US20210308157A1 - Stimulation of an immune response by enantiomers of cationic lipids - Google Patents
Stimulation of an immune response by enantiomers of cationic lipids Download PDFInfo
- Publication number
- US20210308157A1 US20210308157A1 US17/212,051 US202117212051A US2021308157A1 US 20210308157 A1 US20210308157 A1 US 20210308157A1 US 202117212051 A US202117212051 A US 202117212051A US 2021308157 A1 US2021308157 A1 US 2021308157A1
- Authority
- US
- United States
- Prior art keywords
- antigen
- dotap
- cationic lipid
- antigens
- composition
- 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.)
- Pending
Links
- 230000028993 immune response Effects 0.000 title abstract description 47
- 125000002091 cationic group Chemical group 0.000 title description 52
- HBAQYPYDRFILMT-UHFFFAOYSA-N 8-[3-(1-cyclopropylpyrazol-4-yl)-1H-pyrazolo[4,3-d]pyrimidin-5-yl]-3-methyl-3,8-diazabicyclo[3.2.1]octan-2-one Chemical class C1(CC1)N1N=CC(=C1)C1=NNC2=C1N=C(N=C2)N1C2C(N(CC1CC2)C)=O HBAQYPYDRFILMT-UHFFFAOYSA-N 0.000 title description 26
- 230000000638 stimulation Effects 0.000 title description 13
- -1 cationic lipid Chemical class 0.000 claims abstract description 104
- 239000000203 mixture Substances 0.000 claims abstract description 71
- 238000000034 method Methods 0.000 claims abstract description 40
- 108091007433 antigens Proteins 0.000 claims description 208
- 102000036639 antigens Human genes 0.000 claims description 208
- 239000000427 antigen Substances 0.000 claims description 207
- 150000002632 lipids Chemical class 0.000 claims description 58
- KWVJHCQQUFDPLU-YEUCEMRASA-N 2,3-bis[[(z)-octadec-9-enoyl]oxy]propyl-trimethylazanium Chemical compound CCCCCCCC\C=C/CCCCCCCC(=O)OCC(C[N+](C)(C)C)OC(=O)CCCCCCC\C=C/CCCCCCCC KWVJHCQQUFDPLU-YEUCEMRASA-N 0.000 claims description 57
- 108090000623 proteins and genes Proteins 0.000 claims description 29
- 102000004169 proteins and genes Human genes 0.000 claims description 27
- 238000004519 manufacturing process Methods 0.000 claims description 22
- 210000000987 immune system Anatomy 0.000 claims description 18
- 102000004127 Cytokines Human genes 0.000 claims description 16
- 108090000695 Cytokines Proteins 0.000 claims description 16
- 108010012236 Chemokines Proteins 0.000 claims description 15
- 102000019034 Chemokines Human genes 0.000 claims description 15
- 208000015181 infectious disease Diseases 0.000 claims description 15
- 230000000890 antigenic effect Effects 0.000 claims description 14
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 claims description 13
- 230000002163 immunogen Effects 0.000 claims description 13
- 241000124008 Mammalia Species 0.000 claims description 11
- 239000012634 fragment Substances 0.000 claims description 11
- 239000008194 pharmaceutical composition Substances 0.000 claims description 11
- 239000003814 drug Substances 0.000 claims description 9
- 230000001225 therapeutic effect Effects 0.000 claims description 9
- 230000007935 neutral effect Effects 0.000 claims description 8
- 101100346932 Mus musculus Muc1 gene Proteins 0.000 claims description 7
- 229940079593 drug Drugs 0.000 claims description 7
- 230000003308 immunostimulating effect Effects 0.000 claims description 7
- 102000039446 nucleic acids Human genes 0.000 claims description 7
- 108020004707 nucleic acids Proteins 0.000 claims description 7
- 150000007523 nucleic acids Chemical class 0.000 claims description 7
- 235000012000 cholesterol Nutrition 0.000 claims description 6
- MWRBNPKJOOWZPW-CLFAGFIQSA-N dioleoyl phosphatidylethanolamine Chemical compound CCCCCCCC\C=C/CCCCCCCC(=O)OCC(COP(O)(=O)OCCN)OC(=O)CCCCCCC\C=C/CCCCCCCC MWRBNPKJOOWZPW-CLFAGFIQSA-N 0.000 claims description 6
- 229920000642 polymer Polymers 0.000 claims description 5
- SNKAWJBJQDLSFF-NVKMUCNASA-N 1,2-dioleoyl-sn-glycero-3-phosphocholine Chemical compound CCCCCCCC\C=C/CCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCCCCCC\C=C/CCCCCCCC SNKAWJBJQDLSFF-NVKMUCNASA-N 0.000 claims description 4
- 102000007298 Mucin-1 Human genes 0.000 claims description 4
- 108010008707 Mucin-1 Proteins 0.000 claims description 4
- 230000001105 regulatory effect Effects 0.000 claims description 4
- 239000004094 surface-active agent Substances 0.000 claims description 4
- LDGWQMRUWMSZIU-LQDDAWAPSA-M 2,3-bis[(z)-octadec-9-enoxy]propyl-trimethylazanium;chloride Chemical compound [Cl-].CCCCCCCC\C=C/CCCCCCCCOCC(C[N+](C)(C)C)OCCCCCCCC\C=C/CCCCCCCC LDGWQMRUWMSZIU-LQDDAWAPSA-M 0.000 claims description 3
- 230000002708 enhancing effect Effects 0.000 claims description 3
- 150000003904 phospholipids Chemical class 0.000 claims description 3
- RYCNUMLMNKHWPZ-SNVBAGLBSA-N 1-acetyl-sn-glycero-3-phosphocholine Chemical compound CC(=O)OC[C@@H](O)COP([O-])(=O)OCC[N+](C)(C)C RYCNUMLMNKHWPZ-SNVBAGLBSA-N 0.000 claims description 2
- 229920000362 Polyethylene-block-poly(ethylene glycol) Polymers 0.000 claims description 2
- 239000000443 aerosol Substances 0.000 claims description 2
- 239000003937 drug carrier Substances 0.000 claims description 2
- 239000004615 ingredient Substances 0.000 claims description 2
- 238000001361 intraarterial administration Methods 0.000 claims description 2
- 238000007918 intramuscular administration Methods 0.000 claims description 2
- 238000007912 intraperitoneal administration Methods 0.000 claims description 2
- 230000002601 intratumoral effect Effects 0.000 claims description 2
- 238000001990 intravenous administration Methods 0.000 claims description 2
- 239000006193 liquid solution Substances 0.000 claims description 2
- 235000010482 polyoxyethylene sorbitan monooleate Nutrition 0.000 claims description 2
- 229920000053 polysorbate 80 Polymers 0.000 claims description 2
- 238000007920 subcutaneous administration Methods 0.000 claims description 2
- NRWCNEBHECBWRJ-UHFFFAOYSA-M trimethyl(propyl)azanium;chloride Chemical compound [Cl-].CCC[N+](C)(C)C NRWCNEBHECBWRJ-UHFFFAOYSA-M 0.000 claims description 2
- 125000003275 alpha amino acid group Chemical group 0.000 claims 4
- 108091008874 T cell receptors Proteins 0.000 claims 2
- 102000016266 T-Cell Antigen Receptors Human genes 0.000 claims 2
- 210000004899 c-terminal region Anatomy 0.000 claims 2
- 108020001580 protein domains Proteins 0.000 claims 2
- 230000006337 proteolytic cleavage Effects 0.000 claims 2
- 238000000746 purification Methods 0.000 claims 2
- YAMUFBLWGFFICM-PTGWMXDISA-N 1-O-oleoyl-sn-glycero-3-phosphocholine Chemical compound CCCCCCCC\C=C/CCCCCCCC(=O)OC[C@@H](O)COP([O-])(=O)OCC[N+](C)(C)C YAMUFBLWGFFICM-PTGWMXDISA-N 0.000 claims 1
- 108090000765 processed proteins & peptides Proteins 0.000 abstract description 41
- 230000001939 inductive effect Effects 0.000 abstract description 5
- 230000003213 activating effect Effects 0.000 abstract description 4
- 150000002148 esters Chemical class 0.000 abstract description 4
- 150000002430 hydrocarbons Chemical group 0.000 abstract description 4
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 abstract description 2
- 239000004215 Carbon black (E152) Substances 0.000 abstract description 2
- 229910052799 carbon Inorganic materials 0.000 abstract description 2
- 229930195733 hydrocarbon Natural products 0.000 abstract description 2
- 150000002576 ketones Chemical class 0.000 abstract description 2
- 125000001453 quaternary ammonium group Chemical group 0.000 abstract description 2
- 150000004671 saturated fatty acids Chemical class 0.000 abstract description 2
- 235000021122 unsaturated fatty acids Nutrition 0.000 abstract description 2
- 150000004670 unsaturated fatty acids Chemical class 0.000 abstract description 2
- 210000002865 immune cell Anatomy 0.000 abstract 1
- 206010028980 Neoplasm Diseases 0.000 description 65
- 239000002502 liposome Substances 0.000 description 45
- 210000004443 dendritic cell Anatomy 0.000 description 40
- 210000004027 cell Anatomy 0.000 description 33
- 230000000694 effects Effects 0.000 description 24
- 201000010099 disease Diseases 0.000 description 17
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 17
- 229960005486 vaccine Drugs 0.000 description 17
- 210000001744 T-lymphocyte Anatomy 0.000 description 15
- 238000009472 formulation Methods 0.000 description 15
- 244000052769 pathogen Species 0.000 description 14
- 230000003389 potentiating effect Effects 0.000 description 14
- 102000004196 processed proteins & peptides Human genes 0.000 description 14
- 241000700605 Viruses Species 0.000 description 13
- 201000011510 cancer Diseases 0.000 description 13
- 239000002671 adjuvant Substances 0.000 description 11
- 230000000813 microbial effect Effects 0.000 description 11
- 241000699670 Mus sp. Species 0.000 description 10
- 230000004913 activation Effects 0.000 description 10
- 230000002458 infectious effect Effects 0.000 description 10
- 244000005700 microbiome Species 0.000 description 10
- 230000004614 tumor growth Effects 0.000 description 9
- 108010028921 Lipopeptides Proteins 0.000 description 8
- ATUOYWHBWRKTHZ-UHFFFAOYSA-N Propane Chemical compound CCC ATUOYWHBWRKTHZ-UHFFFAOYSA-N 0.000 description 8
- 150000001720 carbohydrates Chemical class 0.000 description 8
- 150000001875 compounds Chemical class 0.000 description 8
- 238000009169 immunotherapy Methods 0.000 description 8
- 241000894007 species Species 0.000 description 8
- 238000011282 treatment Methods 0.000 description 8
- 108090001030 Lipoproteins Proteins 0.000 description 7
- 102000004895 Lipoproteins Human genes 0.000 description 7
- 231100000673 dose–response relationship Toxicity 0.000 description 7
- 230000006870 function Effects 0.000 description 7
- 230000001717 pathogenic effect Effects 0.000 description 7
- 241000894006 Bacteria Species 0.000 description 6
- 229930186217 Glycolipid Natural products 0.000 description 6
- 229940022399 cancer vaccine Drugs 0.000 description 6
- 238000009566 cancer vaccine Methods 0.000 description 6
- 239000003795 chemical substances by application Substances 0.000 description 6
- 239000000839 emulsion Substances 0.000 description 6
- 229920001184 polypeptide Polymers 0.000 description 6
- 230000004044 response Effects 0.000 description 6
- 108020004414 DNA Proteins 0.000 description 5
- 102000003886 Glycoproteins Human genes 0.000 description 5
- 108090000288 Glycoproteins Proteins 0.000 description 5
- 101000914484 Homo sapiens T-lymphocyte activation antigen CD80 Proteins 0.000 description 5
- 241000700584 Simplexvirus Species 0.000 description 5
- 102100027222 T-lymphocyte activation antigen CD80 Human genes 0.000 description 5
- 239000012190 activator Substances 0.000 description 5
- 150000001413 amino acids Chemical group 0.000 description 5
- 230000000259 anti-tumor effect Effects 0.000 description 5
- 235000014633 carbohydrates Nutrition 0.000 description 5
- XMBWDFGMSWQBCA-UHFFFAOYSA-N hydrogen iodide Chemical compound I XMBWDFGMSWQBCA-UHFFFAOYSA-N 0.000 description 5
- 239000002609 medium Substances 0.000 description 5
- 238000002360 preparation method Methods 0.000 description 5
- 239000000047 product Substances 0.000 description 5
- 230000004936 stimulating effect Effects 0.000 description 5
- 238000012384 transportation and delivery Methods 0.000 description 5
- 206010008342 Cervix carcinoma Diseases 0.000 description 4
- 241000233866 Fungi Species 0.000 description 4
- 241000282412 Homo Species 0.000 description 4
- 108010065805 Interleukin-12 Proteins 0.000 description 4
- 102000013462 Interleukin-12 Human genes 0.000 description 4
- 108010002350 Interleukin-2 Proteins 0.000 description 4
- 239000002202 Polyethylene glycol Substances 0.000 description 4
- 241000194017 Streptococcus Species 0.000 description 4
- 230000024932 T cell mediated immunity Effects 0.000 description 4
- 208000006105 Uterine Cervical Neoplasms Diseases 0.000 description 4
- 210000000612 antigen-presenting cell Anatomy 0.000 description 4
- 230000008901 benefit Effects 0.000 description 4
- 230000015572 biosynthetic process Effects 0.000 description 4
- 239000000872 buffer Substances 0.000 description 4
- 201000010881 cervical cancer Diseases 0.000 description 4
- 210000001151 cytotoxic T lymphocyte Anatomy 0.000 description 4
- 238000011161 development Methods 0.000 description 4
- 150000004676 glycans Chemical class 0.000 description 4
- 230000001900 immune effect Effects 0.000 description 4
- 230000005847 immunogenicity Effects 0.000 description 4
- 230000005917 in vivo anti-tumor Effects 0.000 description 4
- 230000005764 inhibitory process Effects 0.000 description 4
- 229910052757 nitrogen Inorganic materials 0.000 description 4
- 229920001223 polyethylene glycol Polymers 0.000 description 4
- 230000036515 potency Effects 0.000 description 4
- 239000001294 propane Substances 0.000 description 4
- 230000000069 prophylactic effect Effects 0.000 description 4
- 210000001519 tissue Anatomy 0.000 description 4
- 210000004881 tumor cell Anatomy 0.000 description 4
- 108010022366 Carcinoembryonic Antigen Proteins 0.000 description 3
- 102100025475 Carcinoembryonic antigen-related cell adhesion molecule 5 Human genes 0.000 description 3
- 201000009030 Carcinoma Diseases 0.000 description 3
- XULFJDKZVHTRLG-JDVCJPALSA-N DOSPA trifluoroacetate Chemical compound [O-]C(=O)C(F)(F)F.CCCCCCCC\C=C/CCCCCCCCOCC(C[N+](C)(C)CCNC(=O)C(CCCNCCCN)NCCCN)OCCCCCCCC\C=C/CCCCCCCC XULFJDKZVHTRLG-JDVCJPALSA-N 0.000 description 3
- 241000700721 Hepatitis B virus Species 0.000 description 3
- 241000701806 Human papillomavirus Species 0.000 description 3
- 101000767631 Human papillomavirus type 16 Protein E7 Proteins 0.000 description 3
- 102000043136 MAP kinase family Human genes 0.000 description 3
- 108091054455 MAP kinase family Proteins 0.000 description 3
- 241000224016 Plasmodium Species 0.000 description 3
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 3
- 229930006000 Sucrose Natural products 0.000 description 3
- 230000006044 T cell activation Effects 0.000 description 3
- 230000006052 T cell proliferation Effects 0.000 description 3
- 125000002252 acyl group Chemical group 0.000 description 3
- 102000013529 alpha-Fetoproteins Human genes 0.000 description 3
- 108010026331 alpha-Fetoproteins Proteins 0.000 description 3
- 239000000306 component Substances 0.000 description 3
- 230000016396 cytokine production Effects 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 230000012010 growth Effects 0.000 description 3
- 229940084986 human chorionic gonadotropin Drugs 0.000 description 3
- 238000001727 in vivo Methods 0.000 description 3
- 230000006698 induction Effects 0.000 description 3
- 206010022000 influenza Diseases 0.000 description 3
- 238000002347 injection Methods 0.000 description 3
- 239000007924 injection Substances 0.000 description 3
- 230000003993 interaction Effects 0.000 description 3
- 210000004698 lymphocyte Anatomy 0.000 description 3
- 230000035800 maturation Effects 0.000 description 3
- 230000007246 mechanism Effects 0.000 description 3
- 244000045947 parasite Species 0.000 description 3
- 229920001282 polysaccharide Polymers 0.000 description 3
- 239000005017 polysaccharide Substances 0.000 description 3
- 230000002265 prevention Effects 0.000 description 3
- 239000003381 stabilizer Substances 0.000 description 3
- 238000003860 storage Methods 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 239000005720 sucrose Substances 0.000 description 3
- 238000003786 synthesis reaction Methods 0.000 description 3
- 230000008685 targeting Effects 0.000 description 3
- OPCHFPHZPIURNA-MFERNQICSA-N (2s)-2,5-bis(3-aminopropylamino)-n-[2-(dioctadecylamino)acetyl]pentanamide Chemical compound CCCCCCCCCCCCCCCCCCN(CC(=O)NC(=O)[C@H](CCCNCCCN)NCCCN)CCCCCCCCCCCCCCCCCC OPCHFPHZPIURNA-MFERNQICSA-N 0.000 description 2
- DSNRWDQKZIEDDB-SQYFZQSCSA-N 1,2-dioleoyl-sn-glycero-3-phospho-(1'-sn-glycerol) Chemical compound CCCCCCCC\C=C/CCCCCCCC(=O)OC[C@H](COP(O)(=O)OC[C@@H](O)CO)OC(=O)CCCCCCC\C=C/CCCCCCCC DSNRWDQKZIEDDB-SQYFZQSCSA-N 0.000 description 2
- WALUVDCNGPQPOD-UHFFFAOYSA-M 2,3-di(tetradecoxy)propyl-(2-hydroxyethyl)-dimethylazanium;bromide Chemical compound [Br-].CCCCCCCCCCCCCCOCC(C[N+](C)(C)CCO)OCCCCCCCCCCCCCC WALUVDCNGPQPOD-UHFFFAOYSA-M 0.000 description 2
- HVCOBJNICQPDBP-UHFFFAOYSA-N 3-[3-[3,5-dihydroxy-6-methyl-4-(3,4,5-trihydroxy-6-methyloxan-2-yl)oxyoxan-2-yl]oxydecanoyloxy]decanoic acid;hydrate Chemical compound O.OC1C(OC(CC(=O)OC(CCCCCCC)CC(O)=O)CCCCCCC)OC(C)C(O)C1OC1C(O)C(O)C(O)C(C)O1 HVCOBJNICQPDBP-UHFFFAOYSA-N 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 241001344923 Aulorhynchidae Species 0.000 description 2
- 102100032367 C-C motif chemokine 5 Human genes 0.000 description 2
- 102100035793 CD83 antigen Human genes 0.000 description 2
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
- 102000011022 Chorionic Gonadotropin Human genes 0.000 description 2
- 108010062540 Chorionic Gonadotropin Proteins 0.000 description 2
- 241000193403 Clostridium Species 0.000 description 2
- 241000701022 Cytomegalovirus Species 0.000 description 2
- 101150029707 ERBB2 gene Proteins 0.000 description 2
- 241000709661 Enterovirus Species 0.000 description 2
- 241000713813 Gibbon ape leukemia virus Species 0.000 description 2
- 108010015899 Glycopeptides Proteins 0.000 description 2
- 102000002068 Glycopeptides Human genes 0.000 description 2
- 241000856850 Goose coronavirus Species 0.000 description 2
- 102000004457 Granulocyte-Macrophage Colony-Stimulating Factor Human genes 0.000 description 2
- 108010017213 Granulocyte-Macrophage Colony-Stimulating Factor Proteins 0.000 description 2
- 241000590002 Helicobacter pylori Species 0.000 description 2
- 101000797762 Homo sapiens C-C motif chemokine 5 Proteins 0.000 description 2
- 101000946856 Homo sapiens CD83 antigen Proteins 0.000 description 2
- 241000725303 Human immunodeficiency virus Species 0.000 description 2
- 108090000978 Interleukin-4 Proteins 0.000 description 2
- 108090001005 Interleukin-6 Proteins 0.000 description 2
- 102000004889 Interleukin-6 Human genes 0.000 description 2
- 108090001007 Interleukin-8 Proteins 0.000 description 2
- 101001056675 Klebsiella pneumoniae Ferric aerobactin receptor Proteins 0.000 description 2
- 102000007330 LDL Lipoproteins Human genes 0.000 description 2
- 108010007622 LDL Lipoproteins Proteins 0.000 description 2
- 241000713821 Mason-Pfizer monkey virus Species 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- 241000713333 Mouse mammary tumor virus Species 0.000 description 2
- 102000015728 Mucins Human genes 0.000 description 2
- 108010063954 Mucins Proteins 0.000 description 2
- 241000714177 Murine leukemia virus Species 0.000 description 2
- 108091028043 Nucleic acid sequence Proteins 0.000 description 2
- 108700020796 Oncogene Proteins 0.000 description 2
- 102000043276 Oncogene Human genes 0.000 description 2
- 206010060862 Prostate cancer Diseases 0.000 description 2
- 108010072866 Prostate-Specific Antigen Proteins 0.000 description 2
- 102100038358 Prostate-specific antigen Human genes 0.000 description 2
- 241000711798 Rabies lyssavirus Species 0.000 description 2
- 241000606701 Rickettsia Species 0.000 description 2
- 241000283984 Rodentia Species 0.000 description 2
- 241001529934 Simian T-lymphotropic virus 3 Species 0.000 description 2
- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical compound [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 description 2
- 101710172711 Structural protein Proteins 0.000 description 2
- DTQVDTLACAAQTR-UHFFFAOYSA-M Trifluoroacetate Chemical compound [O-]C(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-M 0.000 description 2
- 241000700618 Vaccinia virus Species 0.000 description 2
- 241000711975 Vesicular stomatitis virus Species 0.000 description 2
- 108010067390 Viral Proteins Proteins 0.000 description 2
- 210000001015 abdomen Anatomy 0.000 description 2
- 229940024606 amino acid Drugs 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 230000030741 antigen processing and presentation Effects 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
- 210000003719 b-lymphocyte Anatomy 0.000 description 2
- 230000001580 bacterial effect Effects 0.000 description 2
- 230000004071 biological effect Effects 0.000 description 2
- 230000020411 cell activation Effects 0.000 description 2
- 230000001413 cellular effect Effects 0.000 description 2
- 230000014564 chemokine production Effects 0.000 description 2
- WLNARFZDISHUGS-MIXBDBMTSA-N cholesteryl hemisuccinate Chemical compound C1C=C2C[C@@H](OC(=O)CCC(O)=O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 WLNARFZDISHUGS-MIXBDBMTSA-N 0.000 description 2
- 230000000139 costimulatory effect Effects 0.000 description 2
- 239000000287 crude extract Substances 0.000 description 2
- 230000007423 decrease Effects 0.000 description 2
- 230000004069 differentiation Effects 0.000 description 2
- PSLWZOIUBRXAQW-UHFFFAOYSA-M dimethyl(dioctadecyl)azanium;bromide Chemical compound [Br-].CCCCCCCCCCCCCCCCCC[N+](C)(C)CCCCCCCCCCCCCCCCCC PSLWZOIUBRXAQW-UHFFFAOYSA-M 0.000 description 2
- 230000003828 downregulation Effects 0.000 description 2
- 238000012377 drug delivery Methods 0.000 description 2
- 239000002158 endotoxin Substances 0.000 description 2
- 238000001125 extrusion Methods 0.000 description 2
- 150000002270 gangliosides Chemical class 0.000 description 2
- 125000003827 glycol group Chemical group 0.000 description 2
- 230000002519 immonomodulatory effect Effects 0.000 description 2
- 230000006058 immune tolerance Effects 0.000 description 2
- 230000003053 immunization Effects 0.000 description 2
- 238000002649 immunization Methods 0.000 description 2
- 229960001438 immunostimulant agent Drugs 0.000 description 2
- 230000001965 increasing effect Effects 0.000 description 2
- NOESYZHRGYRDHS-UHFFFAOYSA-N insulin Chemical compound N1C(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(NC(=O)CN)C(C)CC)CSSCC(C(NC(CO)C(=O)NC(CC(C)C)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CCC(N)=O)C(=O)NC(CC(C)C)C(=O)NC(CCC(O)=O)C(=O)NC(CC(N)=O)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CSSCC(NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2C=CC(O)=CC=2)NC(=O)C(CC(C)C)NC(=O)C(C)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2NC=NC=2)NC(=O)C(CO)NC(=O)CNC2=O)C(=O)NCC(=O)NC(CCC(O)=O)C(=O)NC(CCCNC(N)=N)C(=O)NCC(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC(O)=CC=3)C(=O)NC(C(C)O)C(=O)N3C(CCC3)C(=O)NC(CCCCN)C(=O)NC(C)C(O)=O)C(=O)NC(CC(N)=O)C(O)=O)=O)NC(=O)C(C(C)CC)NC(=O)C(CO)NC(=O)C(C(C)O)NC(=O)C1CSSCC2NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(N)CC=1C=CC=CC=1)C(C)C)CC1=CN=CN1 NOESYZHRGYRDHS-UHFFFAOYSA-N 0.000 description 2
- 229920006008 lipopolysaccharide Polymers 0.000 description 2
- 210000001165 lymph node Anatomy 0.000 description 2
- 210000002540 macrophage Anatomy 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 230000001404 mediated effect Effects 0.000 description 2
- 201000001441 melanoma Diseases 0.000 description 2
- 210000001806 memory b lymphocyte Anatomy 0.000 description 2
- 210000003071 memory t lymphocyte Anatomy 0.000 description 2
- 229920000609 methyl cellulose Polymers 0.000 description 2
- 239000001923 methylcellulose Substances 0.000 description 2
- 235000010981 methylcellulose Nutrition 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 229940051875 mucins Drugs 0.000 description 2
- 230000035772 mutation Effects 0.000 description 2
- 230000001338 necrotic effect Effects 0.000 description 2
- 210000000440 neutrophil Anatomy 0.000 description 2
- 230000036961 partial effect Effects 0.000 description 2
- 239000002245 particle Substances 0.000 description 2
- 239000013612 plasmid Substances 0.000 description 2
- 239000013641 positive control Substances 0.000 description 2
- 230000019491 signal transduction Effects 0.000 description 2
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 description 2
- 230000004797 therapeutic response Effects 0.000 description 2
- 239000010409 thin film Substances 0.000 description 2
- 241000712461 unidentified influenza virus Species 0.000 description 2
- 239000012646 vaccine adjuvant Substances 0.000 description 2
- 229940124931 vaccine adjuvant Drugs 0.000 description 2
- 239000003981 vehicle Substances 0.000 description 2
- 239000000277 virosome Substances 0.000 description 2
- LNAZSHAWQACDHT-XIYTZBAFSA-N (2r,3r,4s,5r,6s)-4,5-dimethoxy-2-(methoxymethyl)-3-[(2s,3r,4s,5r,6r)-3,4,5-trimethoxy-6-(methoxymethyl)oxan-2-yl]oxy-6-[(2r,3r,4s,5r,6r)-4,5,6-trimethoxy-2-(methoxymethyl)oxan-3-yl]oxyoxane Chemical compound CO[C@@H]1[C@@H](OC)[C@H](OC)[C@@H](COC)O[C@H]1O[C@H]1[C@H](OC)[C@@H](OC)[C@H](O[C@H]2[C@@H]([C@@H](OC)[C@H](OC)O[C@@H]2COC)OC)O[C@@H]1COC LNAZSHAWQACDHT-XIYTZBAFSA-N 0.000 description 1
- LOGFVTREOLYCPF-KXNHARMFSA-N (2s,3r)-2-[[(2r)-1-[(2s)-2,6-diaminohexanoyl]pyrrolidine-2-carbonyl]amino]-3-hydroxybutanoic acid Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)[C@H]1CCCN1C(=O)[C@@H](N)CCCCN LOGFVTREOLYCPF-KXNHARMFSA-N 0.000 description 1
- QNPBWACMHMATDF-WRBBJXAJSA-N (9z,29z)-19-[(dimethylamino)methyl]-20-(2-hydroxyethylamino)octatriaconta-9,29-diene-18,21-dione Chemical compound CCCCCCCC\C=C/CCCCCCCC(=O)C(CN(C)C)C(NCCO)C(=O)CCCCCCC\C=C/CCCCCCCC QNPBWACMHMATDF-WRBBJXAJSA-N 0.000 description 1
