WO2011055324A1 - Synhetic immunogenic glycoconjugate for melanoma immunotherapy - Google Patents
Synhetic immunogenic glycoconjugate for melanoma immunotherapy Download PDFInfo
- Publication number
- WO2011055324A1 WO2011055324A1 PCT/IB2010/055003 IB2010055003W WO2011055324A1 WO 2011055324 A1 WO2011055324 A1 WO 2011055324A1 IB 2010055003 W IB2010055003 W IB 2010055003W WO 2011055324 A1 WO2011055324 A1 WO 2011055324A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- formula
- compound
- carrier
- anyone
- adjuvant
- Prior art date
Links
- 230000002163 immunogen Effects 0.000 title claims abstract description 10
- 201000001441 melanoma Diseases 0.000 title abstract description 19
- 238000009169 immunotherapy Methods 0.000 title description 7
- 150000001875 compounds Chemical class 0.000 claims abstract description 38
- 206010028980 Neoplasm Diseases 0.000 claims abstract description 18
- 230000003053 immunization Effects 0.000 claims abstract description 14
- 238000002649 immunization Methods 0.000 claims abstract description 14
- 238000003745 diagnosis Methods 0.000 claims abstract description 3
- 238000004393 prognosis Methods 0.000 claims abstract description 3
- 210000004027 cell Anatomy 0.000 claims description 35
- 239000002671 adjuvant Substances 0.000 claims description 20
- 150000002270 gangliosides Chemical class 0.000 claims description 8
- 230000015572 biosynthetic process Effects 0.000 claims description 7
- 239000000412 dendrimer Substances 0.000 claims description 7
- 229920000736 dendritic polymer Polymers 0.000 claims description 7
- 239000002105 nanoparticle Substances 0.000 claims description 7
- 102000004169 proteins and genes Human genes 0.000 claims description 7
- 108090000623 proteins and genes Proteins 0.000 claims description 7
- 239000000203 mixture Substances 0.000 claims description 6
- 238000003786 synthesis reaction Methods 0.000 claims description 6
- 150000002596 lactones Chemical class 0.000 claims description 5
- 239000002502 liposome Substances 0.000 claims description 5
- 238000000034 method Methods 0.000 claims description 5
- 125000006850 spacer group Chemical group 0.000 claims description 5
- 229910052717 sulfur Inorganic materials 0.000 claims description 5
- 229910019142 PO4 Inorganic materials 0.000 claims description 4
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical compound C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 claims description 4
- VTJUKNSKBAOEHE-UHFFFAOYSA-N calixarene Chemical class COC(=O)COC1=C(CC=2C(=C(CC=3C(=C(C4)C=C(C=3)C(C)(C)C)OCC(=O)OC)C=C(C=2)C(C)(C)C)OCC(=O)OC)C=C(C(C)(C)C)C=C1CC1=C(OCC(=O)OC)C4=CC(C(C)(C)C)=C1 VTJUKNSKBAOEHE-UHFFFAOYSA-N 0.000 claims description 4
- 210000004443 dendritic cell Anatomy 0.000 claims description 4
- 239000003814 drug Substances 0.000 claims description 4
- 239000010931 gold Substances 0.000 claims description 4
- 229910052737 gold Inorganic materials 0.000 claims description 4
- 239000010452 phosphate Substances 0.000 claims description 4
- 231100000699 Bacterial toxin Toxicity 0.000 claims description 3
- 201000009906 Meningitis Diseases 0.000 claims description 3
- SMWDFEZZVXVKRB-UHFFFAOYSA-N Quinoline Chemical compound N1=CC=CC2=CC=CC=C21 SMWDFEZZVXVKRB-UHFFFAOYSA-N 0.000 claims description 3
- 125000002777 acetyl group Chemical group [H]C([H])([H])C(*)=O 0.000 claims description 3
- 239000000688 bacterial toxin Substances 0.000 claims description 3
- 150000001720 carbohydrates Chemical group 0.000 claims description 3
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 claims description 3
- 125000002887 hydroxy group Chemical group [H]O* 0.000 claims description 3
- 239000004615 ingredient Substances 0.000 claims description 3
- 239000002245 particle Substances 0.000 claims description 3
- 239000008194 pharmaceutical composition Substances 0.000 claims description 3
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 claims description 3
- 230000002265 prevention Effects 0.000 claims description 3
- 125000006239 protecting group Chemical group 0.000 claims description 3
- 125000003236 benzoyl group Chemical group [H]C1=C([H])C([H])=C(C([H])=C1[H])C(*)=O 0.000 claims description 2
- 125000001797 benzyl group Chemical group [H]C1=C([H])C([H])=C(C([H])=C1[H])C([H])([H])* 0.000 claims description 2
- 150000002678 macrocyclic compounds Chemical class 0.000 claims description 2
- 229920001542 oligosaccharide Polymers 0.000 claims description 2
- 150000002482 oligosaccharides Chemical class 0.000 claims description 2
- 125000001181 organosilyl group Chemical group [SiH3]* 0.000 claims description 2
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 claims description 2
- 239000000126 substance Substances 0.000 claims description 2
- 125000004178 (C1-C4) alkyl group Chemical group 0.000 claims 1
- 241001529936 Murinae Species 0.000 claims 1
- 150000001412 amines Chemical group 0.000 claims 1
- 238000010171 animal model Methods 0.000 claims 1
- 125000003588 lysine group Chemical group [H]N([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])(N([H])[H])C(*)=O 0.000 claims 1
- 230000000630 rising effect Effects 0.000 claims 1
- -1 GM3 lactone Chemical class 0.000 abstract description 28
- 239000000427 antigen Substances 0.000 abstract description 14
- 102000036639 antigens Human genes 0.000 abstract description 14
- 108091007433 antigens Proteins 0.000 abstract description 14
- 239000004480 active ingredient Substances 0.000 abstract description 4
- 208000029742 colonic neoplasm Diseases 0.000 abstract description 2
- 229960005486 vaccine Drugs 0.000 abstract description 2
- 206010009944 Colon cancer Diseases 0.000 abstract 1
- 239000006228 supernatant Substances 0.000 description 15
- 125000005647 linker group Chemical group 0.000 description 13
- 238000002965 ELISA Methods 0.000 description 11
- 210000004408 hybridoma Anatomy 0.000 description 11
- 239000000243 solution Substances 0.000 description 10
- 238000012360 testing method Methods 0.000 description 10
- OKKJLVBELUTLKV-MZCSYVLQSA-N Deuterated methanol Chemical compound [2H]OC([2H])([2H])[2H] OKKJLVBELUTLKV-MZCSYVLQSA-N 0.000 description 8
- 241000699666 Mus <mouse, genus> Species 0.000 description 8
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 6
- 241000699670 Mus sp. Species 0.000 description 6
- 230000001173 tumoral effect Effects 0.000 description 6
- 125000004429 atom Chemical group 0.000 description 5
- 230000004927 fusion Effects 0.000 description 5
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 4
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 4
- 239000002202 Polyethylene glycol Substances 0.000 description 4
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 4
- 210000001072 colon Anatomy 0.000 description 4
- 229920001223 polyethylene glycol Polymers 0.000 description 4
- 239000007787 solid Substances 0.000 description 4
- AVWQQPYHYQKEIZ-UHFFFAOYSA-K trisodium;2-dodecylbenzenesulfonate;3-dodecylbenzenesulfonate;4-dodecylbenzenesulfonate Chemical compound [Na+].[Na+].[Na+].CCCCCCCCCCCCC1=CC=C(S([O-])(=O)=O)C=C1.CCCCCCCCCCCCC1=CC=CC(S([O-])(=O)=O)=C1.CCCCCCCCCCCCC1=CC=CC=C1S([O-])(=O)=O AVWQQPYHYQKEIZ-UHFFFAOYSA-K 0.000 description 4
- 101100217502 Caenorhabditis elegans lgg-3 gene Proteins 0.000 description 3
- 229940125898 compound 5 Drugs 0.000 description 3
- 238000002330 electrospray ionisation mass spectrometry Methods 0.000 description 3
- 125000000816 ethylene group Chemical group [H]C([H])([*:1])C([H])([H])[*:2] 0.000 description 3
- 238000001704 evaporation Methods 0.000 description 3
- 230000008020 evaporation Effects 0.000 description 3
- 238000003818 flash chromatography Methods 0.000 description 3
- 210000000987 immune system Anatomy 0.000 description 3
- 230000005847 immunogenicity Effects 0.000 description 3
- 230000002018 overexpression Effects 0.000 description 3
- 239000000741 silica gel Substances 0.000 description 3
- 229910002027 silica gel Inorganic materials 0.000 description 3
- 239000002904 solvent Substances 0.000 description 3
- GLGNXYJARSMNGJ-VKTIVEEGSA-N (1s,2s,3r,4r)-3-[[5-chloro-2-[(1-ethyl-6-methoxy-2-oxo-4,5-dihydro-3h-1-benzazepin-7-yl)amino]pyrimidin-4-yl]amino]bicyclo[2.2.1]hept-5-ene-2-carboxamide Chemical compound CCN1C(=O)CCCC2=C(OC)C(NC=3N=C(C(=CN=3)Cl)N[C@H]3[C@H]([C@@]4([H])C[C@@]3(C=C4)[H])C(N)=O)=CC=C21 GLGNXYJARSMNGJ-VKTIVEEGSA-N 0.000 description 2
- 238000001644 13C nuclear magnetic resonance spectroscopy Methods 0.000 description 2
- 238000005160 1H NMR spectroscopy Methods 0.000 description 2
