible, gel, self-assembling, liquid crystalline, or fluid compositions such as peptide polymers, polysaccharides, synthetic polymers, hydrogel materials, ceramics (e.g., calcium phosphate or hydroxyapatite), proteins, glycoproteins, proteoglycans, metals and metal alloys. The compositions are assembled into cell scaffold structures using methods known in the art, e.g., injection molding, lyophillization of preformed structures, printing, self-assembly, phase inversion, solvent casting, melt processing, gas foaming, fiber forming/processing, particulate leaching or a combination thereof. The assembled devices are then implanted or administered to the body of an individual to be treated.
[0271] The device is assembled in vivo in several ways. The scaffold is made from a gelling material, which is introduced into the body in its ungelled form where it gels in situ. Exemplary methods of delivering device components to a site at which assembly occurs include injection through a needle or other extrusion tool, spraying, painting, or methods of deposit at a tissue site, e.g., delivery using an application device inserted through a cannula. In one example, the ungelled or unformed scaffold material is mixed with bioactive substances and cells prior to introduction into the body or while it is introduced. The resultant in vivo/in situ assembled scaffold contains a mixture of these substances and cells.
[0272] In situ assembly of the scaffold occurs as a result of spontaneous association of polymers or from synergistically or chemically catalyzed polymerization. Synergistic or chemical catalysis is initiated by a number of endogenous factors or conditions at or near the assembly site, e.g., body temperature, ions or pH in the body, or by exogenous factors or conditions supplied by the operator to the assembly site, e.g., photons, heat, electrical, sound, or other radiation directed at the ungelled material after it has been introduced. The energy is directed at the scaffold material by a radiation beam or through a heat or light conductor, such as a wire or fiber optic cable or an ultrasonic transducer. Alternatively, a shear-thinning material, such as an ampliphile, is used which re-cross links after the shear force exerted upon it, for example by its passage through a needle, has been relieved.
[0273] Suitable hydrogels for both in vivo and ex vivo assembly of scaffold devices are well known in the art and described, e.g., in Lee et al., 2001, Chem. Rev. 7:1869-1879. The peptide amphiphile approach to self-assembly assembly is described, e.g., in Hartgerink et al., 2002, Proc. Natl. Acad. Sci. U.S.A 99:5133-5138. A method for reversible gellation following shear thinning is exemplied in Lee et al., 2003, Adv. Mat. 15:1828-1832.
[0274] A multiple compartment device is assembled in vivo by applying sequential layers of similarly or differentially doped gel or other scaffold material to the target site. For example, the device is formed by sequentially injecting the next, inner layer into the center of the previously injected material using a needle, forming concentric spheroids. Non-concentric compartments are formed by injecting material into different locations in a previously injected layer. A multi-headed injection device extrudes compartments in parallel and simultaneously. The layers are made of similar or different scaffolding compositions differentially doped with bioactive substances and different cell types. Alternatively, compartments self-organize based on their hydro-philic/phobic characteristics or on secondary interactions within each compartment.
Compartmentalized Device
[0275] In certain situations, a device containing compartments with distinct chemical and/or physical properties is useful. A compartmentalized device is designed and fabricated using different compositions or concentrations of compositions for each compartment.
[0276] Alternatively, the compartments are fabricated individually, and then adhered to each other (e.g., a sandwich with an inner compartment surrounded on one or all sides with the second compartment). This latter construction approach is accomplished using the intrinsic adhesiveness of each layer for the other, diffusion and interpenetration of polymer chains in each layer, polymerization or cross-linking of the second layer to the first, use of an adhesive (e.g., fibrin glue), or physical entrapment of one compartment in the other. The compartments self-assemble and interface appropriately, either in vitro or in vivo, depending on the presence of appropriate precursors (e.g., temperature sensitive oligopeptides, ionic strength sensitive oligopeptides, block polymers, cross-linkers and polymer chains (or combinations thereof), and precursors containing cell adhesion molecules that allow cell-controlled assembly).
[0277] Alternatively, the compartmentalized device is formed using a printing technology. Successive layers of a scaffold precursor doped with bioactive substances is placed on a substrate then cross linked, for example by self-assembling chemistries. When the cross linking is controlled by chemical-, photo- or heat-catalyzed polymerization, the thickness and pattern of each layer is controlled by a masque, allowing complex three dimensional patterns to be built up when un-cross-linked precursor material is washed away after each catalyzation. (W T Brinkman et al., Photo-cross-linking of type 1 collagen gels in the presence of smooth muscle cells: mechanical properties, cell viability, and function. Biomacromolecules, 2003 July-August; 4(4): 890-895; W. Ryu et al., The construction of three-dimensional micro-fluidic scaffolds of biodegradable polymers by solvent vapor based bonding of micro-molded layers. Biomaterials, 2007 February; 28(6): 1174-1184; Wright, Paul K. (2001). 21st Century manufacturing. New Jersey: Prentice-Hall Inc.) Complex, multi-compartment layers are also built up using an inkjet device which paints different doped-scaffold precursors on different areas of the substrate. Julie Phillippi (Carnegie Mellon University) presentation at the annual meeting of the American Society for Cell Biology on Dec. 10, 2006; Print me a heart and a set of arteries, Aldhouse P., New Scientist 13 Apr. 2006 Issue 2547 p 19; Replacement organs, hot off the press, C. Choi, New Scientist, 25 Jan. 2003, v2379. These layers are built-up into complex, three dimensional compartments. The device is also built using any of the following methods: Jetted Photopolymer, Selective Laser Sintering, Laminated Object Manufacturing, Fused Deposition Modeling, Single Jet Inkjet, Three Dimensional Printing, or Laminated Object Manufacturing.
[0278] The release profiles of bioactive substances from scaffold devices is controlled by both factor diffusion and polymer degradation, the dose of the factor loaded in the system, and the composition of the polymer Similarly, the range of action (tissue distribution) and duration of action, or spatiotemporal gradients of the released factors are regulated by these variables. The diffusion and degradation of the factors in the tissue of interest is optionally regulated by chemically modifying the factors (e.g., PEGylating growth factors). In both cases, the time frame of release determines the time over which effective cell delivery by the device is desired.
[0279] The bioactive substances are added to the scaffold compositions using known methods including surface absorption, physical immobilization, e.g., using a phase change to entrap the substance in the scaffold material. For example, a growth factor is mixed with the scaffold composition while it is in an aqueous or liquid phase, and after a change in environmental conditions (e.g., pH, temperature, ion concentration), the liquid gels or solidifies thereby entrapping the bioactive substance. Alternatively, covalent coupling, e.g., using alkylating or acylating agents, is used to provide a stable, long term presentation of a bioactive substance on the scaffold in a defined conformation. Exemplary reagents for covalent coupling of such substances are provided in the table below.
Methods to Covalently Couple Peptides/Proteins to Polymers
[0280]
TABLE-US-00038 Reacting Functional groups on Group of proteins/ Polymer Coupling reagents and cross-linker peptides OH Cyanogen bromide (CNBr) NH.sub.2 Cyanuric chloride 4-(4,6-Dimethoxy-1,3,5-triazin-2-yl)- 4-methyl-morpholinium chloride (DMT-MM) NH.sub.2 Diisocyanate compounds NH.sub.2 Diisothoncyanate compounds OH Glutaraldehyde Succinic anhydride NH.sub.2 Nitrous Acid NH.sub.2 Hydrazine + nitrous acid SH PhOH NH.sub.2 Carbodiimide compounds (e.g., EDC, DCC)[a] COOH DMT-MM COOH Thionyl chloride NH.sub.2 N-hydroxysuccinimide N-hydroxysulfosuccinimide + EDC SH Disulfide compound SH [a]EDC: 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride; DCC: dicyclohexylcarbodiimide
[0281] Bioactive substances suitable for use in the present invention include, but are not limited to: interferons, interleukins, chemokines, cytokines, colony stimulating factors, chemotactic factors, granulocyte/macrophage colony stimulating factor (GM-CSF). Splice variants of any of the above mentioned proteins, and small molecule agonists or antagonists thereof that may be used advantageously to activate dendritic cells are also contemplated herein.