- PORPENFLTBBHSG-MGBGTMOVSA-N 1,2-dihexadecanoyl-sn-glycerol-3-phosphate Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP(O)(O)=O)OC(=O)CCCCCCCCCCCCCCC PORPENFLTBBHSG-MGBGTMOVSA-N 0.000 description 1
- RUAUPNFNQOGIFF-UHFFFAOYSA-N 1-(4-tert-butyl-2,5-dimethoxyphenyl)propan-2-amine Chemical compound COC1=CC(C(C)(C)C)=C(OC)C=C1CC(C)N RUAUPNFNQOGIFF-UHFFFAOYSA-N 0.000 description 1
- LUNCLNIYHUMRFU-UHFFFAOYSA-N 1-(trimethylazaniumyl)butan-2-yl hydrogen phosphate Chemical compound C[N+](C)(C)CC(CC)OP(O)([O-])=O LUNCLNIYHUMRFU-UHFFFAOYSA-N 0.000 description 1
- FJUOBOJIJWDANZ-UHFFFAOYSA-N 2-[(4-anilinophenyl)iminomethyl]-5-(diethylamino)phenol Chemical compound CCN(CC)C1=CC(=C(C=C1)C=NC2=CC=C(C=C2)NC3=CC=CC=C3)O FJUOBOJIJWDANZ-UHFFFAOYSA-N 0.000 description 1
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-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
- WEVYNIUIFUYDGI-UHFFFAOYSA-N 3-[6-[4-(trifluoromethoxy)anilino]-4-pyrimidinyl]benzamide Chemical compound NC(=O)C1=CC=CC(C=2N=CN=C(NC=3C=CC(OC(F)(F)F)=CC=3)C=2)=C1 WEVYNIUIFUYDGI-UHFFFAOYSA-N 0.000 description 1
- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 description 1
- 102000007469 Actins Human genes 0.000 description 1
- 108010085238 Actins Proteins 0.000 description 1
- 241000701242 Adenoviridae Species 0.000 description 1
- 208000009746 Adult T-Cell Leukemia-Lymphoma Diseases 0.000 description 1
- 208000016683 Adult T-cell leukemia/lymphoma Diseases 0.000 description 1
- 101000689231 Aeromonas salmonicida S-layer protein Proteins 0.000 description 1
- 241000701386 African swine fever virus Species 0.000 description 1
- 102100024321 Alkaline phosphatase, placental type Human genes 0.000 description 1
- GUBGYTABKSRVRQ-XLOQQCSPSA-N Alpha-Lactose Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@H](O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-XLOQQCSPSA-N 0.000 description 1
- QGZKDVFQNNGYKY-UHFFFAOYSA-O Ammonium Chemical compound [NH4+] QGZKDVFQNNGYKY-UHFFFAOYSA-O 0.000 description 1
- 101001057424 Archaeoglobus fulgidus (strain ATCC 49558 / DSM 4304 / JCM 9628 / NBRC 100126 / VC-16) Iron-sulfur flavoprotein AF_1519 Proteins 0.000 description 1
- 241000712892 Arenaviridae Species 0.000 description 1
- 102100024003 Arf-GAP with SH3 domain, ANK repeat and PH domain-containing protein 1 Human genes 0.000 description 1
- 241001167018 Aroa Species 0.000 description 1
- 108010002913 Asialoglycoproteins Proteins 0.000 description 1
- 241000714230 Avian leukemia virus Species 0.000 description 1
- 241000713826 Avian leukosis virus Species 0.000 description 1
- 241000713838 Avian myeloblastosis virus Species 0.000 description 1
- 102100035526 B melanoma antigen 1 Human genes 0.000 description 1
- 208000003950 B-cell lymphoma Diseases 0.000 description 1
- 101150078806 BCAT2 gene Proteins 0.000 description 1
- 241000193830 Bacillus <bacterium> Species 0.000 description 1
- 241001148536 Bacteroides sp. Species 0.000 description 1
- 108060000903 Beta-catenin Proteins 0.000 description 1
- 102000015735 Beta-catenin Human genes 0.000 description 1
- 241000702628 Birnaviridae Species 0.000 description 1
- 241000228405 Blastomyces dermatitidis Species 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 241001227615 Bovine foamy virus Species 0.000 description 1
- 241000714266 Bovine leukemia virus Species 0.000 description 1
- 102100026413 Branched-chain-amino-acid aminotransferase, mitochondrial Human genes 0.000 description 1
- 206010006187 Breast cancer Diseases 0.000 description 1
- 208000026310 Breast neoplasm Diseases 0.000 description 1
- 208000011691 Burkitt lymphomas Diseases 0.000 description 1
- LRHPLDYGYMQRHN-UHFFFAOYSA-N Butanol Natural products CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 description 1
- FERIUCNNQQJTOY-UHFFFAOYSA-M Butyrate Chemical compound CCCC([O-])=O FERIUCNNQQJTOY-UHFFFAOYSA-M 0.000 description 1
- 102100036842 C-C motif chemokine 19 Human genes 0.000 description 1
- 238000011740 C57BL/6 mouse Methods 0.000 description 1
- 108010040471 CC Chemokines Proteins 0.000 description 1
- 102000001902 CC Chemokines Human genes 0.000 description 1
- 108700012434 CCL3 Proteins 0.000 description 1
- 241000589994 Campylobacter sp. Species 0.000 description 1
- 241000222122 Candida albicans Species 0.000 description 1
- 102000003846 Carbonic anhydrases Human genes 0.000 description 1
- 108090000209 Carbonic anhydrases Proteins 0.000 description 1
- 102100027668 Carboxy-terminal domain RNA polymerase II polypeptide A small phosphatase 1 Human genes 0.000 description 1
- 101710134395 Carboxy-terminal domain RNA polymerase II polypeptide A small phosphatase 1 Proteins 0.000 description 1
- 229920002134 Carboxymethyl cellulose Polymers 0.000 description 1
- 108090000538 Caspase-8 Proteins 0.000 description 1
- 102000004091 Caspase-8 Human genes 0.000 description 1
- 102100025064 Cellular tumor antigen p53 Human genes 0.000 description 1
- 101710150820 Cellular tumor antigen p53 Proteins 0.000 description 1
- 108010082161 Chemokine CCL19 Proteins 0.000 description 1
- 102000003805 Chemokine CCL19 Human genes 0.000 description 1
- 102000000013 Chemokine CCL3 Human genes 0.000 description 1
- 102000001326 Chemokine CCL4 Human genes 0.000 description 1
- 108010055165 Chemokine CCL4 Proteins 0.000 description 1
- 108010055166 Chemokine CCL5 Proteins 0.000 description 1
- 102000001327 Chemokine CCL5 Human genes 0.000 description 1
- 241001227713 Chiron Species 0.000 description 1
- 241000606153 Chlamydia trachomatis Species 0.000 description 1
- 241000223205 Coccidioides immitis Species 0.000 description 1
- 241000711573 Coronaviridae Species 0.000 description 1
- 102100028233 Coronin-1A Human genes 0.000 description 1
- 241000186227 Corynebacterium diphtheriae Species 0.000 description 1
- 241000709687 Coxsackievirus Species 0.000 description 1
- 201000007336 Cryptococcosis Diseases 0.000 description 1
- 241000221204 Cryptococcus neoformans Species 0.000 description 1
- 108010025464 Cyclin-Dependent Kinase 4 Proteins 0.000 description 1
- 102000013701 Cyclin-Dependent Kinase 4 Human genes 0.000 description 1
- 101710112752 Cytotoxin Proteins 0.000 description 1
- 108010041986 DNA Vaccines Proteins 0.000 description 1
- 229940021995 DNA vaccine Drugs 0.000 description 1
- 241000710829 Dengue virus group Species 0.000 description 1
- 102100036912 Desmin Human genes 0.000 description 1
- 108010044052 Desmin Proteins 0.000 description 1
- 108010016626 Dipeptides Proteins 0.000 description 1
- 238000012286 ELISA Assay Methods 0.000 description 1
- 241001115402 Ebolavirus Species 0.000 description 1
- 241001466953 Echovirus Species 0.000 description 1
- 101100491986 Emericella nidulans (strain FGSC A4 / ATCC 38163 / CBS 112.46 / NRRL 194 / M139) aromA gene Proteins 0.000 description 1
- 241000194032 Enterococcus faecalis Species 0.000 description 1
- 241001495410 Enterococcus sp. Species 0.000 description 1
- 241000991587 Enterovirus C Species 0.000 description 1
- 208000000832 Equine Encephalomyelitis Diseases 0.000 description 1
- 241000713730 Equine infectious anemia virus Species 0.000 description 1
- 241000186810 Erysipelothrix rhusiopathiae Species 0.000 description 1
- 108050001049 Extracellular proteins Proteins 0.000 description 1
- 241000713800 Feline immunodeficiency virus Species 0.000 description 1
- 241000714165 Feline leukemia virus Species 0.000 description 1
- 241000711950 Filoviridae Species 0.000 description 1
- 241000604777 Flavobacterium columnare Species 0.000 description 1
- 102000001390 Fructose-Bisphosphate Aldolase Human genes 0.000 description 1
- 108010068561 Fructose-Bisphosphate Aldolase Proteins 0.000 description 1
- 206010017553 Furuncle Diseases 0.000 description 1
- 241000605986 Fusobacterium nucleatum Species 0.000 description 1
- 102000000805 Galectin 4 Human genes 0.000 description 1
- 108010001515 Galectin 4 Proteins 0.000 description 1
- 102100031351 Galectin-9 Human genes 0.000 description 1
- 101710121810 Galectin-9 Proteins 0.000 description 1
- 208000005577 Gastroenteritis Diseases 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Chemical compound OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 102100041003 Glutamate carboxypeptidase 2 Human genes 0.000 description 1
- 241001506229 Goose reovirus Species 0.000 description 1
- 102000009465 Growth Factor Receptors Human genes 0.000 description 1
- 108010009202 Growth Factor Receptors Proteins 0.000 description 1
- 229920000084 Gum arabic Polymers 0.000 description 1
- 208000031886 HIV Infections Diseases 0.000 description 1
- 108010088729 HLA-A*02:01 antigen Proteins 0.000 description 1
- 241000606790 Haemophilus Species 0.000 description 1
- 206010061192 Haemorrhagic fever Diseases 0.000 description 1
- 241000150562 Hantaan orthohantavirus Species 0.000 description 1
- 108010006464 Hemolysin Proteins Proteins 0.000 description 1
- 241000711549 Hepacivirus C Species 0.000 description 1
- 241000700739 Hepadnaviridae Species 0.000 description 1
- 208000005176 Hepatitis C Diseases 0.000 description 1
- 208000005331 Hepatitis D Diseases 0.000 description 1
- 241000709721 Hepatovirus A Species 0.000 description 1
- 208000001688 Herpes Genitalis Diseases 0.000 description 1
- 241000228404 Histoplasma capsulatum Species 0.000 description 1
- 101000874316 Homo sapiens B melanoma antigen 1 Proteins 0.000 description 1
- 101000713106 Homo sapiens C-C motif chemokine 19 Proteins 0.000 description 1
- 101000892862 Homo sapiens Glutamate carboxypeptidase 2 Proteins 0.000 description 1
- 101000986086 Homo sapiens HLA class I histocompatibility antigen, A alpha chain Proteins 0.000 description 1
- 101001011441 Homo sapiens Interferon regulatory factor 4 Proteins 0.000 description 1
- 101001131670 Homo sapiens PWWP domain-containing DNA repair factor 3A Proteins 0.000 description 1
- 101000880770 Homo sapiens Protein SSX2 Proteins 0.000 description 1
- 101001012157 Homo sapiens Receptor tyrosine-protein kinase erbB-2 Proteins 0.000 description 1
- 241000714260 Human T-lymphotropic virus 1 Species 0.000 description 1
- 241000701085 Human alphaherpesvirus 3 Species 0.000 description 1
- 241000713673 Human foamy virus Species 0.000 description 1
- 241000701044 Human gammaherpesvirus 4 Species 0.000 description 1
- 241000713772 Human immunodeficiency virus 1 Species 0.000 description 1
- 241000713340 Human immunodeficiency virus 2 Species 0.000 description 1
- 101100540311 Human papillomavirus type 16 E6 gene Proteins 0.000 description 1
- 241000714192 Human spumaretrovirus Species 0.000 description 1
- XQFRJNBWHJMXHO-RRKCRQDMSA-N IDUR Chemical compound C1[C@H](O)[C@@H](CO)O[C@H]1N1C(=O)NC(=O)C(I)=C1 XQFRJNBWHJMXHO-RRKCRQDMSA-N 0.000 description 1
- 108010043496 Immunoglobulin Idiotypes Proteins 0.000 description 1
- 229940124873 Influenza virus vaccine Drugs 0.000 description 1
- 108090001061 Insulin Proteins 0.000 description 1
- 102100023915 Insulin Human genes 0.000 description 1
- 102100030126 Interferon regulatory factor 4 Human genes 0.000 description 1
- 102000003777 Interleukin-1 beta Human genes 0.000 description 1
- 108090000193 Interleukin-1 beta Proteins 0.000 description 1
- 102100033421 Keratin, type I cytoskeletal 18 Human genes 0.000 description 1
- 102100033420 Keratin, type I cytoskeletal 19 Human genes 0.000 description 1
- 102100023972 Keratin, type II cytoskeletal 8 Human genes 0.000 description 1
- 108010066327 Keratin-18 Proteins 0.000 description 1
- 108010066302 Keratin-19 Proteins 0.000 description 1
- 108010070511 Keratin-8 Proteins 0.000 description 1
- 241000588915 Klebsiella aerogenes Species 0.000 description 1
- 241000588747 Klebsiella pneumoniae Species 0.000 description 1
- 102100031413 L-dopachrome tautomerase Human genes 0.000 description 1
- 101710093778 L-dopachrome tautomerase Proteins 0.000 description 1
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 description 1
- 241000589248 Legionella Species 0.000 description 1
- 208000007764 Legionnaires' Disease Diseases 0.000 description 1
- 241000713666 Lentivirus Species 0.000 description 1
- 241000589902 Leptospira Species 0.000 description 1
- 241000186779 Listeria monocytogenes Species 0.000 description 1
- 206010058467 Lung neoplasm malignant Diseases 0.000 description 1
- 206010025323 Lymphomas Diseases 0.000 description 1
- 102000043129 MHC class I family Human genes 0.000 description 1
- 108091054437 MHC class I family Proteins 0.000 description 1
- 108700018351 Major Histocompatibility Complex Proteins 0.000 description 1
- 241000712079 Measles morbillivirus Species 0.000 description 1
- 101001057427 Methanocaldococcus jannaschii (strain ATCC 43067 / DSM 2661 / JAL-1 / JCM 10045 / NBRC 100440) Iron-sulfur flavoprotein MJ1083 Proteins 0.000 description 1
- 241000713862 Moloney murine sarcoma virus Species 0.000 description 1
- 241000711386 Mumps virus Species 0.000 description 1
- 241000186367 Mycobacterium avium Species 0.000 description 1
- 241000186364 Mycobacterium intracellulare Species 0.000 description 1
- 241000186363 Mycobacterium kansasii Species 0.000 description 1
- 235000009421 Myristica fragrans Nutrition 0.000 description 1
- 208000001894 Nasopharyngeal Neoplasms Diseases 0.000 description 1
- 241000588652 Neisseria gonorrhoeae Species 0.000 description 1
- 241000588650 Neisseria meningitidis Species 0.000 description 1
- 208000034176 Neoplasms, Germ Cell and Embryonal Diseases 0.000 description 1
- 208000009277 Neuroectodermal Tumors Diseases 0.000 description 1
- 108091034117 Oligonucleotide Proteins 0.000 description 1
- 241000712464 Orthomyxoviridae Species 0.000 description 1
- 101710116435 Outer membrane protein Proteins 0.000 description 1
- 108010058846 Ovalbumin Proteins 0.000 description 1
- 241001631646 Papillomaviridae Species 0.000 description 1
- 241000711504 Paramyxoviridae Species 0.000 description 1
- 208000002606 Paramyxoviridae Infections Diseases 0.000 description 1
- 241000701945 Parvoviridae Species 0.000 description 1
- 206010034107 Pasteurella infections Diseases 0.000 description 1
- 241000606856 Pasteurella multocida Species 0.000 description 1
- 229930182555 Penicillin Natural products 0.000 description 1
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 1
- 241000713137 Phlebovirus Species 0.000 description 1
- 206010035148 Plague Diseases 0.000 description 1
- 241001505293 Plasmodium ovale Species 0.000 description 1
- 241000223810 Plasmodium vivax Species 0.000 description 1
- 239000004698 Polyethylene Substances 0.000 description 1
- 229920002873 Polyethylenimine Polymers 0.000 description 1
- 241001505332 Polyomavirus sp. Species 0.000 description 1
- 208000024777 Prion disease Diseases 0.000 description 1
- 208000000236 Prostatic Neoplasms Diseases 0.000 description 1
- 229940124158 Protease/peptidase inhibitor Drugs 0.000 description 1
- 102100037686 Protein SSX2 Human genes 0.000 description 1
- 102000052575 Proto-Oncogene Human genes 0.000 description 1
- 108700020978 Proto-Oncogene Proteins 0.000 description 1
- 241000125945 Protoparvovirus Species 0.000 description 1
- 241000589516 Pseudomonas Species 0.000 description 1
- 239000012979 RPMI medium Substances 0.000 description 1
- 102100030086 Receptor tyrosine-protein kinase erbB-2 Human genes 0.000 description 1
- 241000186813 Renibacterium Species 0.000 description 1
- 241000702247 Reoviridae Species 0.000 description 1
- 241000725643 Respiratory syncytial virus Species 0.000 description 1
- 241000712909 Reticuloendotheliosis virus Species 0.000 description 1
- 241000712907 Retroviridae Species 0.000 description 1
- 241000711931 Rhabdoviridae Species 0.000 description 1
- 241000702670 Rotavirus Species 0.000 description 1
- 241000714474 Rous sarcoma virus Species 0.000 description 1
- 241000710799 Rubella virus Species 0.000 description 1
- 241000607142 Salmonella Species 0.000 description 1
- 206010039491 Sarcoma Diseases 0.000 description 1
- 241000978776 Senegalia senegal Species 0.000 description 1
- 241000713656 Simian foamy virus Species 0.000 description 1
- 241000714229 Simian retrovirus 1 Species 0.000 description 1
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 1
- 241000713896 Spleen necrosis virus Species 0.000 description 1
- 241000295644 Staphylococcaceae Species 0.000 description 1
- 241000191967 Staphylococcus aureus Species 0.000 description 1
- 229930182558 Sterol Natural products 0.000 description 1
- 241001478880 Streptobacillus moniliformis Species 0.000 description 1
- 241000193985 Streptococcus agalactiae Species 0.000 description 1
- 241000194049 Streptococcus equinus Species 0.000 description 1
- 241000193996 Streptococcus pyogenes Species 0.000 description 1
- 241001505901 Streptococcus sp. 'group A' Species 0.000 description 1
- 241000193990 Streptococcus sp. 'group B' Species 0.000 description 1
- 238000000692 Student's t-test Methods 0.000 description 1
- 102000004874 Synaptophysin Human genes 0.000 description 1
- 108090001076 Synaptophysin Proteins 0.000 description 1
- 230000005867 T cell response Effects 0.000 description 1
- 208000000389 T-cell leukemia Diseases 0.000 description 1
- 208000028530 T-cell lymphoblastic leukemia/lymphoma Diseases 0.000 description 1
- 101000748795 Thermus thermophilus (strain ATCC 27634 / DSM 579 / HB8) Cytochrome c oxidase polypeptide I+III Proteins 0.000 description 1
- 241000223997 Toxoplasma gondii Species 0.000 description 1
- 241000589886 Treponema Species 0.000 description 1
- 241000589904 Treponema pallidum subsp. pertenue Species 0.000 description 1
- 108060008682 Tumor Necrosis Factor Proteins 0.000 description 1
- 102000044209 Tumor Suppressor Genes Human genes 0.000 description 1
- 108700025716 Tumor Suppressor Genes Proteins 0.000 description 1
- 102100040247 Tumor necrosis factor Human genes 0.000 description 1
- 102000003425 Tyrosinase Human genes 0.000 description 1
- 108060008724 Tyrosinase Proteins 0.000 description 1
- 206010046865 Vaccinia virus infection Diseases 0.000 description 1
- 241000700647 Variola virus Species 0.000 description 1
- 241000251539 Vertebrata <Metazoa> Species 0.000 description 1
- 241000713325 Visna/maedi virus Species 0.000 description 1
- 241000714205 Woolly monkey sarcoma virus Species 0.000 description 1
- 241000120645 Yellow fever virus group Species 0.000 description 1
- 206010048249 Yersinia infections Diseases 0.000 description 1
- 208000025079 Yersinia infectious disease Diseases 0.000 description 1
- HIHOWBSBBDRPDW-PTHRTHQKSA-N [(3s,8s,9s,10r,13r,14s,17r)-10,13-dimethyl-17-[(2r)-6-methylheptan-2-yl]-2,3,4,7,8,9,11,12,14,15,16,17-dodecahydro-1h-cyclopenta[a]phenanthren-3-yl] n-[2-(dimethylamino)ethyl]carbamate Chemical compound C1C=C2C[C@@H](OC(=O)NCCN(C)C)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HIHOWBSBBDRPDW-PTHRTHQKSA-N 0.000 description 1
- 0 [1*]CC([2*])[3*] Chemical compound [1*]CC([2*])[3*] 0.000 description 1
- ATBOMIWRCZXYSZ-XZBBILGWSA-N [1-[2,3-dihydroxypropoxy(hydroxy)phosphoryl]oxy-3-hexadecanoyloxypropan-2-yl] (9e,12e)-octadeca-9,12-dienoate Chemical compound CCCCCCCCCCCCCCCC(=O)OCC(COP(O)(=O)OCC(O)CO)OC(=O)CCCCCCC\C=C\C\C=C\CCCCC ATBOMIWRCZXYSZ-XZBBILGWSA-N 0.000 description 1
- QKAHUWGYVDCLQF-UHFFFAOYSA-N [2-hexadecanoyloxy-3-(1-methylimidazol-4-yl)propyl] hexadecanoate Chemical compound CCCCCCCCCCCCCCCC(=O)OCC(OC(=O)CCCCCCCCCCCCCCC)CC1=CN(C)C=N1 QKAHUWGYVDCLQF-UHFFFAOYSA-N 0.000 description 1
- JLCPHMBAVCMARE-UHFFFAOYSA-N [3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methyl [5-(6-aminopurin-9-yl)-2-(hydroxymethyl)oxolan-3-yl] hydrogen phosphate Polymers Cc1cn(C2CC(OP(O)(=O)OCC3OC(CC3OP(O)(=O)OCC3OC(CC3O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c3nc(N)[nH]c4=O)C(COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3CO)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cc(C)c(=O)[nH]c3=O)n3cc(C)c(=O)[nH]c3=O)n3ccc(N)nc3=O)n3cc(C)c(=O)[nH]c3=O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)O2)c(=O)[nH]c1=O JLCPHMBAVCMARE-UHFFFAOYSA-N 0.000 description 1
- 239000000205 acacia gum Substances 0.000 description 1
- 235000010489 acacia gum Nutrition 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 150000007513 acids Chemical class 0.000 description 1
- 108700010877 adenoviridae proteins Proteins 0.000 description 1
- 230000001464 adherent effect Effects 0.000 description 1
- 230000000240 adjuvant effect Effects 0.000 description 1
- 201000006966 adult T-cell leukemia Diseases 0.000 description 1
- MGSDFCKWGHNUSM-QVPNGJTFSA-N alpha-L-Fucp-(1->2)-beta-D-Galp-(1->3)-beta-D-GlcpNAc Chemical compound O[C@H]1[C@H](O)[C@H](O)[C@H](C)O[C@H]1O[C@H]1[C@H](O[C@@H]2[C@H]([C@H](O)O[C@H](CO)[C@H]2O)NC(C)=O)O[C@H](CO)[C@H](O)[C@@H]1O MGSDFCKWGHNUSM-QVPNGJTFSA-N 0.000 description 1
- AWUCVROLDVIAJX-UHFFFAOYSA-N alpha-glycerophosphate Natural products OCC(O)COP(O)(O)=O AWUCVROLDVIAJX-UHFFFAOYSA-N 0.000 description 1
- 229940037003 alum Drugs 0.000 description 1
- AZDRQVAHHNSJOQ-UHFFFAOYSA-N alumane Chemical class [AlH3] AZDRQVAHHNSJOQ-UHFFFAOYSA-N 0.000 description 1
- 125000003277 amino group Chemical group 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 230000005809 anti-tumor immunity Effects 0.000 description 1
- 230000006907 apoptotic process Effects 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 101150037081 aroA gene Proteins 0.000 description 1
- 244000309743 astrovirus Species 0.000 description 1
- 244000052616 bacterial pathogen Species 0.000 description 1
- 230000002457 bidirectional effect Effects 0.000 description 1
- 230000017531 blood circulation Effects 0.000 description 1
- 239000012503 blood component Substances 0.000 description 1
- OBUOQZXTPAZQNP-UHFFFAOYSA-N butan-1-ol;propane-1,2,3-triol Chemical compound CCCCO.OCC(O)CO OBUOQZXTPAZQNP-UHFFFAOYSA-N 0.000 description 1
- 229940095731 candida albicans Drugs 0.000 description 1
- 239000002775 capsule Substances 0.000 description 1
- 239000001768 carboxy methyl cellulose Substances 0.000 description 1
- 235000010948 carboxy methyl cellulose Nutrition 0.000 description 1
- 239000008112 carboxymethyl-cellulose Substances 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 239000013592 cell lysate Substances 0.000 description 1
- 239000002771 cell marker Substances 0.000 description 1
- 230000012292 cell migration Effects 0.000 description 1
- 230000004663 cell proliferation Effects 0.000 description 1
- 239000002458 cell surface marker Substances 0.000 description 1
- 230000007969 cellular immunity Effects 0.000 description 1
- 230000008614 cellular interaction Effects 0.000 description 1
- 229940032077 cervical cancer vaccine Drugs 0.000 description 1
- 239000002975 chemoattractant Substances 0.000 description 1
- 230000003399 chemotactic effect Effects 0.000 description 1
- 229940038705 chlamydia trachomatis Drugs 0.000 description 1
- 229960001231 choline Drugs 0.000 description 1
- 230000004087 circulation Effects 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 208000029742 colonic neoplasm Diseases 0.000 description 1
- 238000011254 conventional chemotherapy Methods 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
- 230000009089 cytolysis Effects 0.000 description 1
- 231100000599 cytotoxic agent Toxicity 0.000 description 1
- 239000002619 cytotoxin Substances 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 230000002950 deficient Effects 0.000 description 1
- 239000003405 delayed action preparation Substances 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 210000005045 desmin Anatomy 0.000 description 1
- 230000001687 destabilization Effects 0.000 description 1
- XEYBRNLFEZDVAW-ARSRFYASSA-N dinoprostone Chemical compound CCCCC[C@H](O)\C=C\[C@H]1[C@H](O)CC(=O)[C@@H]1C\C=C/CCCC(O)=O XEYBRNLFEZDVAW-ARSRFYASSA-N 0.000 description 1
- 229960002986 dinoprostone Drugs 0.000 description 1
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 1
- 239000006185 dispersion Substances 0.000 description 1
- 239000002552 dosage form Substances 0.000 description 1
- 238000002296 dynamic light scattering Methods 0.000 description 1
- 238000001493 electron microscopy Methods 0.000 description 1
- 206010014599 encephalitis Diseases 0.000 description 1
- 210000002889 endothelial cell Anatomy 0.000 description 1
- 229940092559 enterobacter aerogenes Drugs 0.000 description 1
- 210000003979 eosinophil Anatomy 0.000 description 1
- RTZKZFJDLAIYFH-UHFFFAOYSA-N ether Substances CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 1
- 125000001495 ethyl group Chemical group [H]C([H])([H])C([H])([H])* 0.000 description 1
- 230000001747 exhibiting effect Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000012091 fetal bovine serum Substances 0.000 description 1
- 239000000706 filtrate Substances 0.000 description 1
- 238000000684 flow cytometry Methods 0.000 description 1
- 229940014144 folate Drugs 0.000 description 1
- OVBPIULPVIDEAO-LBPRGKRZSA-N folic acid Chemical compound C=1N=C2NC(N)=NC(=O)C2=NC=1CNC1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 OVBPIULPVIDEAO-LBPRGKRZSA-N 0.000 description 1
- 235000019152 folic acid Nutrition 0.000 description 1