- 238000011725 BALB/c mouse Methods 0.000 description 2
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 2
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 2
- 239000004472 Lysine Substances 0.000 description 2
- 238000005481 NMR spectroscopy Methods 0.000 description 2
- 206010035226 Plasma cell myeloma Diseases 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 description 2
- 238000002835 absorbance Methods 0.000 description 2
- 125000000217 alkyl group Chemical group 0.000 description 2
- 229940098773 bovine serum albumin Drugs 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 229940125758 compound 15 Drugs 0.000 description 2
- OPTASPLRGRRNAP-UHFFFAOYSA-N cytosine Chemical compound NC=1C=CNC(=O)N=1 OPTASPLRGRRNAP-UHFFFAOYSA-N 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 150000002148 esters Chemical class 0.000 description 2
- CCGKOQOJPYTBIH-UHFFFAOYSA-N ethenone Chemical compound C=C=O CCGKOQOJPYTBIH-UHFFFAOYSA-N 0.000 description 2
- 235000019439 ethyl acetate Nutrition 0.000 description 2
- UYTPUPDQBNUYGX-UHFFFAOYSA-N guanine Chemical compound O=C1NC(N)=NC2=C1N=CN2 UYTPUPDQBNUYGX-UHFFFAOYSA-N 0.000 description 2
- 229910052735 hafnium Inorganic materials 0.000 description 2
- 230000028993 immune response Effects 0.000 description 2
- 229920002521 macromolecule Polymers 0.000 description 2
- 201000000050 myeloid neoplasm Diseases 0.000 description 2
- 239000013642 negative control Substances 0.000 description 2
- 229940046166 oligodeoxynucleotide Drugs 0.000 description 2
- 239000000825 pharmaceutical preparation Substances 0.000 description 2
- 230000004962 physiological condition Effects 0.000 description 2
- 239000011541 reaction mixture Substances 0.000 description 2
- 239000011734 sodium Substances 0.000 description 2
- 210000004989 spleen cell Anatomy 0.000 description 2
- 238000010189 synthetic method Methods 0.000 description 2
- 229940104230 thymidine Drugs 0.000 description 2
- AOSZTAHDEDLTLQ-AZKQZHLXSA-N (1S,2S,4R,8S,9S,11S,12R,13S,19S)-6-[(3-chlorophenyl)methyl]-12,19-difluoro-11-hydroxy-8-(2-hydroxyacetyl)-9,13-dimethyl-6-azapentacyclo[10.8.0.02,9.04,8.013,18]icosa-14,17-dien-16-one Chemical compound C([C@@H]1C[C@H]2[C@H]3[C@]([C@]4(C=CC(=O)C=C4[C@@H](F)C3)C)(F)[C@@H](O)C[C@@]2([C@@]1(C1)C(=O)CO)C)N1CC1=CC=CC(Cl)=C1 AOSZTAHDEDLTLQ-AZKQZHLXSA-N 0.000 description 1
- KXLXLSGGQYPWPE-UHFFFAOYSA-N 1-azidohexan-1-ol Chemical compound CCCCCC(O)N=[N+]=[N-] KXLXLSGGQYPWPE-UHFFFAOYSA-N 0.000 description 1
- WDBQJSCPCGTAFG-QHCPKHFHSA-N 4,4-difluoro-N-[(1S)-3-[4-(3-methyl-5-propan-2-yl-1,2,4-triazol-4-yl)piperidin-1-yl]-1-pyridin-3-ylpropyl]cyclohexane-1-carboxamide Chemical compound FC1(CCC(CC1)C(=O)N[C@@H](CCN1CCC(CC1)N1C(=NN=C1C)C(C)C)C=1C=NC=CC=1)F WDBQJSCPCGTAFG-QHCPKHFHSA-N 0.000 description 1
- YRNWIFYIFSBPAU-UHFFFAOYSA-N 4-[4-(dimethylamino)phenyl]-n,n-dimethylaniline Chemical compound C1=CC(N(C)C)=CC=C1C1=CC=C(N(C)C)C=C1 YRNWIFYIFSBPAU-UHFFFAOYSA-N 0.000 description 1
- WHYHCPIPOSTZRU-UHFFFAOYSA-N 6-azidohexan-1-ol Chemical compound OCCCCCCN=[N+]=[N-] WHYHCPIPOSTZRU-UHFFFAOYSA-N 0.000 description 1
- 108010042708 Acetylmuramyl-Alanyl-Isoglutamine Proteins 0.000 description 1
- 241000193830 Bacillus <bacterium> Species 0.000 description 1
- 108010071134 CRM197 (non-toxic variant of diphtheria toxin) Proteins 0.000 description 1
- KXDHJXZQYSOELW-UHFFFAOYSA-M Carbamate Chemical compound NC([O-])=O KXDHJXZQYSOELW-UHFFFAOYSA-M 0.000 description 1
- BVKZGUZCCUSVTD-UHFFFAOYSA-L Carbonate Chemical compound [O-]C([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-L 0.000 description 1
- 229940126657 Compound 17 Drugs 0.000 description 1
- YXQDRIRSAHTJKM-IMJSIDKUSA-N Cys-Ser Chemical compound SC[C@H](N)C(=O)N[C@@H](CO)C(O)=O YXQDRIRSAHTJKM-IMJSIDKUSA-N 0.000 description 1
- BWGNESOTFCXPMA-UHFFFAOYSA-N Dihydrogen disulfide Chemical compound SS BWGNESOTFCXPMA-UHFFFAOYSA-N 0.000 description 1
- 108010067770 Endopeptidase K Proteins 0.000 description 1
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 1
- 108091006905 Human Serum Albumin Proteins 0.000 description 1
- 102000008100 Human Serum Albumin Human genes 0.000 description 1
- 239000004201 L-cysteine Substances 0.000 description 1
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 description 1
- 229930182816 L-glutamine Natural products 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 241001092142 Molina Species 0.000 description 1
- AFVCSFKPYWLSIO-UHFFFAOYSA-N OCCCNC(C(CCNNC(C1CCC(CN(C(C=C2)=O)C2=O)CC1)=O)C(NCCCO)=O)=O Chemical compound OCCCNC(C(CCNNC(C1CCC(CN(C(C=C2)=O)C2=O)CC1)=O)C(NCCCO)=O)=O AFVCSFKPYWLSIO-UHFFFAOYSA-N 0.000 description 1
- 101710116435 Outer membrane protein Proteins 0.000 description 1
- 108700020962 Peroxidase Proteins 0.000 description 1
- 102000003992 Peroxidases Human genes 0.000 description 1
- 241001454523 Quillaja saponaria Species 0.000 description 1
- 235000009001 Quillaja saponaria Nutrition 0.000 description 1
- WQDUMFSSJAZKTM-UHFFFAOYSA-N Sodium methoxide Chemical compound [Na+].[O-]C WQDUMFSSJAZKTM-UHFFFAOYSA-N 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000000890 antigenic effect Effects 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 125000000852 azido group Chemical group *N=[N+]=[N-] 0.000 description 1
- 238000001815 biotherapy Methods 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 239000012267 brine Substances 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 230000007910 cell fusion Effects 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 229940125810 compound 20 Drugs 0.000 description 1
- 230000021615 conjugation Effects 0.000 description 1
- 230000008878 coupling Effects 0.000 description 1
- 238000010168 coupling process Methods 0.000 description 1
- 238000005859 coupling reaction Methods 0.000 description 1
- 230000009260 cross reactivity Effects 0.000 description 1
- 239000012228 culture supernatant Substances 0.000 description 1
- CSEGCHWAMVIXSA-UHFFFAOYSA-L cyclopenta-1,3-diene;hafnium(4+);dichloride Chemical compound [Cl-].[Cl-].[Hf+4].C=1C=C[CH-]C=1.C=1C=C[CH-]C=1 CSEGCHWAMVIXSA-UHFFFAOYSA-L 0.000 description 1
- 229940104302 cytosine Drugs 0.000 description 1
- 238000010511 deprotection reaction Methods 0.000 description 1
- 239000000032 diagnostic agent Substances 0.000 description 1
- 229940039227 diagnostic agent Drugs 0.000 description 1
- 125000002228 disulfide group Chemical group 0.000 description 1
- 238000009510 drug design Methods 0.000 description 1
- 239000000839 emulsion Substances 0.000 description 1
- 239000000706 filtrate Substances 0.000 description 1
- 125000001153 fluoro group Chemical class F* 0.000 description 1
- 150000002339 glycosphingolipids Chemical class 0.000 description 1
- 230000013595 glycosylation Effects 0.000 description 1
- 238000006206 glycosylation reaction Methods 0.000 description 1
- JAXFJECJQZDFJS-XHEPKHHKSA-N gtpl8555 Chemical compound OC(=O)C[C@H](N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](C(C)C)C(=O)N1CCC[C@@H]1C(=O)N[C@H](B1O[C@@]2(C)[C@H]3C[C@H](C3(C)C)C[C@H]2O1)CCC1=CC=C(F)C=C1 JAXFJECJQZDFJS-XHEPKHHKSA-N 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 230000006058 immune tolerance Effects 0.000 description 1
- 238000003365 immunocytochemistry Methods 0.000 description 1
- 238000012744 immunostaining Methods 0.000 description 1
- 229960001438 immunostimulant agent Drugs 0.000 description 1
- 239000003022 immunostimulating agent Substances 0.000 description 1
- 230000003308 immunostimulating effect Effects 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 238000003780 insertion Methods 0.000 description 1
- 230000037431 insertion Effects 0.000 description 1
- 238000007912 intraperitoneal administration Methods 0.000 description 1
- 238000001990 intravenous administration Methods 0.000 description 1
- 238000010253 intravenous injection Methods 0.000 description 1
- 108010045069 keyhole-limpet hemocyanin Proteins 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 230000000873 masking effect Effects 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 210000002752 melanocyte Anatomy 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 239000002207 metabolite Substances 0.000 description 1
- 229910052751 metal Inorganic materials 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- 229910044991 metal oxide Inorganic materials 0.000 description 1
- 150000004706 metal oxides Chemical class 0.000 description 1
- 150000002739 metals Chemical class 0.000 description 1
- 230000001394 metastastic effect Effects 0.000 description 1