[0282] Exemplary bioactive substances suitable for use in, on, or in combination with the vaccine device of the invention include an inhibitor of an immune-inhibitory protein. Exemplary immune-inhibitory proteins include immune checkpoint proteins (e.g., CTLA4, PD1, PDL1, and PDL2). Other exemplary immune inhibitory proteins include B7-H3, B7-H4, LAG3, 2B4, BTLA, TIM3, A2aR, and/or a killer inhibitory receptor. Exemplary inhibitors include small molecules, proteins, peptides, antibodies or fragments thereof, and nucleic acids. For example, an inhibitor is a nucleic acid, protein, antibody, or fragment thereof that binds to CTLA4, PD1, PDL1, PDL2, B7-H3, B7-H4, LAG3, 2B4, BTLA, TIM3, A2aR, and/or a killer inhibitory receptor. For example, an inhibitor is a nucleic acid that binds to a mRNA that encodes CTLA4, PD1, PDL1, PDL2, B7-H3, B7-H4, LAG3, 2B4, BTLA, TIM3, A2aR, and/or a killer inhibitory receptor. In some embodiments, the nucleic acid that binds to a mRNA of the inhibitor downregulates inhibitor expression at the mRNA and/or protein level.
[0283] A small molecule is a low molecular weight compound of less than 1000 Daltons, less than 800 Daltons, or less than 500 Daltons. Antibodies and fragments thereof described herein include, but are not limited to, polyclonal, monoclonal, chimeric, dAb (domain antibody), single chain, Fab, Fab and F(ab)2 fragments, Fv, scFvs. A fragment of an antibody possess the immunological activity of its respective antibody. In some embodiments, a fragment of an antibody contains 1500 or less, 1250 of less, 1000 or less, 900 or less, 800 or less, 700 or less, 600 or less, 500 or less, 400 or less, 300 or less, 200 or less amino acids. For example, a protein or peptide inhibitor contains 1500 or less, 1250 of less, 1000 or less, 900 or less, 800 or less, 700 or less, 600 or less, 500 or less, 400 or less, 300 or less, 200 or less, 100 or less, 80 or less, 70 or less, 60 or less, 50 or less, 40 or less, 30 or less, 25 or less, 20 or less, 10 or less amino acids. For example, a nucleic acid inhibitor of the invention contains 400 or less, 300 or less, 200 or less, 150 or less, 100 or less, 90 or less, 80 or less, 70 or less, 60 or less, 50 or less, 40 or less, 35 or less, 30 or less, 28 or less, 26 or less, 24 or less, 22 or less, 20 or less, 18 or less, 16 or less, 14 or less, 12 or less, 10 or less nucleotides.
[0284] In some cases, a compound (e.g., small molecule) or macromolecule (e.g., nucleic acid, polypeptide, or protein) of the invention is purified and/or isolated. As used herein, an isolated or purified small molecule, nucleic acid molecule, polynucleotide, polypeptide, or protein (e.g., antibody or fragment thereof), is substantially free of other cellular material, or culture medium when produced by recombinant techniques, or chemical precursors or other chemicals when chemically synthesized. Purified compounds are at least 60% by weight (dry weight) the compound of interest. Preferably, the preparation is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight the compound of interest. For example, a purified compound is one that is at least 90%, 91%, 92%, 93%, 94%, 95%, 98%, 99%, or 100% (w/w) of the desired compound by weight. Purity is measured by any appropriate standard method, for example, by column chromatography, thin layer chromatography, or high-performance liquid chromatography (HPLC) analysis. A purified or isolated polynucleotide (ribonucleic acid (RNA) or deoxyribonucleic acid (DNA)) is free of the genes or sequences that flank it in its naturally occurring state. Purified also defines a degree of sterility that is safe for administration to a human subject, e.g., lacking infectious or toxic agents.
[0285] By substantially pure is meant a nucleotide or polypeptide that has been separated from the components that naturally accompany it. Typically, the nucleotides and polypeptides are substantially pure when they are at least 60%, 70%, 80%, 90%, 95%, or even 99%, by weight, free from the proteins and naturally-occurring organic molecules with they are naturally associated.
[0286] For example, a nucleic acid inhibitor is a short interfering RNA, a short hairpin RNA, antisense RNA, aptamers, peptide nucleic acids (PNAs), microRNAs (miRNAs), or locked nucleic acids (LNAs). In some embodiments, the nucleic acid comprises modified oligonucleotides (e.g., 2-o-methyl RNA).
[0287] Examples of cytokines as mentioned above include, but are not limited to IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12, IL-15, IL-17, IL-18, granulocyte-macrophage colony stimulating factor (GM-CSF), granulocyte colony stimulating factor (G-CSF), interferon- (-IFN), IFN-, tumor necrosis factor (TNF), TGF-, FLT-3 ligand, and CD40 ligand.
[0288] Scaffolds of the invention optionally comprise at least one non-viral gene therapy vector such that either the transplanted cells or host cells in the vicinity of the implant would take up and express gene that lead to local availability of the desired factor for a desirable time frame. Such non-viral vectors include, but are not limited to, cationic lipids, polymers, targeting proteins, and calcium phosphate.
Scaffold Fabrication.
[0289] A 85:15, 120 kD copolymer of D,L-lactide and glycolide (PLG) (Alkermes, Cambridge, Mass.) was utilized in a gas-foaming process to form scaffolds with open, interconnected pores (Cohen S., Yoshioka T., Lucarelli, M., Hwang L. H., and Langer R. Pharm. Res. 8, 713-720 (1991); herein incorporated by reference). PLG microspheres encapsulating GM-CSF were made using standard double emulsion (Harris, L. D., Kim, B. S., and Mooney, D. J. J. Biomed. Mater. Res. 42,396-402 (1998); herein incorporated by reference). 16 mg of PLG microspheres were then mixed with 150 mg of the porogens, NaCl or sucrose (sieved to a particle size between 250 mm and 425 m), and compression molded. The resulting disc was allowed to equilibrate within a high-pressure CO.sub.2 environment, and a rapid reduction in pressure causes the polymer particles to expand and fuse into an interconnected structure. The NaCl was leached from the scaffolds by immersion in water yielding scaffolds that were 90% porous. To incorporate tumor lysates into PLG scaffolds, biopsies of B16-F10 tumors, that had grown subcutaneously in the backs of C57BL/6J mice (Jackson Laboratory, Bar Harbor Me.), were digested in collagenase (250 Um) (Worthington, Lakewood, N.J.) and suspended at a concentration equivalent to 10.sup.7 cells per ml after filtration through 40 m cell strainers. The tumor cell suspension was subjected to 4 cycles of rapid freeze in liquid nitrogen and thaw (37 C.) and then centrifuged at 400 rpm for 10 min. The supernatant (1 ml) containing tumor lysates was collected and lyophilized with the PLG microspheres and the resulting mixture was used to make PLG scaffold-based cancer vaccines. To incorporate CpG-ODNs into PLG scaffolds, PEI-CpG-ODN condensate solutions were vortexed with 60 l of 50% (wt/vol) sucrose solution, lyophilized and mixed with dry sucrose to a final weight of 150 mg. The sucrose containing PEI-CpG-ODN condensate was then mixed with blank, GM-CSF and/or tumor lysate loaded PLG microspheres to make PLG cancer vaccines.
[0290] Scaffold compositions of the present invention comprise GM-CSF, Flt3L, and/or CCL20, and CpG-ODN sequences. A range of concentrations of each element are contemplated. In a preferred embodiment, the scaffold composition comprises PLG. With respect to GM-CSF, Flt3L, and/or CCL20, per 40 mg polymeric scaffold composition, 0-100 mg of GM-CSF, Flt3L, and/or CCL20 polypeptide is incorporated into or coated onto the scaffold composition. Alternatively, doses comprising 0-50 g, 0-25 g, 0-10 g, 0-5 g, and 0-3 g of GM-CSF, Flt3L, and/or CCL20 are incorporated into the scaffold composition. In a preferred embodiment, 0-3 mg of GM-CSF, Flt3L, and/or CCL20 are incorporated into the scaffold composition. With respect to CpG-ODN sequences, or PEI-CpG-ODN condensates, per 40 mg polymeric scaffold composition, 0-1000 mg of PEI-CpG-ODN is incorporated into or coated onto the scaffold composition. Alternatively, doses comprising 0-500 g, 0-250 g, 0-100 mg (e.g., 100 g), 0-50 g, 0-25 g, 0-10 g, and 0-5 mg of PEI-CpG-ODN are incorporated into the scaffold composition. In a preferred embodiment, 0-50 mg of PEI-CpG-ODN are incorporated into the scaffold composition.