- 239000011724 folic acid Substances 0.000 description 1
- 208000003512 furunculosis Diseases 0.000 description 1
- 201000011587 gastric lymphoma Diseases 0.000 description 1
- 210000001035 gastrointestinal tract Anatomy 0.000 description 1
- 238000001641 gel filtration chromatography Methods 0.000 description 1
- 108091006104 gene-regulatory proteins Proteins 0.000 description 1
- 102000034356 gene-regulatory proteins Human genes 0.000 description 1
- 201000004946 genital herpes Diseases 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- PEDCQBHIVMGVHV-UHFFFAOYSA-N glycerol Substances OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 1
- 201000010536 head and neck cancer Diseases 0.000 description 1
- 208000014829 head and neck neoplasm Diseases 0.000 description 1
- 239000003228 hemolysin Substances 0.000 description 1
- 208000006454 hepatitis Diseases 0.000 description 1
- 231100000283 hepatitis Toxicity 0.000 description 1
- 208000029570 hepatitis D virus infection Diseases 0.000 description 1
- 229940124724 hepatitis-A vaccine Drugs 0.000 description 1
- 206010073071 hepatocellular carcinoma Diseases 0.000 description 1
- 231100000844 hepatocellular carcinoma Toxicity 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 230000005745 host immune response Effects 0.000 description 1
- 235000020256 human milk Nutrition 0.000 description 1
- 210000004251 human milk Anatomy 0.000 description 1
- 244000052637 human pathogen Species 0.000 description 1
- 230000036571 hydration Effects 0.000 description 1
- 238000006703 hydration reaction Methods 0.000 description 1
- 239000000017 hydrogel Substances 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 230000008105 immune reaction Effects 0.000 description 1
- 230000003832 immune regulation Effects 0.000 description 1
- 230000006028 immune-suppresssive effect Effects 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 230000007235 immunity generation Effects 0.000 description 1
- 229940127121 immunoconjugate Drugs 0.000 description 1
- 239000000568 immunological adjuvant Substances 0.000 description 1
- 239000002955 immunomodulating agent Substances 0.000 description 1
- 229940121354 immunomodulator Drugs 0.000 description 1
- 230000002434 immunopotentiative effect Effects 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000010348 incorporation Methods 0.000 description 1
- 210000004969 inflammatory cell Anatomy 0.000 description 1
- 229960003971 influenza vaccine Drugs 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 230000000977 initiatory effect Effects 0.000 description 1
- 229940125396 insulin Drugs 0.000 description 1
- 229940117681 interleukin-12 Drugs 0.000 description 1
- 229940100601 interleukin-6 Drugs 0.000 description 1
- 238000010255 intramuscular injection Methods 0.000 description 1
- 239000007927 intramuscular injection Substances 0.000 description 1
- 239000000644 isotonic solution Substances 0.000 description 1
- 208000017169 kidney disease Diseases 0.000 description 1
- 229940045505 klebsiella pneumoniae Drugs 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 239000008101 lactose Substances 0.000 description 1
- 230000021633 leukocyte mediated immunity Effects 0.000 description 1
- 239000003446 ligand Substances 0.000 description 1
- 230000000670 limiting effect Effects 0.000 description 1
- 229940124590 live attenuated vaccine Drugs 0.000 description 1
- 229940023012 live-attenuated vaccine Drugs 0.000 description 1
- 210000004072 lung Anatomy 0.000 description 1
- 201000005202 lung cancer Diseases 0.000 description 1
- 208000020816 lung neoplasm Diseases 0.000 description 1
- 210000005210 lymphoid organ Anatomy 0.000 description 1
- 239000001115 mace Substances 0.000 description 1
- 229920002521 macromolecule Polymers 0.000 description 1
- 230000036210 malignancy Effects 0.000 description 1
- 208000025848 malignant tumor of nasopharynx Diseases 0.000 description 1
- 210000002752 melanocyte Anatomy 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 210000004379 membrane Anatomy 0.000 description 1
- 239000000693 micelle Substances 0.000 description 1
- 238000012737 microarray-based gene expression Methods 0.000 description 1
- 238000013508 migration Methods 0.000 description 1
- 230000001617 migratory effect Effects 0.000 description 1
- 230000003278 mimic effect Effects 0.000 description 1
- 150000007522 mineralic acids Chemical class 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 239000003607 modifier Substances 0.000 description 1
- 210000001616 monocyte Anatomy 0.000 description 1
- 150000002772 monosaccharides Chemical class 0.000 description 1
- 238000012243 multiplex automated genomic engineering Methods 0.000 description 1
- 210000003205 muscle Anatomy 0.000 description 1
- DDBRXOJCLVGHLX-UHFFFAOYSA-N n,n-dimethylmethanamine;propane Chemical compound CCC.CN(C)C DDBRXOJCLVGHLX-UHFFFAOYSA-N 0.000 description 1
- ZUHZZVMEUAUWHY-UHFFFAOYSA-N n,n-dimethylpropan-1-amine Chemical compound CCCN(C)C ZUHZZVMEUAUWHY-UHFFFAOYSA-N 0.000 description 1
- PUPNJSIFIXXJCH-UHFFFAOYSA-N n-(4-hydroxyphenyl)-2-(1,1,3-trioxo-1,2-benzothiazol-2-yl)acetamide Chemical compound C1=CC(O)=CC=C1NC(=O)CN1S(=O)(=O)C2=CC=CC=C2C1=O PUPNJSIFIXXJCH-UHFFFAOYSA-N 0.000 description 1
- 201000011216 nasopharynx carcinoma Diseases 0.000 description 1
- 210000000822 natural killer cell Anatomy 0.000 description 1
- 230000003472 neutralizing effect Effects 0.000 description 1
- 231100000590 oncogenic Toxicity 0.000 description 1
- 230000002246 oncogenic effect Effects 0.000 description 1
- 150000007524 organic acids Chemical class 0.000 description 1
- 235000005985 organic acids Nutrition 0.000 description 1
- 229940092253 ovalbumin Drugs 0.000 description 1
- 230000003071 parasitic effect Effects 0.000 description 1
- 229940051027 pasteurella multocida Drugs 0.000 description 1
- 201000005115 pasteurellosis Diseases 0.000 description 1
- 239000008188 pellet Substances 0.000 description 1
- 229940049954 penicillin Drugs 0.000 description 1
- 239000000137 peptide hydrolase inhibitor Substances 0.000 description 1
- 238000010647 peptide synthesis reaction Methods 0.000 description 1
- 239000000546 pharmaceutical excipient Substances 0.000 description 1
- 239000000825 pharmaceutical preparation Substances 0.000 description 1
- 239000002953 phosphate buffered saline Substances 0.000 description 1
- 230000026731 phosphorylation Effects 0.000 description 1
- 238000006366 phosphorylation reaction Methods 0.000 description 1
- 230000000704 physical effect Effects 0.000 description 1
- 108010031345 placental alkaline phosphatase Proteins 0.000 description 1
- 238000007747 plating Methods 0.000 description 1
- 230000010287 polarization Effects 0.000 description 1
- 229920001308 poly(aminoacid) Polymers 0.000 description 1
- 229920000747 poly(lactic acid) Polymers 0.000 description 1
- 229920001481 poly(stearyl methacrylate) Polymers 0.000 description 1
- 229920000515 polycarbonate Polymers 0.000 description 1
- 239000004417 polycarbonate Substances 0.000 description 1
- 229920000728 polyester Polymers 0.000 description 1
- 108040000983 polyphosphate:AMP phosphotransferase activity proteins Proteins 0.000 description 1
- 150000004804 polysaccharides Polymers 0.000 description 1
- 239000011148 porous material Substances 0.000 description 1
- 208000017805 post-transplant lymphoproliferative disease Diseases 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 238000011321 prophylaxis Methods 0.000 description 1
- XEYBRNLFEZDVAW-UHFFFAOYSA-N prostaglandin E2 Natural products CCCCCC(O)C=CC1C(O)CC(=O)C1CC=CCCCC(O)=O XEYBRNLFEZDVAW-UHFFFAOYSA-N 0.000 description 1
- 210000002307 prostate Anatomy 0.000 description 1
- 238000003498 protein array Methods 0.000 description 1
- 238000002731 protein assay Methods 0.000 description 1
- 101150002764 purA gene Proteins 0.000 description 1
- 238000001959 radiotherapy Methods 0.000 description 1
- 230000008707 rearrangement Effects 0.000 description 1
- 108020003175 receptors Proteins 0.000 description 1
- 102000005962 receptors Human genes 0.000 description 1
- 238000009877 rendering Methods 0.000 description 1
- 238000012552 review Methods 0.000 description 1
- 229920006395 saturated elastomer Polymers 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 238000000527 sonication Methods 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 238000010561 standard procedure Methods 0.000 description 1
- 238000007619 statistical method Methods 0.000 description 1
- 150000003432 sterols Chemical class 0.000 description 1
- 235000003702 sterols Nutrition 0.000 description 1
- 229960005322 streptomycin Drugs 0.000 description 1
- 238000010254 subcutaneous injection Methods 0.000 description 1
- 239000007929 subcutaneous injection Substances 0.000 description 1
- 229940031626 subunit vaccine Drugs 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 230000020382 suppression by virus of host antigen processing and presentation of peptide antigen via MHC class I Effects 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 238000012385 systemic delivery Methods 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 210000001550 testis Anatomy 0.000 description 1
- 229940124597 therapeutic agent Drugs 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 229940021747 therapeutic vaccine Drugs 0.000 description 1
- 230000032258 transport Effects 0.000 description 1
- GETQZCLCWQTVFV-UHFFFAOYSA-N trimethylamine Chemical group CN(C)C GETQZCLCWQTVFV-UHFFFAOYSA-N 0.000 description 1
- 230000001573 trophoblastic effect Effects 0.000 description 1
- 208000029387 trophoblastic neoplasm Diseases 0.000 description 1
- 201000008827 tuberculosis Diseases 0.000 description 1
- 230000000381 tumorigenic effect Effects 0.000 description 1
- 241000724775 unclassified viruses Species 0.000 description 1
- 241000701161 unidentified adenovirus Species 0.000 description 1
- 241001529453 unidentified herpesvirus Species 0.000 description 1
- 241001430294 unidentified retrovirus Species 0.000 description 1
- 238000002255 vaccination Methods 0.000 description 1
- 208000007089 vaccinia Diseases 0.000 description 1
- 238000010200 validation analysis Methods 0.000 description 1
- 229920002554 vinyl polymer Polymers 0.000 description 1
- 244000052613 viral pathogen Species 0.000 description 1
- 239000013603 viral vector Substances 0.000 description 1
- 230000003612 virological effect Effects 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Images
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/66—Phosphorus compounds
- A61K31/683—Diesters of a phosphorus acid with two hydroxy compounds, e.g. phosphatidylinositols
- A61K31/685—Diesters of a phosphorus acid with two hydroxy compounds, e.g. phosphatidylinositols one of the hydroxy compounds having nitrogen atoms, e.g. phosphatidylserine, lecithin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
- A61P37/04—Immunostimulants
Definitions
- the present invention generally related to stimulating an immune response, and more particularly to the use of the R and S enantiomers of lipids in stimulating immune responses.
- immunologic modifiers that enhance, direct or promote an immune response
- vaccines may include antigens to stimulate an immune response.
- some potential vaccines that include antigens are weak stimulators of an immune response because the vaccines do not efficiently deliver the antigen to antigen presenting cells (“APC”) of the immune system and/or the antigen is weakly immunogenic.
- APC antigen presenting cells
- an immunomodifier when included as part of a therapeutic vaccine, should at least (1) improve antigen delivery and/or processing in the APC [Wang, R. F., and Wang, H. Y. Enhancement of antitumor immunity by prolonging antigen presentation on dendritic cells .
- the primary class of agents used to enhance the efficacy of antigens such as peptide or protein antigens, in eliciting an immune response are adjuvants such as water-in-oil emulsions, alum, and other chemicals which enhance antigen responses; however, these adjuvants are not immunomodifiers, as described above, because they have no direct immunomodulatory effects themselves [Vogel, F. R., and Powell, M. F. A compendium of vaccine adjuvants and excipients , Pharm Biotechnol 6:141 (1995)1. Several such adjuvants are available for use in animals and some of them have been tested in clinical trials.
- influenza virosomes In addition to traditional adjuvants such as the aluminum salts, products such as influenza virosomes [Gluck, R., and Walti, E. 2000 . Biophysical validation of Epaxal Berna, a hepatitis A vaccine adjuvanted with immunopotentiating reconstituted influenza virosomes ORM . Dev Biol (Basel) 103:189 (2000)1, and Chiron's MF59 [Kahn, J. 0., et al. Clinical and immunologic responses to human immunodeficiency virus ( I ⁇ 1.17) type ISF 2 gp 120 subunit vaccine combined with MF 59 adjuvant with or without mummy!
- Vaccination strategies to prevent genital herpes and neonatal herpes simplex virus (HSV) disease Herpes 11:12 (2004); Minutello, M. et al., Safety and immunogenicity of an inactivated subunit influenza virus vaccine combined with ilifF 59 adjuvant emulsion in elderly subjects, immunized for three consecutive influenza seasons .
- Vaccine 17:99 (1999)1 evidence from animal models suggests that the MF59 adjuvant enhances production of neutralizing antibodies rather than enhancing responses. Thus, new methods of stimulating cell mediated immune responses are needed.
- a weakly immunogenic antigen can be modified to increase its immunogenicity.
- a weakly immunogenic antigen can be coupled to immunogenic peptides, polysaccharides, or lipids to increase its immunogenicity.
- simply coupling weakly immunogenic antigens to these types of compounds may not be sufficient to elicit an immune response.
- the resulting immune response may be directed to immunogenic epitopes on the coupled compound and not the weak antigen, or the coupled antigen may not be efficiently delivered to APC of the immune system.
- additional methods are needed to stimulate immune responses to antigens that are weakly immunogenic.
- This invention is directed to the chirality of cationic lipids and the use of the R and S enantiomers of cationic lipids, which under certain dose and composition conditions act as a novel class of immune-stimulants, to (1) effectively present or deliver an antigen to the immune system and (2) stimulate the immune system to respond to the antigen.
- Liposomes have been extensively used for delivering small molecular weight drugs, plasmid DNA, oligonucleotides, proteins, and peptides. Vaccines using liposomal vehicles as nonviral antigen carriers are preferable compared to traditional immunizations using live attenuated vaccines or viral vectors such as vaccinia or influenza virus.
- U.S. patent application Ser. No. 12/049,957, assigned to the assignee of the present application discloses simple yet effective lipid-based immunotherapies, including a cationic lipid/antigen complex, which has two molecules, a cationic lipid and an antigen, and the effects of the lipid dose on the resulting immune response. The reported results demonstrate that the cationic liposome complexed with an antigen serves to stimulate immune responses and initiate dendritic cell (an APC) interaction with T-cells.
- an APC dendritic cell
- one aspect of the invention provides a composition of at least one enantiomer of a cationic lipid in a dose sufficient to induce an immune response in a subject.
- Another aspect of the invention provides a method of inducing an immune response in a subject by administering a specific enantiomer or a mixture of enantiomers of a cationic lipid to the subject.
- Another aspect of the invention provides a composition of an R or S enantiomer of a cationic lipid in a dose sufficient to induce an immune response in a subject.
- Additional aspects of the invention involve the addition of at least one antigen to the R or S enantiomer to form a cationic lipid/antigen complex in which case the immune response is antigen-specific.
- FIGS. 1A and 1B depict chirality of I,2-dioleoyl-3-trimethylammonium propane (“DOTAP”).
- DOTAP I,2-dioleoyl-3-trimethylammonium propane
- FIG. 2 is a graph depicting activation of human dendritic cells resulting in expression of the co-stimulatory molecule CD 80 by R-DOTAP, S-DOTAP and the racemic mixture RS-DOTAP.
- FIG. 3 is a graph depicting activation of human dendritic cells resulting in expression of the co-stimulatory molecule CD 83 by R-DOTAP, S-DOTAP and the racernic mixture RS-DOTAP.
- FIG. 4 is a graph depicting activation of human dendritic cells resulting in expression of the co-stimulatory molecule CD 86 by R-DOTAP, S-DOTAP and the racemic mixture RS-DOTAP.
- FIG. 5 is a graph depicting stimulation of human dendritic cells resulting in production of the chemokine CCL-3 by R-DOTAP, S-DOTAP and the racemic mixture RS-DOTAP.
- FIG. 6 is a graph depicting stimulation of human dendritic cells resulting in production of the chemokine CCL-4 by R-DOTAP, S-DOTAP and the racemic mixture RS-DOTAP.
- FIG. 7 is a graph depicting stimulation of human dendritic cells resulting in production of the chemokine CCL-5 by R-DOTAP, S-DOTAP and the racemic mixture RS-DOTAP.
- FIG. 8 is a graph depicting stimulation of human dendritic cells resulting in production of the chemokine CCL-19 by R-DOTAP, S-DOTAP and the racemic mixture RS-DOTAP.
- FIG. 9 is a graph depicting stimulation of human dendritic cells resulting in production of the cytokine IL-2 by R-DOTAP, S-DOTAP and the racemic mixture RS-DOTAP.
- FIG. 10 is a graph depicting stimulation of human dendritic cells resulting in production of the cytokine IL-S by R-DOTAP, S-DOTAP and the racemic mixture RS-DOTAP.
- FIG. 11 is a graph depicting stimulation of human dendritic cells resulting in production of the cytokine 1L-12 by R-DOTAP, S-DOTAP and the racemic mixture RS-DOTAP.
- FIG. 12 is a graph demonstrating the in vivo antitumor effect of various doses of a cationic lipid/antigen complex based on tumor size and. time post-injection.
- FIG. 13 is a graph demonstrating the effect of S-DOTAP dose on the in vivo anti tumor efficacy of the cationic lipid/antigen complex.
- FIG. 14 is a graph demonstrating the effect of R-DOTAP dose on the in vivo anti tumor efficacy of the cationic lipid/antigen complex.
- FIG. 15 is a graph depicting the lipid dose response effects of the racemic mixture of DOTAP, R-DOTAP and S-DOTAP on the in vivo anti-tumor immune response of the cationic lipid/antigen complex with antigen dose of 201.1 g. The effect of antigen dose is also demonstrated with the racemic mixture of DOTAP.
- R-DOTAP compared to S-DOTAP: * p ⁇ 0.05, ** p ⁇ 0.01, n 5-6.
- One aspect of the present invention provides an enantiomer of a cationic lipid to stimulate an immune response in a mammal to prevent or treat disease
- the individual chiral lipids can function independently as immunomodulators, in a dose dependent manner, such as for production of chemokines and/or cytokines, by activating various components of the MAP kinase signaling pathway.
- the dose range that effectively induces an immune response is observed to differ between the R and S enantiomers and also within various mammalian species.
- the R-enantiomer of DOTAP effectively attenuates tumor growth over a range of about 30 nmole to about 400 nmole.
- the S-enantiomer of DOTAP is effective over this same range of doses in the same species of rodent, though less so than the R-enantiomer.
- the chiral cationic lipid may be associated with antigens or drugs for presentation to cells of the immune system while simultaneously stimulating a strong antigen-specific immune response.
- the antigen is a lipopeptide.
- the chiral cationic lipid at a dose sufficient to stimulate an immune response, is administered in combination with an antigen or antigens.
- the cationic lipid/antigen combination is capable of generating an immune response that is specific to the antigen(s) delivered in combination with the cationic lipid.
- the response generated may include production of specific cytotoxic T cells, memory T cells, or B cells resulting in the prevention of, or therapeutic response to, the specific disease associated with the antigen(s).
- the chiral cationic lipids of the invention may be in the form of cationic lipid complexes.
- the cationic lipid complex can take the form of various vesicles such as liposomes, micelles, or emulsions.
- the cationic lipid complexes may be unilaminar or multilaminar.
- the antigen may be encapsulated in the cationic lipid complex or may be unencapsulated. Encapsulated is understood to mean that the antigen may be contained within the internal space of the complex and/or incorporated into the lipid walls of the complex.
- Another aspect of the invention relates to a method for producing these complexes, wherein the method may optionally include the step of purifying these formulations from excess individual components.
- the cationic lipid complexes have a net positive charge and/or a positively charged surface at pH 6.0-8.0.
- the optional “antigen” which may be included with cationic lipid complexes of the invention may be nucleic acids, peptides, lipopeptides, proteins, lipoproteins, polysaccharides, and other macromolecules which may be complexed directly with cationic lipids.
- cationic drugs e.g., large cationic protein
- anionic lipid or sequentially complexed first with anionic lipid or polymer followed by the chiral cationic lipid can be directly complexed with an anionic lipid or sequentially complexed first with anionic lipid or polymer followed by the chiral cationic lipid. The use of this process permits delivery of positive or neutral charged drugs to cells by the complexes of the present invention.
- One aspect of the present invention involves the use of the chiral cationic lipid complexes to activate dendritic cells and also to stimulate the production of chemokines and cytokines.
- Chemokines and cytokines are important regulators of immune responses. Chemokines were originally identified as potent chemoattractants for inflammatory cells including neutrophils, eosinophils, and in onocytes/macrophages. Subsequent studies have revealed that chemokines have profound effects on immune reactions by regulating the trafficking of dendritic cells and other lymphocytes into lymphoid organs. Dendritic cells are migratory cells that sample antigens in the tissue, migrate to the draining lymph nodes and mature to stimulate the T cell response.
- CCL2 cytotoxic T lymphocytes
- IL-12 interleukin-12
- IFN-y interferon-y
- CC chemokine Another CC chemokine, CCL-4, has also been shown to recruit and expand dendritic cells in vivo and potentiate the immunogenicity of plasmid DNA vaccines. Recently, it has been shown that chemokines enhance immunity by guiding naive CDS+ T cells to sites of CD4+ T cell-dendritic cell interaction and promote memory CD8+ T cell generation.
- chemokines that may be stimulated by the cationic lipid complexes of the present invention are CCL-2, CCL-3, and CCL-4.
- Examples of cytokines that may be stimulated by the cationic lipid complexes of the present invention are IL-2, IL-8, IL-12 and IFN-y.
- the chiral cationic lipid complexes of the present invention may form liposomes that are optionally mixed with antigen and may contain the chiral cationic lipids alone or chiral cationic lipids in combination with neutral lipids.
- Suitable chiral cationic lipid species include, but are not limited to the R and S enantiomers of: 3- ⁇ [ 4 N-( 1 N, 8 -diguanidino spennidine)-carbaroyl]cholesterol (BGSC); 3- ⁇ 0[N,N-diguanidinoethyl-aminoethane)-carbamoyl] cholesterol (BGTC); N,N 1 N 2 N 3 Tetra-methyttetrapalmitylsperrnine (cellfectin); N-t-butyl-N′-tctraclecyl-3-tetradecyl-aininopropion-amidine (CLONfectin); dimethyldioctadecyl am
- ammonium surfactants 1,2 dioleoyl-3-(4′-trimethylammonio) butanol-sn-glycerol (DOBT) or cholesteryl (4′trimethylammonia) butanoate (ChOTB) where the trim ethylammonium group is connected via a butanol spacer arm to either the double chain (for DOTB) or cholesteryl group (for ChOTB); DORI (DL-1,2-dioleoyl-3-dimethylaminopropyl- ⁇ -hydroxyethylammonium) or DORIE (DL-1,2-O-dioleoyl-3-dimethylaminopropyl-(3-hydroxyethylammonium) (DORIE) or analogs thereof as disclosed in WO 93/03709; 2-dioleoyl-3-succinyl-sn-glycerol choline ester (DOSC); cholesteryl hemisuccinate ester (ChOS
- nonsteroidal chiral cationic lipids having a structure represented by the following formula:
- R 3 is a quaternary ammonium group
- Y 1 is chosen from a hydrocarbon chain, an ester, a ketone, and a peptide
- C* is a chiral carbon
- R 2 and R 3 are independently chosen from a saturated fatty acid, an unsaturated fatty acid, an ester-linked hydrocarbon, phosphor-diesters, and combinations thereof.
- DOTAP, DMTAP, DSTAP, DPTAP, DPEPC, DSEPC, DMEPC, DLEPC, DOEPC, DMKE, DMKD, DOSPA, DOTMA are examples of lipids having this general structure.