- 206010061289 metastatic neoplasm Diseases 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- BSOQXXWZTUDTEL-ZUYCGGNHSA-N muramyl dipeptide Chemical compound OC(=O)CC[C@H](C(N)=O)NC(=O)[C@H](C)NC(=O)[C@@H](C)O[C@H]1[C@H](O)[C@@H](CO)O[C@@H](O)[C@@H]1NC(C)=O BSOQXXWZTUDTEL-ZUYCGGNHSA-N 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
- 239000012074 organic phase Substances 0.000 description 1
- 102000013415 peroxidase activity proteins Human genes 0.000 description 1
- 108040007629 peroxidase activity proteins Proteins 0.000 description 1
- 239000003208 petroleum Substances 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- UEZVMMHDMIWARA-UHFFFAOYSA-M phosphonate Chemical compound [O-]P(=O)=O UEZVMMHDMIWARA-UHFFFAOYSA-M 0.000 description 1
- 229920000962 poly(amidoamine) Polymers 0.000 description 1
- 229920000136 polysorbate Polymers 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 125000001436 propyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- 108010030416 proteoliposomes Proteins 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 239000012047 saturated solution Substances 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 239000012679 serum free medium Substances 0.000 description 1
- 238000004904 shortening Methods 0.000 description 1
- QRUBYZBWAOOHSV-UHFFFAOYSA-M silver trifluoromethanesulfonate Chemical compound [Ag+].[O-]S(=O)(=O)C(F)(F)F QRUBYZBWAOOHSV-UHFFFAOYSA-M 0.000 description 1
- 229910000030 sodium bicarbonate Inorganic materials 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 229910000162 sodium phosphate Inorganic materials 0.000 description 1
- 239000001488 sodium phosphate Substances 0.000 description 1
- HPALAKNZSZLMCH-UHFFFAOYSA-M sodium;chloride;hydrate Chemical compound O.[Na+].[Cl-] HPALAKNZSZLMCH-UHFFFAOYSA-M 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 210000000952 spleen Anatomy 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 125000004434 sulfur atom Chemical group 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 150000007970 thio esters Chemical class 0.000 description 1
- 150000003568 thioethers Chemical class 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 238000004448 titration Methods 0.000 description 1
- 239000003053 toxin Substances 0.000 description 1
- 231100000765 toxin Toxicity 0.000 description 1
- RIOQSEWOXXDEQQ-UHFFFAOYSA-N triphenylphosphine Chemical compound C1=CC=CC=C1P(C=1C=CC=CC=1)C1=CC=CC=C1 RIOQSEWOXXDEQQ-UHFFFAOYSA-N 0.000 description 1
- RYFMWSXOAZQYPI-UHFFFAOYSA-K trisodium phosphate Chemical compound [Na+].[Na+].[Na+].[O-]P([O-])([O-])=O RYFMWSXOAZQYPI-UHFFFAOYSA-K 0.000 description 1
- 210000004881 tumor cell Anatomy 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
- A61K47/549—Sugars, nucleosides, nucleotides or nucleic acids
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/44—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material not provided for elsewhere, e.g. haptens, metals, DNA, RNA, amino acids
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
- A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
- A61K47/643—Albumins, e.g. HSA, BSA, ovalbumin or a Keyhole Limpet Hemocyanin [KHL]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
- A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
- A61K47/646—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent the entire peptide or protein drug conjugate elicits an immune response, e.g. conjugate vaccines
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
Definitions
- the invention relates to the synthetic chemistry domain and in particular the synthesis of immunogenic glycoconjugate derivatives, their synthetic methods and their use for active and passive immunotherapy or for the prognosis of cancers characterized by the overexpression of GM3 ganglioside and GM 3 lactone.
- GM 3 ganglioside 1 ( Figure 1 ), a glycosphingolipid found in essentially all types of mammalians' cells and tissues, is the major ganglioside in normal melanocytes. Ganglioside 1 and especially its metabolite, the GM 3 lactone 2 (Fig.
- Aim of the present invention is to provide a synthetic derivative of GM3 lactone which is hydrolytically stable under physiological conditions and good to raise the immune system to elicit antibodies able to recognize antigens express on tumoral cells which present overexpression of GM3 lactone (i.e. melanoma cells or colon tumoral cells). Description and shortenings
- CRM Cross-Reacting Material 197 (non toxic mutation of difteric toxin)
- BSA Bovine Serum Albumin
- cBSA cationized Bovine Serum Albumin
- HSA Human Serum Albumin
- MPL Membrane-Patched Proteoliposomes
- Pal3CysSer N-a-palmitoyl-S-[2,3-bis(palmitoyloxy)-(2RS)-propyl]"Cys-Ser"OH
- Pal3Cys N-D-palmitoyl-S-[2,3-bis(palmitoyloxy)-(2RS)"propyl]-L-cysteine
- QS-2 Quillaja Saponaria Molina based immunological adjiuvant
- CpG Cytosine linked to a Guanine by a phosphate bond
- X is O, NH, S, CH2;
- L is a linker that is a divalent spacer
- A is an adjuvant or a carrier that is an immunogenic molecule or particle ; and where bonds between linker and carbohydratic portion and between linker and carrier are covalent.
- the present invention relates to compounds of formula (I) for their use as drugs, that is as antigens in active immunization.
- the invention concerns pharmaceutical preparations containing at least one compound of formula (I) as above described and at least another pharmaceutically acceptable ingredient.
- the present invention concerns antibodies obtainable by immunization of mice treated with a compound of formula (1) as above described. These antibodies are specific, that is specifically bind cells which overexpress GM3 ganglioside and GM3 lactone and therefore for a further aspect the invention relates to antibodies as above described, as drugs, that is as active ingredient in pharmaceutical preparations for the passive immunization and as diagnostic and/or prognostic devices.
- FIGURE 1 Structure of the GM3 ganglioside 1 , GM 3 lactone 2, thioether mimetic 3 and GMi.
- FIGURE 2 Synthesis of a glycoconjugate of formula (I) according to the invention, in particular of compound 5 KLH-conjugate.
- FIGURE 3 ELISA tests performed for isotype determination.
- FIGURE 5 Melanoma cell lines expressing (A375) and not expressing (WM-266- 4) GM3 antigen. Signals were obtained using hybridoma population IV1 :4C5C4 supernatant.
- the linker L is a divalent spacer with a structure suitable for maintaining a correct distance between the carbohydratic portion and the carrier/adjuvant avoiding the undesired masking of their antigenic regions: as first choice the linker presents a chemical structure suitable to space the carbohyd ratio portion and the carrier by 7-30 atoms or better by 12-16 atoms when the adjuvant/carrier A is a biomacromoiecule or by 9-30 atoms when the adjuvant/carrier A is a metallic nanoparticle.
- the adjuvant/carrier A is an immunogenic species i.e. able to induce an immune response when administered to an animal subject;
- the latter carrier/adjuvant A can be selected among a macromolecule, a macrocycle, a dendrimer, a liposome, a nanoparticle, an oligosaccharide featuring from two to five monosaccharidic units or cells like dendritic cells (DC).
- DC dendritic cells
- Macromolecule means a molecule containing at least 100 atoms, among these are included the bio-macromoiecules like proteins, lipids and polisaccharides.
- Macrocyctes means compound featuring a seven- (or larger-) membered ring like calixarenes, phthalocyanines, cyclodestrins, porfirines and cyclo-oligopepdides.
- Dendrimers of GO, G1 and G2 are included.
- PAMAM polyethylenamine
- Nanoparticies are particles whose dimensions range between 1 and 100 nm; they are formed by metals or metal oxides selected among Au, Fe, Ti.
- A is a carrier with a modest immunogenicity
- a compound of formula (I) can be associated with another suitable adjuvant able to increase the immunogenicity of the carrier.
- object of the invention is a composition comprising a compound of formula (I) as above described and another adjuvant B able to enhance the immunogenity of carrier A.
- the carrier/adjuvant A and B is selected in the group consisting of KLH, CRM, OMP, BSA, HAS, BCG, MLP, MDP, Pal3CysSer, Pal3Cys, QS21 , trealosio- 6,6'-dimicolato, synthetic CpG , CpG-DNA, Nisseria Meningitis, tetanic toxin, bacterial toxin, liposome, gold nanoparticies, Fe(ll) oxide nanoparticies, calixarenes, dendrimers, RAFT, DC, as single species or combined.