Vaccine Device
[0291] The biocompatible scaffolds are useful as delivery vehicles for cancer vaccines. The cancer vaccine stimulates an endogenous immune response against cancer cells. Currently produced vaccines predominantly activate the humoral immune system (i.e., the antibody dependent immune response). Other vaccines currently in development are focused on activating the cell-mediated immune system including cytotoxic T lymphocytes which are capable of killing tumor cells. Cancer vaccines generally enhance the presentation of cancer antigens to both antigen presenting cells (e.g., macrophages and dendritic cells) and/or to other immune cells such as T cells, B cells, and NK cells. Although cancer vaccines may take one of several forms, their purpose is to deliver cancer antigens and/or cancer associated antigens to antigen presenting cells (APC) in order to facilitate the endogenous processing of such antigens by APC and the ultimate presentation of antigen presentation on the cell surface in the context of MHC class I molecules. One form of cancer vaccine is a whole cell vaccine which is a preparation of cancer cells which have been removed from a subject, treated ex vivo and then reintroduced as whole cells in the subject. These treatments optionally involve cytokine exposure to activate the cells, genetic manipulation to overexpress cytokines from the cells, or priming with tumor specific antigens or cocktails of antigens, and expansion in culture. Dendritic cell vaccines activate antigen presenting cells directly, and their proliferation, activation and migration to lymph nodes is regulated by scaffold compositions to enhance their ability to elicit an immune response. Types of cancers to be treated include central nervous system (CNS) cancers, CNS Germ Cell tumor, lung cancer, Leukemia, Multiple Myeloma, Renal Cancer, Malignant Glioma, Medulloblastoma, and Melanoma.
[0292] For the purpose of eliciting an antigen-specific immune response, a scaffold device is implanted into a mammal. The device is tailored to activate immune cells and prime the cells with a specific antigen thereby enhancing immune defenses and destruction of undesired tissues and targeted microorganisms such as bacterial or viral pathogens. The device attracts appropriate immune cells, such as macrophages, T cells, B cells, NK cells, and dendritic cells, by containing and/or releasing signaling substances such as GM-CSF. These signaling substances are incorporated in the scaffold composition in such a way as to control their release spatially and temporally using the same techniques used to integrate other bioactive compounds in the scaffold composition.
[0293] Once the immune cells are inside the device, the device programs the immune cells to attack or cause other aspects of the immune system to attack undesired tissues (e.g., cancer, adipose deposits, or virus-infected or otherwise diseased cells) or microorganisms. Immune cell activation is accomplished by exposing the resident immune cells to preparations of target-specific compositions, e.g., ligands found on the surface of the undesired tissues or organisms, such as cancer cell surface markers, viral proteins, oligonucleatides, peptide sequences or other specific antigens. For example, useful cancer cell-specific antigens and other tissue or organism-specific proteins are listed in the table below.
[0294] The device optionally contains multiple ligands or antigens in order to create a multivalent vaccine. The compositions are embedded in or coated on the surface of one or more compartments of the scaffold composition such that immune cells migrating through the device are exposed to the compositions in their traverse through the device. Antigens or other immune stimulatory molecules are exposed or become exposed to the cells as the scaffold composition degrades. The device may also contain vaccine adjuvants that program the immune cells to recognize ligands and enhance antigen presentation. Exemplary vaccine adjuvants include chemokines/cytokines, CpG rich oligonucleotides. or antibodies that are exposed concurrently with target cell-specific antigens or ligands.
[0295] The device attracts immune cells to migrate into a scaffold where they are educated in an antigen-specific manner and activated. The programmed immune cells are then induced to egress towards lymph nodes in a number of ways. The recruitment composition and deployment signal/composition, e.g., a lymph node migration inducing substance, is released in one or more bursts, programmed by the method of incorporation and/or release from the scaffold material, or controlled by the sequential degradation of scaffold compartments which contain the attractant. When a burst dissipates, the cells migrate away. Compartments containing repulsive substances are designed to degrade and release the repulsive substance in one or more bursts or steadily over time. Relative concentration of the repulsive substances cause the immune cells to migrate out of the device. Alternatively, cells which have been placed in or have migrated into the device are programmed to release repulsive substances or to change their own behavior. For example, localized gene therapy is carried out by cell exposure to plasmid DNA attached to the scaffold. Useful repulsive substances include chemokines and cytokines. Alternatively, the device may cause immune cells to egress by degrading and releasing them.
[0296] Target disease states, stimulatory molecules and antigens useful in vaccine device construction are listed below.
[0297] Bioactive Factors to Promote Immune Responses
a. Interleukins: IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12 IL-15, IL-17, IL-18 etc.
b. TNF-
c. IFN-
d. IFN-
e. GM-CSF
f. G-CSF
g. Ft1-3 ligand
h. MIP-3 (CCL19)
i. CCL21
j. M-CSF
k. MIF
l. CD40L
m. CD3
n. ICAM
o. Anti-CTLA4 proteins or antibodies or fragments thereof (e.g., ipilimumab or tremelimumab)
p. TGF-
q. CPG rich DNA or oligonucleotides
r. Sugar moieties associated with Bacteria: Lipopolysacharides (LPS) is an example
s. Fas ligand
t. Trail
u. Lymphotactin
v. Mannan (M-FP)
w. Heat Shock Proteins (apg-2, Hsp70 and Hsp 90 are examples)
x. anti-PD1 proteins or antibodies (e.g., MDX-1106, MK3475, CT-011, or AMP-224)
y. anti-PDL1 or anti-PDL2 proteins or antibodies (e.g., MDX-1105)
z. anti-LAG3 proteins or antibodies or fragments thereof
aa. anti-B7-H3 proteins or antibodies or fragments thereof
bb. anti-B7-H4 proteins or antibodies or fragments thereof
cc. anti-TIM3 proteins or antibodies or fragments thereof
dd. anti-BTLA proteins or antibodies or fragments thereof
ee. anti-A2aR proteins or antibodies or fragments thereof
ff. anti-killer inhibitor receptor (KIR) (e.g., killer cell immunoglobulin-like receptor or C-type lectin receptor) proteins or antibodies or fragments thereof
gg. anti-TIM4 proteins or antibodies or fragments thereof
hh. anti-TIM2 proteins or antibodies or fragments thereof
ii. anti-OX40 proteins or antibodies or fragments thereof
jj. anti-4-1BB proteins or antibodies or fragments thereof
kk. anti-phosphatidylserine proteins or antibodies or fragments thereof (e.g., a monoclonal antibody against phosphatidylserine
Diseases and AntigensVaccination Targets
[0298] a. Cancer: antigens and their sources
i. Tumor lysates extracted from biopsies (e.g., from melanoma tumor biopsies)
ii. Irradiated tumor cells (e.g., irradiated melanoma cells)
iii. Melanoma
1. MAGE series of antigens (MAGE-1 is an example)
2. MART-1/melanA
3. Tyrosinase
[0299] 4. ganglioside
5. gp100
6. GD-2
7. O-acetylated GD-3
8. GM-2
[0300] 9. B16-F10 tumor lysate, e.g., from mice challenged with B16-F10 melanoma tumor cells (ATCC, Manassas, N.J.)