- chiral cationic lipids of the invention are lipids in which bonds between the lipophilic group and the amino group are stable in aqueous solution.
- bonds used in the cationic lipids include amide bonds, ester bonds, ether bonds and carbamoyl bonds.
- liposomes comprising two cationic lipid species, lysyl-phosphatidylethanolamine and ⁇ -alanyl cholesterol ester have been disclosed for certain drug delivery applications [Brunette, E. et al., Nucl. Acids Res., 20:1151 (1992)].
- the methods of the invention are not restricted only to the use of the cationic lipids recited above but rather, any lipid composition may be used so long as a cationic liposome is produced and the resulting cationic charge density is sufficient to activate and induce an immune response.
- the complexes of the invention may contain other lipids in addition to the chiral cationic lipids,
- lipids include, but are not limited to, lyso lipids of which lysophosphatidylcholine (1-oleoyl lysophospliatidyleholine) is an example, cholesterol, or neutral phospholipids including dioleoyl phosphatidyl ethanolamine (DOPE) or dioleoyl phosphatidylcholine (DOPC) as well as various lipophilic surfactants, containing polyethylene glycol moieties, of which Tween-80 and PEG-PE are examples.
- DOPE dioleoyl phosphatidyl ethanolamine
- DOPC dioleoyl phosphatidylcholine
- various lipophilic surfactants containing polyethylene glycol moieties, of which Tween-80 and PEG-PE are examples.
- the chiral cationic lipid complexes of the invention may also contain negatively charged lipids as well as cationic lipids so long as the net charge of the complexes formed is positive and/or the surface of the complex is positively charged.
- Negatively charged lipids of the invention are those comprising at least one lipid species having a net negative charge at or near physiological pH or combinations of these. Suitable negatively charged lipid species include, but are not limited to, CHEMS (cholesteryl hemisuccinate), NGPE (N-glutaryl phosphatidlylethanolanine), phosphatidyl glycerol and phosphatidic acid or a similar phospholipid analog.
- Liposomes to be used in the production of the lipid comprising drug delivery complexes of the present invention are known to those of ordinary skill in the art.
- a review of methodologies of liposome preparation may be found in Liposome Technology (CFC Press New York 1984); Liposomes by Ostro (Marcel Dekker, 1987); Methods Biochem Anal, 33:337-462 (1988) and U.S. Pat. No. 5,283,185.
- Such methods include freeze-thaw extrusion and sonication. Both unilamellar liposomes (less than about 200 mu in average diameter) and multilamellar liposomes (greater than about 300 nm in average diameter) may be used as starting components to produce the complexes of this invention.
- the chiral cationic lipid is present in the liposome at from about 10 mole % to about 100 mole % of total liposomal lipid, or from about 20 mole % to about 80 mole %.
- the neutral lipid when included in the liposome, may be present at a concentration of from about 0 mole % to about 90 mole % of the total liposomal lipid, or from about 20 mole % to about 80 mole %, or from 40 mole % to 80 mole %.
- the negatively charged lipid when included in the liposome, may be present at a concentration ranging from about 0 mole % to about 49 mole % of the total liposomal lipid, or from about 0 mole % to about 40 mole %.
- the liposomes contain a chiral cationic and a neutral lipid, in ratios between about 2:8 to about 6:4.
- the complexes of the present invention may contain modified lipids, protein, polycations or receptor ligands which function as a targeting factor directing the complex to a particular tissue or cell type.
- targeting factors include, but are not limited to, asialoglycoprotein, insulin, low density lipoprotein (LDL), folate and monoclonal and polyclonal antibodies directed against cell surface molecules.
- LDL low density lipoprotein
- the positive surface charge can be sterically shielded by incorporating lipophilic surfactants which contain polyethylene glycol moieties.
- the cationic lipid complexes may be stored in isotonic sucrose or dextrose solution upon collection from the sucrose gradient or they may be lyophilized and then reconstituted in an isotonic solution prior to use. In one embodiment, the cationic lipid complexes are stored in solution.
- the stability of the cationic lipid complexes of the present invention is measured by specific assays to determine the physical stability and biological activity of the cationic lipid complexes over time in storage.
- the physical stability of the cationic lipid complexes is measured by determining the diameter and charge of the cationic lipid complexes by methods known to those of ordinary skill in the art, including for example, electron microscopy, gel filtration chromatography or by means of quasi-elastic light scattering using, for example, a Coulter N4SD particle size analyzer as described in the Example,
- the physical stability of the cationic lipid complex is “substantially unchanged” over storage when the diameter of the stored cationic lipid complexes is not increased by more than 100%, or by not more than 50%, or by not more than 30%, over the diameter of the cationic lipid complexes as determined at the time the cationic lipid complexes were purified.
- compositions using the chiral cationic lipid complexes of the invention may comprise the cationic lipid complexes in a physiologically compatible sterile buffer such as, for example, phosphate buffered saline, isotonic saline or low ionic strength buffer such as acetate or Hepes (an exemplary pH being in the range of about 3.0 to about 8.0).
- a physiologically compatible sterile buffer such as, for example, phosphate buffered saline, isotonic saline or low ionic strength buffer such as acetate or Hepes (an exemplary pH being in the range of about 3.0 to about 8.0).
- the chiral cationic lipid complexes may be administered as aerosols or as liquid solutions for intratumoral, intraarterial, intravenous, intratracheal, intraperitoneal, subcutaneous, and intramuscular administration.
- the formulations of the present invention may incorporate any stabilizer known in the art.
- Illustrative stabilizers arc cholesterol and other sterols that may help rigidify the liposome bilayer and prevent disintegration or destabilization of the bilayer.
- agents such as polyethylene glycol, poly-, and monosaccharides may be incorporated into the liposome to modify the liposome surface and prevent it from being destabilized due to interaction with blood-components.
- Other illustrative stabilizers are proteins, saccharides, inorganic acids, or organic acids which may be used either on their own or as admixtures.
- Controlled release preparations may be achieved through the use of polymer complexes such as polyesters, polyamino acids, methylcellulose, polyvinyl, poly(lactic acid), and hydrogels to encapsulate or entrap the cationic lipids and slowly release them. Similar polymers may also be used to adsorb the liposomes.
- the liposomes may be contained in emulsion formulations in order to alter the release profile of the stimulant.
- the duration of the stimulant's presence in the blood circulation may be enhanced by coating the surface of the liposome with compounds such as polyethylene glycol or other polymers and other substances such as saccharides which are capable of enhancing the circulation time or half life of liposomes and emulsions.
- the chiral cationic lipids may be combined with typical pharmaceutical carriers known in the art such as, for example, sucrose, lactose, methylcellulose, carboxymethyl cellulose, or gum Arabic, among others.
- the cationic lipids may also be encapsulated in capsules or tablets for systemic delivery.
- Administration of the chiral cationic lipid of the present invention may be for either a prophylactic or therapeutic purpose.
- the cationic lipid is provided in advance of any evidence or symptoms of illness.
- the cationic lipid is provided at or after the onset of disease.
- the therapeutic administration of the immune-stimulant serves to attenuate or cure the disease.
- the cationic lipid may be administered with an additional therapeutic agent(s) or antigen(s).
- the prophylactic or therapeutic effect may be generated against a specific disease.
- the formulations of the present invention both for veterinary and for human use, comprise a chiral cationic lipid alone as described above, as a mixture of R and S enantiomers, or also optionally, with one or more therapeutic ingredients such as an antigen(s) or drug molecule(s).
- the formulations may conveniently be presented in unit dosage form and may be prepared by any method known in the pharmaceutical art.
- the chiral cationic lipid is administered without any additional agents in order to boost or lower various immune responses, including production of other immune modulators, and to boost the immune response to fighting disease.
- the chiral cationic lipid is administered in combination with an antigen or antigens.
- the objective is to generate an immune response, which is specific to the antigen(s) delivered in combination with the cationic lipid.
- the response generated may include production of specific cytotoxic T-cells, memory T-cells, or B-cells resulting in the prevention of or therapeutic response to the specific disease associated with those antigen(s).
- the antigen can be any tumor-associated antigen or microbial antigen or any other antigen known to one skilled in the art.
- tumor-associated antigen is a molecule or compound (e.g., a protein, peptide, polypeptide, lipoprotein, lipopeptide, glycoprotein, glycopeptides, lipid, glycolipid, carbohydrate, RNA, and/or DNA) associated with a tumor or cancer cell and which is capable of provoking an immune response (humoral and/or cellular) when expressed on the surface of an antigen presenting cell in the context of a major histocompatibility complex (“MHC”) molecule.
- MHC major histocompatibility complex
- Tumor-associated antigens include self antigens, as well as other antigens that may not be specifically associated with a cancer, but nonetheless enhance an immune response to and/or reduce the growth of a tumor or cancer cell when administered to an animal. More specific embodiments are provided herein.
- a “microbial antigen,” as used herein, is an antigen of a microorganism and includes, but is not limited to, infectious virus, infectious bacteria, infectious parasites and infectious fungi.
- Microbial antigens may be intact microorganisms, and natural isolates, fragments, or derivatives thereof, synthetic compounds which are identical to or similar to naturally-occurring microbial antigens and, preferably, induce an immune response specific for the corresponding microorganism (from which the naturally-occurring microbial antigen originated).
- a compound is similar to a naturally-occurring microorganism antigen if it induces an immune response (humoral and/or cellular) similar to a naturally-occurring microorganism antigen.
- a naturally-occurring microorganism antigen is well known to those of ordinary skill in the art such as, for example, a protein, peptide, polypeptide, Lipoprotein, lipopeptide, glycoprotein, glycopeptides, lipid, glycolipid, carbohydrate, RNA, and/or DNA.
- a compound that is similar to a naturally-occurring microorganism antigen is a peptide mimic of a polysaccharide antigen. More specific embodiments are provided herein.
- antigen is further intended to encompass peptide or protein analogs of known or wild-type antigens such as those described in. this specification.
- the analogs may be more soluble or more stable than wild type antigen, and may also contain mutations or modifications rendering the antigen more immunologically active.
- Antigen can be modified in any manner, such as adding lipid or sugar moieties, mutating peptide or protein amino acid sequences, mutating the DNA or RNA sequence, or any other modification known to one skilled in the art.
- Antigens can be modified using standard methods known by one skilled in the art.
- compositions and methods of the present invention are peptides or proteins which have amino acid sequences homologous with a desired antigen's amino acid sequence, where the homologous antigen induces an immune response to the respective tumor, microorganism or infected cell.
- the antigen in the cationic lipid complex comprises an antigen associated with a tumor or cancer, i.e., a tumor-associated antigen, to make a vaccine to prevent or treat a tumor.
- the tumor or cancer vaccines of the present invention further comprise at least one epitope of at least one tumor-associated antigen.
- the tumor or cancer vaccines of the present invention further comprise a plurality of epitopes from one or more tumor-associated antigens.
- the tumor-associated antigens finding use in the cationic lipid complexes and methods of the present invention can be inherently immunogenic, or nonimmunogenic, or slightly immunogenic.
- antigens include, but are not limited to, synthetic, recombinant, foreign, or homologous antigens
- antigenic materials may include but are not limited to proteins, peptides, polypeptides, lipoproteins, lipopeptides, lipids, glycolipids, carbohydrates, RNA and DNA.
- Such vaccines include, but are not limited to, those for the treatment or prevention of breast cancer, head and neck cancer, melanoma, cervical cancer, Lung cancer, prostate cancer, gut carcinoma, or any other cancer known in the art using a cationic lipid in a complex with a tumor-associated antigen(s), It is also possible to formulate the antigen. with the cationic lipid without encapsulating it in the liposome.
- the chiral cationic lipid complexes of the present invention may be used in methods to treat or prevent cancer.
- the mammal to be immunized maybe injected with the pharmaceutical formulation containing the liposome with the encapsulated antigen(s).
- Tumor-associated antigens suitable for use in the present invention include both naturally occurring and modified molecules which may be indicative of single tumor type, shared among several types of tumors, and/or exclusively expressed or overexpressed in tumor cells in comparison with normal cells.
- proteins, glycoproteins, lipoproteins, peptides, and lipopeptides tumor-specific patterns of expression of carbohydrates, gangliosides, glycolipids, and mucins have also been documented.
- Exemplary tumor-associated antigens for use in cancer vaccines include protein products of oncogenes, tumor suppressor genes, and other genes with mutations or rearrangements unique to tumor cells, reactivated embryonic gene products, oncofetal antigens, tissue-specific (but not tumor-specific) differentiation antigens, growth factor receptors, cell surface carbohydrate residues, foreign viral proteins, and a number of other self proteins.
- tumor-associated antigens include, e.g., mutated or modified antigens such as the protein products of the Ras p21 protooncogenes, tumor suppressor p53 and HER-2/neu and BCR-abl oncogenes, as well as CDK4, MUM1, Caspase 8, and Beta catenin; overexpressed antigens such as galectin 4, galectin 9, carbonic anhydrase, Aldolase A, PRAMS, Her2/neu, ErbB-2 and KSA, oncofetal antigens such as alpha fetoprotein (AFP), human chorionic gonadotropin (hCG); self antigens such as carcinoembryonic antigen (CEA) and melanocyte differentiation antigens such as Mart I/Melan A, 0)100, gp75, Tyrosinase, TRP I and TRP2; prostate associated antigens such as PSA, PAP, PSMA, PSM-P1 and PSM-P2; repressed antigen
- Tumor-associated antigens and their respective tumor cell targets include, e.g., cytokeratins, particularly cytokeratin 8, 18 and 19, as antigens for carcinoma.
- Epithelial membrane antigen (EMA), human embryonic antigen (HEA-125), human milk fat globules, MBrl, MBr8, Ber-EP4, 17-1 A, C26 and T16 are also known carcinoma antigens.
- Desmin and muscle-specific actin are antigens of cryogenic sarcomas. Placental alkaline phosphatase, beta-human chorionic gonadotropin, and alpha-fetoprotein are antigens of trophoblastic and germ cell tumors.
- Prostate specific antigen is an antigen of prostatic carcinomas, carcinoembryonic antigen of colon adenocarcinomas.
- EIMB-45 is an antigen of melanomas.
- useful antigens could be encoded by human papilloma virus.
- Chromagranin-A and synaptophysin are antigens of neu pendocrine and neuroectodermal tumors.
- aggressive tumors that form solid tumor masses having necrotic areas. The lysis of such necrotic cells is a rich source of antigens for antigen-presenting cells, and thus the subject therapy may find advantageous use in conjunction with conventional chemotherapy and/or radiation therapy.
- the human papillomavirus HPV antigens are used.
- a specific HPV antigen that used as a tumor-associated antigen is HPV subtype 16 E7.
- HPV E7 antigen-cationic lipid complexes are effective at preventing and treating cervical cancer.
- a genetically engineered E7 protein, i.e., E7 in protein, having antigenic activity, but without tumorigenic activity is an effective tumor-associated antigen.
- Elm-cationic lipid complexes induce cellular immunity to cause complete regression of established tumors and, thus, are useful as potent anti-cervical cancer vaccines.
- Tumor-associated antigens can be prepared by methods well known in the art.
- these antigens can be prepared from cancer cells either by preparing crude extracts of cancer cells (e.g., as described in Cohen et al., Cancer Res., 54:1055 (1994)), by partially purifying the antigens, by recombinant technology, or by de novo synthesis of known antigens.
- the antigen may also be in the form of a nucleic acid encoding an antigenic peptide in a form suitable for expression in a subject and presentation to the immune system of the immunized subject.
- the antigen may be a complete antigen, or it may be a fragment of a complete antigen comprising at least one epitope.
- Antigens derived from pathogens known to predispose to certain cancers may also be advantageously included in the cancer vaccines of the present invention. It is estimated that close to 16% of the worldwide incidence of cancer can be attributed to infectious pathogens; and a number of common malignancies are characterized by the expression of specific viral gene products. Thus, the inclusion of one or more antigens from pathogens implicated in causing cancer may help broaden the host immune response and enhance the prophylactic or therapeutic effect of the cancer vaccine.
- Pathogens of particular interest for use in the cancer vaccines provided herein include the, hepatitis B virus (hepatocellular carcinoma), hepatitis C virus (heptomas), Epstein Barr virus (EMT) (Burkitt lymphoma, nasopharynx cancer, PTLD in immunosuppressed individuals), I-ITLVL (adult T cell leukemia), oncogenic human papilloma viruses types 16, 18, 33, 45 (adult cervical cancer), and the bacterium Helicobacter pylori (B cell gastric lymphoma).
- EMT Epstein Barr virus
- I-ITLVL adult T cell leukemia
- oncogenic human papilloma viruses types 16, 18, 33, 45 adult cervical cancer
- Helicobacter pylori B cell gastric lymphoma
- Other medically relevant microorganisms that may serve as antigens in mammals and more particularly humans are described extensively in the literature, e.g., C. G. A Thomas, Medical Microbiology, Baii
- the antigen in the cationic lipid complex comprises an antigen derived from or associated with a pathogen, i.e., a microbial antigen.
- the pathogen vaccines of the present invention further comprise at least one epitope of at least one microbial antigen.
- Pathogens that may be targeted by the subject vaccines include, but are not limited to, viruses, bacteria, parasites and fungi.
- the pathogen vaccines of the present invention further comprise a plurality of epitopes from one or more microbial antigens.
- the microbial antigens finding use in the cationic lipid complexes and methods may be inherently immunogenic, or nonimmunogenic, or slightly immunogenic.
- Exemplary antigens include, but are not limited to, synthetic, recombinant, foreign, or homologous antigens, and antigenic materials may include but are not limited to proteins, peptides, polypeptides, lipoproteins, lipopeptides, lipids, glycolipids, carbohydrates, RNA, and DNA.
- Exemplary viral pathogens include, but are not limited to, viruses that infect mammals, and more particularly humans.
- virus include, but are not limited to: Retroviridae (e.g., human immunodeficiency viruses), such as HIV-1 (also referred to as HTLV-III, LAV or HTLV-IHiLAV, or HIV-III; and other isolates, such as HIV-LP; Picornaviridac (e.g. polio viruses, hepatitis A virus; enteroviruses, human Coxsackie viruses, rhinoviruses, echoviruses); Calciviridae (e.g. strains that cause gastroenteritis); Togavridae (e.g.
- Flaviridae e.g. dengue viruses, encephalitis viruses, yellow fever viruses
- Coronoviridae e.g. coronaviruses
- Rhabdoviradae e.g. vesicular stomatitis viruses, rabies viruses
- Coronaviridae e.g. coronaviruses
- Rhabdoviridae e.g. vesicular stomatitis viruses, rabies viruses
- Filoviridae e.g. ebola viruses
- Paramyxoviridae e.g.
- Orthomyxoviridae e.g. influenza viruses
- Bungaviridae e.g. Hantaan viruses, bunga viruses, phleboviruses and Naito viruses
- Arena viridae hemorrhagic fever viruses
- Reoviridae e.g.
- reoviruses reoviruses, orbiviurses and rotaviruses
- Birnaviridae Hepadnaviridae (Hepatitis B virus); Parvoviridae (parvoviruses); Papovaviridae (papilloma viruses, polyoma viruses); Adenoviridae (most adenoviruses); Herpesviridae herpes simplex virus (HSV) 1 and 2, varicella zoster virus, cytomegalovirus (CMV), herpes virus; Poxyiridae (variola viruses, vaccinia viruses, pox viruses); and lridoviridae (e.g. African swine fever virus); and unclassified viruses (e.g.
- gram negative and grain positive bacteria may be targeted by the subject compositions and methods in vertebrate animals.
- grain positive bacteria include, but are not limited to Pasteurelkt species, Staphylococci species, and Streptococcus species.
- Gram negative bacteria include, but are not limited to, Escherich fa coli, Pseudomonas species , and Salmonella species.
- infectious bacteria include but are not limited to: Helicobacter pyloris, Borella burgdoiferi, Legionella pneumophilia, Mycobacteria sps (e.g. M. tuberculosis, M. avium, M. intracellulare, M.
- Polypeptides of bacterial pathogens which may find use as sources of microbial antigens in the subject compositions include but are not limited to an iron-regulated outer membrane protein, (“TROMP”), an outer membrane protein (“OMP”), and an A-protein of A eromonis sahnonicida which causes furunculosis, p57 protein of Renibacterium salmonittarum which causes bacterial kidney disease (“BKD”), major surface associated antigen (“msa”), a surface expressed cytotoxin (“mpr”), a surface expressed hemolysin (“ish”), and a flagellar antigen of Yersiniosis; an extracellular protein (“ECP”), an iron-regulated outer membrane protein (“TROMP”), and a structural protein of Pasteurellosis; an OMP and a flagellar protein of Vibrosis anguillarum and V ordalii; a flagellar protein, an OMP protein, aroA, and purA of Edwardsiellosis ic
- pathogens further include, but are not limited to, fungi that infect mammals, and more particularly humans.
- fungi include, but are not limited to: Cryptococcus neoformans, Histoplasma capsulatum, Coccidioides immitis, Blastomyces dermatitidis, Chlamydia trachomatis , and Candida albicans .
- infectious parasites include Plasmodium such as Plasmodium. falciparum, Plasmodium malartae, Plasmodium ovale , and Plasmodium vivax .
- Other infectious organisms i.e. protists
- Polypeptides of a parasitic pathogen include but are not limited to the surface antigens of khthyophthirius.
- compositions and methods of the present invention are useful for treating infections of nonhuman mammals.
- Many vaccines for the treatment of nonhuman mammals are disclosed in Bennett, K. Compendium of Veterinary Products, 3rd ed. North American Compendiums, Inc., 1995; see also WO 02/069369, the disclosure of which is expressly incorporated by reference herein in its entirety.
- nonhuman pathogens include, but are not limited to, mouse mammary tumor virus (“MMTV”), Rous sarcoma virus (“RSV”), avian leukemia virus (“ALV”), avian myeloblastosis virus (“AMV”), murine leukemia virus (“MLV”), feline leukemia virus (“FeLV”), murine sarcoma virus (“MSV”), gibbon ape leukemia virus (“GALV”), spleen necrosis virus (“SNV”), reticuloendotheliosis virus (“RV”), simian sarcoma virus (“SSV”), Mason-Pfizer monkey virus (“MPMV”), simian retrovirus type 1 (“SRV-1”), lentiviruses such as HIV-I, HIV-2, SIV, Visna virus, feline immunodeficiency virus (“Fly”), and equine infectious anemia virus (“EIAY”), T-cell leukemia viruses such as HTLV-1, HTLV
- treatment refers to a prophylactic treatment which increases the resistance of a subject to infection with a pathogen or decreases the likelihood that the subject will become infected with the pathogen; and/or treatment after the subject has become infected in order to fight the infection, e.g., reduce or eliminate the infection or prevent it from becoming worse.
- Microbial antigens can be prepared by methods well known in the art. For example, these antigens can be prepared directly from viral and bacterial cells either by preparing crude extracts, by partially purifying the antigens, or alternatively by recombinant technology or by de MVO synthesis of known antigens.
- the antigen may also be in the form of a nucleic acid encoding an antigenic peptide in a form suitable for expression in a subject and presentation to the immune system of the immunized subject. Further, the antigen may be a complete antigen, or it may be a fragment of a complete antigen comprising at least one epitope.
- the antigen may be conjugated to a lipid chain in order to improve its solubility in the hydrophobic acyl chains of the cationic lipid, while maintaining the antigenic properties of the molecule.
- the lipidated antigen can be a lipoprotein, or a lipopeptide, and combinations thereof.
- the lapidated antigen may have a linker conjugated between the lipid and the antigen such as, for example, an N-terminal a or a-palmitoyl lysine may be connected to antigen via a dipeptide Ser-Ser linker.
- TC-1 cells are C57BL/6 mouse lung endothelial cells that have been transformed with the HPV16 E6 and E7 oncogenes and activated H-ras.
- Cells were grown in RPMI medium (commercially available from Invitrogen of Carlsbad, Calif.) supplemented with 10% fetal bovine serum and 100 U/mI penicillin, and 100 mg/ml streptomycin.
- the MHC class I restricted peptide from the HPV 16 E7 protein (amino acid 11 to 20, YMLDLQPETT [SEQ. ID. NO. I]) was synthesized by the University of Pittsburgh Peptide Synthesis Facility by solid state synthesis using an Advanced ChernTech model 200 peptide synthesizer and purified by HPLC.
- DOTAP DOTAP liposomes
- Merck AG EPROVA
- All other lipids were purchased from Avanti Polar Lipids (Alabaster, Ala.).
- Small unilamellar DOTAP liposomes were prepared by thin film hydration followed by extrusion. The lipid, in chloroform, was dried as a thin layer under a stream of nitrogen in a glass tube.
- the lipid dispersion was sequentially extruded through polycarbonate membranes with pore size of 0.4, 0.2, and 0.1 pan. The un-entrapped peptide was not removed.
- the liposomes were stored at 4° C. until use. E7 peptide association with the liposome was determined by measuring the percentage of liposome-bound peptide.
- unbound E7 peptide from R-DOTAP/E7, S-DOTAP/E7 or RS-DOTAP/E7 complexes was separated by a Microcon® centrifugal filtrate device (Millipore, Bedford, Mass.) and the concentration of unbound peptide was measured by Micro BCATM Protein Assay Kit (Pierce, Rockford, Ill.). The efficiency of peptide association was determined as percent unbound peptide. Other methods used in general liposome preparation that are well known to those skilled in the art may also be used.
- Cationic liposomes were prepared as described above.
- the E7 antigen used in the formulation is the identified human E7 peptide restricted by HLA-A*0201 [HPV-16 E7, amino acids 11-20, YMLDLQPETT (SEQ. ID. NO. 1)1
- the peptide was synthesized by the University of Pittsburgh, Molecular Medicine Institute, Pittsburgh, Pa.
- Human 1-ILA-A2 human dendritic cells were obtained from Lonza (of Walkersville, Md.). Frozen cryovials were thawed and the dendritic cells were cultured in LGM-3 medium (commercially available from Lonza of Walkersville, Md.) supplemented with 50 microgram/mI IL-4 and GM-CSF at 37° C. and 5% CO 2 at an initial plating density of 125,000 cells/cm 2 in 2 ml of medium in 12-well tissue culture dishes. The cells were grown for 3 days in culture and appeared as a mixture of adherent and rounded cells by microscopic examination
- the cells were treated on day 3 with a fresh dose of 50 microgram/ml of IL-4 and GM-CSF (all wells) and test wells were treated with either a mixture of interleukin 1-beta (“IL- ⁇ ”), interleukin 6 (“IL-6”) and TNF-a at 10 ng/ml, and prostaglandin E2 (“PGE-2”) at 10 ⁇ g/ml (positive control for activation), no treatment (negative activation control), and S-DOTAP/E7 at 2.5, 10 and 40 micromolar final concentrations, and R-DOTAP/E7 at 2.5, 10 and 40 micromolar final concentrations.
- the treated dendritic cells were maintained in culture for 24 hours and harvested for cell surface marker staining and flow cytornetry analysis.
- the harvested cells were counted by hernacytometer and 10 pi of the following antibody conjugates were added sequentially to each sample for labeling surface markers: CD80-FITC, CD83-APC, and CD86-PE (BD Biosciences).