- linkage between linker and carrier/adjuvant is chosen in the group of amidic, aminic, iminic, ethereal, thioethereal, disulfide, ester, thioester, phosphate, phosphonate, ureidic, thioureidic, carbamate, carbonate, where at least one atom belongs to the protein.
- the linker is bond to the nanoparticies with a pseudo-covalent linkage though a sulfur atom or a disulfide group.
- A is a protein the linker presents in preference the structure R1-Z-W1-R2-
- A is a protein
- L is a bivalent spacer of structure (CH2)n-NH-CO-(CH2)m-CO-Y where n and/or m are integer ranging from 2 to 8 and Y is an NH which is part of A.
- a preferred compound is the compound 5 (Fig. 2) of structure (I) where X is O, linker L is -(CH2)6NHCO-(CH2)4-CO- and carrier A is KLH linked through lysine - NH2 residues to -COOH residues of the linker.
- G is F, Br or CI; SR, SAr or trichloroacetimide, phosphate where R is Et or C1 -C4 alkyl; Ar is Ph, substituted Ph t Pyridine, chinoline or other aromatic heterocycles; P independently are suitable hydroxyls protecting groups selected among those protecting groups known to those skilled in the art and such as and preferably acetyl, benzoyl, silyl and benzyl; prepared by known synthetic methods for the insertion of a linker L or L-A.
- a compound of formula (II) has groups P all the same.
- a compound of formula (II) as above described is coupled to the linker only it will follow, after optional suitable deprotection of hydroxyl groups, coupling, through known synthetic strategies, with a carrier/adjuvant A,
- Fig, 2 the synthetic scheme for the compound 5 obtained from the compound 15, i.e. a compound of formula (II) where G is F.
- the compound 15 was prepared as already reported in Toma et al. Che BioChem, 2007, 8, 1646.
- the ⁇ -amino glicosyl derivative 4 (Fig. 2) was prepared from the fluoro derivative15 by treatment with azido hexanol under Ley's glycosylation conditions.
- TNBS trinitrobenzensulfonic
- Compounds of formula (l) as above described are artificial antigens, mimetic of the G 3 lactone and therefore useful as active ingredient for vaccines against tumoral cells over-expressing GM3 ganglioside and GM3 lactone, in particular they are useful in the treatment and/or the prevention and/or the diagnosis of melanoma and cancers of colon.
- Compounds of formula (I) as above described can also be useful as diagnostic devices for tumors whose cells over-express GM 3 ganglioside and GM3 lactone.
- the compound in the embodiment 5 when is administrated to mice, produced a strong immune response and the antibodies obtained are able to bind to the GM3 ganglioside overexpressed on melanoma tumoral cells or colon tumoral cell lines.
- mice were immunized with the KLH conjugate.
- Milstein procedure G., Milstein, C, "Preparation of monoclonal antibodies: strategies and procedures" in Methods Enzymol., 73(Pt B), 3-46, (1981 )
- HAT hypoxanthine-aminopterin-thymidine
- NSO mouse myeloma ceil line
- the ensuing hybridomas were screened by testing supernatants for binding to GM3 ganglioside 1 by enzyme-linked immunosorbent assay (ELISA).
- ELISA enzyme-linked immunosorbent assay
- the supernatant displaying the highest absorbance (IV1 :4C5C4) was then chosen for further analyses.
- An ELISA test aimed at isotype determination was also performed.
- the IV1 :4C5C4 supernatant turned out to be composed by two isotypes: IgM and lgG3, with the IgM quantitatively prevailing on the lgG3 (Fig, 3).
- glycoconjugate 5 is a real immunogen "in vivo" since it is capable to induce the formation of specific GM3 antibodies. These antibodies cross react with the native antigen in EL1SA tests as well as with melanoma cell lines.
- the invention relates to specific antibodies against the artificial antigen of formula (I).
- the present invention relates to hybridomas obtainable by immunization of mice with compounds of formula (I).
- the invention relates to chimeric or humanized antibodies including the variable or hyper variable sequences of the monoclonal antibodies achievable by immunization of mice with compounds of formula (I).
- the above said antibodies can be useful active ingredient in pharmaceutical compositions for passive immunization in particular for the immunization against tumours whose cells overexpress GM3 ganglioside and G 3 lactone and in preferred embodiments are useful in the treatment of melanoma and colon cancers.
- the above said antibodies can also be useful as diagnostic agents and/or prognostic of the above mentioned tumours.
- tumours which cells overexpress GM3 ganglioside and GM3 lactone
- antigen-antibody commercially available antibody anti-Glv
- immunocytochemical tests result to be positive to said overexpression.
- the present invention could be better understood upon reading of the following preferred embodiments.
- Thioether-protein ratios were evaluated by determination of free lysine amino groups before and after conjugation by titration with trinitrobenzensulfonic acid. Immunization and fusion.
- mice Two Female, 8 weeks old, BALB/c mice (Ce.S.A.L., Firenze) were immunized with intraperitoneal (mouse A) and intravenous (mouse B) injections with antigen-KLH conjugate solution four times, twice a week.
- the antigen was injected as a 1 :1 (v/v) emulsion in Freund's complete adjuvant (mouse A) to a final volume of 0.2 mL and as a 1 :1 (v/v) solution in sterile PBS (mouse B) to a total volume of 0.2 mL.
- mice were killed by cervical dislocation, spleens were harvested, and 1x10 s cells were collected and used for each fusion.
- Spleen cells were fused with a hypoxanthine-aminopterin-thymidine (HAT) sensitive mouse myeloma cell line, NSO, by the polyethylene glycol (PEG) method (29).
- NSO cells were maintained in DMEM with 10% Fetatclone I (HyClone) and split 1 :2 the day before fusion.
- Hybridoma supernatants were screened for binding to GM 3 ganglioside by enzyme-linked immunosorbent assay (ELISA).
- ELISA enzyme-linked immunosorbent assay
- the ELISA 96 well plate was first treated with 100 pL of GM 3 (30 pg/mL in 100% ethanol) overnight at room temperature.
- 300 pL of PBS containing 0.05% Tween (TPBS) and 3% BSA were added for 1 h, and washed three times with TPBS.
- Hybridoma culture supernatants 100 pL/well were added to the coated plate and incubated at room temperature for 2 h.
- the plate was washed and incubated with secondary antibody (peroxidase-labeled anti-mouse, 1 :500 in TPBS) for 1 h.
- secondary antibody peroxidase-labeled anti-mouse, 1 :500 in TPBS
- TMB peroxidase-labeled anti-mouse
- ICC was performed on B16F1 melanoma.
- Cultured B 6 melanoma cells were grown in DMEM supplemented with 10% FCS and 2 mM L-glutamine. Cells were mounted on polyiysine-coated slides and fixed with formaline solution. After blocking endogenous peroxidases, cells were treated with proteinase K (Roche; 5 g/ml in PBS) and UltraVBIock solution (LabVision) and then incubated with the primary antibody (hybridomas supernatant) and with the anti-GM3 antibody (M2590) diluted 1 :100 in PBS-UitraVBIock (10:1 v/v) overnight at 4°C. Immunostaining was carried out using a commercially available kit (PicTure Plus kit; Zymed). For the negative control hybridomas medium replacing the primary antibody was used.
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Medicinal Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- Veterinary Medicine (AREA)
- Epidemiology (AREA)
- Organic Chemistry (AREA)
- Immunology (AREA)
- Biochemistry (AREA)
- Virology (AREA)
- Genetics & Genomics (AREA)
- Biophysics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Peptides Or Proteins (AREA)
- Medicinal Preparation (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Polysaccharides And Polysaccharide Derivatives (AREA)
Abstract
The present invention refers to compounds of formula (I) as useful melanoma vaccines' active ingredients. As a matter of fact, compounds of formula (1) are immunogenic artificial antigens, mimetics of GM3 and GM3 lactone, well known tumor associated antigens (TAAs). In addition, the present invention refers to specific antibodies for compounds of formula (I) which are able to bind to the TAAs. The hereinabove reported compounds and antibodies are therefore potentially useful respectively for the active and passive immunization against tumors overexpressing GM3 ganglioside, in a preferred embodiment melanoma and colon cancer, and for their diagnosis and prognosis.
Description
SYNHETIC IMMUNOGENIC GLYCOCONJUGATE FOR MELANOMA IMMUNOTHERAPY
Field of the invention
The invention relates to the synthetic chemistry domain and in particular the synthesis of immunogenic glycoconjugate derivatives, their synthetic methods and their use for active and passive immunotherapy or for the prognosis of cancers characterized by the overexpression of GM3 ganglioside and GM3 lactone.
State of the art
Immunotherapy against tumours and against melanoma in particular has a long history but only recently this therapeutic approach has found a reliable scientific rationale. The main target of this biological therapy consists of teaching the tumour bearing host's immune system how to recognize antigens express on tumoral cells and destroy them, without damaging healthy cells. The success of this therapy greatly depends on the choice of the antigens responsible for the growth and proliferation of the tumour. GM3 ganglioside 1 (Figure 1 ), a glycosphingolipid found in essentially all types of mammalians' cells and tissues, is the major ganglioside in normal melanocytes. Ganglioside 1 and especially its metabolite, the GM3 lactone 2 (Fig. 1 ), are overexpressed in melanoma cells with metastatic potential. These antigens are wrongly recognized as "self by patient's immune system and are potential targets for the immunotherapy of this tumours. Although more immunogenic than GM3 ganglioside 1 , GM3 lactone 2 failed as immunostimulant because unstable under physiological conditions, in unsutable to be used in immunotherapy as melanoma's associate antigen. Toma et al. ChemBioChem, 2007, 8, 1646 describes the rational design and the synthesis of the hydrolytically stable mimetic 3 (Fig. 1 ) which is remarkably simpler than the native 2. The mimetic 3, as reported after its publication, has been demonstrated to be scarcely immunogen.