10. tyrosinase-related protein (TRP)-2
11. lung cancer cell lysate or lung cancer cell antigen
12. glioma cancer cell lysate or glioma cancer cell antigen
13. prostate cancer cell lysate or prostate cancer cell antigen
iv. Breast Cancer
1. MUC-1
2. Sosl
[0301] 3. Protein kinase C-binding protein
4. Reverse trascriptase protein
5. AKAP protein
6. VRK1
7. KIAA1735
8. T7-1, T11-3, T11-9
[0302] 9. Her2 (also known as CD340)
v. Other General and Specific Cancer Antigens
1. Homo Sapiens telomerase ferment (hTRT)
2. Cytokeratin-19 (CYFRA21-1)
3. SQUAMOUS CELL CARCINOMA ANTIGEN 1 (SCCA-1), (PROTEIN T4-A)
4. SQUAMOUS CELL CARCINOMA ANTIGEN 2 (SCCA-2)
[0303] 5. Ovarian carcinoma antigen CA125 (1A1-3B) (KIAA0049)
6. MUCIN 1 (TUMOR-ASSOCIATED MUCIN), (CARCINOMA-ASSOCIATED MUCIN), (POLYMORPHIC EPITHELIAL MUCIN), (PEM), (PEMT), (EPISIALIN), (TUMOR-ASSOCIATED EPITHELIAL MEMBRANE ANTIGEN), (EMA), (H23AG), (PEANUT-REACTIVE URINARY MUCIN), (PUM), (BREAST CARCINOMA-ASSOCIATED ANTIGEN DF3)
7. CTCL tumor antigen set-1
8. CTCL tumor antigen se14-3
9. CTCL tumor antigen se20-4
10. CTCL tumor antigen se20-9
11. CTCL tumor antigen se33-1
12. CTCL tumor antigen se37-2
13. CTCL tumor antigen se57-1
14. CTCL tumor antigen se89-1
15. Prostate-specific membrane antigen
16. 5T4 oncofetal trophoblast glycoprotein
17. Orf73 Kaposi's sarcoma-associated herpesvirus
18. MAGE-C1 (cancer/testis antigen CT7)
19. MAGE-B1 ANTIGEN (MAGE-XP ANTIGEN) (DAM10)
20. MAGE-B2 ANTIGEN (DAM6)
21. MAGE-2 ANTIGEN
[0304] 22. MAGE-4a antigen
23. MAGE-4b antigen
24. Colon cancer antigen NY-CO-45
25. Lung cancer antigen NY-LU-12 variant A
26. Cancer associated surface antigen
27. Adenocarcinoma antigen ART1
28. Paraneoplastic associated brain-testis-cancer antigen (onconeuronal antigen MA2; paraneoplastic neuronal antigen)
29. Neuro-oncological ventral antigen 2 (NOVA2)
30. Hepatocellular carcinoma antigen gene 520
31. TUMOR-ASSOCIATED ANTIGEN CO-029
[0305] 32. Tumor-associated antigen MAGE-X2
33. Synovial sarcoma, X breakpoint 2
34. Squamous cell carcinoma antigen recognized by T cell
35. Serologically defined colon cancer antigen 1
36. Serologically defined breast cancer antigen NY-BR-15
37. Serologically defined breast cancer antigen NY-BR-16
38. Chromogranin A; parathyroid secretory protein 1
39. DUPAN-2
40. CA 19-9
41. CA 72-4
42. CA 195
[0306] 43. Carcinoembryonic antigen (CEA)
b. AIDS (HIV Associated Antigens)
i. Gp120
ii. SIV229
iii. SIVE660
iv. SHIV89.6P
v. E92
vi. HCl
vii. OKMS5
viii. FVIIIRAg
ix. HLA-DR (Ia) antigens
x. OKM1
xi. LFA-3
c. General Infectious Diseases and Associated Antigens
i. Tuberculosis
1. Mycobacterium tuberculosis antigen 5
2. Mycobacterium tuberculosis antigen 85
3. ESAT-6
4. CFP-10
5. Rv3871
6. GLU-S
[0307] ii. Malaria
1. CRA
2. RAP-2
3. MSP-2
4. AMA-1
[0308] iii. Possible mutant influenza and meningitis strains
d. Neuro ProtectionProtect Against Neurological Diseases (e.g., Alzheimer's, Parkinsons, Prion disease)
1. Classes of self CNS antigens
2. human alpha-synuclein (Parkinson's)
3. beta amyloid plaques (Alzheimer's)
e. Autoimmune Diseases (multiple sclerosis, Rheumatoid arthritis etc)
i. Disease linked MHC antigens
ii. Different classes of Self antigens
iii. Insulin
iv. Insulin peptide B9-23
v. glutamic acid
vi. decarboxylase 65 (GAD 65)
vii. HSP 60
Disease linked T-cell receptor (TCR)
[0309] Prior vaccines have been largely ineffective for patients with established cancer, as advanced disease requires potent and sustained activation of CD8.sup.+ cytotoxic T lymphocytes (CTLs) to kill tumor cells and clear the disease. Subsets of dendritic cells (DCs) specialize in antigen cross-presentation and in the production of cytokines, which regulate both CTLs and T regulatory (Treg) cells that shut down effector T cell responses. Coordinated regulation of a DC network, and plasmacytoid DCs (pDCs) and CD8.sup.+ DCs in particular, enhances host immunity in mice. Functionalized biomaterials incorporating various combinations of an inflammatory cytokine, immune danger signal, and tumor lysates are used in the vaccines described herein to control the activation and localization of host DC populations in situ.
[0310] Implantable synthetic polymer matrices (antigen-loaded acellular biomaterial device) that spatially and temporally control the in vivo presentation of cytokines, tumor antigens, and danger signals are utilized. GM-CSF is released from these polylactide-co-glycolide (PLG) [a FDA-approved biomaterial] matrices into the surrounding tissue to recruit DC precursors and DCs. CpG-rich oligonucleotides are immobilized on the matrices as danger signals, and antigen (tumor lysates) is released to matrix-resident DCs to program DC development and maturation. These matrices quantitatively regulate DC activation and trafficking in situ and induce prophylactic immunity against inoculations of murine B16-F10 melanoma cells (P. Schnorrer, G. M. Behrens, N. S. Wilson, J. L. Pooley, C. M. Smith, D. El-Sukkari, G. Davey, F. Kupresanin, M. Li, E. Maraskovsky, G. T. Belz, F. R. Carbone, K. Shortman, W. R. Heath, J. A. Villadangos, The dominant role of CD8.sup.+ dendritic cells in cross-presentation is not dictated by antigen capture. Proc. Natl. Acad. Sci. U.S.A. 103, 10729-10734 (2006)). As described herein, this system administered repeatedly over time to controls the recruitment and activation of multiple DC and T cell subsets and is effective as a therapeutic vaccine against established tumors.
Matrix Fabrication
[0311] An exemplary protocol for matrix fabrication is described herein (see, e.g., US 2013-0202707, incorporated herein by reference). An 85:15, 120-kD copolymer of .sub.D,L-lactide and glycolide (PLG) (Alkermes) was utilized in a gas-foaming process to form porous PLG matrices (L. D. Harris, B. S. Kim, D. J. Mooney, Open pore biodegradable matrices formed with gas foaming. J. Biomed. Mater. Res. 42, 396-402 (1998)). PLG microspheres encapsulating GMCSF were first made with standard double emulsion (S. Cohen, T. Yoshioka, M. Lucarelli, L. H. Hwang, R. Langer, Controlled delivery systems for proteins based on poly(lactic/glycolic acid) microspheres. Pharm. Res. 8, 713-720 (1991)). PLG micro-spheres were then mixed with 150 mg of the porogen, sucrose (sieved to a particle size between 250 and 425 mm), and compression molded. The resulting disc was allowed to equilibrate within a high-pressure CO.sub.2 environment, and a rapid reduction in pressure causes the polymer particles to expand and fuse into an interconnected structure. The sucrose was leached from the scaffolds by immersion in water, yielding scaffolds that were 90% porous. To incorporate tumor lysates into PLG scaffolds, the biopsies of B16-F10 tumors that had grown subcutaneously in the backs of C57BL/6J mice (Jackson Laboratory) were digested in collagenase (250 Um) (Worthington) and suspended at a concentration equivalent to 10.sup.7 cells per milliliter after filtration through 40-m cell strainers. The tumor cell suspension was subjected to four cycles of rapid freeze in liquid nitrogen and thaw (37 C.) and then centrifuged at 400 rpm for 10 min. The supernatant (1 ml) containing tumor lysates was collected, incubated with the PLG microspheres, and lyophilized, and the resulting mixture was utilized in the high-pressure CO.sub.2 process to foam macroporous PLG matrices incorporating tumor lysates.
[0312] To incorporate CpG-ODNs into PLG scaffolds, CpG-ODN 1826, 5-tccatgacgttcctgacgtt-3 (Invivogen, San Diego, Calif.; SEQ ID NO: 29) was condensed with poly(ethylenimine) (PET) (M.sub.n60,000; Sigma Aldrich) molecules by dropping ODN 1826 solutions into PEI solution while vortexing the mixture (L. D. Harris, B. S. Kim, D. J. Mooney, Open pore biodegradable matrices formed with gas foaming. J. Biomed. Mater. Res. 42, 396-402 (1998); S. Cohen, T. Yoshioka, M. Lucarelli, L. H. Hwang, R. Langer, Controlled delivery systems for proteins based on poly(lactic/glycolic acid) microspheres. Pharm. Res. 8, 713-720 (1991); Y. C. Huang, M. Connell, Y. Park, D. J. Mooney, K. G. Rice, Fabrication and in vitro testing of polymeric delivery system for condensed DNA. J. Biomed. Mater. Res. A 67, 1384-1392(2003)). The charge ratio between PEI and CpG-ODN (NH.sub.3.sup.+:PO.sub.4.sup.) was kept constant at 7 during condensation. PEI-CpG-ODN condensate solutions were then vortexed with 60 l of 50% (w/v) sucrose solution, lyophilized, and mixed with dry sucrose to a final weight of 150 mg. The sucrose containing PEI-CpG-ODN condensate was then mixed with blank, GM-CSF, and/or tumor lysate-loaded PLG microspheres to make PLG cancer vaccines.
