EP2496265A1 - Vaccine compositions and methods of use - Google Patents
Vaccine compositions and methods of useInfo
- Publication number
- EP2496265A1 EP2496265A1 EP09744895A EP09744895A EP2496265A1 EP 2496265 A1 EP2496265 A1 EP 2496265A1 EP 09744895 A EP09744895 A EP 09744895A EP 09744895 A EP09744895 A EP 09744895A EP 2496265 A1 EP2496265 A1 EP 2496265A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- particle
- phagocytosed
- component
- composition
- buffering
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn
Links
- 239000000203 mixture Substances 0.000 title claims abstract description 53
- 238000000034 method Methods 0.000 title claims abstract description 25
- 229960005486 vaccine Drugs 0.000 title claims abstract description 17
- 239000002245 particle Substances 0.000 claims abstract description 96
- 108090000765 processed proteins & peptides Proteins 0.000 claims abstract description 59
- 102000004196 processed proteins & peptides Human genes 0.000 claims abstract description 53
- 229920001184 polypeptide Polymers 0.000 claims abstract description 49
- 230000003139 buffering effect Effects 0.000 claims abstract description 37
- 210000000612 antigen-presenting cell Anatomy 0.000 claims abstract description 21
- 229920000392 Zymosan Polymers 0.000 claims description 25
- 102000008949 Histocompatibility Antigens Class I Human genes 0.000 claims description 15
- 108010088652 Histocompatibility Antigens Class I Proteins 0.000 claims description 13
- 239000012634 fragment Substances 0.000 claims description 8
- 229920002873 Polyethylenimine Polymers 0.000 claims description 7
- 230000005867 T cell response Effects 0.000 claims description 6
- 230000000890 antigenic effect Effects 0.000 claims description 6
- 210000000172 cytosol Anatomy 0.000 claims description 5
- 230000030741 antigen processing and presentation Effects 0.000 claims description 3
- 230000001159 endocytotic effect Effects 0.000 claims description 3
- 210000004027 cell Anatomy 0.000 abstract description 27
- 108090000623 proteins and genes Proteins 0.000 abstract description 22
- 102000004169 proteins and genes Human genes 0.000 abstract description 22
- 210000004443 dendritic cell Anatomy 0.000 abstract description 18
- 210000002540 macrophage Anatomy 0.000 abstract description 18
- 230000000813 microbial effect Effects 0.000 abstract description 5
- 102000036639 antigens Human genes 0.000 description 40
- 108091007433 antigens Proteins 0.000 description 40
- 239000000427 antigen Substances 0.000 description 39
- 235000018102 proteins Nutrition 0.000 description 20
- 238000006243 chemical reaction Methods 0.000 description 19
- 210000003712 lysosome Anatomy 0.000 description 15
- 230000001868 lysosomic effect Effects 0.000 description 15
- 210000000680 phagosome Anatomy 0.000 description 15
- 239000003153 chemical reaction reagent Substances 0.000 description 12
- 210000001539 phagocyte Anatomy 0.000 description 12
- 108010058846 Ovalbumin Proteins 0.000 description 11
- 239000000872 buffer Substances 0.000 description 11
- 125000005647 linker group Chemical group 0.000 description 10
- 206010028980 Neoplasm Diseases 0.000 description 9
- 241000700605 Viruses Species 0.000 description 9
- 125000002228 disulfide group Chemical group 0.000 description 9
- 210000001616 monocyte Anatomy 0.000 description 9
- 229940092253 ovalbumin Drugs 0.000 description 9
- 125000003277 amino group Chemical group 0.000 description 8
- 150000001413 amino acids Chemical group 0.000 description 7
- 210000000865 mononuclear phagocyte system Anatomy 0.000 description 7
- 210000005253 yeast cell Anatomy 0.000 description 7
- 108091054437 MHC class I family Proteins 0.000 description 6
- -1 epitope Proteins 0.000 description 6
- 238000001727 in vivo Methods 0.000 description 6
- 230000006698 induction Effects 0.000 description 6
- 239000000463 material Substances 0.000 description 6
- 230000037361 pathway Effects 0.000 description 6
- QYEAAMBIUQLHFQ-UHFFFAOYSA-N (2,5-dioxopyrrolidin-1-yl) 6-[3-(pyridin-2-yldisulfanyl)propanoylamino]hexanoate Chemical compound O=C1CCC(=O)N1OC(=O)CCCCCNC(=O)CCSSC1=CC=CC=N1 QYEAAMBIUQLHFQ-UHFFFAOYSA-N 0.000 description 5
- 206010057249 Phagocytosis Diseases 0.000 description 5
- 235000001014 amino acid Nutrition 0.000 description 5
- 210000000805 cytoplasm Anatomy 0.000 description 5
- 125000000524 functional group Chemical group 0.000 description 5
- 230000008782 phagocytosis Effects 0.000 description 5
- 238000002255 vaccination Methods 0.000 description 5
- 102000043129 MHC class I family Human genes 0.000 description 4
- 210000001744 T-lymphocyte Anatomy 0.000 description 4
- 239000012467 final product Substances 0.000 description 4
- 230000028993 immune response Effects 0.000 description 4
- 230000000242 pagocytic effect Effects 0.000 description 4
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 4
- 238000012546 transfer Methods 0.000 description 4
- 210000004881 tumor cell Anatomy 0.000 description 4
- 230000003612 virological effect Effects 0.000 description 4
- 229920000936 Agarose Polymers 0.000 description 3
- 206010039491 Sarcoma Diseases 0.000 description 3
- 230000004913 activation Effects 0.000 description 3
- WQZGKKKJIJFFOK-FPRJBGLDSA-N beta-D-galactose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@H]1O WQZGKKKJIJFFOK-FPRJBGLDSA-N 0.000 description 3
- 108010005774 beta-Galactosidase Proteins 0.000 description 3
- 150000001875 compounds Chemical class 0.000 description 3
- 150000004985 diamines Chemical class 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 210000004408 hybridoma Anatomy 0.000 description 3
- 238000000338 in vitro Methods 0.000 description 3
- 239000012528 membrane Substances 0.000 description 3
- 102000035118 modified proteins Human genes 0.000 description 3
- 108091005573 modified proteins Proteins 0.000 description 3
- 239000000546 pharmaceutical excipient Substances 0.000 description 3
- 229920000642 polymer Polymers 0.000 description 3
- 239000002243 precursor Substances 0.000 description 3
- 238000012545 processing Methods 0.000 description 3
- 230000000069 prophylactic effect Effects 0.000 description 3
- 230000004044 response Effects 0.000 description 3
- 230000001225 therapeutic effect Effects 0.000 description 3
- JWDFQMWEFLOOED-UHFFFAOYSA-N (2,5-dioxopyrrolidin-1-yl) 3-(pyridin-2-yldisulfanyl)propanoate Chemical compound O=C1CCC(=O)N1OC(=O)CCSSC1=CC=CC=N1 JWDFQMWEFLOOED-UHFFFAOYSA-N 0.000 description 2
- JVJGCCBAOOWGEO-RUTPOYCXSA-N (2s)-2-[[(2s)-2-[[(2s)-2-[[(2s)-2-[[(2s)-4-amino-2-[[(2s,3s)-2-[[(2s,3s)-2-[[(2s)-2-azaniumyl-3-hydroxypropanoyl]amino]-3-methylpentanoyl]amino]-3-methylpentanoyl]amino]-4-oxobutanoyl]amino]-3-phenylpropanoyl]amino]-4-carboxylatobutanoyl]amino]-6-azaniumy Chemical compound OC[C@H](N)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@H](C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(O)=O)CC1=CC=CC=C1 JVJGCCBAOOWGEO-RUTPOYCXSA-N 0.000 description 2
- 229920002498 Beta-glucan Polymers 0.000 description 2
- 229920002101 Chitin Polymers 0.000 description 2
- 102000006312 Cyclin D2 Human genes 0.000 description 2
- 108010058544 Cyclin D2 Proteins 0.000 description 2
- 102000004127 Cytokines Human genes 0.000 description 2
- 108090000695 Cytokines Proteins 0.000 description 2
- BWGNESOTFCXPMA-UHFFFAOYSA-N Dihydrogen disulfide Chemical compound SS BWGNESOTFCXPMA-UHFFFAOYSA-N 0.000 description 2
- 241000713772 Human immunodeficiency virus 1 Species 0.000 description 2
- 108090000604 Hydrolases Proteins 0.000 description 2
- 102000004157 Hydrolases Human genes 0.000 description 2
- 102000043131 MHC class II family Human genes 0.000 description 2
- 108091054438 MHC class II family Proteins 0.000 description 2
- 102100028389 Melanoma antigen recognized by T-cells 1 Human genes 0.000 description 2
- QSBINWBNXWAVAK-PSXMRANNSA-N PE-NMe(16:0/16:0) Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP(O)(=O)OCCNC)OC(=O)CCCCCCCCCCCCCCC QSBINWBNXWAVAK-PSXMRANNSA-N 0.000 description 2
- 229920002684 Sepharose Polymers 0.000 description 2
- 125000003172 aldehyde group Chemical group 0.000 description 2
- 230000014102 antigen processing and presentation of exogenous peptide antigen via MHC class I Effects 0.000 description 2
- 230000007503 antigenic stimulation Effects 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
- 230000001580 bacterial effect Effects 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 230000003592 biomimetic effect Effects 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 150000001720 carbohydrates Chemical class 0.000 description 2
- 235000014633 carbohydrates Nutrition 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- 229940028617 conventional vaccine Drugs 0.000 description 2
- 239000003431 cross linking reagent Substances 0.000 description 2
- 210000001151 cytotoxic T lymphocyte Anatomy 0.000 description 2
- ZWLIYXJBOIDXLL-UHFFFAOYSA-N decanedihydrazide Chemical compound NNC(=O)CCCCCCCCC(=O)NN ZWLIYXJBOIDXLL-UHFFFAOYSA-N 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 238000009472 formulation Methods 0.000 description 2
- 125000002485 formyl group Chemical group [H]C(*)=O 0.000 description 2
- 230000004927 fusion Effects 0.000 description 2
- 230000001939 inductive effect Effects 0.000 description 2
- 238000002347 injection Methods 0.000 description 2
- 239000007924 injection Substances 0.000 description 2
- 230000003834 intracellular effect Effects 0.000 description 2
- 208000032839 leukemia Diseases 0.000 description 2
- 230000002132 lysosomal effect Effects 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 244000052769 pathogen Species 0.000 description 2
- 230000001717 pathogenic effect Effects 0.000 description 2
- 230000003389 potentiating effect Effects 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 229940021993 prophylactic vaccine Drugs 0.000 description 2
- 230000001681 protective effect Effects 0.000 description 2
- 230000002797 proteolythic effect Effects 0.000 description 2
- KIDHWZJUCRJVML-UHFFFAOYSA-N putrescine Chemical compound NCCCCN KIDHWZJUCRJVML-UHFFFAOYSA-N 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- JQWHASGSAFIOCM-UHFFFAOYSA-M sodium periodate Chemical compound [Na+].[O-]I(=O)(=O)=O JQWHASGSAFIOCM-UHFFFAOYSA-M 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 230000000638 stimulation Effects 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 238000002560 therapeutic procedure Methods 0.000 description 2
- 229940021747 therapeutic vaccine Drugs 0.000 description 2
- 125000003396 thiol group Chemical group [H]S* 0.000 description 2
- 210000001519 tissue Anatomy 0.000 description 2
- 241001430294 unidentified retrovirus Species 0.000 description 2