Aim of the present invention is to provide a synthetic derivative of GM3 lactone which is hydrolytically stable under physiological conditions and good to raise the immune system to elicit antibodies able to recognize antigens express on tumoral cells which present overexpression of GM3 lactone (i.e. melanoma cells or colon tumoral cells).
Description and shortenings
KLH: Keyhole Limpet Hemocyanin
CRM: Cross-Reacting Material 197 (non toxic mutation of difteric toxin)
OMP: Outer Membrane Protein
BSA: Bovine Serum Albumin
cBSA: cationized Bovine Serum Albumin
HSA: Human Serum Albumin
BCG Bacillus Ca!mette and Guerin
MPL: Membrane-Patched Proteoliposomes
MDP: Muramyl DiPeptide
Pal3CysSer: N-a-palmitoyl-S-[2,3-bis(palmitoyloxy)-(2RS)-propyl]"Cys-Ser"OH
Pal3Cys: N-D-palmitoyl-S-[2,3-bis(palmitoyloxy)-(2RS)"propyl]-L-cysteine
QS-2 : Quillaja Saponaria Molina based immunological adjiuvant
CpG: Cytosine linked to a Guanine by a phosphate bond
RAFT; Regioselectively Addressable Functional Template
ODN: OligoDeoxyNucleotides
Summary of the invention
The present invention solves the hereinbefore mentioned problems thanks to compounds of formula (I)
(I)
wherein:
X is O, NH, S, CH2;
L is a linker that is a divalent spacer;
A is an adjuvant or a carrier that is an immunogenic molecule or particle ;
and where bonds between linker and carbohydratic portion and between linker and carrier are covalent.
Surprisingly, compound of formula (I) as above reported, revealed to be immonogen. Therefore, for an aspect, the present invention relates to compounds of formula (I) for their use as drugs, that is as antigens in active immunization. For another aspect, the invention concerns pharmaceutical preparations containing at least one compound of formula (I) as above described and at least another pharmaceutically acceptable ingredient.
For a further aspect, the present invention concerns antibodies obtainable by immunization of mice treated with a compound of formula (1) as above described. These antibodies are specific, that is specifically bind cells which overexpress GM3 ganglioside and GM3 lactone and therefore for a further aspect the invention relates to antibodies as above described, as drugs, that is as active ingredient in pharmaceutical preparations for the passive immunization and as diagnostic and/or prognostic devices.
Brief description of the figures
FIGURE 1 - Structure of the GM3 ganglioside 1 , GM3 lactone 2, thioether mimetic 3 and GMi.
FIGURE 2 - Synthesis of a glycoconjugate of formula (I) according to the invention, in particular of compound 5 KLH-conjugate.
FIGURE 3 - ELISA tests performed for isotype determination. The hybridoma IV1 :4C5C4 supernatant results to be composed of two isotypes: IgM and lgG3. FIGURE 4 - ELISA test to compare antibody immunoreactivity towards either GM3- or GMi-ganglioside.
FIGURE 5 - Melanoma cell lines expressing (A375) and not expressing (WM-266- 4) GM3 antigen. Signals were obtained using hybridoma population IV1 :4C5C4 supernatant.
Detailed description of the invention
In the scope of the present invention, the linker L is a divalent spacer with a structure suitable for maintaining a correct distance between the carbohydratic portion and the carrier/adjuvant avoiding the undesired masking of their antigenic regions: as first choice the linker presents a chemical structure suitable to space
the carbohyd ratio portion and the carrier by 7-30 atoms or better by 12-16 atoms when the adjuvant/carrier A is a biomacromoiecule or by 9-30 atoms when the adjuvant/carrier A is a metallic nanoparticle.
In the scope of the present invention, the adjuvant/carrier A is an immunogenic species i.e. able to induce an immune response when administered to an animal subject; the latter carrier/adjuvant A can be selected among a macromolecule, a macrocycle, a dendrimer, a liposome, a nanoparticle, an oligosaccharide featuring from two to five monosaccharidic units or cells like dendritic cells (DC).
Macromolecule means a molecule containing at least 100 atoms, among these are included the bio-macromoiecules like proteins, lipids and polisaccharides.
Macrocyctes means compound featuring a seven- (or larger-) membered ring like calixarenes, phthalocyanines, cyclodestrins, porfirines and cyclo-oligopepdides. Dendrimers of GO, G1 and G2 are included. PAMAM (polyethylenamine) is a good example of dendrimer.
Nanoparticies are particles whose dimensions range between 1 and 100 nm; they are formed by metals or metal oxides selected among Au, Fe, Ti.
Optionally, if A is a carrier with a modest immunogenicity a compound of formula (I) can be associated with another suitable adjuvant able to increase the immunogenicity of the carrier. In this case object of the invention is a composition comprising a compound of formula (I) as above described and another adjuvant B able to enhance the immunogenity of carrier A.
Preferably, the carrier/adjuvant A and B is selected in the group consisting of KLH, CRM, OMP, BSA, HAS, BCG, MLP, MDP, Pal3CysSer, Pal3Cys, QS21 , trealosio- 6,6'-dimicolato, synthetic CpG , CpG-DNA, Nisseria Meningitis, tetanic toxin, bacterial toxin, liposome, gold nanoparticies, Fe(ll) oxide nanoparticies, calixarenes, dendrimers, RAFT, DC, as single species or combined.
When the carrier/adjuvant is proteic the linkage between linker and carrier/adjuvant is chosen in the group of amidic, aminic, iminic, ethereal, thioethereal, disulfide, ester, thioester, phosphate, phosphonate, ureidic, thioureidic, carbamate, carbonate, where at least one atom belongs to the protein. When the carrier A is a nanoparticies the linker is bond to the nanoparticies with a pseudo-covalent linkage though a sulfur atom or a disulfide group.
When A is a protein the linker presents in preference the structure R1-Z-W1-R2-
W2 where R1 and R2 independently from each other are a C2-C8- alkyl, C2-C8- alkenyi, C5-C7-cycloalkyl, 1-4-phenyl; Z is O, S, NH; W1 is missing, C=0, C=NH,
S; W2 is missing, C=0, C=NH, S, 1 ,4-succinamide. The above reported alkyls are in preference linear.
Examples of possible linkers are:
Side to be
linked to protein
linked to antigen t
linked to rotein
More preferred are those compounds wherein:
X = 0;
A is a protein;
L is a bivalent spacer of structure (CH2)n-NH-CO-(CH2)m-CO-Y where n and/or m are integer ranging from 2 to 8 and Y is an NH which is part of A.
A preferred compound is the compound 5 (Fig. 2) of structure (I) where X is O, linker L is -(CH2)6NHCO-(CH2)4-CO- and carrier A is KLH linked through lysine - NH2 residues to -COOH residues of the linker.
Compounds of formula (I) as above described can be obtained starting from compounds of structure
where:
G is F, Br or CI; SR, SAr or trichloroacetimide, phosphate where R is Et or C1 -C4 alkyl; Ar is Ph, substituted Pht Pyridine, chinoline or other aromatic heterocycles; P independently are suitable hydroxyls protecting groups selected among those protecting groups known to those skilled in the art and such as and preferably acetyl, benzoyl, silyl and benzyl; prepared by known synthetic methods for the insertion of a linker L or L-A.
In a preferred embodiment a compound of formula (II) has groups P all the same. In the event a compound of formula (II) as above described is coupled to the linker only it will follow, after optional suitable deprotection of hydroxyl groups, coupling, through known synthetic strategies, with a carrier/adjuvant A,
In a particular, as example embodiment, is herein reported (Fig, 2) the synthetic scheme for the compound 5 obtained from the compound 15, i.e. a compound of formula (II) where G is F. The compound 15 was prepared as already reported in Toma et al. Che BioChem, 2007, 8, 1646. The ω-amino glicosyl derivative 4 (Fig. 2) was prepared from the fluoro derivative15 by treatment with azido hexanol under Ley's glycosylation conditions. The pseudo-oo azido derivative 17 obtained after removal of acetyl groups, was reduced under Staudinger's reaction conditions to give 4 which was reacted with the bis-p nitrophenyl adipic ester 18 (Wu, X., Ling, C.-C., Bundle, D. Org. Lett. 2004, 6, 4407) to form the activated carboxylic derivative 19 (87% over two steps) (Scheme 3). Finally, 19 was conjugated to the proteic carrier KLH. The KLH hioether conjugate 5 was prepared by mixing 19 with KLH (phosphate buffer, room temperature, 20h). Saccharide molecule/ KLH loading was determined by trinitrobenzensulfonic (TNBS) test (Habeeb, F.S.A. Anal. Biochem. 1966, 14, 328) to be ~ 27%.
Compounds of formula (l) as above described are artificial antigens, mimetic of the G 3 lactone and therefore useful as active ingredient for vaccines against tumoral cells over-expressing GM3 ganglioside and GM3 lactone, in particular they are useful in the treatment and/or the prevention and/or the diagnosis of melanoma and cancers of colon.