[0313] To achieve controlled GM-CSF and TLR agonist presentation, macroporous, poly-lactide-co-glycolide (PLG) matrices quickly release GM-CSF (Ali et al., 2009 Nat Mater, 2: 151-8); e.g., approximately 60% of the protein was released by day 10 (US 2013-0202707, incorporated herein by reference), to induce the recruitment of DCs or their precursors. GM-CSF loaded PLG scaffolds were also modified to present TLR-activating, CpG-ODN, MPLA and P(I:C) molecules, as danger signals. Presentation of the TLR agonists was designed to provide a long-term, local signal to activate DCs. Importantly, the relatively high molecular
[0314] weight and composition of the particular PLG chosen to fabricate scaffolds results in slow scaffold degradation, allowing for long-term analysis of the vaccine site and its regulation over DC activation and T cell immunity.
[0315] The vaccine system of the invention is capable of generating prophylactic immunity against poorly immunogenic B16-F10 melanoma (0. A. Ali, N. Huebsch, L. Cao, G. Dranoff, D. J. Mooney, Infection-mimicking materials to program dendritic cells in situ. Nat. Mater. 8, 151-158 (2009) and US 2013-0202707, incorporated herein by reference). As described in US 2013-0202707, incorporated herein by reference, the vaccine system promotes and extends CTL responses through nave T cell differentiation induced by pDCs and CD8.sup.+ DCs, the corresponding production of type 1 IFNs and IL-12, and inhibition of negative feedback mechanisms.
[0316] As described in US 2013-0202707, incorporated herein by reference, vaccine formulations containing various TLR agonists produce significant and systemic anti-melanoma CTLs in correlation with the activation of specific DC subsets and reduce tumor burden. Inclusion of TLR agonists was activates DCs, in general, increasing their surface expression of MHCII and the costimulatory molecule, CD86, indicating an enhanced capacity to present antigen and activate T cell populations. In particular, appropriate TLR signaling enhanced the generation of CD8(+) and pDC subsets at the vaccine site and stimulated the production of IFNs and the potent T cell growth factor, IL-12.
[0317] In some embodiments, three different types of pathogen associated molecular patterns (PAMPs) are incorporated into or onto structural polymeric devices such as PLG disc structures/scaffolds to act as adjuvants in vaccines (3 types; a short oligonucleotide (CpG-ODN); a synthetic RNA-(Poly(I:C); P(I:C)), a synthetic lipid (monophosphoryl lipid A; MPLA). Such vaccine formulations recruit and activate dendritic cells in situ.
[0318] Vaccine-dependent survival in an aggressive melanoma cancer model correlates strongly with the ability of the vaccine to specifically activate 2 subsets of dendritic cellsCD8(+) DCs and plasmacytoid DCsregardless of the adjuvant utilized in the vaccine system. This correlation has been confirmed utilizing 4 different vaccine adjuvants in the PLG vaccine. These vaccines induce potent tumor rejection in a therapeutic model of melanoma, by activating specific T cell responses that have been detected at the vaccine site and at tumors. These findings demonstrate the PLG vaccine system's versatility in incorporating different types of agonists that stimulate different pathways in innate and adaptive immune responses.
The Role of Dendritic Cells in the Immune Response
[0319] Dendritic cells (DCs) orchestrate immune responses to infection and tumors by priming and propagating specific, cytotoxic T lymphocyte (CTL) responses. Immature DCs residing in peripheral tissue detect foreign substances (i.e., antigens) unique to invading pathogens, and are activated by stimuli, such as pathogen associated molecular patterns (PAMPs) or products of dying cells (i.e., danger signals), originating during pathogen induced inflammatory responses. Maturing DCs mature both process and present antigens on major histocompatibility complexes (MHC) receptors, and express the costimulatory molecules CD80 and CD86, both of which are required for effector T-cell stimulation. Another important result of DC maturation by danger signaling, is that DCs acquire the ability to home to the lymph nodes to engage and activate naive T-cells, enabling the T cells to recognize the antigens DCs are presenting.
[0320] The ability of particular DCs to initiate and control immune responses is a consequence of both their localization within tissues and their specialized capacity for mobilization. DCs originate from pluripotent stem cells in the bone marrow, enter the blood stream and localize into almost all organs. Based on the relative expression of a series of surface markers, different subsets of DCs or DC precursors can be identified in peripheral blood, including plasmacytoid DCs (pDCs) and conventional DCs (cDCs)2. pDCs are major type I interferon (IFN) producers, and specialize in activating adaptive immune responses to virus challenge via cytokine signaling. CD11c(+) cDCs, such as epidermal DCs, are especially adept at antigen presentation and co-stimulation of T cells.
[0321] Upon microbial invasion and inflammation, DCs rapidly migrate into the draining lymph nodes and primary sites of infection at rates that vastly outnumber other APCs, such as macrophages. The production of most DC subsets, including (pDCs) is controlled in the steady state by the cytokine Fms-related tyrosine kinase 3 ligand ligand (FL). Other cytokines, such as GM-CSF and CCL20, released by damaged or infected cells, actively recruit and localize cDCs to the sites of inflammation. In inflammatory models, both in vivo and in vitro, these inflammatory cytokines have been shown to also enhance DC migration and proliferation and may regulate DC activation state. The quantity of DCs activated during infection or within tumors is correlated with the strength of the subsequent immune response and disease prognosis.
[0322] To generate sufficient numbers of dendritic cells (DCs) for immunotherapy, laboratory-based culture of DC precursors with inflammatory cytokines, such as granulocyte macrophage-colony stimulating factor (GM-CSF) and FL (Flt3) has often been used. DCs modified in vitro to present tumor antigens are capable of eliciting antitumor effects in murine models upon transplantation. Initial clinical testing of ex vivo DC-based vaccines has revealed the induction of tumor regression in a subset of cancer patients, but little survival benefit. Protocols involving the ex vivo manipulation of DCs are limited by the quantities and types of DCs that can be produced, poor engraftment efficiency and LN homing, and loss of DC activation upon injection in the in vivo environment.
[0323] To address these limitations, infection-mimicking materials of the device present inflammatory cytokines in combination with a danger signal to recruit and activate DCs in vivo. Also, nanoparticles containing cytosine-guanosine (CpG) rich oligonucleotide (CpG-ODN) sequences were immobilized onto scaffolds, as CpG-ODN are expressed in bacterial DNA, and are potent danger signals that can stimulate activation of matrix resident DCs.
[0324] CD141+ DCs and plasmacytoid DCs are critical for successful cancer vaccination (prophylactic and therapeutic). Plasmacytoid DCs look like plasma cells, but have certain characteristics similar to myeloid dendritic cells, can produce high amounts of interferon-alpha, and are characterized by TLR7 and TLR9. The TLR agonist, CpG, binds to TLR9. CD8+ DCs in mice are equivalent to CD141+ dendritic cells. CD141+ DCs are found in human lymph nodes, bone marrow, tonsil, and blood. They are characterized by high expression of toll-like receptor 3 (TLR3), production of IL-12p70 and IFN-, and superior capacity to induce T helper 1 cell responses, when compared with the more commonly studied CD1c+ DC subset.
[0325] Polyinosine-polycytidylic acid (poly I:C)-activated CD141+ DCs have a superior capacity to cross-present antigens to CD8+ cytotoxic T lymphocytes than poly I:C-activated CD1c+ DCs. Thus, CD141+DC subset represents an important functionally distinct human DC subtype with characteristics similar to those of the mouse CD8a+ DC subset. CD141+ DCs play a role in the induction of cytotoxic T lymphocyte responses and their activation is important for vaccination against cancers, viruses, and other pathogens.
[0326] p(I:C) in the vaccine device stimulates CD141+ DCs in humans (CD8+ DCs in mice) and CpG stimulates plasmacytoid DCs. Devices with one or both of these TLR agonists lead to potent DC activation and the generation of significant prophylactic and therapeutic anti-tumor immune responses. A combination of different TLR agonists, e.g., a combination of p(I:C) and CpG, in a device leads to a synergistic effect in the activation of a DC immune response against tumors.
[0327] PLG vaccines incorporating CpG-ODN and P(I:C) act synergistically to generate significant tumor inhibition, reduced tumor burden, and to generate improved anti-tumor immune responses.
Controlled Release of Cytokines and In Vivo DC Recruitment
[0328] Macroporous, poly-lactide-co-glycolide (PLG) matrices were designed to provide long-term and sustained release of GM-CSF, FL, and CCL20 and to house DCs for activation. These PLG scaffolds were 80-90% porous with an average pore size between 125-200 um to facilitate dendritic cell infiltration. The in vitro release kinetics for the three cytokines were similar, as the matrices quickly released protein with a burst over the first 5 days followed by sustained release over the next several weeks (US 2013-0202707, incorporated herein by reference).
In Vivo DC Activation
[0329] PLG scaffolds were modified to present nanoparticles containing TLR-activating, CpG-ODN, as an infection-mimicking danger signal in concert with delivery with inflammatory cytokines. This dramatically enhanced DC activation in situ over control conditions lacking cytokine signaling.