- the surface labeled cells were subsequently analyzed by flow cytometry using a BD FACxcaliber flow cytometer, and the co-stimulatory dendritic cell marker molecules CD80, CD83, and CD86 which are produced upon activation, were monitored. As seen in FIGS.
- Human HLA-A 2 dendritic cells (Lonza, Walkersville, Md.), were treated and grown in culture as described above. On day 3 the cells were treated with 40 micromolar DOTAP/E7 complex or the potent immunostimulator lipopolysaccharide (LPS) at 50 micromolar concentrations (positive control). Medium from assay wells was removed and centrifuged at 1300 rpm in a microfuge for 5 minutes to pellet unattached dendritic cells. The supernatants were removed and treated with 10 microliters per ml of Calbiochem (La Jolla, Calif.) protease inhibitor cocktail set I (Cat. No. 539131) and stored frozen prior to analysis. Samples were analyzed for chemokine and cytokine expression by Searchlight Protein Array Multiplex ELISA assay [Pierce Biotechnology (Woburn, Mass.)].
- LPS potent immunostimulator lipopolysaccharide
- FIGS. 5-11 which illustrate the ability of R-DOTAP/E7 and S-DOTAP/E7 to induce production of CCL3, CCL4, CCL5, CCL-19, IL-2, IL-8 and 1L-12
- the figures illustrate that the DOTAP/E7 complex containing the individual enantiomers of DOTAP induce cytokine and chemokine production by human dendritic cells. Both enantiorners however activate the immune system to different extents with the R-enantiomer exhibiting higher potency.
- mice were subcutaneously injected with TC-1 cells on day 0 in order to induce the growth of HPV-positive tumors
- the DOTAP/E7 compositions were comprised of racemic mixtures of DOTAP.
- the mice received DOTAP/E7 compositions containing 10 11.g E7 peptide subcutaneously on the opposite side of the abdomen on. day 6.
- DOTAP lipid concentration in the complex varied from 3 to 600 nmole (3, 15, 30, 75, 150, 300, and 600 nmole),
- Low dose of DOTAP (15 nmole) showed partial tumor inhibition effect (P ⁇ 0.05) compared to the untreated control on day 23, while DOTAP at 30, 150 or 300 nmole exhibited an enhanced efficacy (P ⁇ 0.01).
- DOTAP at 75 nmole showed the most significant tumor regression effect (P ⁇ 0.001). Again, mice given a high dose of DOTAP (600 nmole) did not show anti-tumor activity, confirming that DOTAP liposomes at a high dose might have induced a negative regulation to the immune response. In addition, DOTAP liposomes at the 100 nmole dose, but without E7 peptide, did not show significant inhibition of tumor growth, indicating that the anti-tumor effect was antigen specific. Further, liposomes of 1,2-dioleoyl-sn-glycero-3-phosphoglycerol (DOPG), an anionic lipid, administered at the 150 nmole dose with antigen failed to significantly inhibit tumor growth.
- DOPG 1,2-dioleoyl-sn-glycero-3-phosphoglycerol
- mice were subcutaneously injected with TC-1 cells on day 0 in order to induce the growth of HPV-positive tumors.
- the mice received R and S-DOTAP/E7 compositions containing 20 E7 peptide subcutaneously on the opposite side of the abdomen on day 6.
- R or S-DOTAP lipid concentrations in the complex varied from 3 to 600 nmole (3, 15, 30, 75, 100, 125, 150, 300, and 600 nmole).
- S-DOTAP complexes did not exhibit the ability to inhibit tumor growth and no tumor regression was observed ( FIG. 13 ).
- FIG. 15 shows the lipid dose-response curves for the tumor regression efficacy of the various cationic lipid/E7 antigen complexes DOTAP, S-DOTAP, and R-DOTAP at 20 ⁇ g of the antigen and DOTAP at 10 ⁇ g of the antigen.
- DOTAP-mediated T cell activation was further confirmed by ERK phosphorylation in T cells.
- DOTAP-induced expression of the costimulatory molecule, CD86 on human T lymphocytes was also observed.
- Those results suggested that DOTAP has a direct impact on T cell activation via a MAP kinase mediated cell proliferation.
- a broad class of cationic lipids can act as potent immunostimulators together with an antigen to generate antigen specific immune responses in the treatment of disease.
- U.S. Pat. No. 7,303,881 discloses that liposomes comprised of cationic lipids activate dendritic cells as demonstrated by the stimulation by cationic lipids of the expression of costimulatory molecules CD80/CD86 on DC2A dendritic cells ( FIGS. 14A and 14B ).
- FIG. 14A of U.S. Pat. No. 7,303,881 the ability to stimulate the expression of CD80/CD86 on DC2.4 cells by different cationic liposomes varies greatly.
- Lipofectamine® a 3:1 (w/w) liposome formulation of the polycationic lipid 2,3-dioleyloxy-N-[2(spen-ninecarboxamido)ethyll-N,N-dimethyl-1-propanarninium trifluoroacetate (DOSPA) and the neutral lipid dioleoyl phosphatidylethanolamine (DOPE), and liposomes prepared from 0,0 1 -dirnyristyl-N-lysyl aspartate (DMKE) and 0,0′-dimyristyl-N-lysyl-glutamate (DMKD), strongly stimulated the expression of CD80/CD86 by CD2.4 cells.
- DOSPA polycationic lipid 2,3-dioleyloxy-N-[2(spen-ninecarboxamido)ethyll-N,N-dimethyl-1-propanarninium trifluoroacetate
- DOPE neutral lipid dioleoyl phosphat
- the ability of different cationic lipids to stimulate the expression of CD 80 on DC 2.4 cells varied. Both hydrophilic head and the lipophilic tail of the lipids have significant effect on this ability.
- the DXEPC lipids with the ethyl phosphocholine (EPC) head groups appear, in general, to be more potent than the DXTAP lipids with trimethylammonium propane (TAP) head group.
- EPC ethyl phosphocholine
- cationic lipids are not only efficient targeting and delivery vehicles for antigens to APC of the immune system, but also function as potent adjuvants under low dose composition ranges to directly influence immune system function through activation of MAP kinase dependent signaling pathways with resultant production of immune system regulatory molecules including cytokines and chemokines.
- a clear dose-response effect of cationic lipid on the immunostimulatory capabilities of the formulations have been demonstrated. It was demonstrated that upon receiving the lipid/antigen complex, the particles were mainly taken up by dendritic cells, the major professional antigen presenting cells.
- the initiation of dendritic cell activation and migration to the draining lymph node facilitates immune responses against antigen specific TC-1 tumors as demonstrated.
- Functional CD8 + T lymphocytes were generated in mice upon receiving a DOTAP/E7 injection and tumor sizes decreased and exhibited enhanced apoptosis, owing to the increasing number of infiltrating T cells in the tumor microenvironment.
- the resulting bell-shaped (activity decreases above and below the optimal dose) cationic lipid dose response curve demonstrated activity at very low doses, indicating that the activity of the cationic lipids as adjuvants or immunostimulators is so potent that the EC 50 is as low as about 15 nmole per injection.
- High doses of cationic lipids eliminate the immunostimulatory activity.
- An important characteristic of an immunostimulator capable of inducing cellular immune responses to disease is its ability to induce the production of critical chemokines and cytokines. As reported in the Example, significant differences were observed between the R and S enantiomers of DOTAP in their ability to induce chetnokine and cytokine production. R-DOTAP was observed to be a more potent immune activator than S-DOTAP. In all cases the potency of R-DOTAP was equivalent to or higher than that of the DOATP racemic mixture.
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Pharmacology & Pharmacy (AREA)
- Medicinal Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Immunology (AREA)
- Organic Chemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Epidemiology (AREA)
- Oncology (AREA)
- Communicable Diseases (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Virology (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Medicinal Preparation (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Peptides Or Proteins (AREA)
Abstract
Description
- This application is a continuation of U.S. patent application Ser. No. 16/899,763 filed Jun. 12, 2020, which is a continuation U.S. patent application Ser. No. 15/702,063 filed Sep. 12, 2017, now issued as U.S. Pat. No. 10,702,541 on Jul. 7, 2020, which is a divisional of U.S. patent application Ser. No. 12/988,236 filed Dec. 23, 2010, now issued as U.S. Pat. No. 9,789,129 on Oct. 17, 2017, which is a 371 of International Patent Application. No. PCT/US2009/040500 filed Apr. 14, 2009, which claims benefit of U.S. Provisional Application No. 61/045,837 filed Apr. 17, 2008, the disclosures of which are incorporated by reference herein in their entirety.
- The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Jun. 16, 2021, is named PDS-16-1033WO-US-DC-CON_SL.txt and is 648 bytes in size.
- The present invention generally related to stimulating an immune response, and more particularly to the use of the R and S enantiomers of lipids in stimulating immune responses.
- This section is intended to introduce the reader to various aspects of art that may be related to various aspects of the present invention, which are described and/or claimed below. This discussion is believed to be helpful in providing the reader with background information to facilitate a better understanding of various aspects of the present invention. Accordingly, it should be understood that these statements are to be read in this light, and not as admissions of prior art.
- Development of safe and effective immunotherapies for human use remains an urgent medical need for patients worldwide. In order to elicit appropriate immune responses, immunologic modifiers (“immunomodifiers”) that enhance, direct or promote an immune response can be used in vaccine design or immunotherapy [Gregoriadis, G., Immunological adjuvants: a role for liposomes, Immunol Today 11:89 (1990)]. For example, vaccines may include antigens to stimulate an immune response. However, some potential vaccines that include antigens are weak stimulators of an immune response because the vaccines do not efficiently deliver the antigen to antigen presenting cells (“APC”) of the immune system and/or the antigen is weakly immunogenic. Thus, immunotherapies that effectively deliver antigens to APC, and also stimulate the immune system to respond to the antigen, are needed. Immunomodifiers have the potential to function as such an immunotherapy. Such immunotherapies may have these and other benefits. For example, when included as part of a therapeutic vaccine, an immunomodifier should at least (1) improve antigen delivery and/or processing in the APC [Wang, R. F., and Wang, H. Y. Enhancement of antitumor immunity by prolonging antigen presentation on dendritic cells. Nat Biotechnol 20:149 (2002)1, (2) induce the production of immunomodulatory cytokines that favor the development of immune responses to the vaccine antigen, thus promoting cell mediated immunity, including cytotoxic T-Lymphocytes (“CTL”), (3) reduce the number of immunizations or the amount of antigen required for an effective vaccine [Vogel, F. R. Improving vaccine performance with adjuvants. Clin Infect Dis 30 Suppl 3:S266 (2000)], (4) increase the biological or immunological half-life of the vaccine antigen, and (5) overcome immune tolerance to antigen by inhibiting immune suppressive factors [Baecher-Allan, C., and Anderson, D. E. Immune regulation in tailor-bearing hosts. Curr Opin Immunol 18:214 (2006)].
- Presently, the primary class of agents used to enhance the efficacy of antigens, such as peptide or protein antigens, in eliciting an immune response are adjuvants such as water-in-oil emulsions, alum, and other chemicals which enhance antigen responses; however, these adjuvants are not immunomodifiers, as described above, because they have no direct immunomodulatory effects themselves [Vogel, F. R., and Powell, M. F. A compendium of vaccine adjuvants and excipients, Pharm Biotechnol 6:141 (1995)1. Several such adjuvants are available for use in animals and some of them have been tested in clinical trials. In addition to traditional adjuvants such as the aluminum salts, products such as influenza virosomes [Gluck, R., and Walti, E. 2000. Biophysical validation of Epaxal Berna, a hepatitis A vaccine adjuvanted with immunopotentiating reconstituted influenza virosomes ORM. Dev Biol (Basel) 103:189 (2000)1, and Chiron's MF59 [Kahn, J. 0., et al. Clinical and immunologic responses to human immunodeficiency virus (I −1.17) type ISF2 gp120 subunit vaccine combined with MF59 adjuvant with or without mummy! tripeptide dipalinitoylphosphatidylethanolamine in non-I − IIV-ittfected human volunteers. J Infect Dis 170:1288 (1994)], which have intrinsic immune effects, are being marketed. For example, MF59, which is a submicron emulsion based adjuvant, is internalized by dendritic cells [Dupuis, M., et al., Dendritic cells internalize vaccine adjuvant after intramuscular injection. Cell Immunol 186:18 (1998)]. However, according to clinical trial reports on HSV and influenza vaccines [Jones, C. A., and Cunningham, A. L. Vaccination strategies to prevent genital herpes and neonatal herpes simplex virus (HSV) disease. Herpes 11:12 (2004); Minutello, M. et al., Safety and immunogenicity of an inactivated subunit influenza virus vaccine combined with ilifF59 adjuvant emulsion in elderly subjects, immunized for three consecutive influenza seasons. Vaccine 17:99 (1999)1, evidence from animal models suggests that the MF59 adjuvant enhances production of neutralizing antibodies rather than enhancing responses. Thus, new methods of stimulating cell mediated immune responses are needed.
- Further, as mentioned above, some antigens are weak stimulators of an immune response. Thus, in addition to co-administering antigen with substances that stimulate immune responses, as described above, a weakly immunogenic antigen can be modified to increase its immunogenicity. For example, a weakly immunogenic antigen can be coupled to immunogenic peptides, polysaccharides, or lipids to increase its immunogenicity. However, simply coupling weakly immunogenic antigens to these types of compounds may not be sufficient to elicit an immune response. For example, the resulting immune response may be directed to immunogenic epitopes on the coupled compound and not the weak antigen, or the coupled antigen may not be efficiently delivered to APC of the immune system. Thus, additional methods are needed to stimulate immune responses to antigens that are weakly immunogenic.
- Certain exemplary aspects of the invention are set forth below. Tt should be understood that these aspects arc presented merely to provide the reader with a brief summary of certain forms the invention might take and that these aspects are not intended to limit the scope of the invention. Indeed, the invention may encompass a variety of aspects that may not be explicitly set forth below.
- This invention is directed to the chirality of cationic lipids and the use of the R and S enantiomers of cationic lipids, which under certain dose and composition conditions act as a novel class of immune-stimulants, to (1) effectively present or deliver an antigen to the immune system and (2) stimulate the immune system to respond to the antigen.
- Liposomes have been extensively used for delivering small molecular weight drugs, plasmid DNA, oligonucleotides, proteins, and peptides. Vaccines using liposomal vehicles as nonviral antigen carriers are preferable compared to traditional immunizations using live attenuated vaccines or viral vectors such as vaccinia or influenza virus. U.S. patent application Ser. No. 12/049,957, assigned to the assignee of the present application, discloses simple yet effective lipid-based immunotherapies, including a cationic lipid/antigen complex, which has two molecules, a cationic lipid and an antigen, and the effects of the lipid dose on the resulting immune response. The reported results demonstrate that the cationic liposome complexed with an antigen serves to stimulate immune responses and initiate dendritic cell (an APC) interaction with T-cells.
- In the present invention, additional studies performed with the two enantiomers of a selected cationic lipid have led to the discovery that differences exist in the ability of the R and S enantiomers of the cationic lipids to act as potent immune activators under various conditions. In combination with an antigen, the cationic lipid/antigen complex containing the R enantiomer, under various dose conditions (including low dose conditions), induces strong immune responses specific to the antigen formulated in the complex and results in tumor regression. Complexes consisting of S-DOTAP and the antigen however were able to induce only limited tumor regression, and not at all doses at which R-DOTAP was effective. Both enantiomers of DOTAP are however equally effective at inducing maturation and activation of dendritic cells, which is the first step in inducing a cellular immune response.
- Thus, one aspect of the invention provides a composition of at least one enantiomer of a cationic lipid in a dose sufficient to induce an immune response in a subject.
- Another aspect of the invention provides a method of inducing an immune response in a subject by administering a specific enantiomer or a mixture of enantiomers of a cationic lipid to the subject.
- Another aspect of the invention provides a composition of an R or S enantiomer of a cationic lipid in a dose sufficient to induce an immune response in a subject.
- Additional aspects of the invention involve the addition of at least one antigen to the R or S enantiomer to form a cationic lipid/antigen complex in which case the immune response is antigen-specific.
- Various features, aspects, and advantages of the present invention will become better understood when the following detailed description is read with reference to the accompanying figures, wherein:
-
FIGS. 1A and 1B depict chirality of I,2-dioleoyl-3-trimethylammonium propane (“DOTAP”). -
FIG. 2 is a graph depicting activation of human dendritic cells resulting in expression of theco-stimulatory molecule CD 80 by R-DOTAP, S-DOTAP and the racemic mixture RS-DOTAP. -
FIG. 3 is a graph depicting activation of human dendritic cells resulting in expression of the co-stimulatory molecule CD 83 by R-DOTAP, S-DOTAP and the racernic mixture RS-DOTAP. -
FIG. 4 is a graph depicting activation of human dendritic cells resulting in expression of the co-stimulatory molecule CD 86 by R-DOTAP, S-DOTAP and the racemic mixture RS-DOTAP. -
FIG. 5 is a graph depicting stimulation of human dendritic cells resulting in production of the chemokine CCL-3 by R-DOTAP, S-DOTAP and the racemic mixture RS-DOTAP. -
FIG. 6 is a graph depicting stimulation of human dendritic cells resulting in production of the chemokine CCL-4 by R-DOTAP, S-DOTAP and the racemic mixture RS-DOTAP. -
FIG. 7 is a graph depicting stimulation of human dendritic cells resulting in production of the chemokine CCL-5 by R-DOTAP, S-DOTAP and the racemic mixture RS-DOTAP. -
FIG. 8 is a graph depicting stimulation of human dendritic cells resulting in production of the chemokine CCL-19 by R-DOTAP, S-DOTAP and the racemic mixture RS-DOTAP. -
FIG. 9 is a graph depicting stimulation of human dendritic cells resulting in production of the cytokine IL-2 by R-DOTAP, S-DOTAP and the racemic mixture RS-DOTAP. -
FIG. 10 is a graph depicting stimulation of human dendritic cells resulting in production of the cytokine IL-S by R-DOTAP, S-DOTAP and the racemic mixture RS-DOTAP. -
FIG. 11 is a graph depicting stimulation of human dendritic cells resulting in production of the cytokine 1L-12 by R-DOTAP, S-DOTAP and the racemic mixture RS-DOTAP. -
FIG. 12 is a graph demonstrating the in vivo antitumor effect of various doses of a cationic lipid/antigen complex based on tumor size and. time post-injection. -
FIG. 13 is a graph demonstrating the effect of S-DOTAP dose on the in vivo anti tumor efficacy of the cationic lipid/antigen complex. -
FIG. 14 is a graph demonstrating the effect of R-DOTAP dose on the in vivo anti tumor efficacy of the cationic lipid/antigen complex. -
FIG. 15 is a graph depicting the lipid dose response effects of the racemic mixture of DOTAP, R-DOTAP and S-DOTAP on the in vivo anti-tumor immune response of the cationic lipid/antigen complex with antigen dose of 201.1 g. The effect of antigen dose is also demonstrated with the racemic mixture of DOTAP. R-DOTAP compared to S-DOTAP: * p<0.05, ** p<0.01, n=5-6. - One or more specific embodiments of the present invention will be described below. In an effort to provide a concise description of these embodiments, all features of an actual implementation may not be described in the specification. It should be appreciated that in the development of any such actual implementation numerous implementation-specific decisions must be made to achieve the developers' specific goals, which may vary from one implementation to another. Moreover, it. should be appreciated that such a development effort might be complex and time consuming, but would nevertheless be a routine undertaking for those of ordinary skill having the benefit of this disclosure.
- When introducing elements of the present invention (e.g., the exemplary embodiments(s) thereof), the articles “a”, “an”, “the” and “said” are intended to mean that there are one or more of the elements. The terms “comprising”, “including” and “having” are intended to be inclusive and mean that there may be additional elements other than the listed elements.
- One aspect of the present invention provides an enantiomer of a cationic lipid to stimulate an immune response in a mammal to prevent or treat disease The individual chiral lipids can function independently as immunomodulators, in a dose dependent manner, such as for production of chemokines and/or cytokines, by activating various components of the MAP kinase signaling pathway. The dose range that effectively induces an immune response is observed to differ between the R and S enantiomers and also within various mammalian species. For example, in the rodent species the R-enantiomer of DOTAP effectively attenuates tumor growth over a range of about 30 nmole to about 400 nmole. In contrast, the S-enantiomer of DOTAP is effective over this same range of doses in the same species of rodent, though less so than the R-enantiomer. In another aspect, the chiral cationic lipid may be associated with antigens or drugs for presentation to cells of the immune system while simultaneously stimulating a strong antigen-specific immune response. In some aspects of the invention, the antigen is a lipopeptide.
- U.S. Pat. No. 7,303,881, incorporated by reference herein in its entirety, discloses that multiple cationic lipids complexed with disease-associated antigens were shown to stimulate a prophylactic immune response that prevented the specific disease (e.g., HPV-positive cancer) and also a therapeutic immune response that killed cells expressing the particular antigen and resulted in an effective treatment of the disease. Presently, studies were performed to further understand the effects of chirality on the immunostimulatory capability of cationic lipids by using the R and S enantiomers of DOTAP. (The R and S enantiomers of DOTAP are shown in FIGS. I A and 113). These studies have led to the discovery that individual enantiomers of cationic lipids can function independently as immunodulators to stimulate an immune response with (or without) antigens. Further, when enantiomers of cationic lipids are complexed with an antigen, an antigen specific immune response is generated. The extent of the disease specific immune response differs significantly between the R and S enantiomers of the cationic lipid.
- In another aspect, the chiral cationic lipid, at a dose sufficient to stimulate an immune response, is administered in combination with an antigen or antigens. In this case the cationic lipid/antigen combination is capable of generating an immune response that is specific to the antigen(s) delivered in combination with the cationic lipid. The response generated may include production of specific cytotoxic T cells, memory T cells, or B cells resulting in the prevention of, or therapeutic response to, the specific disease associated with the antigen(s).
- The chiral cationic lipids of the invention may be in the form of cationic lipid complexes. The cationic lipid complex can take the form of various vesicles such as liposomes, micelles, or emulsions. The cationic lipid complexes may be unilaminar or multilaminar. When an antigen is included, the antigen may be encapsulated in the cationic lipid complex or may be unencapsulated. Encapsulated is understood to mean that the antigen may be contained within the internal space of the complex and/or incorporated into the lipid walls of the complex.
- Another aspect of the invention relates to a method for producing these complexes, wherein the method may optionally include the step of purifying these formulations from excess individual components.
- In certain embodiments, the cationic lipid complexes have a net positive charge and/or a positively charged surface at pH 6.0-8.0.
- The optional “antigen” which may be included with cationic lipid complexes of the invention may be nucleic acids, peptides, lipopeptides, proteins, lipoproteins, polysaccharides, and other macromolecules which may be complexed directly with cationic lipids. However, cationic drugs (e.g., large cationic protein) can be directly complexed with an anionic lipid or sequentially complexed first with anionic lipid or polymer followed by the chiral cationic lipid. The use of this process permits delivery of positive or neutral charged drugs to cells by the complexes of the present invention.
- One aspect of the present invention involves the use of the chiral cationic lipid complexes to activate dendritic cells and also to stimulate the production of chemokines and cytokines. Chemokines and cytokines are important regulators of immune responses. Chemokines were originally identified as potent chemoattractants for inflammatory cells including neutrophils, eosinophils, and in onocytes/macrophages. Subsequent studies have revealed that chemokines have profound effects on immune reactions by regulating the trafficking of dendritic cells and other lymphocytes into lymphoid organs. Dendritic cells are migratory cells that sample antigens in the tissue, migrate to the draining lymph nodes and mature to stimulate the T cell response. CCL2, a member of the CC chemolcines was originally identified as a chemotactic and activating factor for monocytes/macrophages. Subsequent studies showed that it can also affect the function of T cells, natural killer cells, and neutrophils. Further exploration found that CCL2 was the most potent activator of CD8+ cytotoxic T lymphocytes (“CTL”) activity, when in the presence of the Thl cytokines, interleukin-12 (“IL-12”) and interferon-y (IFN-y”). This can be explained by a positive bidirectional interaction between CCL2 and IFN-y systems. An absence of either the cytokine or chemokine may interfere with Thl polarization and subsequent specific tumor immunity generation. Another CC chemokine, CCL-4, has also been shown to recruit and expand dendritic cells in vivo and potentiate the immunogenicity of plasmid DNA vaccines. Recently, it has been shown that chemokines enhance immunity by guiding naive CDS+ T cells to sites of CD4+ T cell-dendritic cell interaction and promote memory CD8+ T cell generation. A few examples of chemokines that may be stimulated by the cationic lipid complexes of the present invention are CCL-2, CCL-3, and CCL-4. Examples of cytokines that may be stimulated by the cationic lipid complexes of the present invention are IL-2, IL-8, IL-12 and IFN-y. The inventors contemplate that the cationic lipid complexes of the present invention may stimulate chemokines and cytokines in addition to those disclosed in this specification.
- Lipids
- The chiral cationic lipid complexes of the present invention may form liposomes that are optionally mixed with antigen and may contain the chiral cationic lipids alone or chiral cationic lipids in combination with neutral lipids. Suitable chiral cationic lipid species include, but are not limited to the R and S enantiomers of: 3-β[4N-(1N,8-diguanidino spennidine)-carbaroyl]cholesterol (BGSC); 3-β0[N,N-diguanidinoethyl-aminoethane)-carbamoyl] cholesterol (BGTC); N,N1N2N3Tetra-methyttetrapalmitylsperrnine (cellfectin); N-t-butyl-N′-tctraclecyl-3-tetradecyl-aininopropion-amidine (CLONfectin); dimethyldioctadecyl ammonium bromide (DDAB); 1,2-dimyristyloxypropyl-3-dimethyl-hydroxy ethyl ammonium bromide (DMRIE); 2,3-dioleoyloxy-N-[2(spenninecarboxamido)ethyl]-N,N-dimethyl-l-propanaminium trifluoracetate) (DOSPA); 1,3-dioleoyloxy-2-(6-carboxyspennyl)-propyl amide (DOSPER); 4-(2,3-bis-palmitoyloxy-propyl)-1-methyl-1H-imidazole (DPIM) N,N,1\11,Ni-tetramethyl-N,N′-bis(2-hydroxyethyl)-2,3 dioleoyloxy-1,4-butanediammonium iodide) (Tfx-50); N-I-(2,3-dioleoyloxy) propyl-N,N,N-trimethyl ammonium chloride (DOTMA) or other N—(N,N-1-dialkoxy)-alkyl-N,N,N-trisubstituted. ammonium surfactants; 1,2 dioleoyl-3-(4′-trimethylammonio) butanol-sn-glycerol (DOBT) or cholesteryl (4′trimethylammonia) butanoate (ChOTB) where the trim ethylammonium group is connected via a butanol spacer arm to either the double chain (for DOTB) or cholesteryl group (for ChOTB); DORI (DL-1,2-dioleoyl-3-dimethylaminopropyl-β-hydroxyethylammonium) or DORIE (DL-1,2-O-dioleoyl-3-dimethylaminopropyl-(3-hydroxyethylammonium) (DORIE) or analogs thereof as disclosed in WO 93/03709; 2-dioleoyl-3-succinyl-sn-glycerol choline ester (DOSC); cholesteryl hemisuccinate ester (ChOSC); lipopolyamines such as dioctadecylamidoglycylspermine (DOGS) and dipalinitoyl phospliatidylethanolamylspennine (DPPES) or the cationic lipids disclosed in U.S. Pat. No. 5,283,185, cholesteryl-3 β-carboxyl-amido-ethylenetrimethylammonium iodide, 1-dimethylamino-3-trimethylammonio-DL-2-propyl-cholesteryl carboxylate iodide, cholesteryl-3-O-carboxyamidoethyleneamine, cholesteryl-3-β-oxysuccinamido-ethylenetrimethylammonium iodide, 1-dimethylamino-3-trimethylammonio-DL-2-propyl-cholesteryl-3-β-oxosuccinate iodide, 2-(2-trimethylammonio)-ethylmethylamino ethyl-cholesteryl-3-β-oxysuccinate iodide, 3-β-N-(1\1′,N′-dimethylaminoethane) carbamoyl cholesterol (DC-chol), and 3-β-N-(polyethyleneimine)-carbamoylcholesterol; 0,0′-dirnyristyl-N-lysyl aspartate (DMKE); 0,0′-dirnyristyl-N-lysyl-glutamate (DMKD); 1,2-climyristyloxypropyl-3-dintethyl-hydroxy ethyl ammonium bromide (DMRIE); 1,2-dilauroyl-sn-glycero-3-ethylphosphocholine (DMEPC); 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (DMEPC); 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (DOEPC); 1,2-clipalmitoyl-sn-glycero-3-ethylphosphocholine (DPEPC); 1,2-distearoyl-sn-glycero-3-ethylphosphocholine (DMEPC); 1,2-dioleoyl-3-trimethylammonium propane (DOTAP); dioleoyl dim ethylaminopropane (DODAP); 1,2-pahnitoyl-3-trimethylammonium propane (DSTAP); 1,2-distearoyl-3-trimetbylam onium propane (DSTAP), 1,2-nlyristoyl-3-trimethylammonium propane (DMTAP); and sodium dodecyl sulfate (SDS). The present invention contemplates the use of structural variants and derivatives of the cationic lipids disclosed in this application.