- FYGDTMLNYKFZSV-URKRLVJHSA-N (2s,3r,4s,5s,6r)-2-[(2r,4r,5r,6s)-4,5-dihydroxy-2-(hydroxymethyl)-6-[(2r,4r,5r,6s)-4,5,6-trihydroxy-2-(hydroxymethyl)oxan-3-yl]oxyoxan-3-yl]oxy-6-(hydroxymethyl)oxane-3,4,5-triol Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1OC1[C@@H](CO)O[C@@H](OC2[C@H](O[C@H](O)[C@H](O)[C@H]2O)CO)[C@H](O)[C@H]1O FYGDTMLNYKFZSV-URKRLVJHSA-N 0.000 description 1
- ASNTZYQMIUCEBV-UHFFFAOYSA-N 2,5-dioxo-1-[6-[3-(pyridin-2-yldisulfanyl)propanoylamino]hexanoyloxy]pyrrolidine-3-sulfonic acid Chemical compound O=C1C(S(=O)(=O)O)CC(=O)N1OC(=O)CCCCCNC(=O)CCSSC1=CC=CC=N1 ASNTZYQMIUCEBV-UHFFFAOYSA-N 0.000 description 1
- HEBDGRTWECSNNT-UHFFFAOYSA-N 2-methylidenepentanoic acid Chemical compound CCCC(=C)C(O)=O HEBDGRTWECSNNT-UHFFFAOYSA-N 0.000 description 1
- LKKMLIBUAXYLOY-UHFFFAOYSA-N 3-Amino-1-methyl-5H-pyrido[4,3-b]indole Chemical compound N1C2=CC=CC=C2C2=C1C=C(N)N=C2C LKKMLIBUAXYLOY-UHFFFAOYSA-N 0.000 description 1
- 208000030507 AIDS Diseases 0.000 description 1
- YZXBAPSDXZZRGB-DOFZRALJSA-M Arachidonate Chemical compound CCCCC\C=C/C\C=C/C\C=C/C\C=C/CCCC([O-])=O YZXBAPSDXZZRGB-DOFZRALJSA-M 0.000 description 1
- 208000035143 Bacterial infection Diseases 0.000 description 1
- 206010006187 Breast cancer Diseases 0.000 description 1
- 208000026310 Breast neoplasm Diseases 0.000 description 1
- 102100040840 C-type lectin domain family 7 member A Human genes 0.000 description 1
- 102000012406 Carcinoembryonic Antigen Human genes 0.000 description 1
- 108010022366 Carcinoembryonic Antigen Proteins 0.000 description 1
- 208000005623 Carcinogenesis Diseases 0.000 description 1
- 102100025064 Cellular tumor antigen p53 Human genes 0.000 description 1
- VEXZGXHMUGYJMC-UHFFFAOYSA-M Chloride anion Chemical compound [Cl-] VEXZGXHMUGYJMC-UHFFFAOYSA-M 0.000 description 1
- 206010009944 Colon cancer Diseases 0.000 description 1
- 102100041003 Glutamate carboxypeptidase 2 Human genes 0.000 description 1
- 101710154606 Hemagglutinin Proteins 0.000 description 1
- 101000721661 Homo sapiens Cellular tumor antigen p53 Proteins 0.000 description 1
- 101000892862 Homo sapiens Glutamate carboxypeptidase 2 Proteins 0.000 description 1
- 101000578784 Homo sapiens Melanoma antigen recognized by T-cells 1 Proteins 0.000 description 1
- 101000831567 Homo sapiens Toll-like receptor 2 Proteins 0.000 description 1
- 101000669406 Homo sapiens Toll-like receptor 6 Proteins 0.000 description 1
- 241000725303 Human immunodeficiency virus Species 0.000 description 1
- 206010020751 Hypersensitivity Diseases 0.000 description 1
- 206010025323 Lymphomas Diseases 0.000 description 1
- 239000004472 Lysine Substances 0.000 description 1
- 108010010995 MART-1 Antigen Proteins 0.000 description 1
- 241001529936 Murinae Species 0.000 description 1
- KJCNTPGGZJVZSD-UHFFFAOYSA-N OC(=O)CCCCCN(N1C(=O)CCC1=O)C(=O)CCSSc1ccccn1 Chemical compound OC(=O)CCCCCN(N1C(=O)CCC1=O)C(=O)CCSSc1ccccn1 KJCNTPGGZJVZSD-UHFFFAOYSA-N 0.000 description 1
- 101710093908 Outer capsid protein VP4 Proteins 0.000 description 1
- 101710135467 Outer capsid protein sigma-1 Proteins 0.000 description 1
- 208000030852 Parasitic disease Diseases 0.000 description 1
- 101710173835 Penton protein Proteins 0.000 description 1
- 108010072866 Prostate-Specific Antigen Proteins 0.000 description 1
- 101710176177 Protein A56 Proteins 0.000 description 1
- 239000005700 Putrescine Substances 0.000 description 1
- 206010038389 Renal cancer Diseases 0.000 description 1
- 208000006265 Renal cell carcinoma Diseases 0.000 description 1
- 108700008625 Reporter Genes Proteins 0.000 description 1
- 230000006044 T cell activation Effects 0.000 description 1
- 230000024932 T cell mediated immunity Effects 0.000 description 1
- 108010017842 Telomerase Proteins 0.000 description 1
- 102000002689 Toll-like receptor Human genes 0.000 description 1
- 108020000411 Toll-like receptor Proteins 0.000 description 1
- 102100024333 Toll-like receptor 2 Human genes 0.000 description 1
- 102100039387 Toll-like receptor 6 Human genes 0.000 description 1
- 101800001690 Transmembrane protein gp41 Proteins 0.000 description 1
- 108060008682 Tumor Necrosis Factor Proteins 0.000 description 1
- 102100040247 Tumor necrosis factor Human genes 0.000 description 1
- 208000036142 Viral infection Diseases 0.000 description 1
- INAPMGSXUVUWAF-GCVPSNMTSA-N [(2r,3s,5r,6r)-2,3,4,5,6-pentahydroxycyclohexyl] dihydrogen phosphate Chemical compound OC1[C@H](O)[C@@H](O)C(OP(O)(O)=O)[C@H](O)[C@@H]1O INAPMGSXUVUWAF-GCVPSNMTSA-N 0.000 description 1
- 208000009956 adenocarcinoma Diseases 0.000 description 1
- 108700025771 adenovirus penton Proteins 0.000 description 1
- IBVAQQYNSHJXBV-UHFFFAOYSA-N adipic acid dihydrazide Chemical compound NNC(=O)CCCCC(=O)NN IBVAQQYNSHJXBV-UHFFFAOYSA-N 0.000 description 1
- 239000002671 adjuvant Substances 0.000 description 1
- 239000013566 allergen Substances 0.000 description 1
- 208000026935 allergic disease Diseases 0.000 description 1
- 230000007815 allergy Effects 0.000 description 1
- 239000003708 ampul Substances 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 239000008135 aqueous vehicle Substances 0.000 description 1
- 229940114078 arachidonate Drugs 0.000 description 1
- 230000002238 attenuated effect Effects 0.000 description 1
- 208000022362 bacterial infectious disease Diseases 0.000 description 1
- 239000011324 bead Substances 0.000 description 1
- 230000001588 bifunctional effect Effects 0.000 description 1
- 201000008275 breast carcinoma Diseases 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 230000036952 cancer formation Effects 0.000 description 1
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 1
- 231100000504 carcinogenesis Toxicity 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 239000013592 cell lysate Substances 0.000 description 1
- 229940030156 cell vaccine Drugs 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 239000003638 chemical reducing agent Substances 0.000 description 1
- 230000024203 complement activation Effects 0.000 description 1
- 230000008878 coupling Effects 0.000 description 1
- 238000010168 coupling process Methods 0.000 description 1
- 238000005859 coupling reaction Methods 0.000 description 1
- 125000000151 cysteine group Chemical group N[C@@H](CS)C(=O)* 0.000 description 1
- 238000011266 cytolytic assay Methods 0.000 description 1
- 230000001461 cytolytic effect Effects 0.000 description 1
- 230000001086 cytosolic effect Effects 0.000 description 1
- 108010025838 dectin 1 Proteins 0.000 description 1
- 230000007123 defense Effects 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 230000000593 degrading effect Effects 0.000 description 1
- 238000002716 delivery method Methods 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 230000001066 destructive effect Effects 0.000 description 1
- 235000014113 dietary fatty acids Nutrition 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 231100000676 disease causative agent Toxicity 0.000 description 1
- 208000035475 disorder Diseases 0.000 description 1
- 239000002270 dispersing agent Substances 0.000 description 1
- 239000002552 dosage form Substances 0.000 description 1
- 239000000839 emulsion Substances 0.000 description 1
- 239000002158 endotoxin Substances 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 239000000194 fatty acid Substances 0.000 description 1
- 229930195729 fatty acid Natural products 0.000 description 1
- 150000004665 fatty acids Chemical class 0.000 description 1
- 230000000799 fusogenic effect Effects 0.000 description 1
- 238000001415 gene therapy Methods 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- 239000000185 hemagglutinin Substances 0.000 description 1
- 239000003228 hemolysin Substances 0.000 description 1
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 1
- 210000000987 immune system Anatomy 0.000 description 1
- 230000005847 immunogenicity Effects 0.000 description 1
- 238000000099 in vitro assay Methods 0.000 description 1
- 238000010874 in vitro model Methods 0.000 description 1
- 239000012678 infectious agent Substances 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 230000002757 inflammatory effect Effects 0.000 description 1
- 230000028709 inflammatory response Effects 0.000 description 1
- 230000004941 influx Effects 0.000 description 1
- 238000001802 infusion Methods 0.000 description 1
- 238000003780 insertion Methods 0.000 description 1
- 230000037431 insertion Effects 0.000 description 1
- 238000010255 intramuscular injection Methods 0.000 description 1
- 238000010253 intravenous injection Methods 0.000 description 1
- 231100000518 lethal Toxicity 0.000 description 1
- 230000001665 lethal effect Effects 0.000 description 1
- 230000000670 limiting effect Effects 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 239000002502 liposome Substances 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 230000035800 maturation Effects 0.000 description 1
- 230000001404 mediated effect Effects 0.000 description 1
- 201000001441 melanoma Diseases 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 210000002864 mononuclear phagocyte Anatomy 0.000 description 1
- 238000010172 mouse model Methods 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 229960003104 ornithine Drugs 0.000 description 1
- 230000003204 osmotic effect Effects 0.000 description 1
- 244000045947 parasite Species 0.000 description 1
- 238000007911 parenteral administration Methods 0.000 description 1
- KHIWWQKSHDUIBK-UHFFFAOYSA-N periodic acid Chemical compound OI(=O)(=O)=O KHIWWQKSHDUIBK-UHFFFAOYSA-N 0.000 description 1
- 150000003904 phospholipids Chemical class 0.000 description 1
- 239000013612 plasmid Substances 0.000 description 1
- 229920000768 polyamine Polymers 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 239000003755 preservative agent Substances 0.000 description 1
- 230000002335 preservative effect Effects 0.000 description 1
- 150000003141 primary amines Chemical class 0.000 description 1
- 230000000770 proinflammatory effect Effects 0.000 description 1
- 230000009822 protein phosphorylation Effects 0.000 description 1
- 108020003175 receptors Proteins 0.000 description 1
- 102000005962 receptors Human genes 0.000 description 1
- 230000002829 reductive effect Effects 0.000 description 1
- 201000010174 renal carcinoma Diseases 0.000 description 1
- BEOOHQFXGBMRKU-UHFFFAOYSA-N sodium cyanoborohydride Chemical compound [Na+].[B-]C#N BEOOHQFXGBMRKU-UHFFFAOYSA-N 0.000 description 1
- 239000003381 stabilizer Substances 0.000 description 1
- 230000000087 stabilizing effect Effects 0.000 description 1
- 230000004936 stimulating effect Effects 0.000 description 1
- 238000010254 subcutaneous injection Methods 0.000 description 1
- 239000007929 subcutaneous injection Substances 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- MCHSLXDOQURRLX-UHFFFAOYSA-N sulfo 6-[(2,5-dioxopyrrolidin-1-yl)-[3-(pyridin-2-yldisulfanyl)propanoyl]amino]hexanoate Chemical compound C1(CCC(N1N(C(CCSSC1=NC=CC=C1)=O)CCCCCC(=O)OS(=O)(=O)O)=O)=O MCHSLXDOQURRLX-UHFFFAOYSA-N 0.000 description 1