[0330] Controlled mobilization and activation of DCs and DC precursors is of particular interest in the development of ex vivo DC based vaccines, and more generally the design of material systems that activate the immune system in vivo. As described herein, polymers which mimic key aspects of microbial infection effectively recruit DCs for cancer vaccination. PLG scaffolds engineered to release GM-CSF. FL, and CCL20 led to significant numbers of resident DCs, and the co-presentation of danger signals led to DC maturation. Even though all vaccine formulations were capable of inducing tumor protection in a therapeutic model of B16-F10 melanoma, GM-CSF and FL vaccines produced more antigen specific CTLs, higher levels of Th1 priming cytokines, and greater survival rates when compared to CCL20.
[0331] pDCs, and their cDC counterparts are targeted to exploit their specialized abilities to mediate anti-tumor T cell responses. In contrast to nanoparticle targeting systems, the polymer systems described herein not only serve as a antigen delivery devices to recruit and activate DCs, but also serve as a physical structure where DCs temporarily reside while they are activated.
[0332] The systems described herein demonstrated significant anti-tumor activity. In addition to the polymers, e.g., PLG, described herein, matrices are optionally fabricated from other more inflammatory polymers to boost immune responses and DC mobilization. Another important aspect of subsequent T cell priming by these cells is LN homing. The exit or dispersement of DCs after antigen exposure is optimized by incorporating different adjuvants into the material to activate migratory function. Alternatively, other matrix properties, including degradation kinetics and porosity are altered to promote further control over DC trafficking.
[0333] FL, CCL20 and GM-CSF are utilized in biomaterial systems to mimic infection-induced recruitment of DCs in situ. As described in US 2013-0202707, e.g., at page 111, line 17-page 113, line 17 (incorporated herein by reference), infection-mimicking porous devices are effective as therapeutic cancer vaccines.
Antibodies
[0334] As used herein, the term antibody refers to immunoglobulin molecules and immunologically active portions of immunoglobulin (Ig) molecules, i.e., molecules that contain an antigen binding site that specifically binds (immunoreacts with) an antigen. By specifically binds or immunoreacts with is meant that the antibody reacts with one or more antigenic determinants of the desired antigen and does not significantly react with other antigens. Antibodies include, but are not limited to, polyclonal, monoclonal, chimeric, dAb (domain antibody), single chain, F.sub.ab, F.sub.ab and F.sub.(ab)2 fragments, scFvs, and F.sub.ab expression libraries.
[0335] A single chain Fv (scFv) polypeptide molecule is a covalently linked V.sub.H::V.sub.L heterodimer, which can be expressed from a gene fusion including V.sub.H- and V.sub.L-encoding genes linked by a peptide-encoding linker. (See Huston et al. (1988) Proc Nat Acad Sci USA 85(16):5879-5883). A number of methods have been described to discern chemical structures for converting the naturally aggregated, but chemically separated, light and heavy polypeptide chains from an antibody V region into an scFv molecule, which will fold into a three dimensional structure substantially similar to the structure of an antigen-binding site. See, e.g., U.S. Pat. Nos. 5,091,513; 5,132,405; and 4,946,778.
[0336] The term antigen-binding site, or binding portion refers to the part of the immunoglobulin molecule that participates in antigen binding. The antigen binding site is formed by amino acid residues of the N-terminal variable (V) regions of the heavy (H) and light (L) chains. Three highly divergent stretches within the V regions of the heavy and light chains, referred to as hypervariable regions, are interposed between more conserved flanking stretches known as framework regions, or FRs. Thus, the term FR refers to amino acid sequences which are naturally found between, and adjacent to, hypervariable regions in immunoglobulins. In an antibody molecule, the three hypervariable regions of a light chain and the three hypervariable regions of a heavy chain are disposed relative to each other in three dimensional space to form an antigen-binding surface. The antigen-binding surface is complementary to the three-dimensional surface of a bound antigen, and the three hypervariable regions of each of the heavy and light chains are referred to as complementarity-determining regions, or CDRs.
[0337] As used herein, the term epitope includes any protein determinant capable of specific binding to an immunoglobulin, an scFv, or a T-cell receptor. Epitopic determinants consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and have specific three dimensional structural characteristics, as well as specific charge characteristics. For example, antibodies may be raised against N-terminal or C-terminal peptides of a polypeptide, linear or non-linear peptide sequences of a protein, as well as epitopes that comprise amino acids of a first antigen and those of a second antigen.
[0338] As used herein, the terms immunological binding, and immunological binding properties refer to the non-covalent interactions of the type which occur between an immunoglobulin molecule and an antigen for which the immunoglobulin is specific. The strength, or affinity of immunological binding interactions can be expressed in terms of the dissociation constant (K.sub.d) of the interaction, wherein a smaller K.sub.d represents a greater affinity. Immunological binding properties of selected polypeptides can be quantified using methods well known in the art. One such method entails measuring the rates of antigen-binding site/antigen complex formation and dissociation, wherein those rates depend on the concentrations of the complex partners, the affinity of the interaction, and geometric parameters that equally influence the rate in both directions. Thus, both the on rate constant (K.sub.on) and the off rate constant (K.sub.off) can be determined by calculation of the concentrations and the actual rates of association and dissociation. (Nature 361:186-87 (1993)). The ratio of K.sub.off/K.sub.on enables the cancellation of all parameters not related to affinity, and is equal to the dissociation constant K.sub.d. Davies et al. (1990) Annual Rev Biochem 59:439-473). An antibody of the present invention is said to specifically bind to an antigen or epitope described herein (e.g., a CTLA, PD1, PDL1, or other immune inhibitory protein and/or tumor antigen) when the equilibrium binding constant (K.sub.d) is 1 M, preferably 100 nM, more preferably 10 nM, more preferably 1 nM, and most preferably 100 pM to about 1 pM, as measured by assays such as radioligand binding assays or similar assays known to those skilled in the art.
Routes of Administration
[0339] A pharmaceutical composition of the invention (e.g., an inhibitor described herein) is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intraperitoneal, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (i.e., topical), transmucosal, and rectal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates or phosphates, and agents for the adjustment of tonicity such as sodium chloride or dextrose. The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
[0340] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringeability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as manitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.
[0341] Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, methods of preparation are vacuum drying and freeze-drying that yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
[0342] Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.
[0343] For administration by inhalation, the compounds are delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.
[0344] Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.
[0345] The compounds can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.
[0346] In one embodiment, the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as sustained/controlled release formulations, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art.
[0347] For example, the active ingredients can be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly-(methylmethacrylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles, and nanocapsules) or in macroemulsions.
[0348] Sustained-release preparations can be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as the LUPRON DEPOT (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-()-3-hydroxybutyric acid. While polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid enable release of molecules for over 100 days, certain hydrogels release proteins for shorter time periods.
[0349] The materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) and can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,811, incorporated herein by reference.
[0350] It is especially advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the invention are dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals.
[0351] The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.
Dosages
[0352] The methods of the invention include administering one or more inhibitors of an immune-inhibitory protein described herein at a dosage of 0.01-10 mg/kg (e.g., 0.1-5 mg/kg) bodyweight. For example, the inhibitor is administered at a dosage of 0.01, 0.02, 0.05, 0.1, 0.3, 0.5, 1, 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 mg/kg. In some embodiments, the inhibitor is administered every day, every other day, every 2 days, every 3 days, every 4 days, every 5 days, or every 6 days. In other embodiments, the inhibitor is administered every 1-10 weeks (e.g., every 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 weeks). For example, the inhibitor is administered for a total of 7 days to 3 years (e.g., 7 days, 14 days, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 24 weeks, 36 weeks, 1 year, 1.5 years, 2 years, 2.5 years, or 3 years). For example, the inhibitor is administered indefinitely (e.g., at least 3 years). In some embodiments, the inhibitor is provided in an amount of 0.01-50 mg (e.g., 0.05-30 mg) per dose. For example, the inhibitor is administered in an amount of 0.01, 0.02, 0.05 mg, 0.1 mg, 0.2 mg, 0.4 mg, 0.8 mg, 2 mg, 5 mg, 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 40 mg, or 50 mg) per administration (e.g., per injection). In some cases, the inhibitor is administered biweekly. For example, the inhibitor is administered every other week for a total of 1-20 times (e.g., 1, 2, 4, 6, 8, 10, 15, or 20 times).