- Certain aspects of the present invention include nonsteroidal chiral cationic lipids having a structure represented by the following formula:
- wherein in R3 is a quaternary ammonium group, Y1 is chosen from a hydrocarbon chain, an ester, a ketone, and a peptide, C* is a chiral carbon, R2 and R3 are independently chosen from a saturated fatty acid, an unsaturated fatty acid, an ester-linked hydrocarbon, phosphor-diesters, and combinations thereof. DOTAP, DMTAP, DSTAP, DPTAP, DPEPC, DSEPC, DMEPC, DLEPC, DOEPC, DMKE, DMKD, DOSPA, DOTMA, are examples of lipids having this general structure.
- In one embodiment, chiral cationic lipids of the invention are lipids in which bonds between the lipophilic group and the amino group are stable in aqueous solution. Thus, an attribute of the complexes of the invention is their stability during storage (i.e., their ability to maintain a small diameter and retain biological activity over time following their formation). Such bonds used in the cationic lipids include amide bonds, ester bonds, ether bonds and carbamoyl bonds. Those of skill in the art would readily understand that liposomes containing more than one cationic lipid species maybe used to produce the complexes of the present invention. For example, liposomes comprising two cationic lipid species, lysyl-phosphatidylethanolamine and β-alanyl cholesterol ester have been disclosed for certain drug delivery applications [Brunette, E. et al., Nucl. Acids Res., 20:1151 (1992)].
- It is to be further understood that in considering chiral cationic liposomes suitable for use in the invention and optionally mixing with antigen, the methods of the invention are not restricted only to the use of the cationic lipids recited above but rather, any lipid composition may be used so long as a cationic liposome is produced and the resulting cationic charge density is sufficient to activate and induce an immune response.
- Thus, the complexes of the invention may contain other lipids in addition to the chiral cationic lipids, These lipids include, but are not limited to, lyso lipids of which lysophosphatidylcholine (1-oleoyl lysophospliatidyleholine) is an example, cholesterol, or neutral phospholipids including dioleoyl phosphatidyl ethanolamine (DOPE) or dioleoyl phosphatidylcholine (DOPC) as well as various lipophilic surfactants, containing polyethylene glycol moieties, of which Tween-80 and PEG-PE are examples.
- The chiral cationic lipid complexes of the invention may also contain negatively charged lipids as well as cationic lipids so long as the net charge of the complexes formed is positive and/or the surface of the complex is positively charged. Negatively charged lipids of the invention are those comprising at least one lipid species having a net negative charge at or near physiological pH or combinations of these. Suitable negatively charged lipid species include, but are not limited to, CHEMS (cholesteryl hemisuccinate), NGPE (N-glutaryl phosphatidlylethanolanine), phosphatidyl glycerol and phosphatidic acid or a similar phospholipid analog.
- Methods for producing the liposomes to be used in the production of the lipid comprising drug delivery complexes of the present invention are known to those of ordinary skill in the art. A review of methodologies of liposome preparation may be found in Liposome Technology (CFC Press New York 1984); Liposomes by Ostro (Marcel Dekker, 1987); Methods Biochem Anal, 33:337-462 (1988) and U.S. Pat. No. 5,283,185. Such methods include freeze-thaw extrusion and sonication. Both unilamellar liposomes (less than about 200 mu in average diameter) and multilamellar liposomes (greater than about 300 nm in average diameter) may be used as starting components to produce the complexes of this invention.
- In the cationic liposomes utilized to produce the cationic lipid complexes of this invention, the chiral cationic lipid is present in the liposome at from about 10 mole % to about 100 mole % of total liposomal lipid, or from about 20 mole % to about 80 mole %. The neutral lipid, when included in the liposome, may be present at a concentration of from about 0 mole % to about 90 mole % of the total liposomal lipid, or from about 20 mole % to about 80 mole %, or from 40 mole % to 80 mole %. The negatively charged lipid, when included in the liposome, may be present at a concentration ranging from about 0 mole % to about 49 mole % of the total liposomal lipid, or from about 0 mole % to about 40 mole %. In one embodiment, the liposomes contain a chiral cationic and a neutral lipid, in ratios between about 2:8 to about 6:4.
- It is further understood that the complexes of the present invention may contain modified lipids, protein, polycations or receptor ligands which function as a targeting factor directing the complex to a particular tissue or cell type. Examples of targeting factors include, but are not limited to, asialoglycoprotein, insulin, low density lipoprotein (LDL), folate and monoclonal and polyclonal antibodies directed against cell surface molecules. Furthermore, to modify the circulatory half-life of the complexes, the positive surface charge can be sterically shielded by incorporating lipophilic surfactants which contain polyethylene glycol moieties.
- The cationic lipid complexes may be stored in isotonic sucrose or dextrose solution upon collection from the sucrose gradient or they may be lyophilized and then reconstituted in an isotonic solution prior to use. In one embodiment, the cationic lipid complexes are stored in solution. The stability of the cationic lipid complexes of the present invention is measured by specific assays to determine the physical stability and biological activity of the cationic lipid complexes over time in storage. The physical stability of the cationic lipid complexes is measured by determining the diameter and charge of the cationic lipid complexes by methods known to those of ordinary skill in the art, including for example, electron microscopy, gel filtration chromatography or by means of quasi-elastic light scattering using, for example, a Coulter N4SD particle size analyzer as described in the Example, The physical stability of the cationic lipid complex is “substantially unchanged” over storage when the diameter of the stored cationic lipid complexes is not increased by more than 100%, or by not more than 50%, or by not more than 30%, over the diameter of the cationic lipid complexes as determined at the time the cationic lipid complexes were purified.
- While it is possible for the chiral cationic lipid to be administered in a pure or substantially pure form, it is preferable to present it as a pharmaceutical composition, formulation or preparation. Pharmaceutical formulations using the chiral cationic lipid complexes of the invention may comprise the cationic lipid complexes in a physiologically compatible sterile buffer such as, for example, phosphate buffered saline, isotonic saline or low ionic strength buffer such as acetate or Hepes (an exemplary pH being in the range of about 3.0 to about 8.0). The chiral cationic lipid complexes may be administered as aerosols or as liquid solutions for intratumoral, intraarterial, intravenous, intratracheal, intraperitoneal, subcutaneous, and intramuscular administration.
- The formulations of the present invention may incorporate any stabilizer known in the art. Illustrative stabilizers arc cholesterol and other sterols that may help rigidify the liposome bilayer and prevent disintegration or destabilization of the bilayer. Also agents such as polyethylene glycol, poly-, and monosaccharides may be incorporated into the liposome to modify the liposome surface and prevent it from being destabilized due to interaction with blood-components. Other illustrative stabilizers are proteins, saccharides, inorganic acids, or organic acids which may be used either on their own or as admixtures.
- A number of pharmaceutical methods may be employed to control, modify, or prolong the duration of immune stimulation. Controlled release preparations may be achieved through the use of polymer complexes such as polyesters, polyamino acids, methylcellulose, polyvinyl, poly(lactic acid), and hydrogels to encapsulate or entrap the cationic lipids and slowly release them. Similar polymers may also be used to adsorb the liposomes. The liposomes may be contained in emulsion formulations in order to alter the release profile of the stimulant. Alternatively, the duration of the stimulant's presence in the blood circulation may be enhanced by coating the surface of the liposome with compounds such as polyethylene glycol or other polymers and other substances such as saccharides which are capable of enhancing the circulation time or half life of liposomes and emulsions.
- When oral preparations are required, the chiral cationic lipids may be combined with typical pharmaceutical carriers known in the art such as, for example, sucrose, lactose, methylcellulose, carboxymethyl cellulose, or gum Arabic, among others. The cationic lipids may also be encapsulated in capsules or tablets for systemic delivery.
- Administration of the chiral cationic lipid of the present invention may be for either a prophylactic or therapeutic purpose. When provided prophylactically, the cationic lipid is provided in advance of any evidence or symptoms of illness. When provided therapeutically, the cationic lipid is provided at or after the onset of disease. The therapeutic administration of the immune-stimulant serves to attenuate or cure the disease. For both purposes, the cationic lipid may be administered with an additional therapeutic agent(s) or antigen(s). When the cationic lipids are administered with an additional therapeutic agent or antigen, the prophylactic or therapeutic effect may be generated against a specific disease.
- The formulations of the present invention, both for veterinary and for human use, comprise a chiral cationic lipid alone as described above, as a mixture of R and S enantiomers, or also optionally, with one or more therapeutic ingredients such as an antigen(s) or drug molecule(s). The formulations may conveniently be presented in unit dosage form and may be prepared by any method known in the pharmaceutical art.
- Antigens
- In one embodiment, the chiral cationic lipid is administered without any additional agents in order to boost or lower various immune responses, including production of other immune modulators, and to boost the immune response to fighting disease. In another embodiment, the chiral cationic lipid is administered in combination with an antigen or antigens. In this case the objective is to generate an immune response, which is specific to the antigen(s) delivered in combination with the cationic lipid. The response generated may include production of specific cytotoxic T-cells, memory T-cells, or B-cells resulting in the prevention of or therapeutic response to the specific disease associated with those antigen(s). The antigen can be any tumor-associated antigen or microbial antigen or any other antigen known to one skilled in the art.
- A “tumor-associated antigen,” as used herein is a molecule or compound (e.g., a protein, peptide, polypeptide, lipoprotein, lipopeptide, glycoprotein, glycopeptides, lipid, glycolipid, carbohydrate, RNA, and/or DNA) associated with a tumor or cancer cell and which is capable of provoking an immune response (humoral and/or cellular) when expressed on the surface of an antigen presenting cell in the context of a major histocompatibility complex (“MHC”) molecule. Tumor-associated antigens include self antigens, as well as other antigens that may not be specifically associated with a cancer, but nonetheless enhance an immune response to and/or reduce the growth of a tumor or cancer cell when administered to an animal. More specific embodiments are provided herein.
- A “microbial antigen,” as used herein, is an antigen of a microorganism and includes, but is not limited to, infectious virus, infectious bacteria, infectious parasites and infectious fungi. Microbial antigens may be intact microorganisms, and natural isolates, fragments, or derivatives thereof, synthetic compounds which are identical to or similar to naturally-occurring microbial antigens and, preferably, induce an immune response specific for the corresponding microorganism (from which the naturally-occurring microbial antigen originated). In a preferred embodiment, a compound is similar to a naturally-occurring microorganism antigen if it induces an immune response (humoral and/or cellular) similar to a naturally-occurring microorganism antigen. Compounds or antigens that are similar to a naturally-occurring microorganism antigen are well known to those of ordinary skill in the art such as, for example, a protein, peptide, polypeptide, Lipoprotein, lipopeptide, glycoprotein, glycopeptides, lipid, glycolipid, carbohydrate, RNA, and/or DNA. Another nonlimiting example of a compound that is similar to a naturally-occurring microorganism antigen is a peptide mimic of a polysaccharide antigen. More specific embodiments are provided herein.
- The term “antigen” is further intended to encompass peptide or protein analogs of known or wild-type antigens such as those described in. this specification. The analogs may be more soluble or more stable than wild type antigen, and may also contain mutations or modifications rendering the antigen more immunologically active. Antigen can be modified in any manner, such as adding lipid or sugar moieties, mutating peptide or protein amino acid sequences, mutating the DNA or RNA sequence, or any other modification known to one skilled in the art. Antigens can be modified using standard methods known by one skilled in the art.
- Also useful in the compositions and methods of the present invention are peptides or proteins which have amino acid sequences homologous with a desired antigen's amino acid sequence, where the homologous antigen induces an immune response to the respective tumor, microorganism or infected cell.
- In one embodiment, the antigen in the cationic lipid complex comprises an antigen associated with a tumor or cancer, i.e., a tumor-associated antigen, to make a vaccine to prevent or treat a tumor. As such, in one embodiment, the tumor or cancer vaccines of the present invention further comprise at least one epitope of at least one tumor-associated antigen. In another preferred embodiment, the tumor or cancer vaccines of the present invention further comprise a plurality of epitopes from one or more tumor-associated antigens. The tumor-associated antigens finding use in the cationic lipid complexes and methods of the present invention can be inherently immunogenic, or nonimmunogenic, or slightly immunogenic. As demonstrated herein, even tumor-associated self antigens maybe advantageously employed in the subject vaccines for therapeutic effect, since the subject compositions are capable of breaking immune tolerance against such antigens. Exemplary antigens include, but are not limited to, synthetic, recombinant, foreign, or homologous antigens, and antigenic materials may include but are not limited to proteins, peptides, polypeptides, lipoproteins, lipopeptides, lipids, glycolipids, carbohydrates, RNA and DNA. Examples of such vaccines include, but are not limited to, those for the treatment or prevention of breast cancer, head and neck cancer, melanoma, cervical cancer, Lung cancer, prostate cancer, gut carcinoma, or any other cancer known in the art using a cationic lipid in a complex with a tumor-associated antigen(s), It is also possible to formulate the antigen. with the cationic lipid without encapsulating it in the liposome. Thus, the chiral cationic lipid complexes of the present invention may be used in methods to treat or prevent cancer. In. such a case, the mammal to be immunized maybe injected with the pharmaceutical formulation containing the liposome with the encapsulated antigen(s).
- Tumor-associated antigens suitable for use in the present invention include both naturally occurring and modified molecules which may be indicative of single tumor type, shared among several types of tumors, and/or exclusively expressed or overexpressed in tumor cells in comparison with normal cells. In addition to proteins, glycoproteins, lipoproteins, peptides, and lipopeptides, tumor-specific patterns of expression of carbohydrates, gangliosides, glycolipids, and mucins have also been documented. Exemplary tumor-associated antigens for use in cancer vaccines include protein products of oncogenes, tumor suppressor genes, and other genes with mutations or rearrangements unique to tumor cells, reactivated embryonic gene products, oncofetal antigens, tissue-specific (but not tumor-specific) differentiation antigens, growth factor receptors, cell surface carbohydrate residues, foreign viral proteins, and a number of other self proteins.
- Specific embodiments of tumor-associated antigens include, e.g., mutated or modified antigens such as the protein products of the Ras p21 protooncogenes, tumor suppressor p53 and HER-2/neu and BCR-abl oncogenes, as well as CDK4, MUM1,
Caspase 8, and Beta catenin; overexpressed antigens such asgalectin 4, galectin 9, carbonic anhydrase, Aldolase A, PRAMS, Her2/neu, ErbB-2 and KSA, oncofetal antigens such as alpha fetoprotein (AFP), human chorionic gonadotropin (hCG); self antigens such as carcinoembryonic antigen (CEA) and melanocyte differentiation antigens such as Mart I/Melan A, 0)100, gp75, Tyrosinase, TRP I and TRP2; prostate associated antigens such as PSA, PAP, PSMA, PSM-P1 and PSM-P2; reactivated embryonic gene products such as MA.GE 1,MAGE 3,MACE 4,GAGE 1,GAGE 2, BAGE, RAGE, and other cancer testis antigens such as NY-ES01, SSX2 and SCP1; mucins such as Muc-1 and Muc-2; gangliosides such as GM2, GD2 and GD3, neutral glycolipids and glycoproteins such as Lewis (y) and globo-H; and glycoproteins such as To, Thompson-Freidenreich antigen (TF) and sTn. Also included as tumor-associated antigens herein are whole cell and tumor cell lysates as well as immunogenic portions thereof, as well as immunoglobulin idiotypes expressed on monoclonal proliferations of B lymphocytes for use against B cell lymphomas. - Tumor-associated antigens and their respective tumor cell targets include, e.g., cytokeratins, particularly
cytokeratin - In one embodiment, the human papillomavirus HPV antigens are used. A specific HPV antigen that used as a tumor-associated antigen is
HPV subtype 16 E7. HPV E7 antigen-cationic lipid complexes are effective at preventing and treating cervical cancer. In addition, a genetically engineered E7 protein, i.e., E7 in protein, having antigenic activity, but without tumorigenic activity, is an effective tumor-associated antigen. Elm-cationic lipid complexes induce cellular immunity to cause complete regression of established tumors and, thus, are useful as potent anti-cervical cancer vaccines. - Tumor-associated antigens can be prepared by methods well known in the art. For example, these antigens can be prepared from cancer cells either by preparing crude extracts of cancer cells (e.g., as described in Cohen et al., Cancer Res., 54:1055 (1994)), by partially purifying the antigens, by recombinant technology, or by de novo synthesis of known antigens. The antigen may also be in the form of a nucleic acid encoding an antigenic peptide in a form suitable for expression in a subject and presentation to the immune system of the immunized subject. Further, the antigen may be a complete antigen, or it may be a fragment of a complete antigen comprising at least one epitope.
- Antigens derived from pathogens known to predispose to certain cancers may also be advantageously included in the cancer vaccines of the present invention. It is estimated that close to 16% of the worldwide incidence of cancer can be attributed to infectious pathogens; and a number of common malignancies are characterized by the expression of specific viral gene products. Thus, the inclusion of one or more antigens from pathogens implicated in causing cancer may help broaden the host immune response and enhance the prophylactic or therapeutic effect of the cancer vaccine. Pathogens of particular interest for use in the cancer vaccines provided herein include the, hepatitis B virus (hepatocellular carcinoma), hepatitis C virus (heptomas), Epstein Barr virus (EMT) (Burkitt lymphoma, nasopharynx cancer, PTLD in immunosuppressed individuals), I-ITLVL (adult T cell leukemia), oncogenic human papilloma viruses types 16, 18, 33, 45 (adult cervical cancer), and the bacterium Helicobacter pylori (B cell gastric lymphoma). Other medically relevant microorganisms that may serve as antigens in mammals and more particularly humans are described extensively in the literature, e.g., C. G. A Thomas, Medical Microbiology, Baiiliero Tindall, Great Britain 1983, the entire contents of which is hereby incorporated by reference.
- In another embodiment, the antigen in the cationic lipid complex comprises an antigen derived from or associated with a pathogen, i.e., a microbial antigen. As such, in one embodiment, the pathogen vaccines of the present invention further comprise at least one epitope of at least one microbial antigen. Pathogens that may be targeted by the subject vaccines include, but are not limited to, viruses, bacteria, parasites and fungi. In another embodiment, the pathogen vaccines of the present invention further comprise a plurality of epitopes from one or more microbial antigens.
- The microbial antigens finding use in the cationic lipid complexes and methods may be inherently immunogenic, or nonimmunogenic, or slightly immunogenic. Exemplary antigens include, but are not limited to, synthetic, recombinant, foreign, or homologous antigens, and antigenic materials may include but are not limited to proteins, peptides, polypeptides, lipoproteins, lipopeptides, lipids, glycolipids, carbohydrates, RNA, and DNA.
- Exemplary viral pathogens include, but are not limited to, viruses that infect mammals, and more particularly humans. Examples of virus include, but are not limited to: Retroviridae (e.g., human immunodeficiency viruses), such as HIV-1 (also referred to as HTLV-III, LAV or HTLV-IHiLAV, or HIV-III; and other isolates, such as HIV-LP; Picornaviridac (e.g. polio viruses, hepatitis A virus; enteroviruses, human Coxsackie viruses, rhinoviruses, echoviruses); Calciviridae (e.g. strains that cause gastroenteritis); Togavridae (e.g. equine encephalitis viruses, rubella viruses); Flaviridae (e.g. dengue viruses, encephalitis viruses, yellow fever viruses); Coronoviridae (e.g. coronaviruses); Rhabdoviradae (e.g. vesicular stomatitis viruses, rabies viruses); Coronaviridae (e.g. coronaviruses); Rhabdoviridae (e.g. vesicular stomatitis viruses, rabies viruses); Filoviridae (e.g. ebola viruses); Paramyxoviridae (e.g. parainfluenza viruses, mumps virus, measles virus, respiratory syncytial virus); Orthomyxoviridae (e.g. influenza viruses); Bungaviridae (e.g. Hantaan viruses, bunga viruses, phleboviruses and Naito viruses); Arena viridae (hemorrhagic fever viruses); Reoviridae (e.g. reoviruses, orbiviurses and rotaviruses); Birnaviridae; Hepadnaviridae (Hepatitis B virus); Parvoviridae (parvoviruses); Papovaviridae (papilloma viruses, polyoma viruses); Adenoviridae (most adenoviruses); Herpesviridae herpes simplex virus (HSV) 1 and 2, varicella zoster virus, cytomegalovirus (CMV), herpes virus; Poxyiridae (variola viruses, vaccinia viruses, pox viruses); and lridoviridae (e.g. African swine fever virus); and unclassified viruses (e.g. the etiological agents of Spongiform encephalopathies, the agent of delta hepatitis (thought to be a defective satellite of hepatitis B virus), the agents of non-A, non-B hepatitis (class 1.−-internally transmitted; class 2=parenterally transmitted (i.e. Hepatitis C); Norwalk and related viruses, and astroviruses).
- Also, gram negative and grain positive bacteria may be targeted by the subject compositions and methods in vertebrate animals. Such grain positive bacteria include, but are not limited to Pasteurelkt species, Staphylococci species, and Streptococcus species. Gram negative bacteria include, but are not limited to, Escherich fa coli, Pseudomonas species, and Salmonella species. Specific examples of infectious bacteria include but are not limited to: Helicobacter pyloris, Borella burgdoiferi, Legionella pneumophilia, Mycobacteria sps (e.g. M. tuberculosis, M. avium, M. intracellulare, M. kansaii, M., gordonae), Staphylococcus aureus, Neisseria gonorrhoeae, Neisseria meningitidis, Listeria monocytogenes, Streptococcus pyogenes (Group A Streptococcus), Streptococcus agalactiae (Group B Streptococcus), Streptococcus (viridans group), Streptococcus faecalis, Streptococcus bovis, Streptococcus (anaerobic sps.), Streptococcus pneuinoniae, pathogenic Campylobacter sp., Enterococcus sp., Haemophilus infitenzae, Bacillus antracis, Corynebacterium diphtheriae, coiynebacteriuin sp., Erysipelothrix rhusiopathiae, Clostridium perfringers, Clostridium letani, Enterobacter aerogenes, Klebsiella pneumoniae, Pasteurella multocida, Bacteroides sp., Fusobacterium nucleatum, Streptobacillus moniliformis, Treponema pallidium, Treponema pertenue, Leptospira, Rickettsia, and Actinoinyces.
- Polypeptides of bacterial pathogens which may find use as sources of microbial antigens in the subject compositions include but are not limited to an iron-regulated outer membrane protein, (“TROMP”), an outer membrane protein (“OMP”), and an A-protein of A eromonis sahnonicida which causes furunculosis, p57 protein of Renibacterium salmonittarum which causes bacterial kidney disease (“BKD”), major surface associated antigen (“msa”), a surface expressed cytotoxin (“mpr”), a surface expressed hemolysin (“ish”), and a flagellar antigen of Yersiniosis; an extracellular protein (“ECP”), an iron-regulated outer membrane protein (“TROMP”), and a structural protein of Pasteurellosis; an OMP and a flagellar protein of Vibrosis anguillarum and V ordalii; a flagellar protein, an OMP protein, aroA, and purA of Edwardsiellosis ictaluri and E, tarda; and surface antigen of chthyophthirius; and a structural and regulatory protein of Cytophaga columnari; and a structural and rectulatory protein of Rickettsia. Such antigens can be isolated or prepared recombinantly or by any other means known in the art.
- Examples of pathogens further include, but are not limited to, fungi that infect mammals, and more particularly humans. Examples of fungi include, but are not limited to: Cryptococcus neoformans, Histoplasma capsulatum, Coccidioides immitis, Blastomyces dermatitidis, Chlamydia trachomatis, and Candida albicans. ‘Examples of infectious parasites include Plasmodium such as Plasmodium. falciparum, Plasmodium malartae, Plasmodium ovale, and Plasmodium vivax. Other infectious organisms (i.e. protists) include Toxoplasma gondii. Polypeptides of a parasitic pathogen include but are not limited to the surface antigens of khthyophthirius.
- Other medically relevant microorganisms that serve as antigens in mammals and more particularly humans are described extensively in the literature (e.g., see C. G. A Thomas, Medical Microbiology, Bailliere Tindall, Great Britain 1983). In addition to the treatment of infectious human diseases and human pathogens, the compositions and methods of the present invention are useful for treating infections of nonhuman mammals. Many vaccines for the treatment of nonhuman mammals are disclosed in Bennett, K. Compendium of Veterinary Products, 3rd ed. North American Compendiums, Inc., 1995; see also WO 02/069369, the disclosure of which is expressly incorporated by reference herein in its entirety.
- Exemplary nonhuman pathogens include, but are not limited to, mouse mammary tumor virus (“MMTV”), Rous sarcoma virus (“RSV”), avian leukemia virus (“ALV”), avian myeloblastosis virus (“AMV”), murine leukemia virus (“MLV”), feline leukemia virus (“FeLV”), murine sarcoma virus (“MSV”), gibbon ape leukemia virus (“GALV”), spleen necrosis virus (“SNV”), reticuloendotheliosis virus (“RV”), simian sarcoma virus (“SSV”), Mason-Pfizer monkey virus (“MPMV”), simian retrovirus type 1 (“SRV-1”), lentiviruses such as HIV-I, HIV-2, SIV, Visna virus, feline immunodeficiency virus (“Fly”), and equine infectious anemia virus (“EIAY”), T-cell leukemia viruses such as HTLV-1, HTLY-II, simian T-cell leukemia virus (“STLV”), and bovine leukemia virus (“III,V”), and foamy viruses such as human foamy virus (“HFV”), simian foamy virus (“SFV”) and bovine foamy virus (“B17V”).