- 230000001629 suppression Effects 0.000 description 1
- 239000000375 suspending agent Substances 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 230000008961 swelling Effects 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 230000003614 tolerogenic effect Effects 0.000 description 1
- 238000001890 transfection Methods 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 241000701161 unidentified adenovirus Species 0.000 description 1
- 241001529453 unidentified herpesvirus Species 0.000 description 1
- 241000712461 unidentified influenza virus Species 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
- A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
- A61K9/51—Nanocapsules; Nanoparticles
- A61K9/5107—Excipients; Inactive ingredients
- A61K9/5176—Compounds of unknown constitution, e.g. material from plants or animals
- A61K9/5184—Virus capsids or envelopes enclosing drugs
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
- A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
- A61K47/34—Macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyesters, polyamino acids, polysiloxanes, polyphosphazines, copolymers of polyalkylene glycol or poloxamers
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/46—Ingredients of undetermined constitution or reaction products thereof, e.g. skin, bone, milk, cotton fibre, eggshell, oxgall or plant extracts
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
- A61K47/61—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule the organic macromolecular compound being a polysaccharide or a derivative thereof
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
- A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
- A61K9/1605—Excipients; Inactive ingredients
- A61K9/1629—Organic macromolecular compounds
- A61K9/1652—Polysaccharides, e.g. alginate, cellulose derivatives; Cyclodextrin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
- A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
- A61K9/1605—Excipients; Inactive ingredients
- A61K9/1664—Compounds of unknown constitution, e.g. material from plants or animals
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
- A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
- A61K9/5005—Wall or coating material
- A61K9/5063—Compounds of unknown constitution, e.g. material from plants or animals
- A61K9/5068—Cell membranes or bacterial membranes enclosing drugs
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
- A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
- A61K9/51—Nanocapsules; Nanoparticles
- A61K9/5107—Excipients; Inactive ingredients
- A61K9/5176—Compounds of unknown constitution, e.g. material from plants or animals
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
- A61K2039/6087—Polysaccharides; Lipopolysaccharides [LPS]
Definitions
- the present invention generally relates to compositions and methods for delivering a protein, peptide, epitope, or antigen to an antigen presenting cell, such as a macrophage or dendritic cell.
- the compositions and methods disclosed herein are particularly useful in making prophylactic and therapeutic vaccines.
- Antigen presenting cells including macrophages and other cells of the mononuclear phagocyte system actively phagocytose particles and play a central role in the immune response.
- Macrophages are cells within the tissues that are derived from monocytes. These monocytes/macrophages phagocytose microbes, for example, which are then digested to smaller antigenic portions in the lysosome/phagosome. The resultant antigens are cycled back to the surface for presentation to the humoral and cellular arms of the immune system. Accordingly, monocytes/macrophages are of particular interest because they play an important role in both nonspecific and specific defenses in the host against pathogens.
- Dendritic cells are also antigen presenting cells that express MHC class I and class II molecules.
- An ideal vaccine mimics the rapid uptake and transfer of pathogenic structures without actually establishing an infection and without causing suppression of the MHC class I pathway.
- the present invention relates to a composition comprising a modified particle to be phagocytosed that is avidly and specifically taken up by professional phagocytic cells, and methods for delivering cargo molecules to the cytoplasm of antigen presenting cells for presentation on MHC class I molecules.
- Dkt. No.: 096630-0103 exhibit "cross presentation" phenomena, whereby dendritic cells present exogenous antigens on a class I molecule, the localized concentration of soluble, exogenous antigen must be very high (approximately 100 or even 1000 fold higher than the present invention) for such cross presentation to occur and is therefore inefficient.
- the compositions of the present invention can efficiently deliver exogenous proteins, epitopes, antigens, and peptides, for presentation on class I molecules with only a very low amount of exogenous material.
- One embodiment of the present invention is an antigenic composition
- a polypeptide component comprising (i) a polypeptide component, (ii) a buffering component, and (iii) a particle that can be phagocytosed, wherein the polypeptide component or a fragment thereof is ultimately presented on a class I MHC molecule.
- Another embodiment of the invention is a method for efficient delivery of a polypeptide component to an antigen presenting cell comprising administering a composition comprising (i) a polypeptide component, (ii) a buffering component, and (iii) a particle that can be phagocytosed, wherein the polypeptide component, following administration, enters the cytosol from an endocytotic vesicle, and the polypeptide or a fragment thereof is presented on a MHC class I molecule.
- compositions for exogenous antigen presentation on class I MHC molecules comprising (i) a polypeptide component, (ii) a buffering component, and (iii) a particle that can be phagocytosed, and a vaccine composition comprising (i) a polypeptide component, (ii) a buffering component, and (iii) a particle that can be phagocytosed, wherein the polypeptide component or a fragment thereof is ultimately presented on a class I MHC molecule.
- the polypeptide component is also delivered in an amount sufficient to provoke a CD8 T cell response, in the case of dendritic cells.
- the particle that can be phagocytosed is a biodegradable particle, such as zymosan particle, chitin particle, agarose particle or sepharose particle
- the buffering component can be any buffer with a buffering capacity in the range of about pH 6 to about pH 8, such as RCONHNH 2 , polyethyleneimine, oligohistidine, oligoornithine, and oligolysine.
- the present invention provides a composition for delivery to an antigen presenting cell, and a related method of use.
- the composition according to the present invention comprises a polypeptide component, attached to a particle that can be phagocytosed and a buffering component.
- the present invention takes advantage of the phagocytic property of certain antigen presenting cells in that the polypeptide component is provided on a substrate that "looks" like a microbe. Thus, the particle attracts phagocytic cells.
- compositions of the present invention do not contain liposomes (vesicles with an aqueous interior enclosed by one or more phospholipid bilayers), nor are the compositions disclosed herein taken up by macrophages, dendritic, or other antigen presenting cells based on an agent that permeabilizes the extracellular membrane of these cells.
- the compositions of the present invention also do not utilize haemolysin to permeabilize the phagosomal membrane.
- the particle to be phagocytosed is a biodegradable particle, such as one derived from natural sources.
- the particle to be phagocytosed is of microbial origin, and is preferably a
- yeast cell wall particle is a zymosan particle.
- Zymosan also referred to as Zymosan A
- Natural particles, such as zymosan are better tolerated by macrophages, for example, than magnetic beads and particles from other sources.
- Zymosan is an insoluble polysaccharide component of yeast cell wall.
- Prior publications uncovered zymosan's involvements in (i) induction of the release of cytokines or proinflammatory cytokines, (ii) induction of protein phosphorylation and inositol phosphate formation, (iii) arachidonate mobilization, (iv) activation of the alternative complement pathway; and (v) raise of cyclin D2 levels, suggesting a role of cyclin D2 in macrophage activation (Miyasato et al, Int. Arch. Allergy Immunol. 104: 24-26, 1994).
- zymosan particles are capable of inducing inflammatory signals in macrophages through Toll- like receptors, e.g. TLR2 and TLR6, and dectin-1, which is a receptor that binds ⁇ -glucans and is important for macrophage phagocytosis.
- Zymosan is also involved in inducing inflammatory responses, such as TNF-a production and NF-KB activation in macrophages (Underhill, Journal of Endotoxin Research, 9: 176-180, 2003; Sato et al, J. Immunol, 111 : 417-425, 2003; Dillon et al. , J. Clin. Invest. 116: 916-928, 2006).
- a preferred size for the particle that can be phagocytosed is one that approximates the size of microbial structures that cells of the mononuclear phagocyte system and other phagocytic cells typically ingest.
- the particle will be about 0.05 to about 5.0 ⁇ , about 0.05 to about 2.5 ⁇ , about 0.1 to about 2.5 ⁇ , about 1.0 to about 2.5 ⁇ , about 1.0 to about 2.0 ⁇ , or about 1.0 to about 1.5 ⁇ .
- the term "about” in this context refers to +/- 0.25 ⁇ .
- Zymosan is typically about 2.0 ⁇ in size.
- the particle that can be phagocytosed is not limited by any particular size, a preferred size for the particle that can be phagocytosed is one that approximates the size of microbial structures that cells of the mononuclear phagocyte lineage (e.g., monocytes, macrophages, dendritic cells, dendritic cell precursors (immature dendritic cells), or other antigen presenting cells, typically ingest.
- the particle will be about 0.05 to about 5.0 ⁇ , about 0.05 to about 2.5 ⁇ , about 0.1 to about 2.5 ⁇ , about 1.0 to about 2.5 ⁇ , about 1.0 to about 2.0 ⁇ , or about 1.0 to about 1.5 ⁇ .
- the term "about” in this context refers to +/- 0.25 ⁇ .
- Zymosan is typically about 2.0 ⁇ in size.
- the particle that can be phagocytosed is also not limited by shape or material.
- the particle can be of any shape or material that allows the composition of the present invention to be phagocytized by cells of the mononuclear phagocyte system, such as monocytes/macrophages, dendritic cells, immature dendritic cells (have a high capacity for phagocytosis, then undergo maturation), or other phagocytic cells.