Compounds of formula (I) as above described can also be useful as diagnostic devices for tumors whose cells over-express GM3 ganglioside and GM3 lactone.
Surprisingly indeed, the compound in the embodiment 5, when is administrated to mice, produced a strong immune response and the antibodies obtained are able to bind to the GM3 ganglioside overexpressed on melanoma tumoral cells or colon tumoral cell lines.
To determine the imrnunogenicity of the mimetic 5, BALB/c mice were immunized with the KLH conjugate. Following Milstein procedure (Galfre, G., Milstein, C, "Preparation of monoclonal antibodies: strategies and procedures" in Methods Enzymol., 73(Pt B), 3-46, (1981 )) for the production of monoclonal antibodies, after sequential immunizations spleen cells from immunized mice were collected and fused with a hypoxanthine-aminopterin-thymidine (HAT) sensitive mouse myeloma ceil line (NSO). The ensuing hybridomas were screened by testing supernatants for binding to GM3 ganglioside 1 by enzyme-linked immunosorbent assay (ELISA). The supernatant displaying the highest absorbance (IV1 :4C5C4) was then chosen for further analyses. An ELISA test aimed at isotype determination was also performed. The IV1 :4C5C4 supernatant turned out to be composed by two isotypes: IgM and lgG3, with the IgM quantitatively prevailing on the lgG3 (Fig, 3).
The immunoreactivity of the IV1 :4C5C4 supernatant towards either GM3- or GMr ganglioside was then screened through an ELISA test. Results (Figure 4) clearly indicated that the supernatant indeed binds GM3 whereas no appreciable binding to GMi was observed, Finally, to evaluate the cross-reactivity of the polyclonal serum for GM3 expressed on melanoma cells, immunocytochemistry with B16F1 melanoma cells treated with the hybridoma population IV1 :4C5C4 supernatant was performed. Reference samples were treated with the commercially available anti-GM3 monoclonal antibody (M2590) (positive control) and with an hybridoma supernatant resulted negative to the first ELISA test (taken as negative control). Results indicated a clear positivity of the cell line tested evidenced by the brown staining. Note that there is no detectable signal at the nuclear level but only at the cytoplasmatic membrane level. It is evident (Fig. 5) that the IV1 :4C5C4 supernatant is able to recognize the GM3 ganglioside and the GM3 lactone expressed on melanoma cells better than the antibody anti-G 3 2590.
All together, data herein reported indicate that the glycoconjugate 5 is a real immunogen "in vivo" since it is capable to induce the formation of specific GM3 antibodies. These antibodies cross react with the native antigen in EL1SA tests as well as with melanoma cell lines.
Two main points must be considered: compounds of formula (I) as hereinabove described, and in particular the KLH-conjugate 5, overcomes the problem of the low immunogenicity typical of gangliosides and droops the immunotolerance generally preserved by the GM3 ganglioside covalently linked to carriers such as the KLH (Bitten, R.J., et l, Oncology Report 2002, 9, 267; Gabri, M.R.; et al. Clin. Cancer Res. 2006, 12, 7092).
These results prompt to further clonings of the IVV.4C5C4 hybridoma and purification of the IV1 :4C5C4 supernatant to obtain the monoclonal antibody anti GM3 and GM3 lactone to be used in the future for the passive immunotherapy of tumours whose cells overexpress G 3 ganglioside and GM3 ganglioside lactone, such as melanoma cells and colon tumour cell lines.
In addition the invention relates to specific antibodies against the artificial antigen of formula (I). For an aspect the present invention relates to hybridomas obtainable by immunization of mice with compounds of formula (I).
For another aspect the invention relates to chimeric or humanized antibodies including the variable or hyper variable sequences of the monoclonal antibodies achievable by immunization of mice with compounds of formula (I).
The above said antibodies can be useful active ingredient in pharmaceutical compositions for passive immunization in particular for the immunization against tumours whose cells overexpress GM3 ganglioside and G 3 lactone and in preferred embodiments are useful in the treatment of melanoma and colon cancers.
The above said antibodies can also be useful as diagnostic agents and/or prognostic of the above mentioned tumours.
The term "tumours whose cells overexpress GM3 ganglioside and GM3 lactone" as used herein means those tumours which analyzed by antigen-antibody (commercially available antibody anti-Glv ) and by immunocytochemical tests result to be positive to said overexpression.
The present invention could be better understood upon reading of the following preferred embodiments.
Experimental section
Synthesis of compound 20:
To a mixture of 15 (160 mg, 0.25 mmol), bis(cyclopentadienyl)hafnium(IV) dichloride (Cp2HfCI2) (170 mg, 0.45 mmol), AgOTf (250 mg, 0.97 mmol) and 6- azido-1-hexanol (70 mg, 0.5 mmol) cooled to -40°C, dry CH2CI2 (30 mL) was added. After 40 min at -40°C the solution was neutralized with NEt3 and filtered through a pad of Celite® and the filtrate was washed with a saturated solution of NaHC03 (1 x 50 mL) then with brine (1 x 50 mL). The organic phase was dried over Na2S04 and concentrated to dryness to give 200 mg of crude. The crude was purified by flash column chromatography on silica gel (petroleum ether/EtOAc 1 :1 ) to give 20 (150 mg, 80%), as a glassy solid. 1H NMR (400MHz, CDCI3): δ 5.37 (d, 1 H, Jd-e = 2.4 Hz), 5.32 (bs, 1 H), 5.26-5.21 (m, 1 H), 5.12-5.09 (m,1 H), 4.97 (s, 1 H), 4.50-4.46 (m, 1 H), 4.25-4. 6 (m, 2H), 4.18-4.14 (X part of an AXY system, 1 H, JXA = 6.4 Hz, JXY= .6 Hz), 4. 1-4.06 (Y part of an AXY system, 1 H, JYA = 7.6 Hz, Jyx = 1 .6 Hz), 3.96 (ad, H, J5-6 = 10.0 Hz), 3.75-3.69 (m, 1 H), 3.53-3.47 (m H), 3.24 (t, 2H, J = 7.2 Hz), 3.02-2.98 (A part of an AB system, 1 H, JAB = 13.0 Hz), 2.97- 2.94 (B part of an AB system, 1 H, JBA = 13.0 Hz), 2.15 (s, 3H), 2.10 (s, 3H), 2.07 (s, 3H), 2.02 (s, 3H), 2.00 (s, 3H), 1.99-1.98 (m 2H), 1.95 (s, 3H), 1.62-1.56 (m, 4H), 1.43-1.36 (m, 4H). 3C NMR (50MHz, CDCI3): δ 170.8, 170.2, 170.1 , 169.6, 169.5 (2C), 137.4, 109.0, 95.7, 93.0, 68.6, 68.0, 67.4, 67.3, 66.4, 64.4 (2C), 62.0, 61.7, 51.3, 33.8, 33.6, 29.3, 28.7, 26.4, 25.7, 20.7 (3C), 20.6 (3C). [a]D 23 +22.31 (c 1.09, CH2CI2). Elem. Anal, for C^t^O^S: Calc. C 50.59, H 5.97, N 5.53. Found: C 50.62, H 6.01 , N 5.42; ESI-MS 782.2 [M+Naf, 798.2 [M+K]+.
Synthesis of compound 17:
To a stirred solution of 20 (120 mg, 0.158 mmol) in CH3OH (3.2 mL), MeONa (2 mg, 0.032 mmol) was added. The mixture was stirred at RT for 2.5 h then the pH adjusted to neutrality with HCI (10% in MeOH). Evaporation of the solvent under vacuum gave a crude which was purified by flash chromatography on silica gel (EtOAc/CH3OH 5:1 ) to give 17 (72 mg, 90%) as glassy solid. H NMR (400MHz, CD3OD): δ 4.97 (s, 1 H), 4.17-4.12 (m, 1 H), 4.09-4.06 (m, 1 H), 4.02 (dd, 1 H, J4.3 = 3.2 Hz, J4.5 = 1 .2 Hz), 3.94 (dd, 1 H, Js.e = 9.6 Hz, J5-4 = 1 .2 Hz), 3.84-3.79 (A part of an ABX2 system, 1 H, JA.B = 10.0 Hz, JA.X = 6.4 Hz), 3.77-3.62 (m, 6H), 3.52- 3.47 (B part of an ABX2 system, 1 H, JB.A = 10.0 Hz, JB.x - 6.4 Hz), 3.31-3.28 (m, 2H), 3.05-3.01 (A part of an AB system, H, JAB = 13.0 Hz), 2.99-2.96 (B part of an AB system, 1 H, JBA = 13.0 Hz), 1.94-1 .92 (m, 2H), 1 .64-1 .57 (m, 4H), 1.46-1 .41 (m, 4H). 13C NMR (50MHz, CD3OD): δ 141 .6, 106.4, 96.3, 92.3, 72.6, 70.9, 68.5, 67.8, 66.5, 66.3, 64.6, 62.8, 61 .1 , 51 .1 , 36.3, 33.4, 29.2, 28-6, 26.3, 25.6. [a]D 23 +51.64 (c 0.25, CHgOH). ESI-MS 530.3 [M+Na]+.