- In some embodiments, “treatment,” “treat,” and “treating,” as used herein with reference to infectious pathogens, refer to a prophylactic treatment which increases the resistance of a subject to infection with a pathogen or decreases the likelihood that the subject will become infected with the pathogen; and/or treatment after the subject has become infected in order to fight the infection, e.g., reduce or eliminate the infection or prevent it from becoming worse.
- Microbial antigens can be prepared by methods well known in the art. For example, these antigens can be prepared directly from viral and bacterial cells either by preparing crude extracts, by partially purifying the antigens, or alternatively by recombinant technology or by de MVO synthesis of known antigens. The antigen may also be in the form of a nucleic acid encoding an antigenic peptide in a form suitable for expression in a subject and presentation to the immune system of the immunized subject. Further, the antigen may be a complete antigen, or it may be a fragment of a complete antigen comprising at least one epitope.
- In order to improve incorporation of the antigen into the chiral cationic lipid vesicles and also to improve delivery to the cells of the immune system, the antigen may be conjugated to a lipid chain in order to improve its solubility in the hydrophobic acyl chains of the cationic lipid, while maintaining the antigenic properties of the molecule. The lipidated antigen can be a lipoprotein, or a lipopeptide, and combinations thereof. The lapidated antigen may have a linker conjugated between the lipid and the antigen such as, for example, an N-terminal a or a-palmitoyl lysine may be connected to antigen via a dipeptide Ser-Ser linker. U.S. application Ser. No. 12/049,957 discloses that the DOTAP/E7-lipopeptide complex exhibited an enhanced functional antigen-specific CD8− T lymphocyte responses in vivo compared to the DOTAP/E7 formulation, and therefore provided superior anti-tumor efficacy.
- The present invention will be further appreciated in light of the following example.
- Effective Stimulation of the Immune System by Enantiomers of Cationic Lipids
- I. Cell Lines and Peptides
- TC-1 cells are C57BL/6 mouse lung endothelial cells that have been transformed with the HPV16 E6 and E7 oncogenes and activated H-ras. Cells were grown in RPMI medium (commercially available from Invitrogen of Carlsbad, Calif.) supplemented with 10% fetal bovine serum and 100 U/mI penicillin, and 100 mg/ml streptomycin. The MHC class I restricted peptide from the
HPV 16 E7 protein (amino acid 11 to 20, YMLDLQPETT [SEQ. ID. NO. I]) was synthesized by the University of Pittsburgh Peptide Synthesis Facility by solid state synthesis using anAdvanced ChernTech model 200 peptide synthesizer and purified by HPLC. - 2. Preparation of Lipid/Antigen Complexes and Determination of Physical Properties
- The enantiomers of DOTAP were supplied by Merck AG (EPROVA), Switzerland. All other lipids were purchased from Avanti Polar Lipids (Alabaster, Ala.). Small unilamellar DOTAP liposomes were prepared by thin film hydration followed by extrusion. The lipid, in chloroform, was dried as a thin layer under a stream of nitrogen in a glass tube. The thin film was vacuum desiccated for 2-3 h and then re-hydrated in cell culture grade water (commercially available from Cambrex of Walkersville, Md.) or buffer (such buffers are well known to those skilled in the art) containing E7 peptide to a final concentration of 0.7 mg lipids and 0.1 mg E7 per mL (molar ratio=11:1). The lipid dispersion was sequentially extruded through polycarbonate membranes with pore size of 0.4, 0.2, and 0.1 pan. The un-entrapped peptide was not removed. The liposomes were stored at 4° C. until use. E7 peptide association with the liposome was determined by measuring the percentage of liposome-bound peptide. In brief, unbound E7 peptide from R-DOTAP/E7, S-DOTAP/E7 or RS-DOTAP/E7 complexes was separated by a Microcon® centrifugal filtrate device (Millipore, Bedford, Mass.) and the concentration of unbound peptide was measured by Micro BCA™ Protein Assay Kit (Pierce, Rockford, Ill.). The efficiency of peptide association was determined as percent unbound peptide. Other methods used in general liposome preparation that are well known to those skilled in the art may also be used.
- 3. Statistical Analysis
- Data are presented as mean±SD of at least 3 independent experiments. Two-tailed Student's t tests were used to assess statistical significance for differences in means. Significance was set at p<0.05.
- 4. Individual R and S Enantiorners of Cationic Lipid/E7 Complexes Activate Human Dendritic Cells Similarly to the DOATP Racemic Mixture.
- Cationic liposomes were prepared as described above. The E7 antigen used in the formulation is the identified human E7 peptide restricted by HLA-A*0201 [HPV-16 E7, amino acids 11-20, YMLDLQPETT (SEQ. ID. NO. 1)1 The peptide was synthesized by the University of Pittsburgh, Molecular Medicine Institute, Pittsburgh, Pa. Human 1-ILA-A2 human dendritic cells were obtained from Lonza (of Walkersville, Md.). Frozen cryovials were thawed and the dendritic cells were cultured in LGM-3 medium (commercially available from Lonza of Walkersville, Md.) supplemented with 50 microgram/mI IL-4 and GM-CSF at 37° C. and 5% CO2 at an initial plating density of 125,000 cells/cm2 in 2 ml of medium in 12-well tissue culture dishes. The cells were grown for 3 days in culture and appeared as a mixture of adherent and rounded cells by microscopic examination.
- The cells were treated on
day 3 with a fresh dose of 50 microgram/ml of IL-4 and GM-CSF (all wells) and test wells were treated with either a mixture of interleukin 1-beta (“IL-β”), interleukin 6 (“IL-6”) and TNF-a at 10 ng/ml, and prostaglandin E2 (“PGE-2”) at 10 μg/ml (positive control for activation), no treatment (negative activation control), and S-DOTAP/E7 at 2.5, 10 and 40 micromolar final concentrations, and R-DOTAP/E7 at 2.5, 10 and 40 micromolar final concentrations. The treated dendritic cells were maintained in culture for 24 hours and harvested for cell surface marker staining and flow cytornetry analysis. The harvested cells were counted by hernacytometer and 10 pi of the following antibody conjugates were added sequentially to each sample for labeling surface markers: CD80-FITC, CD83-APC, and CD86-PE (BD Biosciences). The surface labeled cells were subsequently analyzed by flow cytometry using a BD FACxcaliber flow cytometer, and the co-stimulatory dendritic cell marker molecules CD80, CD83, and CD86 which are produced upon activation, were monitored. As seen inFIGS. 2, 3 and 4 primary human dendritic cells treated with the both enantiomers of the cationic lipid/E7 complex up-regulated the expression of all three co-stimulatory markers of dendritic cell activation evaluated and required for successful antigen presentation to T-cells, similarly to what was observed with the racemic mixture (RS-DOTAP) of the cationic lipid and reported in U.S. application Ser. No. 12/049,957, assigned to the assignee of the present application. - 5. Cationic Lipid/E7 Complexes Consisting of Individual R and S Enantiomers Exhibit Different Potencies in Activating Human Dendritic Cells to Induce Chemokine and Cytokine Production
- Human HLA-
A 2 dendritic cells (Lonza, Walkersville, Md.), were treated and grown in culture as described above. Onday 3 the cells were treated with 40 micromolar DOTAP/E7 complex or the potent immunostimulator lipopolysaccharide (LPS) at 50 micromolar concentrations (positive control). Medium from assay wells was removed and centrifuged at 1300 rpm in a microfuge for 5 minutes to pellet unattached dendritic cells. The supernatants were removed and treated with 10 microliters per ml of Calbiochem (La Jolla, Calif.) protease inhibitor cocktail set I (Cat. No. 539131) and stored frozen prior to analysis. Samples were analyzed for chemokine and cytokine expression by Searchlight Protein Array Multiplex ELISA assay [Pierce Biotechnology (Woburn, Mass.)]. - Production of selected chemokines known to be essential in the cellular immune response, CCL3, CCL4, CCL5, and CCL19 was evaluated, and production of IL-2, 11-8 and IL-12 was evaluated (
FIGS. 5-11 , which illustrate the ability of R-DOTAP/E7 and S-DOTAP/E7 to induce production of CCL3, CCL4, CCL5, CCL-19, IL-2, IL-8 and 1L-12), The figures illustrate that the DOTAP/E7 complex containing the individual enantiomers of DOTAP induce cytokine and chemokine production by human dendritic cells. Both enantiorners however activate the immune system to different extents with the R-enantiomer exhibiting higher potency. - 6. Kinetics of TC-.1 HPV-Positive Tumor Growth in Mice Treated with DOTAP/E7 Compositions at Varying Doses of Racemic Mixtures of DOTAP.
- In
FIG. 12 , mice were subcutaneously injected with TC-1 cells onday 0 in order to induce the growth of HPV-positive tumors, The DOTAP/E7 compositions were comprised of racemic mixtures of DOTAP. The mice received DOTAP/E7 compositions containing 10 11.g E7 peptide subcutaneously on the opposite side of the abdomen on.day 6. DOTAP lipid concentration in the complex varied from 3 to 600 nmole (3, 15, 30, 75, 150, 300, and 600 nmole), Low dose of DOTAP (15 nmole) showed partial tumor inhibition effect (P<0.05) compared to the untreated control onday 23, while DOTAP at 30, 150 or 300 nmole exhibited an enhanced efficacy (P<0.01). DOTAP at 75 nmole showed the most significant tumor regression effect (P<0.001). Again, mice given a high dose of DOTAP (600 nmole) did not show anti-tumor activity, confirming that DOTAP liposomes at a high dose might have induced a negative regulation to the immune response. In addition, DOTAP liposomes at the 100 nmole dose, but without E7 peptide, did not show significant inhibition of tumor growth, indicating that the anti-tumor effect was antigen specific. Further, liposomes of 1,2-dioleoyl-sn-glycero-3-phosphoglycerol (DOPG), an anionic lipid, administered at the 150 nmole dose with antigen failed to significantly inhibit tumor growth. - 7. Kinetics of TC-1 HPV-Positive Tumor Growth in Mice Treated with R-DOTAP/E7 and S-DOTAP Compositions at Varying Doses of R and S DOTAP.
- In
FIGS. 13 and 14 , mice were subcutaneously injected with TC-1 cells onday 0 in order to induce the growth of HPV-positive tumors. The mice received R and S-DOTAP/E7 compositions containing 20 E7 peptide subcutaneously on the opposite side of the abdomen onday 6. R or S-DOTAP lipid concentrations in the complex varied from 3 to 600 nmole (3, 15, 30, 75, 100, 125, 150, 300, and 600 nmole). Unlike the racemic mixture of DOTAP (FIG. 12 ), S-DOTAP complexes did not exhibit the ability to inhibit tumor growth and no tumor regression was observed (FIG. 13 ). A dose response effect was however observed, and S-DOTAP closes of 600 nmole induced the slowest tumor growth (P<0.05) compared to untreated control onday 23. Referring toFIG. 14 the anti-tumor effect of complexes containing R-DOTAP and antigen were similar to the effect observed in the race/tile mixture (FIG. 12 ). 75-150 nmole doses of R-DOTAP showed partial tumor inhibition effect (P<0.001) compared to the untreated control onday 23, while R-DOTAP at 300 nmole exhibited the most significant tumor regression efficacy (P<0.0001). Again, mice given a high dose of R-DOTAP (600 nmole) did not show significant anti-tumor activity, confirming that R-DOTAP liposomes at a high dose might have induced a negative regulation to the immune response. E7 peptide alone, did not show any inhibition of tumor growth (not shown).FIG. 15 shows the lipid dose-response curves for the tumor regression efficacy of the various cationic lipid/E7 antigen complexes DOTAP, S-DOTAP, and R-DOTAP at 20 μg of the antigen and DOTAP at 10 μg of the antigen. - 8. Induction of T Cell Proliferation by S-DOTAP and R-DOT.AP Compositions.
- We have previously demonstrated that in U.S. Provisional Application No. 60/983,799, assigned to the assignee of the present application, that DOTAP/E7 interacts directly with human T lymphocytes, leading to clonal expansion and T cell activation. Those studies examined the ability of racemic mixtures of DOTAP to stimulate clonal expansion of T cells. In those studies, enriched human lymphocytes obtained from an I-ILA-A2t healthy donor were directly stimulated by medium, DOTAP alone, peptide alone or DOTAP/hE7. The stimulation was repeated three times with a 7-day interval. Three days after the third stimulation, lymphocytes treated with DOTAP or DOTAP/E7 showed extensive expansion of T cell colonies in culture in contrast to no clonal expansion in medium control. The expanded T-cells also demonstrated significant CTL activity.
- In those studies, the DOTAP-mediated T cell activation was further confirmed by ERK phosphorylation in T cells. DOTAP-induced expression of the costimulatory molecule, CD86 on human T lymphocytes was also observed. Those results suggested that DOTAP has a direct impact on T cell activation via a MAP kinase mediated cell proliferation.
- In the present studies, the induction of human T-cell proliferation by the R and S enantiomers of DOTAP was investigated and confirmed using purified T-cells obtained from Lonza, Mass. R-DOTAP induced more T-cell proliferation than S-DOTAP and was similar in activity to the DOTAP racemic mixture.
- Discussion
- As described in U.S. Pat. No. 7,303,881, a broad class of cationic lipids can act as potent immunostimulators together with an antigen to generate antigen specific immune responses in the treatment of disease. For example, U.S. Pat. No. 7,303,881 discloses that liposomes comprised of cationic lipids activate dendritic cells as demonstrated by the stimulation by cationic lipids of the expression of costimulatory molecules CD80/CD86 on DC2A dendritic cells (
FIGS. 14A and 14B ). As shown in FIG. 14A of U.S. Pat. No. 7,303,881, the ability to stimulate the expression of CD80/CD86 on DC2.4 cells by different cationic liposomes varies greatly. Lipofectamine®, a 3:1 (w/w) liposome formulation of thepolycationic lipid 2,3-dioleyloxy-N-[2(spen-ninecarboxamido)ethyll-N,N-dimethyl-1-propanarninium trifluoroacetate (DOSPA) and the neutral lipid dioleoyl phosphatidylethanolamine (DOPE), and liposomes prepared from 0,01-dirnyristyl-N-lysyl aspartate (DMKE) and 0,0′-dimyristyl-N-lysyl-glutamate (DMKD), strongly stimulated the expression of CD80/CD86 by CD2.4 cells. - As further disclosed in U.S. Pat. No. 7,303,881, the ability of different cationic lipids to stimulate the expression of
CD 80 on DC 2.4 cells varied. Both hydrophilic head and the lipophilic tail of the lipids have significant effect on this ability. For example, the DXEPC lipids with the ethyl phosphocholine (EPC) head groups appear, in general, to be more potent than the DXTAP lipids with trimethylammonium propane (TAP) head group. Within the lipids bearing one particular head group structure, lipids with shorter (1,2-dilauroyl-sn-glycero-3-ethylphosphocholine (DXEPC-12:0), 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (DMEPC-14:0)) or unsaturated (1,2-dioleoyl-sn-glycero-3-ethylphosphocholinc (DOEPC-18:1)) acyl chains appear to be more potent than those with longer (1,2-dipalmitoyl-sn-glycero-3-ethylphosphocholine (DXEPC-16:0)) or saturated (1,2-distearoyl-sn-glycero-3-ethylphosphocholine (DSEPC-16:0)) acyl chains, These data however, demonstrated that multiple cationic lipids were capable of stimulating the activation of dendritic cells. Studies reported in U.S. application Ser. No. 12/049,957 highlight the mechanism by which cationic lipids act as immunostimulators. - Data from the abovementioned studies have led to the observation that the cationic lipids are not only efficient targeting and delivery vehicles for antigens to APC of the immune system, but also function as potent adjuvants under low dose composition ranges to directly influence immune system function through activation of MAP kinase dependent signaling pathways with resultant production of immune system regulatory molecules including cytokines and chemokines. A clear dose-response effect of cationic lipid on the immunostimulatory capabilities of the formulations have been demonstrated. It was demonstrated that upon receiving the lipid/antigen complex, the particles were mainly taken up by dendritic cells, the major professional antigen presenting cells. The initiation of dendritic cell activation and migration to the draining lymph node facilitates immune responses against antigen specific TC-1 tumors as demonstrated. Functional CD8+ T lymphocytes were generated in mice upon receiving a DOTAP/E7 injection and tumor sizes decreased and exhibited enhanced apoptosis, owing to the increasing number of infiltrating T cells in the tumor microenvironment. The resulting bell-shaped (activity decreases above and below the optimal dose) cationic lipid dose response curve demonstrated activity at very low doses, indicating that the activity of the cationic lipids as adjuvants or immunostimulators is so potent that the EC50 is as low as about 15 nmole per injection. High doses of cationic lipids eliminate the immunostimulatory activity. We have also demonstrated that when an antigen such as, for example, ovalbumin, is incorporated into the cationic liposomes and administered in a single subcutaneous injection, effective antibodies against the antigen are produced. Cationic liposomes can also induce expression of the co-stimulatory molecules CD80 and CD83 and activate human dendritic cells. It is clear that at optimal dose compositions, the cationic lipids and cationic lipid/antigen complexes in addition to effective delivery to the dendritic cells are potent activators of the immune system and provide simple, safe, and very efficient immunotherapies useful in preventing and treating diseases.
- Based on an understanding of the mechanism of immunostimulation, further studies were performed to evaluate the effect of chirality in cationic lipids and the immunostimulatory capability of cationic lipids, To this effect pure synthesized R and S enantiomers of DOTAP were utilized and compared with the commonly utilized racemic mixture. Both R and S enantiomers of DOTAP were demonstrated to possess similar ability to the racemic DOTAP with regards to activation and maturation of dendritic cells. All three lipids induced dendritic cells to express the
co-stimulatory molecules CD 80 CD 83 and CD 86. - An important characteristic of an immunostimulator capable of inducing cellular immune responses to disease is its ability to induce the production of critical chemokines and cytokines. As reported in the Example, significant differences were observed between the R and S enantiomers of DOTAP in their ability to induce chetnokine and cytokine production. R-DOTAP was observed to be a more potent immune activator than S-DOTAP. In all cases the potency of R-DOTAP was equivalent to or higher than that of the DOATP racemic mixture.
- In order to determine if the in-vitro potency in cytokine induction would translate to in-vivo therapeutic efficacy, three formulations, R-DOTAP/E7, S-DOTAP/E7 and DOTAP/E7 (racemic mixture) were evaluated for their ability to eradicate HPV-E7 positive tumors in tumor-bearing mice. Each formulation was evaluated at multiple lipid doses. As demonstrated in
FIGS. 12-15 , both R-DOTAP and DOTAP containing formulations exhibited a bell-shaped lipid-dose response with strong E7 specific activity leading to tumor regression within specific optimal dose ranges. S-DOTAP containing formulations did not induce tumor regression under any condition observed, although high lipid formulations slowed tumor growth. - It is therefore evident that the R enantiomer of DOTAP is responsible for the majority of the observed adjuvant effect of DOTAP. However, both enantiomers are potent activators of dendritic cells leading to maturation.
- The studies reported above identify specific unique compositions and applications of cationic lipids consisting of chiral lipid or mixtures of chiral lipids, which can be exploited to develop simple, cost effective, and much needed immunotherapies for several debilitating diseases.
- As various changes could be made in the above-described aspects and exemplary embodiments without departing from the scope of the invention, it is intended that all matter contained in the above description shall be interpreted as illustrative and not in a limiting sense.
- To that end, while the examples primarily discuss enantiomers of the cationic lipid DOTAP, those skilled in the art will recognize that this cationic lipids is merely exemplary and that the methods and mechanisms are applicable to other cationic lipids.