- the particle to be phagocytosed can be made of chitin, a synthetic beta glucan polymer, agarose, sepharose, etc., so long as the particle contains a carbohydrate or other moiety that permits attachment of the polypeptide component of the present invention.
- the polypeptide component of the present invention is attached to the particle to be phagocytosed. Following administration of the polypeptide component attached to the particle, the polypeptide component, or fragment thereof is released from the particle, enters the cytosol from an endocytotic vesicle (e.g., phagosome) and, as a result of normal "processing", is presented on a MHC class I molecule.
- an endocytotic vesicle e.g., phagosome
- the polypeptide component for delivery to a phagocytic cell comprises an amino acid sequence, and can be at least one peptide such as at least one epitope (approximately 8-12 amino acids in length), at least one small peptide (e.g., approximately ⁇ 30 amino acids in length), at least one large peptide (approximately >30 amino acids in length), at least one full length protein, or a combination thereof.
- the cargo can be composed of at least one tumor antigen, protein, protein fragment or a combination thereof.
- the cargo may also be a tumor cell lysate; the large antigen capacity of the particle to be phagocytosed allows for the coupling of complex protein mixtures derived from a patient's lysed tumor tissues.
- the polypeptide component is an exogenous polypeptide ⁇ i.e., exogenous relative to the phagocytic cell).
- suitable cargo for use in the present invention can be allergens, viral antigens, bacterial antigens and antigens derived from parasites.
- Preferred antigens include tumor associated antigens, with which the artisan will be familiar ⁇ e.g., carcinoembryonic antigen, prostate-specific membrane antigen, melanoma antigen, adenocarcinoma antigen, leukemia antigen, lymphoma antigen, sarcoma antigen, MAGE-1,
- viral antigens include HIV, EBV, Herpes virus, and a linear gp41 epitope insertion (LLELDKWASL), which has been identified as a useful construct for improving HIV-1 Env immunogenicity (Liang, et al, Vaccine, 16; 17(22):2862-72, July 1999).
- compositions of the present invention employ a buffering component, which allows the polypeptide component to evade the lysosome and enter the cytosol of the antigen presenting cell.
- a cell of the mononuclear phagocyte system such as a monocyte (or monocyte derived cell), macrophage, dendritic cell or dendritic cell precursor, ingests an antigen, a phagocytic vesicle (phagosome) which engulfs the antigen is formed.
- phagosome a phagocytic vesicle
- a specialized lysosome contained in the phagocytic cell fuses with the newly formed phagosome.
- the phagocytosed antigen is exposed to several highly reactive molecules as well as a concentrated mixture of lysosomal hydrolases. These highly reactive molecules and lysosomal hydrolases digest the contents of the phagosome/lysosome.
- the polypeptide component that is also attached to the particle escapes digestion by the materials in the phagosome/lysosome and enters the cytoplasm of the phagocytic cell because the buffering component is localized in the phagosome and causes a continued influx of protons, accompanied by chloride ions into the phagosome, and therefore osmotic swelling and ultimately rupture of the endosomal membrane.
- the phagosome bursts fusion between the phagosome and lysosome does not occur.
- the inventors of the present invention surprisingly discovered how to efficiently deliver an exogenously produced antigen, epitope, protein, peptide or other amino acid sequence 8 amino acids in length or greater, even in very small amounts, to a phagocytic
- Dkt. No.: 096630-0103 cell such as a cell of the mononuclear phagocyte system (e.g., monocyte/macrophage, dendritic cell, dendritic cell precursor), for cell surface expression by class I MHC molecules.
- a cell of the mononuclear phagocyte system e.g., monocyte/macrophage, dendritic cell, dendritic cell precursor
- class I MHC molecules for cell surface expression by class I MHC molecules.
- the buffering component has a buffering capacity in a pH range of about pH 6 to about pH 8.
- An example of a buffering component suitable for use in the present invention is RCONHNH 2 .
- Another example of a buffering component is an oligo- histidine, oligo-lysine, polyamine, polyethylenimine (e.g., a low molecular weight PEI, either branched or linear), and oligo-ornithine.
- the buffering component does not form a complex with the polypeptide component as one of the buffering components exemplified herein can form with nucleic acid.
- the buffering component may, however, be used as a way to attach the polypeptide component. But in that way, the "buffering component" does not have a buffering capacity in a pH range of about 6-8 and is therefore not acting as a buffer; it is acting as a chemical linkage instead.
- compositions of the present invention may optionally contain an additional component that evades the degrading environment of the phagosome/lysosome.
- an additional “lysosome evading component” can be added to the compositions of the present invention and includes any number of amino acids, carbohydrates, lipids, fatty acids, biomimetic polymers, microorganisms and combinations thereof.
- Preferred lysosome evading components include proteins, viruses or parts of viruses.
- the adenovirus penton protein for example, is a well known complex that enables the virus to evade/disrupt the lysosome/phagosome.
- either the intact adenovirus or the isolated penton protein, or a portion thereof can be utilized as the lysosome evading component.
- Fusogenic peptides derived from N-terminal sequences of the influenza virus hemagglutinin subunit HA-2 may also be used as the lysosome evading component (Wagner, et al, Proc. Natl. Acad. Sci. USA, 89:7934-7938, 1992).
- lysosome evading components include biomimetic polymers such as Poly (2 -propyl acrylic acid) (PPAAc), which has been shown to enhance cell transfection efficiency due to enhancement of the endosomal release of a conjugate containing a plasmid of interest (see Lackey et al., Abstracts of Scientific Presentations: The Third Annual Meeting of the American Society of Gene Therapy, Abstract No. 33, May 31, 2000-Jun. 4, 2000, Denver, Colo.). Examples of other lysosome evading components envisioned by the present invention are discussed by Stayton, et al. J. Control Release, l;65(l-2):203-20, 2000.
- PPAAc Poly (2 -propyl acrylic acid)
- Any number of methods can be used to attach the cargo to the particle to be phagocytosed.
- Attachment of the components discussed above to the particle to be phagocytosed can be done by any number of means.
- the target protein can be linked to a particle to be phagocytosed, such as a biodegradable particle, either directly or indirectly.
- Direct attachment typically requires the biodegradable particle and polypeptide component to present appropriate functional groups such that a direct link between the particle and polypeptide component can be formed via reaction of these groups, and then the polypeptide component can be readily cleaved off so as to release the polypeptide component.
- the polypeptide component may contain a sulfhydryl group that ultimately allows for attachment to the particle, or can be modified by the addition of a cysteine residue at its N or C terminus.
- Indirect methods for attachment typically utilize well-known and rich chemistry of linkers suitable for this purpose and, as with direct attachment, the methods establish a cleavable bond that gives rise to free polypeptide component.
- the particle to be phagocytosed possesses free amino groups or it can be modified with reagents to present such groups, as described in more detail below.
- linking moieties are useful not only for presenting appropriate combinations of functional groups for linking the particle and protein, but also for possessing buffering properties in a physiological environment. The latter property is believed to allow the linked
- Suitable buffers in this regard should confer a pH of about 6 - to about 8 to the final product.
- biodegradable particle in this context is zymosan because it presents convenient functional groups for modification, although any other biodegradable particle that similarly presents suitable functional groups can be adapted to the synthetic methodologies described herein.
- direct or indirect attachment methods are well-known.
- the particle contains amino groups that can be incorporated directly into the synthetic schemes below.
- the particles do not present amino groups but can be modified to exploit advantageous properties of one or more linking moieties that do contain amino groups.
- reaction (i) zymosan was reacted with a source of periodate, such as sodium periodate, to yield aldehyde moieties in intermediate A as shown in reaction (i) below:
- linker can also possess a buffering property, or a linker and buffer can be distinct moieties.
- linker and buffer possess compatible functional groups so as to ultimately present a disulfide moiety that is highly useful for attaching an appropriately modified protein, as described in more detail below.
- aldehyde groups in intermediate A were reacted with a convenient cross-linking reagent, adipic acid dihydrazide (ADH), in the presence of reductant
- ADH isophthalic dihydrazide
- SDH sebacic dihydrazide
- intermediate B is depicted as being doubly substituted with ADH moieities only for illustrative purposes.
- a reagent such as ADH to introduce at least one ADH or other similar moiety having an amino group.
- reaction between B and LC-SPDP or another suitable source of a disulfide moiety may result in fewer than all amino moieties participating in the reaction, as shown above. Less than complete reaction is acceptable. What is important is that at least one amino moiety reacts so as to install a disulfide moiety, thereby allowing attachment to an appropriately modified protein.
- Heterobifunctional cross-linking reagents other than LC-SPDP are well known and are suitable for use in reaction (iii) above, and they also provide a disulfide unit. These include sulfo N-succinimidyl-6-(3'-(2-pyridyldithio)-propionamido)-hexanoate (sulfo-LC- SPDP) and N-succinimidyl-3-(2-pyridyldithio)-propionate (SPDP). It is not necessary to use these particular reagents, so long as a protein ultimately can be attached to the particle. However, the reagents described above are convenient sources of disulfide moieties that are well-adapted for use in protein chemistry.
- intermediate C was treated with a protein that has been reduced to display at least one sulfhydryl group, -SH, for reaction with the disulfide moiety in C, as shown in reaction (iv) below, to yield the final product D:
- the particle is derivatized with moieties that separately confer buffering and linking capacities, respectively.
- reaction (v) illustrates how intermediate A was reacted with an oligo-histidine and 1,4-diaminobutane to yield the mixed addition product E:
- the oligo-histidine can be various lengths. What is important is that it confers buffering properties to the final product. Because the oligo-histidine terminates in a carboxyl group, it is not well-adapted for use in the synthetic methodologies described above for further reaction with reagents containing disulfide moieties. For this reason, the bifunctional diamine serves as a linker. In principle, any diamine is suitable for this purpose because it contains a requisite amino group to react with intermediate A as well as an amino group for further reaction with the disulfide-containing reagents. Typical diamines are primary amines because they are the most reactive.
- intermediate E was then treated with LC-SPDP to give intermediate F that contains a disulfide moiety, as illustrated in reaction (vi):
- oligo-histidine buffer effectively reduces the number of attachment points to the particle that will be available to an appropriately modified protein.
- reaction (vii) reaction of intermediate F with a sulfhydryl-modified polypeptide component yielded final product G, as depicted in reaction (vii) below:
- the polypeptide component is released intracellularly from the phagocytosed particle into the cytoplasm.
- compositions of the present invention may be formulated for mucosal administration (e.g., intranasal and inhalational administration) or percutaneous administration.
- the compositions of the invention can also be formulated for parenteral administration (e.g., intramuscular, intravenous, or subcutaneous injection), and injected directly into the patient and target cells of monocytic origin, like macrophages and dendritic cells.
- parenteral administration e.g., intramuscular, intravenous, or subcutaneous injection
- the compositions of the present invention may be administered just like a conventional vaccine. This also substantially reduces cost because of the lower level of skill required.