[0353] In some examples, the inhibitor (e.g., antibody described herein) is incorporated into or onto the vaccine device. In such cases, 0-100 mg (e.g., 5-100 mg, 10-100 mg, 20-100 mg, 30-100 mg, 40-100 mg, 50-100 mg, 60-100 mg, 70-100 mg, 80-100 mg, 90-100 mg, 1-95 mg, 1-90 mg, 5-95 mg, 5-90 mg, 5-80 mg, 5-70 mg, 5-60 mg, 5-50 mg, 5-40 mg, 5-30 mg, or 5-20 mg) of the inhibitor (e.g., antibody described herein) is present in the device. For example, the inhibitor (e.g., antibody described herein) is present in the device at a weight/weight concentration of at least 5% (e.g., at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or more).
[0354] For example, an inhibitor (e.g., anti-CTLA4 antibody or anti-PD1 antibody) is administered (e.g., systemically) at a dosage of 0.5-5 mg/kg (e.g., 3 mg/kg) body weight, e.g., 43 mg-435 mg per dose for a subject having a body weight of about 87 kg (e.g., 260 mg per dose on average). In some examples, the inhibitor (e.g., anti-CTLA4 antibody or anti-PD1 antibody) is administered (e.g., systemically) for 4 doses, e.g., at 0.5-5 mg/kg per dose (e.g., 3 mg/kg per dose), with a total dose of about 1000 mg after 4 doses. In other examples, the inhibitor (e.g., anti-CTLA4 antibody or anti-PD1 antibody) is administered in more than one dose (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more doses). In some embodiments, the time interval between doses is at least 1 day (e.g., 1, 2, 3, 4, 5, 6, 7 days or more, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 weeks or more, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months or more, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 years or more).
[0355] In some embodiments, ipilimumab is administered to a subject in need thereof at a dosage of 0.5-5 mg/kg (e.g., 3 mg/kg) body weight. For example, ipilimumab is administered every day, every other day, every 2 days, every 3 days, every 4 days, every 5 days, or every 6 days. For example, ipilimumab is administered once every 1-6 weeks (e.g., once every 3 weeks). For example, ipilimumab is administered to the subject for a total of 12 weeks or more. Ipilimumab is administered by routes such as injection or infusion. Ipilimumab is administered to a subject in need thereof for a total of 4 doses. For example, ipilimumab is administered at a dosage of 3 mg/kg body weight intravenously over 90 minutes every 3 weeks for a total of 4 doses. In some embodiments, ipilimumab is administered in combination (e.g., simultaneously or sequentially) with a vaccine device described herein.
[0356] In some cases, tremelimumab is administered to a subject in need thereof at a dosage of 1-20 mg/kg (e.g., 15 mg/kg) body weight. For example, tremelimumab is administered once every day, every other day, every 2 days, every 3 days, every 4 days, every 5 days, every 6 days, or every week. For example, tremelimumab is administered once every 10-100 days (e.g., 90 days).
[0357] In some instances, MDX-1106 is administered to a subject in need thereof at a dosage of 0.01-10 mg/kg (e.g., 0.1-10 mg/kg) body weight (e.g., 0.01-1 mg/kg, 0.5-8 mg/kg, 1-10 mg/kg, or 2-8 mg/kg). For example, MDX-1106 is administered at a dosage of 10 mg/kg. MDX-1106 is administered, e.g., intravenously. In some cases, MDX-1106 is administered once every day, every other day, every 2 days, every 3 days, every 4 days, every 5 days, every 6 days, or every week. In other cases, MDX-1106 is administered every 2 weeks, every 3 weeks, or every 4 weeks. For example, MDX-1106 is administered for a total period of at least 6 months (e.g., 6 months, 1 year, 2 years, 3 years or more).
[0358] The invention also contemplates administering MK3475 to a subject in need thereof at a dosage of 0.5 mg/kg, 1 mg/kg, 2 mg/kg 5 mg/kg, or 10 mg/kg bodyweight. MK3475 is administered once every day, every other day, every 2 days, every 3 days, every 4 days, every 5 days, every 6 days, or every week. In another embodiment, MK3475 is administered every other week, every 2 weeks, or every 3 weeks.
[0359] In some cases, CT-011 is administered to a subject in need thereof at a dosage of 0.05-6 mg/kg (e.g., 0.2-6.0 mg/kg) body weight.
[0360] In some embodiments, MDX-1105 is administered to a subject in need thereof at a dosage of 0.01-10 mg/kg (e.g., 0.1-10 mg/kg) body weight (e.g., 0.01, 0.05, 0.1, 0.3, 1, 3, or 10 mg/kg). For example, MDX-1105 is administered once every day, every other day, every 2 days, every 3 days, every 4 days, every 5 days, every 6 days, or every week. In other cases, MDX-1105 is administered every other week, every 2 weeks, or every 3 weeks. In a preferred embodiment, MDX-1105 is administered every 14 days for a total of at least 42 days.
[0361] The invention also provides for the administration of IMP321 to a subject in need thereof at a dosage of 0.01-30 mg (e.g., 0.050-30 mg, or 0.01, 0.05, 0.25, 1.25, 6.25, or 30 mg) per administration (e.g., per injection). For example, IMP321 is administered biweekly (e.g., for a total of at least 6 weeks, or at least 12 weeks). In other cases, IMP321 is administered once every day, every other day, every 2 days, every 3 days, every 4 days, every 5 days, every 6 days, or every week. In some cases, IMP321 is administered at a dosage of 5 mg/kg. IMP321 is administered by routes, such as subcutaneous injection.
[0362] In some embodiments, the inhibitor(s) described herein is administered in combination (e.g., simultaneously or sequentially) with a vaccine device described herein. For example, the inhibitor(s) is delivered systemically, while the vaccine is delivered locally. In some embodiments, the inhibitor(s) is included in or on the vaccine device. For example, the inhibitor(s) and the vaccine are delivered locally.
[0363] In other examples, the inhibitor is administered at least 6 hours (e.g., at least 6 hours, at least 12 hours, at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 6 months, at least 8 months, at least 1 year, at least 2 years, at least 3 years, at least 4 years, at least 6 years, at least 8 years, or more) prior to administration of the vaccine device. In other embodiments, the vaccine device is administered at least 6 hours (e.g., at least 6 hours, at least 12 hours, at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 6 months, at least 1 year, at least 2 years, at least 3 years, at least 4 years, at least 6 years, at least 8 years, or more) prior to administration of the inhibitor(s).
[0364] For example, an inhibitor (e.g., antibody described herein) is administered systemically prior to administration (e.g., implantation) of the vaccine device. In some cases, the inhibitor (e.g., antibody) causes debulking of a tumor (i.e., regression). For example, the debulking of the tumor occurs prior to, during, and/or after administration of the vaccine device.
[0365] As used herein, the term, about, is plus or minus 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 12%, or 15%.
[0366] The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims.
EXAMPLES
Example 1: Treatment of Tumor Bearing Mice with Anti-CTLA4 and Anti-PD1 Antibodies
[0367] Mouse models of melanoma tumors were used to determine the effect of blockade antibodies (anti-CTLA4 or anti-PD1 antibodies) on tumor growth and survival. To establish melanoma tumors, mice were inoculated with 510.sup.5 B16-F10 melanoma cells and allowed to develop for 9 days.
[0368] Mice bearing established melanoma tumors were treated with intraperitoneal (i.p.) injections of anti-CTLA4 or anti-PD1 antibodies. Antibody treatments were administered every 3 days and initiated on Day 3 of tumor challenge.
[0369] Tumor growth and survival of the mice were compared between untreated mice versus antibody-treated mice. Mice that were treated with either anti-CTLA4 antibody or anti-PD1 antibody had smaller tumor sizes (
[0370] The anti-CTLA4 antibody (9D9, catalog # BE0086) and anti-PD1 antibody (RMP1-14, catalog # BE0146**) were purchased from Bioxcell.
Example 2: Tumor Protection and T Cell Activity Induced by Therapeutic PLG Vaccination in Combination with Blockade Antibodies
[0371] The effect of combining therapeutic PLG vaccination with an anti-PD1 or anti-CTLA4 antibody was determined using mouse models of melanoma tumors. To establish melanoma tumors, mice were inoculated with 510.sup.5 B16-F10 melanoma cells and allowed to develop for 9 days.