To a stirred solution of 17 (13.2 mg, 0.026 mmol) in a mixture of dry THF (1 mL) and dry DMF (0.1 mL), Ph3P (13.6 mg, 0.052 mmol) and Η2θ (2.3 μΐ, 0.13 mmol)
were added. The reaction mixture was warmed to 40 °C and stirred for 18 h. Evaporation of the solvent under vacuum gave the crude 4 which was used
To a stirred solution of 4 in dry DMF (1 mL), 18 (50 mg, 0.130 mmol) was added and the reaction mixture was stirred at RT for 6 h. Evaporation of the solvent under vacuum gave a crude which was purified by flash chromatography on silica gel (CH2CI2/CH3OH 2:1 ) to give 19 (16.5 mg, 87%) as glassy solid.
1H NMR (400MHz, CD3OD): δ 8.30-8.29 (ΑΑ' part of an AA'MM' system, 2H, JA.M = 4.8 Hz, Ph), 7.38-7.37 (MM' part of an AA'MM' system, 2H, JM-A = 4.8 Hz, Ph), 4.96 (s, 1 H, Ha), 4.15 (adt, 1 H, J3.4 = 2.8 Hz, J3- 2 = J3.2'= 8.8 Hz, H3), 4.09-4.05 (m, H, He), 4.03-4.02 (dd, 1 H, = 2.8 Hz, J4.5 = 0-8 Hz, H4), 3.94 (dd, H, J5.E = 9.6 Hz, Js. 4 - 0.8 Hz, H5), 3.82-3.77 (A part of an ABX2 system, 1 H, JA-B = 10-0 Hz, JA-X = 6.4 Hz, Hx), 3.76-3.61 (m, 6H, Hd, H6, H7, H7>, Hf, Hf), 3-51-3.45 (B part of an ABX2 system, 1 H, JB.A = 10.0 Hz, JB-x = 6.4 Hz, Ηχ·), 3.184 (t, 2H, J = 7.0 Hz, CH¾,), 3.03-3.00 (A part of an AB system, 1 H, JA.B = 3.0 Hz, Hi¾), 2.98-2.95 (B part of an AB system, 1 H, JB-A = 13.0 Hz, H ), 2.67 (t, 2H, J = 7.0 Hz, CH2CO), 2.25 (t, 2H, J = 7.0 Hz, CH2CO), 1.93-1.91 (m, 2H, H2, H2.), 1.78-1.72 (m, 4H, CH2CH2), 1.62-1.49 (m, 4H, CH2CH2), 1.44-1.33 (m, 4H, CH2CH2). 13C NMR (50MHz, CD3OD): 5 174.2, 171.1 , 155.7, 141.7, 124.7 (2C), 122.5 (2C), 106.4, 96.3, 92.3, 72.5, 70.8, 68.4, 67.8, 66.4, 66.2, 64.5, 62.7, 60.9, 38.9, 36.1 , 35.2, 33.2, 33.1 , 29.2, 28.9, 26.3, 25.5, 24.8, 23.8. Elem. Anal, for C-^eNaOisS: Calc. C 52.59, H 6.34, N 3.83. Found: C 52.79, H 6.61 , N 5.51 ; ESI-MS 753.3 [M+Naf.
Synthesis of compound 5:
To a solution of KLH (20 mg) in 20 mL of buffer (0.1 M sodium phosphate and 0.15 M sodium chloride, pH 7.2) 19 (15 mg, 0.020 mmol) was added and the mixture was stirred overnight at RT. The solution was lyophilized and the solid was dialysed to give 18.5 mg of glycoconjugate 5.
Thioether-protein ratios were evaluated by determination of free lysine amino groups before and after conjugation by titration with trinitrobenzensulfonic acid. Immunization and fusion.
Two Female, 8 weeks old, BALB/c mice (Ce.S.A.L., Firenze) were immunized with intraperitoneal (mouse A) and intravenous (mouse B) injections with antigen-KLH conjugate solution four times, twice a week. In the first immunization the antigen was injected as a 1 :1 (v/v) emulsion in Freund's complete adjuvant (mouse A) to a final volume of 0.2 mL and as a 1 :1 (v/v) solution in sterile PBS (mouse B) to a total volume of 0.2 mL. The second and third immunizations were performed in the way, except that an incomplete Freund's adjuvant was used in mouse A. The final immunization was given 3 days prior to cell fusion, through intravenous injection in both mouse A and B. On the day of fusion mice were killed by cervical dislocation, spleens were harvested, and 1x10s cells were collected and used for each fusion.
Spleen cells were fused with a hypoxanthine-aminopterin-thymidine (HAT) sensitive mouse myeloma cell line, NSO, by the polyethylene glycol (PEG) method (29). NSO cells were maintained in DMEM with 10% Fetatclone I (HyClone) and split 1 :2 the day before fusion. 1 x10s NSO cells were mixed in serum-free DMEM, centrifuged and, after removing the supernatant, were placed in a water bath at 37°C and 1 ml_ of pre-warmed PEG (Sigma-Aldrich) were added, "drop to drop". At the end of PEG addition, pre-warmed serum-free medium was added and the tube was centrifuged to remove supernatant. The fusion product was resuspended in DMEM containing 20% Fetalclone I and 1X HAT, and the ensuing cell suspension was plated in 24 well plates; cells were incubated at 37°C in a C02 incubator.
ELISA tests
Hybridoma supernatants were screened for binding to GM3 ganglioside by enzyme-linked immunosorbent assay (ELISA). The ELISA 96 well plate (Corning) was first treated with 100 pL of GM3 (30 pg/mL in 100% ethanol) overnight at room temperature. To minimize nonspecific adsorption 300 pL of PBS containing 0.05% Tween (TPBS) and 3% BSA were added for 1 h, and washed three times with TPBS. Hybridoma culture supernatants (100 pL/well) were added to the coated plate and incubated at room temperature for 2 h. Subsequently, the plate was washed and incubated with secondary antibody (peroxidase-labeled anti-mouse, 1 :500 in TPBS) for 1 h. The plate was washed three times with TPBS, then 00 pL tetramethylbenzidine (TMB, Sigma) were added to each well. The plate was further incubated for 5 min, and hence the reaction was stopped with HCI 0.5 M. Absorbance was measured using a microplate reader (ELX800, Bio-Tek Instruments, Inc.) at 450 nm.
Immunocitochemistry (ICC)
ICC was performed on B16F1 melanoma. Cultured B 6 melanoma cells were grown in DMEM supplemented with 10% FCS and 2 mM L-glutamine. Cells were mounted on polyiysine-coated slides and fixed with formaline solution. After blocking endogenous peroxidases, cells were treated with proteinase K (Roche; 5 g/ml in PBS) and UltraVBIock solution (LabVision) and then incubated with the primary antibody (hybridomas supernatant) and with the anti-GM3 antibody
(M2590) diluted 1 :100 in PBS-UitraVBIock (10:1 v/v) overnight at 4°C. Immunostaining was carried out using a commercially available kit (PicTure Plus kit; Zymed). For the negative control hybridomas medium replacing the primary antibody was used.
Claims
1. A compound of formula (l)
X is O, NH, S, CH2;
L is a linker that is a divalent spacer;
A is an adjuvant or carrier that is an immunogenic molecule or particle;
and wherein bonds between linker and saccharide moiety and between linker and carrier are of covalent type.
2. Compound of formula (I) according to claim 1 wherein the linker L presents a chemical structure suitable to space the saccharide moyety and the carrier by 7-30 atoms when the adjuvant/carrier A is a biomacromolecule or by 19-30 atoms when the adjuvant/carrier A is a metallic nanoparticle.
3. Compound according to anyone of claims 1-2 wherein said carrier/adjuvant A can be selected among a macromoiecule, a macrocycle, a dendrimer, a liposome, a nanoparticle, an oligosaccharide featuring from two to five monosaccharidic units or cells like dendritic cells.
4. Compound according to claim 3 wherein carrier/adjuvant A is selected in the group consisting of KLH, CRM, OMP, BSA, HAS, BCG, MLP, MDP, Pal3CysSer, Pal3Cys, QS21 , trealo$io-6,6'~dimicolato, synthetic CpG , CpG- DNA, Nisseria Meningitis, tetanic toxin, bacterial toxin, liposome, gold nanopartlcles, Fe(ll) oxide nanoparticles, calixarenes, dendrimers, RAFT, DC.
5. Compound according to anyone of claims 1 -4 wherein:
X is 0;
A is a protein;
L is a divalent spacer of formula (CH2)n-NH-CO-(CH2)m-CO-Y wherein n and m independently from each other are integer numbers in the range 2-8 and Y is NH coming from A.
6. Compound according to claims 5 wherein:
X is O;
L is -(CH2)6NHCO(CH2)4-CO-; and
A is KLH linked by means of amidic bond through the amine functions of the protein lysine residues and the carboxylic group of the linker.
7. Compound of formula (I) according to anyone of claims 1-6 for use as a medicament.
8. Compound of formula (I) according to anyone of claims 1-6 for use in the treatment and/or prevention of tumors whose cells hyper-express GM-3 ganglioside and/or GM-3 lactone.
9. A pharmaceutical composition containing at least a compound of formula (I) according to anyone of claims 1-6 and at least another pharmaceutically acceptable ingredient.
10. Composition according to claim 9 wherein is present a further carrier/adjuvant B selected in the group consisting of KLH, CRM, OMP, BSA, HAS, BCG, MLP, MDP, Pal3CysSer, Pal3Cys, QS21 , trealosio-6,6'-dimicolato, synthetic CpG , CpG-DNA, Nisseria Meningitis, tetanic toxin, bacterial toxin, liposome, gold nanoparticles, Fe(ll) oxide nanoparticles, calixarenes, dendrimers, RAFT, DC.