- Formulations for injection may be presented in unit dosage form, e.g., in ampules or in multi-dose containers, optionally with an added preservative.
- the compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
- the composition of the present invention may also be formulated using a pharmaceutically acceptable excipient.
- excipients are well known in the art, but typically will be a physiologically tolerable aqueous solution.
- Physiologically tolerable solutions are those which are essentially non-toxic.
- Preferred excipients will either be inert or enhancing, but a suppressive compound may also be used to achieve a tolerogenic response.
- compositions of the present invention attract phagocytic cells, such as cells of the mononuclear phagocyte system, including monocytes, macrophages, dendritic cells or immature dendritic cells.
- phagocytic cells such as cells of the mononuclear phagocyte system, including monocytes, macrophages, dendritic cells or immature dendritic cells.
- cells of the mononuclear phagocyte system are considered "professional" antigen presenting cells and, thus, are the ideal target for vaccine delivery. It is well known that presentation of an antigen within an APC is vastly more effective in generating a strong cellular immune response than expression of this same antigen within any other cell type. Therefore, the ability of the compositions of the present invention to present a polypeptide component for display on an antigen presenting cell via class I MHC molecules dramatically enhances the efficacy of such a vaccine.
- compositions of the present invention can be used to develop CD8 T cell vaccines against viral, bacterial and parasitic infections, as well as cancer.
- the polypeptide component in the compositions of the present invention is delivered in an amount sufficient to provoke a CD8 T cell response.
- modified particles that can be phagocytosed including yeast cell wall particles, present a number of advantages over conventional vaccine methodologies.
- compositions disclosed herein would obviate the need for attenuated live vaccines to obtain protective CD8 T cell immune responses without infectious agents because transfer of inactivated pathogen structures to the MHC-I pathway is mediated by the particle to be phagocytosed.
- the modified particle can accommodate a large amount of different cargo molecules, which together with the avid phagocytosis, ensures very effective transfer of antigens to the MHC-I pathway of professional antigen presenting cells. Furthermore, there is no restriction on the molecular size of antigenic structures, as the particle to be phagocytosed can accommodate whole inactivated virus particles. Additionally, the large surface area of the particle to be phagocytosed, such as zymosan, would allow for the attachment of compounds that potentiate the phagocyte response to the antigen, such as CpG motifs.
- zymosan for example, due to its inherent phagocyte stimulating capacity as a microbial compound, in itself should have potent adjuvant properties, which is a perquisite for the induction of a primary CD8 T cell response.
- compositions of the present invention can be used in both a prophylactic context as well as a therapeutic one.
- a prophylactic vaccine the polypeptide component as part of the compositions disclosed herein that is delivered to an antigen presenting cell is designed to trigger an immune response against the polypeptide/antigen.
- a therapeutic vaccine is also designed to provoke an immune response, but in individuals already affected with the disease or disorder.
- the present invention contemplates both prophylactic and therapeutic uses of the compositions disclosed herein.
- compositions of the present invention come into contact with phagocytic cells either in vivo or in vitro. Hence, both in vivo and ex vivo methods are contemplated.
- compositions of the present invention are generally administered parenterally, usually intravenously, intramuscularly, subcutaneously or intradermally. They may be administered, e.g., by bolus injection or continuous infusion.
- monocytic cells are contacted outside the body and the contacted cells are then parenterally administered to the patient.
- the phagocytosis carrier system is based on yeast cell wall particles that have been chemically modified to allow escape from the phagosome and release of covalently attached cargo molecules into the cytoplasm.
- OVA ovalbumin
- B3Z reporter MHC-I restricted CD8 T cell hybridoma line
- Results indicate that neither the soluble OVA nor the modified zymosan carrier alone caused stimulation of the B3Z hybridoma in the presence of MHC-I matched IC-21 presenting cells. Efficient antigen presentation, however, was observed with the zymosan- coupled OVA peptide (see induction of ⁇ -galactosidease reporter gene for T cell activation). Stimulation of B3Z cells was dependent on both phagocytosis and correct proteolytic processing of the zymosan-attached OVA because neither soluble nor zymosan coupled OVA induced ⁇ -gal reporter activity in the absence of IC-21 presenting cells. See Figure 1.
- Three principal experimental mouse model systems can be used to analyze the efficacy of zymosan-particle based vaccines.
- a simple defined model protein antigen like OVA or ⁇ -gal is coupled to modified zymosan, induction of a cytolytic CD 8 T cell response is the readout, and established OVA or ⁇ -gal transfected, syngeneic tumor cell lines are the target cells for an in vitro assay of cytolytic T cell activity and an in vivo protection against tumor challenge.
- This model system allows the investigator to define the most basic parameters of vaccination, including dosage and vaccination schedule.
- the second system model system is for vaccinating with complete viruses coupled to a modified yeast cell wall particle.
- Moloney viruses would be attractive candidates because these retroviruses are known to rapidly cause sarcomas in various mouse strains which, after initial regression, prove lethal in most cases.
- As a readout system for this model various established murine Moloney virus-transformed leukemia cell lines are used as model target cells both for in vitro cytolytic assays as well as in vivo tumor protection or tumor therapy assays. Furthermore, direct protection against virus-induced sarcoma formation may be used to assay vaccine efficacy in this model.
- This model system is also particularly attractive because retroviruses have been found to be involved in oncogenesis
- the third model system is for tumor vaccination with lysed tumor cells as the model antigen coupled to a modified yeast cell wall particle.
- the experimental readout system is in vitro cytolytic T cell activity against the tumor cells and both tumor protective and therapeutic efficacy in vivo.
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Pharmacology & Pharmacy (AREA)
- Epidemiology (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Medicinal Chemistry (AREA)
- Botany (AREA)
- Zoology (AREA)
- Virology (AREA)
- Optics & Photonics (AREA)
- Molecular Biology (AREA)
- Microbiology (AREA)
- Immunology (AREA)
- Mycology (AREA)
- Physics & Mathematics (AREA)
- Biomedical Technology (AREA)
- Nanotechnology (AREA)
- Biophysics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Oil, Petroleum & Natural Gas (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Inorganic Chemistry (AREA)
- Cell Biology (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
Described are a method and a composition for delivery of a protein to an antigen presenting cell. The composition is composed of a polypeptide component, a buffering component and a particle to be phagocytized. In one embodiment, the antigen presenting cell is aa macrophage or a dendritic cell and the particle to be phagocytized is from a natural source, such as from a microbial source. The composition itself, or cells pretreated with the composition, are useful for strategies in vaccine development.
Description
. Dkt. No.: 096630-0103
VACCINE COMPOSITIONS AND METHODS OF USE FIELD OF THE INVENTION
The present invention generally relates to compositions and methods for delivering a protein, peptide, epitope, or antigen to an antigen presenting cell, such as a macrophage or dendritic cell. The compositions and methods disclosed herein are particularly useful in making prophylactic and therapeutic vaccines.
BACKGROUND OF THE INVENTION
Antigen presenting cells, including macrophages and other cells of the mononuclear phagocyte system actively phagocytose particles and play a central role in the immune response. Macrophages are cells within the tissues that are derived from monocytes. These monocytes/macrophages phagocytose microbes, for example, which are then digested to smaller antigenic portions in the lysosome/phagosome. The resultant antigens are cycled back to the surface for presentation to the humoral and cellular arms of the immune system. Accordingly, monocytes/macrophages are of particular interest because they play an important role in both nonspecific and specific defenses in the host against pathogens.
Dendritic cells are also antigen presenting cells that express MHC class I and class II molecules. An ideal vaccine mimics the rapid uptake and transfer of pathogenic structures without actually establishing an infection and without causing suppression of the MHC class I pathway. For this purpose, the present invention relates to a composition comprising a modified particle to be phagocytosed that is avidly and specifically taken up by professional phagocytic cells, and methods for delivering cargo molecules to the cytoplasm of antigen presenting cells for presentation on MHC class I molecules.
Before the present invention, delivery of exogenous antigens, peptides, or proteins to an antigen presenting cell for presentation on class I MHC molecules was difficult because following degradation in vesicular intracellular compartments, such antigens would be loaded on MHC class II molecules for presentation. Indeed, the only antigens that normally activate the MHC class I pathway are those that are derived from cytosolic antigens (e.g., endogenous ly produced within an antigen presenting cell). Although dendritic cells can
WASH_6546625.1
. Dkt. No.: 096630-0103 exhibit "cross presentation" phenomena, whereby dendritic cells present exogenous antigens on a class I molecule, the localized concentration of soluble, exogenous antigen must be very high (approximately 100 or even 1000 fold higher than the present invention) for such cross presentation to occur and is therefore inefficient. On the other hand, the compositions of the present invention can efficiently deliver exogenous proteins, epitopes, antigens, and peptides, for presentation on class I molecules with only a very low amount of exogenous material.
Thus, while some proteins that escape the phagosome and enter into the cytosol of an antigen presenting cell could also activate the MHC class I pathway, a cost effective and efficient delivery method of exogenous proteins, epitopes, antigens and/or peptides for association with MHC class I molecules could not be purposefully accomplished before the present invention.
SUMMARY OF THE INVENTION
One embodiment of the present invention is an antigenic composition comprising (i) a polypeptide component, (ii) a buffering component, and (iii) a particle that can be phagocytosed, wherein the polypeptide component or a fragment thereof is ultimately presented on a class I MHC molecule.
Another embodiment of the invention is a method for efficient delivery of a polypeptide component to an antigen presenting cell comprising administering a composition comprising (i) a polypeptide component, (ii) a buffering component, and (iii) a particle that can be phagocytosed, wherein the polypeptide component, following administration, enters the cytosol from an endocytotic vesicle, and the polypeptide or a fragment thereof is presented on a MHC class I molecule.
Also described herein is a composition for exogenous antigen presentation on class I MHC molecules comprising (i) a polypeptide component, (ii) a buffering component, and (iii) a particle that can be phagocytosed, and a vaccine composition comprising (i) a polypeptide component, (ii) a buffering component, and (iii) a particle that can be phagocytosed, wherein the polypeptide component or a fragment thereof is ultimately presented on a class I MHC molecule. The polypeptide component is also delivered in an amount sufficient to provoke a CD8 T cell response, in the case of dendritic cells.
WASH_6546625.1
. Dkt. No.: 096630-0103
In the compositions and methods of the present invention, the particle that can be phagocytosed is a biodegradable particle, such as zymosan particle, chitin particle, agarose particle or sepharose particle, and the buffering component can be any buffer with a buffering capacity in the range of about pH 6 to about pH 8, such as RCONHNH2, polyethyleneimine, oligohistidine, oligoornithine, and oligolysine.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1. Antigenic stimulation of MHC-I (H-2Kb) restricted B3Z hybridoma reporter cells specific for the internal OVA epitope SIINFEKL in the absence or presence of IC-21 (H-2Kb) antigen presenting cells.