[0372] The melanoma tumor bearing mice were either untreated, treated with PLG vaccines alone, or treated with PLG vaccines in combination with anti-PD1 or anti-CTLA4 antibodies. The antibody treatments were initiated on Day 3 after tumor challenge (with B16-F10 cells, as described above) and injected i.p. every 3 days for 24 days after tumor challenge. PLG vaccination was performed 9 days after tumor challenge. Tumor size (area in mm.sup.2) and survival were determined for each treatment group. The tumor area is the product of the two longest diameters of the tumor. Tumor diameters were measured using standard methods (e.g., with calipers). Mice treated with vaccine alone survived longer and had tumors with smaller area than untreated mice (
Example 3: Engineered Vaccines in Combination with Blockade Antibodies Enhances Intratumoral Effector T Cell Activity
[0373] The total number of CD3.sup.+CD8.sup.+ tumor infiltrating T cells in each treatment group (i.e., untreated, vaccine alone, vaccine+anti-PD1 antibody, or vaccine+anti-CTLA4 antibody) was determined from B16 (a type of melanoma cell) tumors isolated from the mice. CD3, also called the T cell co-receptor, is a marker for T cells, as it is expressed on the surface of all mature T cells and is required for T cell activation. CD8 is a marker for cytotoxic T lymphocytes (CTLs). An increase in the number of CD3.sup.+CD8.sup.+ T cells that have infiltrated the tumor indicates an increased immune response against the tumor.
[0374] Also, the ratio of CD3.sup.+CD8.sup.+ T cells to CD3.sup.+FoxP3.sup.+ T regulatory (Treg) cells isolated from the B16 tumors of the mice was determined in each treatment group. FoxP3 is a marker for Treg cells, which modulate (e.g., suppress) the immune response. Thus, the ratio provides a measure of strength of the CTL versus Treg response. A higher ratio indicates an increased immune response against the tumor.
[0375] Antibodies were administered i.p. every 3 days starting on Day 3 after tumor challenge (with 510.sup.5 B16-F10 cells) and vaccination was initiated 9 days after tumor challenge. The B16 tumors were extracted at day 20 to determine the types of T cells that have infiltrated the tumor. The number of tumor infiltrating CD3.sup.+CD8.sup.+ T cells was significantly higher in the vaccine treated mice than untreated mice (
[0376] Taken together, these results indicate that the combination of PLG vaccines with anti-PD1 or anti-CTLA4 antibodies synergistically decreases tumor size, extends survival time, and enhances the T effector cell activity relative to Treg cell activity in the population of T cells isolated from tumors.
[0377] In addition, scaffold infiltrating leukocytes, specifically, the percentage of CTLs, were compared by flow cytometry among the treatment groups (i.e., blank matrices, PLG vaccines alone, or vaccines in combination with anti-PD1 or anti-CTLA4 antibodies) at 14 days post-implantation in mice. The antibody treatments were administered on days 0, 3, 6, 9 and 12 after vaccination. Single cell suspensions were prepared from scaffolds at Day 14 and stained for activated, CTL markers, CD8 and CD107a. The percentage of the cells in the scaffold that were positive for both markers was greater in the combination therapy than the vaccine alone treated mice (
[0378] Also, the fold increase (relative to blank controls) of CD8.sup.+, scaffold-infiltrating T cells positive for both IFN and CD107a was compared in blank matrices, PLG vaccines alone, and vaccines in combination with anti-PD1 or anti-CTLA4 antibodies at 14 days post-implantation in mice. The antibody treatments were administered on days 0, 3, 6, 9 and 12 after vaccination. The vaccines were implanted 7 days after tumor challenge. CD107a is a marker for CTLs, and IFN is a cytokine involved in the immune response against tumors, and viral and bacterial infections. The fold increase in activated CD8+ T cells positive for IFN and CD107a was significantly greater in scaffolds from mice treated with the combination vaccine+antibody than vaccine alone (
[0379] Thus, the vaccine works synergistically with the blockade antibodies to enhance T effector cell activity locally, i.e., at the site of the implanted vaccine or within a vaccine. The vaccine plus blockade antibody combination also works synergistically to enhance the infiltration of tumors by activated CD8.sup.+ T cells (e.g., CTLs) and to enhance the T cell activity at tumor sites.
Example 4: Engineered PLG Vaccine in Combination with Blockade Antibodies Enhances Local T Effector Cell Activity
[0380] The effect of the combination of a PLG vaccine with a blockade antibody on local T effector cell activity (i.e., at the site of vaccine scaffold implantation) was determined. Mice were treated with PLG vaccines alone or PLG vaccines in combination with an anti-CTLA4 antibody for 14 days. A subset of mice were treated with antibody and vaccine without tumor challenge to analyze the effects at the vaccine site. Another subset of mice were challenged with 500,000 B16 tumor cells. Vaccines were administered 7 days after tumor challenge. The antibody treatments were administered on days 0, 3, 6, 9 and 12 after vaccination.
[0381] Effects at the tumor site were examined, specifically the numbers of cytotoxic T cells, interferon gamma expression, CD107a expression, and Treg cell numbers. Flow cytometry was used to determine the number of CD3.sup.+ T cell infiltrates into the implanted vaccine scaffolds that were isolated from the two treatment groups. Mice treated with the combination (vaccine+anti-CTLA4 antibody) had more T cell infiltrates in the scaffolds than mice treated with vaccine alone (
[0382] The total number of CD3.sup.+CD8.sup.+ T effector cells was also determined in mice implanted with blank matrices without vaccine, PLG vaccines alone, or vaccines in combination with anti-PD1 or anti-CTLA4 antibodies for 14 days. The antibody treatments were administered on days 0, 3, 6, 9 and 12 after vaccination. Surprisingly, the combination treatments led to a significantly higher number of CD3.sup.+CD8.sup.+ T effector cells infiltrated into the scaffolds than vaccine alone (
Example 5: Engineered PLG Vaccine in Combination with CTLA-4 Maintains Local T Cell Activity
[0383] The amount of T cell infiltration into the PLG vaccines implanted in mice for 14 days was determined. Flow cytometry was used to determine the phenotypes (i.e., CD4.sup.+CD8.sup.+ versus CD4.sup.+FoxP3.sup.+) of T cell infiltrates isolated from PLG implants in mice treated with PLG vaccines alone (Vax) or in combination with anti-CTLA4 antibody (Vax+CTLA4) or with anti-PD1 antibody (Vax+PD1) (
Example 6: Effector T Cell Activity is Greater than Regulatory T Cell Activity in Vaccine Draining Lymph Nodes
[0384] Mice were treated with vaccine alone or in combination with anti-CTLA4 or anti-PD1 antibodies. The antibody treatments were administered on days 0, 3, 6, 9, and 12 after vaccination. The vaccine draining lymph nodes were then extracted at day 14 to measure the degree of T cell infiltration. Flow cytometry was used to quantify the percentage of CD8.sup.+ T cells and FoxP3.sup.+ Treg cells in the vaccine draining lymph nodes. The ratio of CD3.sup.+CD8.sup.+ T cells to CD3.sup.+FoxP3.sup.+ Treg cells was also determined.
[0385] By flow cytometry, the percentage of CD8.sup.+ T effector cells in the lymph nodes of mice treated with a combination of vaccine+antibody was greater than those from vaccine alone treated mice (
[0386] Thus, the vaccine works synergistically with the blockade antibodies, in particular, the anti-CTLA4 antibody, to increase the proportion of T effector cells and decrease the proportion of Treg cells in the vaccine draining lymph nodes.
Example 7: Combining Anti-PD1 Antibody and Anti-CTLA4 Antibody with PLG Vaccination Enhances T Cell Activation and Tumor Inhibition in Melanoma Models
[0387] Described herein is data related to structural vaccines in combination with checkpoint antibodies. Combining checkpoint blockade inhibitors, -PD-1 and -CTLA4, with PLG vaccination had a significant effect on tumor growth in comparison to vaccination with either antibody alone (
Example 8: Combining Anti-PD1 Antibody and Anti-CTLA4 Antibody with PLG Vaccination Enhances Cytotoxic T Cell Response
[0388] As described in detail below, combining blockade antibodies with PLG vaccination significantly skewed the tumor infiltrating leukocyte (TIL) response toward active, cytotoxic T cells, relative to suppressive Tregs (
[0389] PLG Vaccination at day 9 after tumor challenge induced significant levels of CD3(+)CD8(+) T cell infiltration into 20-day-old B16 tumors, resulting in approximately 3,500 cytotoxic T cells per mm.sup.2 of tumor (
[0390] All tumors were pretreated with antibody blockade prior to vaccination because this sequence likely reflects the clinical setting where these antibodies are used to initially treat tumors as they become standards of care. However, if antibody administration is ceased after vaccination, the effects on tumor inhibition are lost (
Other Embodiments
[0391] While the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.
[0392] The patent and scientific literature referred to herein establishes the knowledge that is available to those with skill in the art. All United States patents and published or unpublished United States patent applications cited herein are incorporated by reference. All published foreign patents and patent applications cited herein are hereby incorporated by reference. Genbank and NCBI submissions indicated by accession number cited herein are hereby incorporated by reference. All other published references, documents, manuscripts and scientific literature cited herein are hereby incorporated by reference.
[0393] While this invention has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.