11. A process for the synthesis of compounds of formula (I) according to anyone rising the use of a compound of formula (II)
wherein
G is F, SR, SAr, trichloroacetimide, phosphate wherein R is Et, or C1-C4 alkyl; Ar is Ph, Ph substituted, pyridine, quinoline or other aromatic hetero-cycles; P each other independently are suitable hydroxy protecting groups chosen among those known to the man skilled in the art such as preferably acetyl, benzoyl, silyl or benzyl.
12. Specific antibodies against a compound of formula (I) according to anyone of claims 1-6.
3. Antibodies according to claim 12 obtainable by means of immunization of experimental animal of murine genus by means of administration of compounds of formula (I) according to anyone of claims 1 -6.
4. Antibodies according to any of claims 12-13 for use as medicament.
15. Antibodies according to any of claims 12-13 for use in the treatment and/or prevention of tumors whose cells hyper-express GM-3 ganglioside and/or GM- 3 lactone.
16. Antibodies according to any of claims 12-13 for use in diagnosis and/or prognosis of tumors whose cells hyper-express GM-3 ganglioside and/or GM- 3 lactone.
17. Pharmaceutical compositions containing at least an antibody according to anyone of claims 12-13 and at least another pharmaceutically acceptable ingredient.
Priority Applications (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
EP10798607A EP2496266A1 (en) | 2009-11-04 | 2010-11-04 | Synhetic immunogenic glycoconjugate for melanoma immunotherapy |
US13/505,844 US8680043B2 (en) | 2009-11-04 | 2010-11-04 | Synthetic immunogenic glycoconjugate for melanoma immunotherapy |
US14/183,072 US9255156B2 (en) | 2009-11-04 | 2014-02-18 | Immunogenic synthetic glycoconjugate for the immunotherapy of melanoma |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
IT000230A ITFI20090230A1 (en) | 2009-11-04 | 2009-11-04 | SYNTHETIC GLYCOCOCOUGATE IMMUNOUS USEFUL FOR THE MELANOMA IMMUNOTHERAPY. |
ITFI2009A000230 | 2009-11-04 |
Related Child Applications (2)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US13/505,844 A-371-Of-International US8680043B2 (en) | 2009-11-04 | 2010-11-04 | Synthetic immunogenic glycoconjugate for melanoma immunotherapy |
US14/183,072 Division US9255156B2 (en) | 2009-11-04 | 2014-02-18 | Immunogenic synthetic glycoconjugate for the immunotherapy of melanoma |
Publications (1)
Publication Number | Publication Date |
---|---|
WO2011055324A1 true WO2011055324A1 (en) | 2011-05-12 |
Family
ID=42110141
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/IB2010/055003 WO2011055324A1 (en) | 2009-11-04 | 2010-11-04 | Synhetic immunogenic glycoconjugate for melanoma immunotherapy |
Country Status (4)
Country | Link |
---|---|
US (2) | US8680043B2 (en) |
EP (1) | EP2496266A1 (en) |
IT (1) | ITFI20090230A1 (en) |
WO (1) | WO2011055324A1 (en) |
Family Cites Families (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
DE60331292D1 (en) * | 2003-05-14 | 2010-04-01 | Kenta Biotech Ag | Human monoclonal antibody specific for lipopolysaccharides (LPS) of the serotype IATS O6 of Pseudomonas aeruginosa |
-
2009
- 2009-11-04 IT IT000230A patent/ITFI20090230A1/en unknown
-
2010
- 2010-11-04 US US13/505,844 patent/US8680043B2/en not_active Expired - Fee Related
- 2010-11-04 EP EP10798607A patent/EP2496266A1/en not_active Withdrawn
- 2010-11-04 WO PCT/IB2010/055003 patent/WO2011055324A1/en active Application Filing
-
2014
- 2014-02-18 US US14/183,072 patent/US9255156B2/en not_active Expired - Fee Related
Non-Patent Citations (9)
Title |
---|
BITTON, R.J. ET AL., ONCOLOGY REPORT, vol. 9, 2002, pages 267 |
G. T. HERMANSON: "Bioconjugate Techniques", 1 January 1995, ACADEMIC PRESS, San Diego, article "Preparation of Hapten-Carrier Immunogen Conjugates", pages: 419 - 429, XP002627387 * |
GABRI, M.R. ET AL., CLIN. CANCER RES., vol. 12, 2006, pages 7092 |
GAIFRE, G.; MILSTEIN, C.: "Preparation of monoolonal antibodies: strategies and procedures", METHODS ENZYMOL., vol. 73, 1981, pages 3 - 46, XP009100436, DOI: doi:10.1016/0076-6879(81)73054-4 |
HABEEB, F.S.A., ANAL. BIOCHEM., vol. 14, 1966, pages 328 |
RISTORI S ET AL: "Structural study of liposomes loaded with a GM3 lactone analogue for the targeting of tumor epitopes", BIOCHIMICA ET BIOPHYSICA ACTA. BIOMEMBRANES, AMSTERDAM, NL LNKD- DOI:10.1016/J.BBAMEM.2009.10.005, vol. 1788, no. 12, 23 October 2008 (2008-10-23), pages 2518 - 2525, XP026764786, ISSN: 0005-2736, [retrieved on 20091023] * |
TOMA ET AL., CHEMBIOCHEM, vol. 8, 2007, pages 1646 |
TOMA ET AL.: "Synthesis, Conformational studies, Binding assessment and Liposome Insertion of a Thioether-Bridged Mimetic of the Antigen GM3 Ganglioside Lactone", CHEMBIOCHEM, vol. 8, 24 September 2007 (2007-09-24) - 24 September 2007 (2007-09-24), pages 1646 - 1649, XP002579807 * |
WU, X.; LING, C.-C.; BUNDLE, D., ORG. LETT., vol. 6, 2004, pages 4407 |
Also Published As
Publication number | Publication date |
---|---|
ITFI20090230A1 (en) | 2011-05-05 |
EP2496266A1 (en) | 2012-09-12 |
US20140161806A1 (en) | 2014-06-12 |
US8680043B2 (en) | 2014-03-25 |
US9255156B2 (en) | 2016-02-09 |
US20120263735A1 (en) | 2012-10-18 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN107889493B (en) | anti-CD 123 antibodies and conjugates and derivatives thereof | |
TWI237022B (en) | Novel acyl-dipeptide-like compounds bearing an accessory functional side chain spacer, a method for preparing the same and pharmaceutical compositions containing such products | |
AU2015305332B2 (en) | Novel glycan conjugates and use thereof | |
KR20200121317A (en) | Glypican 3 antibody and conjugates thereof | |
Danussi et al. | A newly generated functional antibody identifies Tn antigen as a novel determinant in the cancer cell–lymphatic endothelium interaction | |
US20220241423A1 (en) | Camptothecine antibody-drug conjugates and methods of use thereof | |
KR20180053599A (en) | Vaccines with higher carbohydrate antigen density and novel saponin adjuvant | |
CA1337403C (en) | Methods for the production of antibodies and induction of immune responses to tumor-associated gangliosides by immunization with ganglioside lactones | |
JP7204132B2 (en) | Conjugates of plasmin-cleavable anti-insoluble fibrin antibodies and drugs | |
EP3773735B1 (en) | Conjugate of cytotoxic drug and prodrug form of said conjugate | |
EP3795590A1 (en) | Complex having anti-human muc1 antibody fab fragment, peptide linker and/or ligand | |
Trabbic et al. | Production of a mouse monoclonal IgM antibody that targets the carbohydrate Thomsen-nouveau cancer antigen resulting in in vivo and in vitro tumor killing | |
EP2032164B1 (en) | Method for the preparation of specific antibodies against saccharidic antigens | |
WO2011158973A1 (en) | Novel antifibrin antibody | |
US20190345255A1 (en) | Monoclonal igm antibodies from entirely carbohydrate constructs | |
US8680043B2 (en) | Synthetic immunogenic glycoconjugate for melanoma immunotherapy | |
CN116761824B (en) | Engineered anti-TROP 2 antibodies and antibody-drug conjugates thereof | |
TW202333798A (en) | Antibody-drug conjugate targeting claudin 18.2 | |
CN116120381A (en) | Monophosphate A (MPLA) conjugated saccharide antigen Tn anti-tumor vaccine and application thereof | |
KR20230133294A (en) | Compounds or salts thereof, and antibodies obtained therefrom | |
CN115779075A (en) | Monophosphoryl ester A (MPLA) conjugated saccharide antigen STn anti-tumor vaccine and application thereof | |
EA043080B1 (en) | A CYTOTOXIC DRUG CONJUGATE AND A PRODRUG FORM OF THE SPECIFIED CONJUGATE | |
Lunghi | Immunotherapy of Tumours: Synthesis of a Mimetic of GM3 Ganglioside Antigen | |
Tong et al. | A synthetic GD2 carbohydrate tetramer vaccine elicits tumor-killing antibodies and is therapeutic against GD2+ tumors |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 10798607 Country of ref document: EP Kind code of ref document: A1 |
|
NENP | Non-entry into the national phase |
Ref country code: DE |
|
REEP | Request for entry into the european phase |
Ref document number: 2010798607 Country of ref document: EP |
|
WWE | Wipo information: entry into national phase |
Ref document number: 2010798607 Country of ref document: EP |
|
WWE | Wipo information: entry into national phase |
Ref document number: 13505844 Country of ref document: US |