DETAILED DESCRIPTION OF THE INVENTION
Introduction
The present invention provides a composition for delivery to an antigen presenting cell, and a related method of use. The composition according to the present invention comprises a polypeptide component, attached to a particle that can be phagocytosed and a buffering component. The present invention takes advantage of the phagocytic property of certain antigen presenting cells in that the polypeptide component is provided on a substrate that "looks" like a microbe. Thus, the particle attracts phagocytic cells.
The compositions of the present invention do not contain liposomes (vesicles with an aqueous interior enclosed by one or more phospholipid bilayers), nor are the compositions disclosed herein taken up by macrophages, dendritic, or other antigen presenting cells based on an agent that permeabilizes the extracellular membrane of these cells. The compositions of the present invention also do not utilize haemolysin to permeabilize the phagosomal membrane.
Particle to be Phagocytosed
In one embodiment of the present invention, the particle to be phagocytosed ("particle") is a biodegradable particle, such as one derived from natural sources. In one embodiment, the particle to be phagocytosed is of microbial origin, and is preferably a
WASH 6546625.1
. Dkt. No.: 096630-0103 particle from a yeast cell wall. In another embodiment, the yeast cell wall particle is a zymosan particle. Zymosan (also referred to as Zymosan A) is commercially available from various companies such as Sigma-Aldrich. Natural particles, such as zymosan, are better tolerated by macrophages, for example, than magnetic beads and particles from other sources.
Zymosan is an insoluble polysaccharide component of yeast cell wall. Prior publications uncovered zymosan's involvements in (i) induction of the release of cytokines or proinflammatory cytokines, (ii) induction of protein phosphorylation and inositol phosphate formation, (iii) arachidonate mobilization, (iv) activation of the alternative complement pathway; and (v) raise of cyclin D2 levels, suggesting a role of cyclin D2 in macrophage activation (Miyasato et al, Int. Arch. Allergy Immunol. 104: 24-26, 1994). For example, it has been reported that zymosan particles are capable of inducing inflammatory signals in macrophages through Toll- like receptors, e.g. TLR2 and TLR6, and dectin-1, which is a receptor that binds β-glucans and is important for macrophage phagocytosis. Zymosan is also involved in inducing inflammatory responses, such as TNF-a production and NF-KB activation in macrophages (Underhill, Journal of Endotoxin Research, 9: 176-180, 2003; Sato et al, J. Immunol, 111 : 417-425, 2003; Dillon et al. , J. Clin. Invest. 116: 916-928, 2006).
A preferred size for the particle that can be phagocytosed is one that approximates the size of microbial structures that cells of the mononuclear phagocyte system and other phagocytic cells typically ingest. In one embodiment, the particle will be about 0.05 to about 5.0 μιη, about 0.05 to about 2.5 μιη, about 0.1 to about 2.5 μιη, about 1.0 to about 2.5 μιη, about 1.0 to about 2.0 μιη, or about 1.0 to about 1.5 μιη. The term "about" in this context refers to +/- 0.25 μιη. Zymosan is typically about 2.0 μιη in size.
Although the particle that can be phagocytosed is not limited by any particular size, a preferred size for the particle that can be phagocytosed is one that approximates the size of microbial structures that cells of the mononuclear phagocyte lineage (e.g., monocytes, macrophages, dendritic cells, dendritic cell precursors (immature dendritic cells), or other antigen presenting cells, typically ingest. In one embodiment, the particle will be about 0.05 to about 5.0 μιη, about 0.05 to about 2.5 μιη, about 0.1 to about 2.5 μιη, about 1.0 to about 2.5 μιη, about 1.0 to about 2.0 μιη, or about 1.0 to about 1.5 μιη. The term "about" in this context refers to +/- 0.25 μιη. Zymosan is typically about 2.0 μιη in size.
WASH 6546625.1
. Dkt. No.: 096630-0103
The particle that can be phagocytosed is also not limited by shape or material. In general, the particle can be of any shape or material that allows the composition of the present invention to be phagocytized by cells of the mononuclear phagocyte system, such as monocytes/macrophages, dendritic cells, immature dendritic cells (have a high capacity for phagocytosis, then undergo maturation), or other phagocytic cells. For example, in addition to zymosan, the particle to be phagocytosed can be made of chitin, a synthetic beta glucan polymer, agarose, sepharose, etc., so long as the particle contains a carbohydrate or other moiety that permits attachment of the polypeptide component of the present invention.
Polypeptide Component
The polypeptide component of the present invention is attached to the particle to be phagocytosed. Following administration of the polypeptide component attached to the particle, the polypeptide component, or fragment thereof is released from the particle, enters the cytosol from an endocytotic vesicle (e.g., phagosome) and, as a result of normal "processing", is presented on a MHC class I molecule.
The polypeptide component for delivery to a phagocytic cell ("cargo") comprises an amino acid sequence, and can be at least one peptide such as at least one epitope (approximately 8-12 amino acids in length), at least one small peptide (e.g., approximately <30 amino acids in length), at least one large peptide (approximately >30 amino acids in length), at least one full length protein, or a combination thereof. For example, the cargo can be composed of at least one tumor antigen, protein, protein fragment or a combination thereof. The cargo may also be a tumor cell lysate; the large antigen capacity of the particle to be phagocytosed allows for the coupling of complex protein mixtures derived from a patient's lysed tumor tissues. The polypeptide component is an exogenous polypeptide {i.e., exogenous relative to the phagocytic cell).
In one embodiment, suitable cargo for use in the present invention can be allergens, viral antigens, bacterial antigens and antigens derived from parasites. Preferred antigens include tumor associated antigens, with which the artisan will be familiar {e.g., carcinoembryonic antigen, prostate-specific membrane antigen, melanoma antigen, adenocarcinoma antigen, leukemia antigen, lymphoma antigen, sarcoma antigen, MAGE-1,
WASH_6546625.1
. Dkt. No.: 096630-0103
MAGE-2, MART-1, Melan-A, p53, gplOO, antigen associated with colonic carcinoma, antigen associated with breast carcinoma, Mucl, Trp-2, telomerase, PSA and antigen associated with renal carcinoma). Whole inactivated viruses, portions of the virus, and viral antigens are also suitable polypeptide components for the present invention. In another embodiment, viral antigens include HIV, EBV, Herpes virus, and a linear gp41 epitope insertion (LLELDKWASL), which has been identified as a useful construct for improving HIV-1 Env immunogenicity (Liang, et al, Vaccine, 16; 17(22):2862-72, July 1999).
Buffering Component
The compositions of the present invention employ a buffering component, which allows the polypeptide component to evade the lysosome and enter the cytosol of the antigen presenting cell.
More specifically, when a cell of the mononuclear phagocyte system, such as a monocyte (or monocyte derived cell), macrophage, dendritic cell or dendritic cell precursor, ingests an antigen, a phagocytic vesicle (phagosome) which engulfs the antigen is formed. Next, a specialized lysosome contained in the phagocytic cell fuses with the newly formed phagosome. Upon fusion, the phagocytosed antigen is exposed to several highly reactive molecules as well as a concentrated mixture of lysosomal hydrolases. These highly reactive molecules and lysosomal hydrolases digest the contents of the phagosome/lysosome. Therefore, by covalently attaching a buffering component to the particle to be phagocytosed, the polypeptide component that is also attached to the particle escapes digestion by the materials in the phagosome/lysosome and enters the cytoplasm of the phagocytic cell because the buffering component is localized in the phagosome and causes a continued influx of protons, accompanied by chloride ions into the phagosome, and therefore osmotic swelling and ultimately rupture of the endosomal membrane. When the phagosome bursts, fusion between the phagosome and lysosome does not occur.
Accordingly, the inventors of the present invention surprisingly discovered how to efficiently deliver an exogenously produced antigen, epitope, protein, peptide or other amino acid sequence 8 amino acids in length or greater, even in very small amounts, to a phagocytic
WASH 6546625.1
. Dkt. No.: 096630-0103 cell, such as a cell of the mononuclear phagocyte system (e.g., monocyte/macrophage, dendritic cell, dendritic cell precursor), for cell surface expression by class I MHC molecules.
In one embodiment, the buffering component has a buffering capacity in a pH range of about pH 6 to about pH 8. An example of a buffering component suitable for use in the present invention is RCONHNH2. Another example of a buffering component is an oligo- histidine, oligo-lysine, polyamine, polyethylenimine (e.g., a low molecular weight PEI, either branched or linear), and oligo-ornithine. Several exemplary buffers can be found in a "pKa data compilation by R. Williams", available at http://research.chem.psu.edu/brpgroup/pKa_compilation.pdf, which is incorporated by reference herein in its entirety. The buffering component does not form a complex with the polypeptide component as one of the buffering components exemplified herein can form with nucleic acid. The buffering component may, however, be used as a way to attach the polypeptide component. But in that way, the "buffering component" does not have a buffering capacity in a pH range of about 6-8 and is therefore not acting as a buffer; it is acting as a chemical linkage instead.
In addition to the buffering component described herein, the compositions of the present invention may optionally contain an additional component that evades the degrading environment of the phagosome/lysosome. Such an additional "lysosome evading component" can be added to the compositions of the present invention and includes any number of amino acids, carbohydrates, lipids, fatty acids, biomimetic polymers, microorganisms and combinations thereof.
Preferred lysosome evading components include proteins, viruses or parts of viruses. The adenovirus penton protein, for example, is a well known complex that enables the virus to evade/disrupt the lysosome/phagosome. Thus, either the intact adenovirus or the isolated penton protein, or a portion thereof (see, for example, Bal et al., Eur J Biochem 267:6074-81 (2000)), can be utilized as the lysosome evading component. Fusogenic peptides derived from N-terminal sequences of the influenza virus hemagglutinin subunit HA-2 may also be used as the lysosome evading component (Wagner, et al, Proc. Natl. Acad. Sci. USA, 89:7934-7938, 1992).
WASH_6546625.1
. Dkt. No.: 096630-0103
Other preferred lysosome evading components include biomimetic polymers such as Poly (2 -propyl acrylic acid) (PPAAc), which has been shown to enhance cell transfection efficiency due to enhancement of the endosomal release of a conjugate containing a plasmid of interest (see Lackey et al., Abstracts of Scientific Presentations: The Third Annual Meeting of the American Society of Gene Therapy, Abstract No. 33, May 31, 2000-Jun. 4, 2000, Denver, Colo.). Examples of other lysosome evading components envisioned by the present invention are discussed by Stayton, et al. J. Control Release, l;65(l-2):203-20, 2000.
Method for Attaching the Polypeptide Component to the Particle to be Phagocytosed
Any number of methods can be used to attach the cargo to the particle to be phagocytosed.
Attachment of the components discussed above to the particle to be phagocytosed can be done by any number of means. In principle, the target protein can be linked to a particle to be phagocytosed, such as a biodegradable particle, either directly or indirectly. Direct attachment, for instance, typically requires the biodegradable particle and polypeptide component to present appropriate functional groups such that a direct link between the particle and polypeptide component can be formed via reaction of these groups, and then the polypeptide component can be readily cleaved off so as to release the polypeptide component. For example, the polypeptide component may contain a sulfhydryl group that ultimately allows for attachment to the particle, or can be modified by the addition of a cysteine residue at its N or C terminus.