Claims (22)
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US17/212,051 US20210308157A1 (en) | 2008-04-17 | 2021-03-25 | Stimulation of an immune response by enantiomers of cationic lipids |
Applications Claiming Priority (6)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US4583708P | 2008-04-17 | 2008-04-17 | |
PCT/US2009/040500 WO2009129227A1 (en) | 2008-04-17 | 2009-04-14 | Stimulation of an immune response by enantiomers of cationic lipids |
US98823610A | 2010-12-23 | 2010-12-23 | |
US15/702,063 US10702541B2 (en) | 2008-04-17 | 2017-09-12 | Stimulation of an immune response by enantiomers of cationic lipids |
US16/899,763 US11801257B2 (en) | 2008-04-17 | 2020-06-12 | Stimulation of an immune response by enantiomers of cationic lipids |
US17/212,051 US20210308157A1 (en) | 2008-04-17 | 2021-03-25 | Stimulation of an immune response by enantiomers of cationic lipids |
Related Parent Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US16/899,763 Continuation US11801257B2 (en) | 2008-04-17 | 2020-06-12 | Stimulation of an immune response by enantiomers of cationic lipids |
Publications (1)
Publication Number | Publication Date |
---|---|
US20210308157A1 true US20210308157A1 (en) | 2021-10-07 |
Family
ID=41199446
Family Applications (4)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US12/988,236 Active 2031-05-04 US9789129B2 (en) | 2008-04-17 | 2009-04-14 | Stimulation of an immune response by enantiomers of cationic lipids |
US15/702,063 Active US10702541B2 (en) | 2008-04-17 | 2017-09-12 | Stimulation of an immune response by enantiomers of cationic lipids |
US16/899,763 Active US11801257B2 (en) | 2008-04-17 | 2020-06-12 | Stimulation of an immune response by enantiomers of cationic lipids |
US17/212,051 Pending US20210308157A1 (en) | 2008-04-17 | 2021-03-25 | Stimulation of an immune response by enantiomers of cationic lipids |
Family Applications Before (3)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US12/988,236 Active 2031-05-04 US9789129B2 (en) | 2008-04-17 | 2009-04-14 | Stimulation of an immune response by enantiomers of cationic lipids |
US15/702,063 Active US10702541B2 (en) | 2008-04-17 | 2017-09-12 | Stimulation of an immune response by enantiomers of cationic lipids |
US16/899,763 Active US11801257B2 (en) | 2008-04-17 | 2020-06-12 | Stimulation of an immune response by enantiomers of cationic lipids |
Country Status (13)
Country | Link |
---|---|
US (4) | US9789129B2 (en) |
EP (1) | EP2276495B1 (en) |
JP (1) | JP5971945B2 (en) |
CN (2) | CN102137675A (en) |
AU (1) | AU2009236306B2 (en) |
BR (1) | BRPI0910464B1 (en) |
CA (1) | CA2721366C (en) |
ES (1) | ES2712505T3 (en) |
IL (1) | IL208713A (en) |
PL (1) | PL2276495T3 (en) |
RU (1) | RU2530555C2 (en) |
TW (1) | TWI449534B (en) |
WO (1) | WO2009129227A1 (en) |
Families Citing this family (27)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
DE10347710B4 (en) | 2003-10-14 | 2006-03-30 | Johannes-Gutenberg-Universität Mainz | Recombinant vaccines and their use |
DE102005046490A1 (en) | 2005-09-28 | 2007-03-29 | Johannes-Gutenberg-Universität Mainz | New nucleic acid molecule comprising promoter, a transcriptable nucleic acid sequence, a first and second nucleic acid sequence for producing modified RNA with transcriptional stability and translational efficiency |
US8877206B2 (en) | 2007-03-22 | 2014-11-04 | Pds Biotechnology Corporation | Stimulation of an immune response by cationic lipids |
CN102137675A (en) | 2008-04-17 | 2011-07-27 | Pds生物科技公司 | Stimulation of an immune response by enantiomers of cationic lipids |
WO2011109294A1 (en) * | 2010-03-01 | 2011-09-09 | Dicerna Pharmaceuticals, Inc. | Lipid delivery formulations |
JP2011184344A (en) * | 2010-03-08 | 2011-09-22 | Kao Corp | p21 EXPRESSION PROMOTER |
BR112013000391B8 (en) | 2010-07-06 | 2022-10-04 | Novartis Ag | COMPOSITION OF CATIONIC OIL IN WATER EMULSION AND ITS USE |
HUE057608T2 (en) | 2011-05-24 | 2022-06-28 | BioNTech SE | Individualized vaccines for cancer |
US9655845B2 (en) | 2011-07-06 | 2017-05-23 | Glaxosmithkline Biologicals, S.A. | Oil-in-water emulsions that contain nucleic acids |
US9636410B2 (en) | 2011-07-06 | 2017-05-02 | Glaxosmithkline Biologicals Sa | Cationic oil-in-water emulsions |
PL2755680T3 (en) * | 2011-09-12 | 2019-03-29 | Pds Biotechnology Corporation | Particulate vaccine formulations |
WO2013143555A1 (en) | 2012-03-26 | 2013-10-03 | Biontech Ag | Rna formulation for immunotherapy |
EP3632464A1 (en) * | 2012-06-15 | 2020-04-08 | PDS Biotechnology Corporation | Cationic lipid vaccine compositions and methods of use |
US20150250872A1 (en) | 2012-09-21 | 2015-09-10 | Frank Bedu-Addo | Vaccine compositions and methods of use |
CA2892391C (en) | 2012-11-28 | 2023-10-17 | Biontech Rna Pharmaceuticals Gmbh | Individualized vaccines for cancer |
WO2014180490A1 (en) | 2013-05-10 | 2014-11-13 | Biontech Ag | Predicting immunogenicity of t cell epitopes |
WO2015090572A1 (en) * | 2013-12-16 | 2015-06-25 | Merck Patent Gmbh | Survivin-directed cancer vaccine therapy |
WO2016045732A1 (en) | 2014-09-25 | 2016-03-31 | Biontech Rna Pharmaceuticals Gmbh | Stable formulations of lipids and liposomes |
WO2016128060A1 (en) | 2015-02-12 | 2016-08-18 | Biontech Ag | Predicting t cell epitopes useful for vaccination |
WO2017059902A1 (en) | 2015-10-07 | 2017-04-13 | Biontech Rna Pharmaceuticals Gmbh | 3' utr sequences for stabilization of rna |
US11612652B2 (en) | 2015-11-13 | 2023-03-28 | Pds Biotechnology Corporation | Lipids as synthetic vectors to enhance antigen processing and presentation ex-vivo in dendritic cell therapy |
JP2019500021A (en) * | 2015-11-13 | 2019-01-10 | ピーディーエス バイオテクノロジー コーポレイションPds Biotechnology Corporation | Lipids as synthetic vectors to enhance ex vivo antigen processing and presentation in dendritic cell therapy |
US20180221475A1 (en) * | 2016-10-05 | 2018-08-09 | Pds Biotechnology Corporation | Methods to alter the tumor microenvironment for effective cancer immunotherapy |
WO2018080253A1 (en) * | 2016-10-31 | 2018-05-03 | 아이진 주식회사 | Immunity enhancing composition including immune response regulator and cationic liposome, and use of same |
TW201924722A (en) * | 2017-12-05 | 2019-07-01 | 美商Pds生技公司 | Methods and compositions comprising cationic lipids for stimulating type I interferon genes |
EP4247493A1 (en) * | 2020-11-20 | 2023-09-27 | PDS Biotechnology Corporation | Methods and compositions comprising cationic lipids for immunotherapy by direct tumor injection |
WO2024086290A2 (en) * | 2022-10-19 | 2024-04-25 | Pds Biotechnology Corporation | Novel t cell activating immunotherapeutic for treatment of mucin 1 protein expressing human cancers |
Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2001057068A1 (en) * | 2000-02-01 | 2001-08-09 | The Austin Research Institute | Mucin-1 derived antigens and their use in immunotherapy |
WO2003003985A2 (en) * | 2001-07-06 | 2003-01-16 | Sloan-Kettering Institute For Cancer Research | Polyvalent conjugate vaccine for cancer |
WO2004089413A1 (en) * | 2003-04-14 | 2004-10-21 | Mallen Huang | Nucleotide and cellular vaccine composition |
WO2005000889A1 (en) * | 2003-06-05 | 2005-01-06 | The Government Of The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services | Immunogenic peptides for the treatment of prostate and breast cancer |
Family Cites Families (119)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US2167480A (en) | 1927-11-02 | 1939-07-25 | Rca Corp | Signaling |
US4703004A (en) | 1984-01-24 | 1987-10-27 | Immunex Corporation | Synthesis of protein with an identification peptide |
JPS6440808A (en) | 1987-08-05 | 1989-02-13 | Canon Kk | Zoom lens |
US6214804B1 (en) | 1989-03-21 | 2001-04-10 | Vical Incorporated | Induction of a protective immune response in a mammal by injecting a DNA sequence |
US5703055A (en) | 1989-03-21 | 1997-12-30 | Wisconsin Alumni Research Foundation | Generation of antibodies through lipid mediated DNA delivery |
US5264618A (en) | 1990-04-19 | 1993-11-23 | Vical, Inc. | Cationic lipids for intracellular delivery of biologically active molecules |
US6183745B1 (en) | 1990-12-12 | 2001-02-06 | The University Of Queensland | Subunit papilloma virus vaccine and peptides for use therein |
WO1993003709A1 (en) | 1991-08-16 | 1993-03-04 | Vical, Inc. | Composition and method for treating cystic fibrosis |
HUT68510A (en) * | 1991-08-26 | 1995-06-28 | Cytel Corp | Process for producing hla-restricted hapatitis b virus ctl epitopes and pharmaceutical compositions containing them |
US6419931B1 (en) | 1991-08-26 | 2002-07-16 | Epimmune Inc. | Compositions and methods for eliciting CTL immunity |
US5283185A (en) | 1991-08-28 | 1994-02-01 | University Of Tennessee Research Corporation | Method for delivering nucleic acids into cells |
IL105554A (en) | 1992-05-05 | 1999-08-17 | Univ Leiden | Peptides of human papilloma virus for use in human t cell response inducing compositions |
WO1993024640A2 (en) | 1992-06-04 | 1993-12-09 | The Regents Of The University Of California | Methods and compositions for in vivo gene therapy |
US7105574B1 (en) | 1999-03-26 | 2006-09-12 | Vical Incorporated | Adjuvant compositions and methods for enhancing immune responses to polynucleotide-based vaccines |
US5334761A (en) | 1992-08-28 | 1994-08-02 | Life Technologies, Inc. | Cationic lipids |
US9266930B1 (en) | 1993-03-05 | 2016-02-23 | Epimmune Inc. | Inducing cellular immune responses to Plasmodium falciparum using peptide and nucleic acid compositions |
EP0721341A4 (en) | 1993-08-06 | 1998-04-22 | Cytel Corp | Cloning and characterization of the complete mage-1 gene |
AUPM500494A0 (en) | 1994-04-12 | 1994-05-05 | Minister For Agriculture & Rural Affairs For The State Of New South Wales, The | Composition for use in increasing mucosal immunity |
FR2726764B1 (en) | 1994-11-14 | 1997-01-31 | Pasteur Merieux Serums Vacc | ADJUVANT FOR VACCINE COMPOSITION |
US6008202A (en) | 1995-01-23 | 1999-12-28 | University Of Pittsburgh | Stable lipid-comprising drug delivery complexes and methods for their production |
FR2732895B1 (en) | 1995-04-11 | 1997-05-16 | Pasteur Merieux Serums Vacc | USE OF A CATIONIC AMPHIPATHIC COMPOUND AS A TRANSFECTING AGENT, AS A VACCINE ADDITIVE, OR AS A MEDICINAL PRODUCT |
CA2227065A1 (en) | 1995-07-21 | 1997-02-06 | Rhone-Poulenc Rorer Pharmaceuticals Inc. | Adeno-associated viral liposomes and their use in transfecting dendritic cells to stimulate specific immunity |
EP0910343A1 (en) | 1996-07-03 | 1999-04-28 | University Of Pittsburgh | Emulsion formulations for hydrophilic active agents |
US7001614B2 (en) | 1996-08-19 | 2006-02-21 | The United States Of America As Represented By The Department Of Health And Human Services | Liposome complexes for increased systemic delivery |
US7288266B2 (en) | 1996-08-19 | 2007-10-30 | United States Of America As Represented By The Secretary, Department Of Health And Human Services | Liposome complexes for increased systemic delivery |
US20030229040A1 (en) | 1997-03-21 | 2003-12-11 | Georgetown University | Cationic liposomal delivery system and therapeutic use thereof |
US6977074B2 (en) | 1997-07-10 | 2005-12-20 | Mannkind Corporation | Method of inducing a CTL response |
US20030022854A1 (en) | 1998-06-25 | 2003-01-30 | Dow Steven W. | Vaccines using nucleic acid-lipid complexes |
US6693086B1 (en) | 1998-06-25 | 2004-02-17 | National Jewish Medical And Research Center | Systemic immune activation method using nucleic acid-lipid complexes |
JP2002536344A (en) | 1999-02-05 | 2002-10-29 | ジエンザイム コーポレイション | Use of cationic lipids to generate anti-tumor immunity |
EP2286792A1 (en) | 1999-02-26 | 2011-02-23 | Novartis Vaccines and Diagnostics, Inc. | Microemulsions with an adsorbent surface, comprising a microdroplet emulsion |
US6586409B1 (en) | 1999-03-26 | 2003-07-01 | Vical Incorporated | Adjuvant compositions and methods for enhancing immune responses to polynucleotide-based vaccines |
CA2370690A1 (en) | 1999-04-20 | 2000-10-26 | The University Of British Columbia | Cationic peg-lipids and methods of use |
US6852334B1 (en) | 1999-04-20 | 2005-02-08 | The University Of British Columbia | Cationic peg-lipids and methods of use |
US6649170B1 (en) | 1999-05-12 | 2003-11-18 | Statens Serum Institut | Adjuvant combinations for immunization composition and vaccines |
FR2794370B1 (en) | 1999-06-03 | 2003-10-17 | Biovector Therapeutics | POLYEPITOPIC PROTEIN FRAGMENTS, THEIR OBTAINMENT AND THEIR USES IN PARTICULAR IN VACCINATION |
KR100768114B1 (en) | 1999-06-10 | 2007-10-17 | 메리알 | Dna vaccines for pets and sport animals |
CA2309313A1 (en) | 1999-08-05 | 2001-02-05 | Board Of Trustees Of The University Of Arkansas | Stimulation of the immune response by human dendritic cell manipulation |
EP2100620A1 (en) | 1999-09-16 | 2009-09-16 | Eisai Corporation of North America | Nucleic acids encoding polyepitope polypeptides |
US20030008813A1 (en) | 1999-12-17 | 2003-01-09 | Felgner Philip L. | Intracellular protein delivery compositions and methods of use |
CA2394831A1 (en) | 2000-01-11 | 2001-07-19 | Maxygen, Inc. | Monocyte-derived dendritic cell subsets |
AU2001254548B2 (en) | 2000-04-20 | 2006-09-28 | The University Of British Columbia | Enhanced stabilised plasmid-lipid particle-mediated transfection using endosomal membrane |
ES2307568T3 (en) | 2000-12-08 | 2008-12-01 | Coley Pharmaceutical Gmbh | CPG TYPE NUCLEIC ACIDS AND SAME USE METHODS. |
JP2004537501A (en) | 2001-02-01 | 2004-12-16 | ボード オブ リージェンツ, ザ ユニバーシティ オブ テキサス システム | Stabilized polymer aerosol for gene delivery to the lung |
WO2002077012A2 (en) | 2001-03-23 | 2002-10-03 | The Government Of The United States Of America, Represented By The Secretary, Department Of Health And Human Services | Human papilloma virus immunoreative peptides |
FR2824326B1 (en) | 2001-05-04 | 2004-03-19 | Commissariat Energie Atomique | MIXTURE OF PEPTIDES FROM PAPILLOMAVIRUS E6 AND / OR E7 PROTEINS AND THEIR APPLICATIONS |
JP4323949B2 (en) | 2001-05-30 | 2009-09-02 | ザ・スクリプス・リサーチ・インステイチユート | Nucleic acid delivery system |
CA2489019A1 (en) * | 2001-06-15 | 2002-12-27 | Nordic Vaccine Technology A/S | Therapeutical vaccination |
WO2003002142A1 (en) | 2001-06-29 | 2003-01-09 | Chugai Seiyaku Kabushiki Kaisha | Cancer vaccine containing cancer antigen based on tumor suppressor gene wt1 product and cationic liposomes |
GB0118517D0 (en) * | 2001-07-30 | 2001-09-19 | Mitsubishi Tokyo Pharm Inc | Compound |
DE60238864D1 (en) | 2001-11-07 | 2011-02-17 | Mankind Corp | FOR EPITOPES OF ANTIGENIC ENCODING EXPRESSION VECTORS AND METHOD FOR THEIR DESIGN |
US7598421B2 (en) | 2002-05-08 | 2009-10-06 | Ucl Biomedica Plc | Materials for the delivery of biologically-active material to cells |
WO2004004758A1 (en) | 2002-07-05 | 2004-01-15 | Lipoxen Technologies Limited | Method to enhance an immune response of nucleic acid vaccination |
CN1688605B (en) | 2002-08-12 | 2011-01-12 | 昆士兰医学研究所理事会 | Novel immunogenic lipopeptides comprising T-helper and cytotoxic T lymphocyte (CTL) epitopes |
US20050031587A1 (en) | 2002-10-04 | 2005-02-10 | Yamanouchi Pharmaceutical Co., Ltd. | Immune response induction method |
ATE465753T1 (en) | 2002-10-21 | 2010-05-15 | Eisai Inc | COMPOSITIONS AND METHODS FOR TREATING DISEASES CAUSED BY THE HUMAN PAPILLOMA VIRUS |
WO2004110493A2 (en) | 2003-05-30 | 2004-12-23 | Alza Corporation | Method of pulmonary administration of an agent |
US7906122B2 (en) | 2003-06-18 | 2011-03-15 | Yissum Research Development Company Of The Hebrew University Of Jersusalem | Sphingoid polyalkylamine conjugates for Hepatitis B virus vaccination |
JP4695075B2 (en) | 2003-06-18 | 2011-06-08 | イッスム・リサーチ・ディベロップメント・カンパニー・オブ・ザ・ヘブルー・ユニバーシティ・オブ・エルサレム | Sphingolipid polyalkylamine conjugates for use in transfection |
US8592197B2 (en) | 2003-07-11 | 2013-11-26 | Novavax, Inc. | Functional influenza virus-like particles (VLPs) |
EP1646427A1 (en) | 2003-07-22 | 2006-04-19 | Cytos Biotechnology AG | Cpg-packaged liposomes |
JP5097400B2 (en) | 2003-09-03 | 2012-12-12 | デンドリセラピューティクス、インク. | Combined vaccine |
CN1609617B (en) * | 2003-09-29 | 2012-02-15 | 香港中文大学 | Composition and method for diagnosing and preventing serious acute respiratory syndrome(SARS) |
CN1893981B (en) | 2003-12-10 | 2011-03-23 | 坎基股份有限公司 | Methods and compositions for treatment of interferon-resistant tumors |
CN1559607A (en) | 2004-02-20 | 2005-01-05 | �й�ũҵ��ѧ | Nucleic acid and protein composition and its production method and application in immune regulation |
EP1742659B1 (en) | 2004-04-05 | 2013-03-13 | Pah Usa 15 Llc | Microfluidized oil-in-water emulsions and vaccine compositions |
GB0716992D0 (en) | 2007-08-31 | 2007-10-10 | Immune Targeting Systems Its L | Influenza antigen delivery vectors and constructs |
US7303881B2 (en) | 2004-04-30 | 2007-12-04 | Pds Biotechnology Corporation | Antigen delivery compositions and methods of use |
AU2005251403B2 (en) | 2004-06-07 | 2011-09-01 | Arbutus Biopharma Corporation | Cationic lipids and methods of use |
EP1778283A2 (en) * | 2004-06-30 | 2007-05-02 | ID Biomedical Corporation of Quebec | Vaccine compositions for treating coronavirus infection |
US20060051405A1 (en) | 2004-07-19 | 2006-03-09 | Protiva Biotherapeutics, Inc. | Compositions for the delivery of therapeutic agents and uses thereof |
DE102004057303A1 (en) | 2004-11-26 | 2006-06-01 | Merck Patent Gmbh | Stable crystal modifications of DOTAP chloride |
US8106092B2 (en) | 2004-12-13 | 2012-01-31 | Leo Laboratories Limited | Treatment of solid cancers |
WO2006065960A2 (en) * | 2004-12-14 | 2006-06-22 | Applera Corporation | Cationic liposomes comprising a charge neutral compound and a cationic phospholipid |
EP1838353A2 (en) | 2005-01-21 | 2007-10-03 | Introgen Therapeutics, Inc. | Topical administration permitting prolonged exposure of target cells to therapeutic and prophylactic nucleic acids |
WO2007149062A2 (en) | 2005-03-14 | 2007-12-27 | Board Of Regents, The University Of Texas System | Bioactive fus1 peptides and nanoprticle-polypeptide complexes |
WO2006097099A1 (en) | 2005-03-18 | 2006-09-21 | Widex A/S | Remote control system for a hearing aid |
MX2007012221A (en) | 2005-04-08 | 2008-03-18 | Argos Therapeutics Inc | Dendritic cell compositions and methods. |
WO2006113214A2 (en) | 2005-04-11 | 2006-10-26 | The Government Of The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services, Centers For Disease Control And Prevention | Vaccine against pandemic strains of influenza viruses |
US20060275777A1 (en) | 2005-06-03 | 2006-12-07 | Waelti Ernst R | Novel strategies for protein vaccines |
US20070059318A1 (en) | 2005-08-15 | 2007-03-15 | Balu-Iyer Sathy V | Lipid nano particulates containing antigens as cancer vaccines |
US20110305713A1 (en) | 2005-10-21 | 2011-12-15 | Medical College Of Georgia Research Institute, Inc | Methods and compositions to enhance vaccine efficacy by reprogramming regulatory t cells |
JP5215865B2 (en) | 2005-11-22 | 2013-06-19 | ノバルティス ヴァクシンズ アンド ダイアグノスティクス インコーポレイテッド | Norovirus and sapovirus antigens |
WO2007106073A2 (en) * | 2006-03-02 | 2007-09-20 | University Of Massachusetts | Modified pathogens for use as vaccines |
JP2007238559A (en) | 2006-03-10 | 2007-09-20 | Nagoya City Univ | Immature dendritic cell-activating agent and use thereof |
BRPI0710238A2 (en) | 2006-04-21 | 2011-08-09 | Transgene Sa | use of a composition |
US20080014251A1 (en) | 2006-07-14 | 2008-01-17 | Advanced Vascular Dynamics | Hemostatic compound and its use |
EP1894941A1 (en) | 2006-09-01 | 2008-03-05 | Institut Pasteur | Treatment of cervical carcinoma with a recombinant adenylate cyclase carrying HPV antigens |
US8129184B2 (en) | 2006-09-26 | 2012-03-06 | Cedars-Sinai Medical Center | Cancer stem cell antigen vaccines and methods |
US20100099745A1 (en) | 2006-10-18 | 2010-04-22 | Suryaprakash Sambhara | Enhancing disease resistance against rna viral infections with intracytoplasmic pathogen sensors |
KR100817024B1 (en) | 2006-11-09 | 2008-03-26 | 재단법인 목암생명공학연구소 | Composite for specifically transporting a nucleic acid or a drug to liver and pharmaceutical composition comprising the same |
US8877206B2 (en) | 2007-03-22 | 2014-11-04 | Pds Biotechnology Corporation | Stimulation of an immune response by cationic lipids |
CA2688346A1 (en) | 2007-05-23 | 2008-12-04 | Vical Incorporated | Compositions and methods for enhancing immune responses to vaccines |
US7832751B2 (en) * | 2007-06-29 | 2010-11-16 | Harley-Davidson Motor Company Group, Inc. | Rear fender assembly |
AU2008318320B2 (en) | 2007-11-02 | 2014-11-27 | The Johns Hopkins University | Multitype HPV peptide compositions and methods for treatment or prevention of human papillomavirus infection |
CN102137675A (en) | 2008-04-17 | 2011-07-27 | Pds生物科技公司 | Stimulation of an immune response by enantiomers of cationic lipids |
CA2724408A1 (en) | 2008-05-19 | 2009-11-26 | The University Of North Carolina At Chapel Hill | Methods and compositions comprising novel cationic lipids |
US20100086584A1 (en) | 2008-09-18 | 2010-04-08 | Juvaris Biotherapeutics, Inc. | VACCINE COMPOSITIONS OF M2e, HA0 AND BM2 MULTIPLE ANTIGENIC PEPTIDES |
JP2012519484A (en) | 2009-03-06 | 2012-08-30 | マウント・シナイ・スクール・オヴ・メディシン | Live attenuated influenza virus vaccine containing a microRNA response element |
DK2429585T3 (en) | 2009-05-15 | 2018-07-30 | Irx Therapeutics Inc | VACCINE IMMUNOTHERAPY |
WO2011109294A1 (en) | 2010-03-01 | 2011-09-09 | Dicerna Pharmaceuticals, Inc. | Lipid delivery formulations |
CN102939103A (en) | 2010-03-30 | 2013-02-20 | 西奈山医学院 | Influenza virus vaccines and uses thereof |
KR101773643B1 (en) | 2010-04-28 | 2017-08-31 | 교와 핫꼬 기린 가부시키가이샤 | Cationic lipid |
AU2012286690A1 (en) | 2011-07-27 | 2014-03-06 | Baylor Research Institute | Dendritic cell (DC)-vaccine therapy for pancreatic cancer |
PL2755680T3 (en) | 2011-09-12 | 2019-03-29 | Pds Biotechnology Corporation | Particulate vaccine formulations |
EP2638896A1 (en) | 2012-03-14 | 2013-09-18 | Bioneer A/S | Cationic liposomal drug delivery system for specific targeting of human cd14+ monocytes in whole blood |
EP2639299A1 (en) | 2012-03-16 | 2013-09-18 | Invectys | Universal cancer peptides derived from telomerase |
EP3632464A1 (en) | 2012-06-15 | 2020-04-08 | PDS Biotechnology Corporation | Cationic lipid vaccine compositions and methods of use |
US20150250872A1 (en) | 2012-09-21 | 2015-09-10 | Frank Bedu-Addo | Vaccine compositions and methods of use |
UA120595C2 (en) | 2013-08-21 | 2020-01-10 | Куревак Аг | Composition and vaccine for treating lung cancer |
JP6788500B2 (en) | 2013-10-23 | 2020-11-25 | アメリカ合衆国 | HLA-A24 Agonist Epitope and Composition of MUC1-C Cancer Protein and Method of Use |
CN104189897A (en) | 2014-05-21 | 2014-12-10 | 深圳先进技术研究院 | Preparation method for dendritic cells capable of loading antigens efficiently |
CA2979493A1 (en) | 2015-03-16 | 2016-09-22 | Max-Delbruck-Centrum Fur Molekulare Medizin In Der Helmholtz-Gemeinschaft | Method of detecting new immunogenic t cell epitopes and isolating new antigen-specific t cell receptors by means of an mhc cell library |
CN105920599B (en) | 2015-09-17 | 2020-02-11 | 武汉生物制品研究所有限责任公司 | Vaccine using cationic liposome DOTAP as adjuvant and preparation method thereof |
US11612652B2 (en) | 2015-11-13 | 2023-03-28 | Pds Biotechnology Corporation | Lipids as synthetic vectors to enhance antigen processing and presentation ex-vivo in dendritic cell therapy |
KR20190073416A (en) | 2016-10-05 | 2019-06-26 | 피디에스 바이오테크놀러지 코퍼레이션 | Novel HPV16 HLA-unrestricted T-cell vaccines, compositions and methods for their use |
US20180221475A1 (en) | 2016-10-05 | 2018-08-09 | Pds Biotechnology Corporation | Methods to alter the tumor microenvironment for effective cancer immunotherapy |
TW201924722A (en) | 2017-12-05 | 2019-07-01 | 美商Pds生技公司 | Methods and compositions comprising cationic lipids for stimulating type I interferon genes |
CN111217918B (en) * | 2020-03-04 | 2020-11-10 | 中山大学 | Novel coronavirus S protein double-region subunit nano vaccine based on 2, 4-dioxotetrahydropteridine synthase |
-
2009
- 2009-04-14 CN CN200980121761XA patent/CN102137675A/en active Pending
- 2009-04-14 PL PL09733034T patent/PL2276495T3/en unknown
- 2009-04-14 CA CA2721366A patent/CA2721366C/en active Active
- 2009-04-14 CN CN201811312211.3A patent/CN110075113A/en active Pending
- 2009-04-14 BR BRPI0910464-0A patent/BRPI0910464B1/en active IP Right Grant
- 2009-04-14 JP JP2011505132A patent/JP5971945B2/en active Active
- 2009-04-14 US US12/988,236 patent/US9789129B2/en active Active
- 2009-04-14 ES ES09733034T patent/ES2712505T3/en active Active
- 2009-04-14 RU RU2010146655/15A patent/RU2530555C2/en active
- 2009-04-14 WO PCT/US2009/040500 patent/WO2009129227A1/en active Application Filing
- 2009-04-14 EP EP09733034.4A patent/EP2276495B1/en active Active
- 2009-04-14 AU AU2009236306A patent/AU2009236306B2/en active Active
- 2009-04-17 TW TW098112878A patent/TWI449534B/en active
-
2010
- 2010-10-14 IL IL208713A patent/IL208713A/en active IP Right Grant
-
2017
- 2017-09-12 US US15/702,063 patent/US10702541B2/en active Active
-
2020
- 2020-06-12 US US16/899,763 patent/US11801257B2/en active Active
-
2021
- 2021-03-25 US US17/212,051 patent/US20210308157A1/en active Pending
Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2001057068A1 (en) * | 2000-02-01 | 2001-08-09 | The Austin Research Institute | Mucin-1 derived antigens and their use in immunotherapy |
WO2003003985A2 (en) * | 2001-07-06 | 2003-01-16 | Sloan-Kettering Institute For Cancer Research | Polyvalent conjugate vaccine for cancer |
WO2004089413A1 (en) * | 2003-04-14 | 2004-10-21 | Mallen Huang | Nucleotide and cellular vaccine composition |
WO2005000889A1 (en) * | 2003-06-05 | 2005-01-06 | The Government Of The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services | Immunogenic peptides for the treatment of prostate and breast cancer |
Also Published As
Publication number | Publication date |
---|---|
RU2530555C2 (en) | 2014-10-10 |
EP2276495A1 (en) | 2011-01-26 |
JP5971945B2 (en) | 2016-08-17 |
BRPI0910464A2 (en) | 2018-03-27 |
US20180015114A1 (en) | 2018-01-18 |
TW201000124A (en) | 2010-01-01 |
US20200330491A1 (en) | 2020-10-22 |
EP2276495A4 (en) | 2013-05-15 |
CN102137675A (en) | 2011-07-27 |
JP2011518170A (en) | 2011-06-23 |
EP2276495B1 (en) | 2018-11-21 |
IL208713A0 (en) | 2010-12-30 |
AU2009236306B2 (en) | 2015-04-02 |
TWI449534B (en) | 2014-08-21 |
US9789129B2 (en) | 2017-10-17 |
BRPI0910464B1 (en) | 2021-08-10 |
PL2276495T3 (en) | 2019-05-31 |
IL208713A (en) | 2016-10-31 |
US10702541B2 (en) | 2020-07-07 |
WO2009129227A1 (en) | 2009-10-22 |
RU2010146655A (en) | 2012-05-27 |
CA2721366A1 (en) | 2009-10-22 |
CA2721366C (en) | 2017-06-06 |
ES2712505T3 (en) | 2019-05-13 |
CN110075113A (en) | 2019-08-02 |
US20110110972A1 (en) | 2011-05-12 |
AU2009236306A1 (en) | 2009-10-22 |
US11801257B2 (en) | 2023-10-31 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US20210308157A1 (en) | Stimulation of an immune response by enantiomers of cationic lipids | |
US20220031650A1 (en) | Stimulation of an immune response by cationic lipids | |
US20080131455A1 (en) | Antigen Delivery Compositions And Methods Of Use | |
US11638753B2 (en) | Lipids as synthetic vectors to enhance antigen processing and presentation ex-vivo in dendritic cell therapy |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
AS | Assignment |
Owner name: PDS BIOTECHNOLOGY CORPORATION, NEW JERSEY Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:TONEY, KENYA;CONN, GREGORY;BEDU-ADDO, FRANK;SIGNING DATES FROM 20090327 TO 20090406;REEL/FRAME:055713/0161 Owner name: PDS BIOTECHNOLOGY CORPORATION, NEW JERSEY Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNOR:UNIVERSITY OF NORTH CAROLINA, CHAPEL HILL;REEL/FRAME:055713/0009 Effective date: 20090403 Owner name: UNIVERSITY OF NORTH CAROLINA, CHAPEL HILL, NORTH CAROLINA Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:VASIEVICH, ELIZABETH;CHEN, WEIHSU;HUANG, LEAF;SIGNING DATES FROM 20090327 TO 20090401;REEL/FRAME:055712/0926 |
|
STPP | Information on status: patent application and granting procedure in general |
Free format text: DOCKETED NEW CASE - READY FOR EXAMINATION |
|
STPP | Information on status: patent application and granting procedure in general |
Free format text: NON FINAL ACTION MAILED |
|
STPP | Information on status: patent application and granting procedure in general |
Free format text: RESPONSE TO NON-FINAL OFFICE ACTION ENTERED AND FORWARDED TO EXAMINER |
|
STPP | Information on status: patent application and granting procedure in general |
Free format text: NON FINAL ACTION MAILED |
|
STPP | Information on status: patent application and granting procedure in general |
Free format text: FINAL REJECTION MAILED |
|
STPP | Information on status: patent application and granting procedure in general |
Free format text: DOCKETED NEW CASE - READY FOR EXAMINATION |
|
STPP | Information on status: patent application and granting procedure in general |
Free format text: NON FINAL ACTION MAILED |
|
STPP | Information on status: patent application and granting procedure in general |
Free format text: RESPONSE TO NON-FINAL OFFICE ACTION ENTERED AND FORWARDED TO EXAMINER |
|
STPP | Information on status: patent application and granting procedure in general |
Free format text: FINAL REJECTION MAILED |