Indirect methods for attachment typically utilize well-known and rich chemistry of linkers suitable for this purpose and, as with direct attachment, the methods establish a cleavable bond that gives rise to free polypeptide component. In either case, the particle to be phagocytosed possesses free amino groups or it can be modified with reagents to present such groups, as described in more detail below.
In particular, without wishing to be bound by any particular theory, the inventors believe that such linking moieties are useful not only for presenting appropriate combinations of functional groups for linking the particle and protein, but also for possessing buffering properties in a physiological environment. The latter property is believed to allow the linked
WASH_6546625.1
. Dkt. No.: 096630-0103 protein and particle to withstand the otherwise destructive action of intracellular lysosomes and/or phagosomes, thereby preserving the protein in its entirety until it can be cleaved from the particle-linker. Suitable buffers in this regard should confer a pH of about 6 - to about 8 to the final product.
An illustrative biodegradable particle in this context is zymosan because it presents convenient functional groups for modification, although any other biodegradable particle that similarly presents suitable functional groups can be adapted to the synthetic methodologies described herein. As described above, direct or indirect attachment methods are well-known. In some embodiments, the particle contains amino groups that can be incorporated directly into the synthetic schemes below. In other embodiments that are explicitly set forth below, the particles do not present amino groups but can be modified to exploit advantageous properties of one or more linking moieties that do contain amino groups.
To illustrate, zymosan was reacted with a source of periodate, such as sodium periodate, to yield aldehyde moieties in intermediate A as shown in reaction (i) below:
A
Many particles to be phagocytosed are suitable for use in the invention. The examples herein utilize zymosan, but any number of other well-known particles can be used such as, for instance, agarose.
The next steps introduce appropriate linkers and buffers consistent with the general requirements discussed above. In general, a linker can also possess a buffering property, or a linker and buffer can be distinct moieties. In addition, the basic chemical requirement is that the combination of linker and buffer possess compatible functional groups so as to ultimately present a disulfide moiety that is highly useful for attaching an appropriately modified protein, as described in more detail below.
Thus, in one alternative, the aldehyde groups in intermediate A were reacted with a convenient cross-linking reagent, adipic acid dihydrazide (ADH), in the presence of reductant
WASH 6546625.1
Atty. Dkt. No.: 096630-0103 sodium cyanoborohydride in order to introduce reactive amino groups in intermediate B as shown in reaction (ii) below:
(ϋ)
Many other reagents that are well known to those who are skilled in organic chemistry can transform aldehydes into amino moieties. These reagents can be used instead of or in addition to ADH. In this instance, the inventors discovered that the ADH moiety conveniently possesses buffering properties, which simplified the chemistry because no further linker or modification was necessary to introduce a buffer, as explained more fully below. Alternatives to ADH include, for instance, isophthalic dihydrazide (IDH) and sebacic dihydrazide (SDH). Thus, other synthetic strategies that present amino moieties, as in reaction (ii) above, should account for the need for a buffer, as detailed in a further embodiment below.
Finally, it should be noted that intermediate B is depicted as being doubly substituted with ADH moieities only for illustrative purposes. In practice, at least one and any number of additional aldehyde groups can be present for reaction with a reagent such as ADH to introduce at least one ADH or other similar moiety having an amino group.
Intermediate B was then treated with the well-known reagent N-succinimidyl-6-(3'- (2-pyridyldithio)-propionamido)-hexanoate, LC-SPDP, in order to introduce a convenient source of a disulfide unit in intermediate C that is capable of reacting with a thiol-substituted protein. The transformation is illustrated by reaction (iii) below:
WASH 6546625.1
. Dkt. No.: 096630-0103
The reaction between B and LC-SPDP or another suitable source of a disulfide moiety may result in fewer than all amino moieties participating in the reaction, as shown above. Less than complete reaction is acceptable. What is important is that at least one amino moiety reacts so as to install a disulfide moiety, thereby allowing attachment to an appropriately modified protein.
Heterobifunctional cross-linking reagents other than LC-SPDP are well known and are suitable for use in reaction (iii) above, and they also provide a disulfide unit. These include sulfo N-succinimidyl-6-(3'-(2-pyridyldithio)-propionamido)-hexanoate (sulfo-LC- SPDP) and N-succinimidyl-3-(2-pyridyldithio)-propionate (SPDP). It is not necessary to use these particular reagents, so long as a protein ultimately can be attached to the particle. However, the reagents described above are convenient sources of disulfide moieties that are well-adapted for use in protein chemistry.
Finally, intermediate C was treated with a protein that has been reduced to display at least one sulfhydryl group, -SH, for reaction with the disulfide moiety in C, as shown in reaction (iv) below, to yield the final product D:
D
(iv)
WASH 6546625.1
. Dkt. No.: 096630-0103
As mentioned above, it is not necessary to link the particle and protein with reagents that serve simultaneously as linkers and buffers, as illustrated by reactions (i) - (iv) above. It is sufficient that a protein ultimately is linked to the particle and that the product is buffered to pH of about 6 - to about pH 8, as mentioned above.
Thus, in another embodiment, the particle is derivatized with moieties that separately confer buffering and linking capacities, respectively. For instance, reaction (v) below illustrates how intermediate A was reacted with an oligo-histidine and 1,4-diaminobutane to yield the mixed addition product E:
(v)
The oligo-histidine can be various lengths. What is important is that it confers buffering properties to the final product. Because the oligo-histidine terminates in a carboxyl group, it is not well-adapted for use in the synthetic methodologies described above for further reaction with reagents containing disulfide moieties. For this reason, the bifunctional diamine serves as a linker. In principle, any diamine is suitable for this purpose because it contains a requisite amino group to react with intermediate A as well as an amino group for further reaction with the disulfide-containing reagents. Typical diamines are primary amines because they are the most reactive.
In accordance with the general guidelines above, intermediate E was then treated with LC-SPDP to give intermediate F that contains a disulfide moiety, as illustrated in reaction (vi):
WASH 6546625.1
. Dkt. No.: 096630-0103
In some embodiments, as described above, other disulfide-containing reagents are used instead of LC-SPDP. Regardless of which reagent is selected, it follows from these methodologies that the presence of the oligo-histidine buffer effectively reduces the number of attachment points to the particle that will be available to an appropriately modified protein.
Thus, reaction of intermediate F with a sulfhydryl-modified polypeptide component yielded final product G, as depicted in reaction (vii) below:
(vii)
Ultimately, the polypeptide component is released intracellularly from the phagocytosed particle into the cytoplasm.
WASH 6546625.1
. Dkt. No.: 096630-0103
Formulation
The compositions of the present invention may be formulated for mucosal administration (e.g., intranasal and inhalational administration) or percutaneous administration. The compositions of the invention can also be formulated for parenteral administration (e.g., intramuscular, intravenous, or subcutaneous injection), and injected directly into the patient and target cells of monocytic origin, like macrophages and dendritic cells. Thus, the compositions of the present invention may be administered just like a conventional vaccine. This also substantially reduces cost because of the lower level of skill required.
Formulations for injection may be presented in unit dosage form, e.g., in ampules or in multi-dose containers, optionally with an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. The composition of the present invention may also be formulated using a pharmaceutically acceptable excipient. Such excipients are well known in the art, but typically will be a physiologically tolerable aqueous solution. Physiologically tolerable solutions are those which are essentially non-toxic. Preferred excipients will either be inert or enhancing, but a suppressive compound may also be used to achieve a tolerogenic response.
Therapeutic Methods
The compositions of the present invention attract phagocytic cells, such as cells of the mononuclear phagocyte system, including monocytes, macrophages, dendritic cells or immature dendritic cells. In the field of vaccination, cells of the mononuclear phagocyte system are considered "professional" antigen presenting cells and, thus, are the ideal target for vaccine delivery. It is well known that presentation of an antigen within an APC is vastly more effective in generating a strong cellular immune response than expression of this same antigen within any other cell type. Therefore, the ability of the compositions of the present invention to present a polypeptide component for display on an antigen presenting cell via class I MHC molecules dramatically enhances the efficacy of such a vaccine.
WASH_6546625.1
. Dkt. No.: 096630-0103
The compositions of the present invention can be used to develop CD8 T cell vaccines against viral, bacterial and parasitic infections, as well as cancer. The polypeptide component in the compositions of the present invention is delivered in an amount sufficient to provoke a CD8 T cell response.
The use of modified particles that can be phagocytosed, including yeast cell wall particles, present a number of advantages over conventional vaccine methodologies.
First, the compositions disclosed herein would obviate the need for attenuated live vaccines to obtain protective CD8 T cell immune responses without infectious agents because transfer of inactivated pathogen structures to the MHC-I pathway is mediated by the particle to be phagocytosed.
Second, the modified particle can accommodate a large amount of different cargo molecules, which together with the avid phagocytosis, ensures very effective transfer of antigens to the MHC-I pathway of professional antigen presenting cells. Furthermore, there is no restriction on the molecular size of antigenic structures, as the particle to be phagocytosed can accommodate whole inactivated virus particles. Additionally, the large surface area of the particle to be phagocytosed, such as zymosan, would allow for the attachment of compounds that potentiate the phagocyte response to the antigen, such as CpG motifs.
Third, zymosan for example, due to its inherent phagocyte stimulating capacity as a microbial compound, in itself should have potent adjuvant properties, which is a perquisite for the induction of a primary CD8 T cell response.
Fourth, because antigen uptake happens via the phagocytic route, it is to be expected that part of the antigenic material would be presented simultaneously via the MHC class II pathway, which would ensure induction of a concomitant CD4 T cell helper response, which in turn is required for a productive CD8 T cell response.
Lastly, ready to use vaccines may be prepared within a short period of time without the requirement for specialized equipment, provided appropriate antigenic material is available.
WASH_6546625.1
. Dkt. No.: 096630-0103
The compositions of the present invention can be used in both a prophylactic context as well as a therapeutic one. For a prophylactic vaccine, the polypeptide component as part of the compositions disclosed herein that is delivered to an antigen presenting cell is designed to trigger an immune response against the polypeptide/antigen. A therapeutic vaccine is also designed to provoke an immune response, but in individuals already affected with the disease or disorder. The present invention contemplates both prophylactic and therapeutic uses of the compositions disclosed herein.
The compositions of the present invention come into contact with phagocytic cells either in vivo or in vitro. Hence, both in vivo and ex vivo methods are contemplated.
As for in vivo methods, the compositions of the present invention are generally administered parenterally, usually intravenously, intramuscularly, subcutaneously or intradermally. They may be administered, e.g., by bolus injection or continuous infusion. In ex vivo methods, monocytic cells are contacted outside the body and the contacted cells are then parenterally administered to the patient.
* * * EXAMPLES
The following non-limiting examples are given by way of illustration only and are not to be considered limitations of this invention. There are many apparent variations within the scope of this invention.
Example 1. Antigenic stimulation of B3Z cells
The phagocytosis carrier system is based on yeast cell wall particles that have been chemically modified to allow escape from the phagosome and release of covalently attached cargo molecules into the cytoplasm. The technical feasibility of this approach is exemplified in an in vitro model system for the presentation of a model peptide structure derived from ovalbumin (OVA) to a reporter MHC-I restricted CD8 T cell hybridoma line (B3Z), recognizing the internal OVA peptide sequence SIINFEKL. See, Shastri, N., and Gonzalez, F. 1993. J. Immunol. 150:2724.
WASH_6546625.1
. Dkt. No.: 096630-0103
Results indicate that neither the soluble OVA nor the modified zymosan carrier alone caused stimulation of the B3Z hybridoma in the presence of MHC-I matched IC-21 presenting cells. Efficient antigen presentation, however, was observed with the zymosan- coupled OVA peptide (see induction of β-galactosidease reporter gene for T cell activation). Stimulation of B3Z cells was dependent on both phagocytosis and correct proteolytic processing of the zymosan-attached OVA because neither soluble nor zymosan coupled OVA induced β-gal reporter activity in the absence of IC-21 presenting cells. See Figure 1.
These data demonstrate that efficient transfer of zymosan-coupled OVA to the cytoplasm as well as correct proteolytic processing via the proteosome and loading onto MHC-I molecules.
Example 2. Experimental model systems for testing vaccination efficacy
Three principal experimental mouse model systems can be used to analyze the efficacy of zymosan-particle based vaccines.
In a first model system, a simple defined model protein antigen like OVA or β-gal is coupled to modified zymosan, induction of a cytolytic CD 8 T cell response is the readout, and established OVA or β-gal transfected, syngeneic tumor cell lines are the target cells for an in vitro assay of cytolytic T cell activity and an in vivo protection against tumor challenge. This model system allows the investigator to define the most basic parameters of vaccination, including dosage and vaccination schedule.
The second system model system is for vaccinating with complete viruses coupled to a modified yeast cell wall particle. As a model antigen, Moloney viruses would be attractive candidates because these retroviruses are known to rapidly cause sarcomas in various mouse strains which, after initial regression, prove lethal in most cases. As a readout system for this model, various established murine Moloney virus-transformed leukemia cell lines are used as model target cells both for in vitro cytolytic assays as well as in vivo tumor protection or tumor therapy assays. Furthermore, direct protection against virus-induced sarcoma formation may be used to assay vaccine efficacy in this model. This model system is also particularly attractive because retroviruses have been found to be involved in oncogenesis
WASH_6546625.1
. Dkt. No.: 096630-0103 both in experimental animal systems and in humans, and in the case of HIV-1 are the causative agent of AIDS.
The third model system is for tumor vaccination with lysed tumor cells as the model antigen coupled to a modified yeast cell wall particle. The experimental readout system is in vitro cytolytic T cell activity against the tumor cells and both tumor protective and therapeutic efficacy in vivo.
WASH 6546625.1
Claims
1. An antigenic composition comprising (i) a polypeptide component, (ii) a buffering component, and (iii) a particle that can be phagocytosed, wherein the polypeptide component or a fragment thereof is ultimately presented on a class I MHC molecule.
2. The composition of claim 1, wherein the particle that can be phagocytosed is a biodegradable particle.
3. The composition of claim 2, wherein the particle that can be phagocytosed is a zymosan particle.
4. The composition of claim 1, wherein the buffering component is RCONHNH2, oligohistidine, or polyethyleneimine.
5. A vaccine composition comprising (i) a polypeptide component, (ii) a buffering component, and (iii) a particle that can be phagocytosed, wherein the polypeptide component or a fragment thereof is delivered in an amount sufficient to provoke a CD 8 T cell response, and the polypeptide component is ultimately presented on a class I MHC molecule.
6. The vaccine of claim 5, wherein the particle that can be phagocytosed is a biodegradable particle.
7. The vaccine of claim 6, wherein the particle that can be phagocytosed is a zymosan particle.
8. The vaccine of claim 5, wherein the buffering component has a buffering capacity in the range of about pH 6 to about pH 8.
9. The vaccine of claim 5, wherein the buffering component is RCONHNH2, oligohistidine, or polyethyleneimine.
10. A method for efficient delivery of a polypeptide component to an antigen presenting cell comprising administering a composition comprising (i) a polypeptide component, (ii) a buffering component, and (iii) a particle that can be phagocytosed, wherein
WASH_6546625.1
. Dkt. No.: 096630-0103 the polypeptide component, following administration, enters the cytosol from an endocytotic vesicle, and the polypeptide or a fragment thereof is presented on a MHC class I molecule.
11. The method of claim 10, wherein the particle that can be phagocytosed is a biodegradable particle.
12. The method of claim 11, wherein the particle that can be phagocytosed is a zymosan particle.
13. The method of claim 10, wherein the buffering component is RCONHNH2, oligohistidine, or polyethyleneimine.
14. A composition for exogenous antigen presentation on class I MHC molecules comprising (i) a polypeptide component, (ii) a buffering component, and (iii) a particle that can be phagocytosed.
15. The composition of claim 14, wherein the particle that can be phagocytosed is a biodegradable particle.
16. The composition of claim 15, wherein the particle that can be phagocytosed is a zymosan particle.
17. The composition of claim 14, wherein the buffering component is RCONHNH2, oligohistidine, or polyethyleneimine.
WASH 6546625.1
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
PCT/US2009/062949 WO2011053331A1 (en) | 2009-11-02 | 2009-11-02 | Vaccine compositions and methods of use |
Publications (1)
Publication Number | Publication Date |
---|---|
EP2496265A1 true EP2496265A1 (en) | 2012-09-12 |
Family
ID=42738896
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
EP09744895A Withdrawn EP2496265A1 (en) | 2009-11-02 | 2009-11-02 | Vaccine compositions and methods of use |
Country Status (3)
Country | Link |
---|---|
EP (1) | EP2496265A1 (en) |
CA (1) | CA2779601A1 (en) |
WO (1) | WO2011053331A1 (en) |
Families Citing this family (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP6494511B2 (en) * | 2013-09-05 | 2019-04-03 | オービス ヘルス ソリューションズ エルエルシー | Particles filled with tumor lysate |
US20140065173A1 (en) * | 2012-09-06 | 2014-03-06 | Orbis Health Solutions Llc | Tumor lysate loaded particles |
AU2020203845C1 (en) * | 2012-09-06 | 2022-08-11 | Orbis Health Solutions, Llc | Tumor lysate loaded particles |
JP6797022B2 (en) | 2014-03-05 | 2020-12-09 | オービス ヘルス ソリューションズ エルエルシー | Vaccine delivery system using yeast cell wall particles |
ES2950653T3 (en) * | 2017-06-22 | 2023-10-11 | Neogap Therapeutics Ab | T cell expansion method and uses |
Family Cites Families (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20080171059A1 (en) * | 2006-08-07 | 2008-07-17 | Shanshan Wu Howland | Methods and compositions for increased priming of t-cells through cross-presentation of exogenous antigens |
WO2008134487A1 (en) * | 2007-04-25 | 2008-11-06 | Ghc Research Development Corporation | Macrophage transfection method |
-
2009
- 2009-11-02 EP EP09744895A patent/EP2496265A1/en not_active Withdrawn
- 2009-11-02 CA CA2779601A patent/CA2779601A1/en not_active Abandoned
- 2009-11-02 WO PCT/US2009/062949 patent/WO2011053331A1/en active Application Filing
Non-Patent Citations (1)
Title |
---|
See references of WO2011053331A1 * |
Also Published As
Publication number | Publication date |
---|---|
CA2779601A1 (en) | 2011-05-05 |
WO2011053331A1 (en) | 2011-05-05 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Singha et al. | Nanoparticles for immune stimulation against infection, cancer, and autoimmunity | |
Nam et al. | Modularly programmable nanoparticle vaccine based on polyethyleneimine for personalized cancer immunotherapy | |
Zhao et al. | Polyelectrolyte-based platforms for the delivery of peptides and proteins | |
Bershteyn et al. | Robust IgG responses to nanograms of antigen using a biomimetic lipid-coated particle vaccine | |
EP2341897B1 (en) | Nanoparticles for immunotherapy | |
Chikh et al. | Liposomal delivery of CTL epitopes to dendritic cells | |
JP2018030837A (en) | Synthetic nano carrier that generates humoral and cytotoxic t lymphocyte (ctl) immune response | |
US20110229556A1 (en) | Lipid-coated polymer particles for immune stimulation | |
HU224410B1 (en) | Pharmaceutical composition for immunomodulation based on peptides and adjuvants | |
Kapadia et al. | Reduction sensitive PEG hydrogels for codelivery of antigen and adjuvant to induce potent CTLs | |
Simón-Vázquez et al. | Polymeric nanostructure vaccines: applications and challenges | |
WO2011053331A1 (en) | Vaccine compositions and methods of use | |
CN105792843B (en) | Adjuvant composition, vaccine composition containing same, and method for producing same | |
US20100111985A1 (en) | Vaccine compositions and methods of use | |
US20090117658A1 (en) | Macrophage transfection method | |
Orosco et al. | Navigating the landscape of adjuvants for subunit vaccines: Recent advances and future perspectives | |
Ariawan et al. | Recent progress in synthetic self-adjuvanting vaccine development | |
Noble et al. | Potent immune stimulation from nanoparticle carriers relies on the interplay of adjuvant surface density and adjuvant mass distribution | |
Li et al. | Self-adjuvant Astragalus polysaccharide-based nanovaccines for enhanced tumor immunotherapy: a novel delivery system candidate for tumor vaccines | |
Ge et al. | MAGE-1/Heat shock protein 70/MAGE-3 fusion protein vaccine in nanoemulsion enhances cellular and humoral immune responses to MAGE-1 or MAGE-3 in vivo | |
Liang et al. | The orientation of CpG conjugation on aluminum oxyhydroxide nanoparticles determines the immunostimulatory effects of combination adjuvants | |
Li et al. | Virus envelope-like self-assembled nanoparticles based on α-CD/PEG for antigens targeting to dendritic cells | |
Savla et al. | Nanoparticles in the development of therapeutic cancer vaccines | |
JP2004528321A (en) | Polynucleotide binding complexes containing sterols and saponins | |
Gautam et al. | Emerging trends in vaccine delivery systems |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PUAI | Public reference made under article 153(3) epc to a published international application that has entered the european phase |
Free format text: ORIGINAL CODE: 0009012 |
|
17P | Request for examination filed |
Effective date: 20120522 |
|
AK | Designated contracting states |
Kind code of ref document: A1 Designated state(s): AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO SE SI SK SM TR |
|
DAX | Request for extension of the european patent (deleted) | ||
17Q | First examination report despatched |
Effective date: 20150803 |
|
STAA | Information on the status of an ep patent application or granted ep patent |
Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN |
|
18D | Application deemed to be withdrawn |
Effective date: 20151215 |