CN112220931B - 用于肿瘤主动靶向治疗的亲和体-细胞毒素偶联物及其纳米颗粒、制备方法、应用 - Google Patents
用于肿瘤主动靶向治疗的亲和体-细胞毒素偶联物及其纳米颗粒、制备方法、应用 Download PDFInfo
- Publication number
- CN112220931B CN112220931B CN202011114498.6A CN202011114498A CN112220931B CN 112220931 B CN112220931 B CN 112220931B CN 202011114498 A CN202011114498 A CN 202011114498A CN 112220931 B CN112220931 B CN 112220931B
- Authority
- CN
- China
- Prior art keywords
- conjugate
- affibody
- cytotoxin
- tumor
- nanoparticle
- 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.)
- Active
Links
- 239000002105 nanoparticle Substances 0.000 title claims abstract description 83
- 239000002619 cytotoxin Substances 0.000 title claims abstract description 66
- 206010028980 Neoplasm Diseases 0.000 title claims abstract description 61
- 238000002360 preparation method Methods 0.000 title claims abstract description 12
- 238000002626 targeted therapy Methods 0.000 title claims description 17
- 230000008685 targeting Effects 0.000 claims abstract description 57
- 231100000599 cytotoxic agent Toxicity 0.000 claims abstract description 33
- 101710112752 Cytotoxin Proteins 0.000 claims abstract description 30
- 238000005859 coupling reaction Methods 0.000 claims abstract description 11
- 230000008878 coupling Effects 0.000 claims abstract description 9
- 238000010168 coupling process Methods 0.000 claims abstract description 9
- 230000002209 hydrophobic effect Effects 0.000 claims abstract description 4
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims description 58
- 239000003814 drug Substances 0.000 claims description 17
- 150000003384 small molecules Chemical class 0.000 claims description 16
- -1 cyano, acetyl Chemical group 0.000 claims description 9
- 125000003545 alkoxy group Chemical group 0.000 claims description 8
- 238000000034 method Methods 0.000 claims description 8
- 239000007864 aqueous solution Substances 0.000 claims description 7
- 125000004209 (C1-C8) alkyl group Chemical group 0.000 claims description 6
- 125000000217 alkyl group Chemical group 0.000 claims description 6
- 239000003960 organic solvent Substances 0.000 claims description 6
- 125000000008 (C1-C10) alkyl group Chemical group 0.000 claims description 4
- QXRSDHAAWVKZLJ-OXZHEXMSSA-N Epothilone B Natural products O=C1[C@H](C)[C@H](O)[C@@H](C)CCC[C@@]2(C)O[C@H]2C[C@@H](/C(=C\c2nc(C)sc2)/C)OC(=O)C[C@H](O)C1(C)C QXRSDHAAWVKZLJ-OXZHEXMSSA-N 0.000 claims description 4
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 4
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 claims description 4
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 claims description 4
- HESCAJZNRMSMJG-HGYUPSKWSA-N epothilone A Natural products O=C1[C@H](C)[C@H](O)[C@H](C)CCC[C@H]2O[C@H]2C[C@@H](/C(=C\c2nc(C)sc2)/C)OC(=O)C[C@H](O)C1(C)C HESCAJZNRMSMJG-HGYUPSKWSA-N 0.000 claims description 4
- QXRSDHAAWVKZLJ-PVYNADRNSA-N epothilone B Chemical compound C/C([C@@H]1C[C@@H]2O[C@]2(C)CCC[C@@H]([C@@H]([C@@H](C)C(=O)C(C)(C)[C@@H](O)CC(=O)O1)O)C)=C\C1=CSC(C)=N1 QXRSDHAAWVKZLJ-PVYNADRNSA-N 0.000 claims description 4
- 238000002560 therapeutic procedure Methods 0.000 claims description 4
- 239000002245 particle Substances 0.000 claims description 3
- 125000006552 (C3-C8) cycloalkyl group Chemical group 0.000 claims description 2
- GAWIXWVDTYZWAW-UHFFFAOYSA-N C[CH]O Chemical group C[CH]O GAWIXWVDTYZWAW-UHFFFAOYSA-N 0.000 claims description 2
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 claims description 2
- 150000001263 acyl chlorides Chemical group 0.000 claims description 2
- 239000012736 aqueous medium Substances 0.000 claims description 2
- 125000000000 cycloalkoxy group Chemical group 0.000 claims description 2
- ZMXDDKWLCZADIW-UHFFFAOYSA-N dimethylformamide Substances CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 claims description 2
- AMRJKAQTDDKMCE-UHFFFAOYSA-N dolastatin Chemical compound CC(C)C(N(C)C)C(=O)NC(C(C)C)C(=O)N(C)C(C(C)C)C(OC)CC(=O)N1CCCC1C(OC)C(C)C(=O)NC(C=1SC=CN=1)CC1=CC=CC=C1 AMRJKAQTDDKMCE-UHFFFAOYSA-N 0.000 claims description 2
- 229930188854 dolastatin Natural products 0.000 claims description 2
- 150000003883 epothilone derivatives Chemical class 0.000 claims description 2
- 229910052736 halogen Inorganic materials 0.000 claims description 2
- 125000001475 halogen functional group Chemical group 0.000 claims description 2
- 150000002367 halogens Chemical class 0.000 claims description 2
- 229910052739 hydrogen Inorganic materials 0.000 claims description 2
- 239000001257 hydrogen Substances 0.000 claims description 2
- 125000002887 hydroxy group Chemical group [H]O* 0.000 claims description 2
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 claims description 2
- 238000001338 self-assembly Methods 0.000 claims description 2
- 125000001424 substituent group Chemical group 0.000 claims description 2
- 229910021642 ultra pure water Inorganic materials 0.000 claims description 2
- 239000012498 ultrapure water Substances 0.000 claims description 2
- 101500025419 Homo sapiens Epidermal growth factor Proteins 0.000 claims 1
- 108090000723 Insulin-Like Growth Factor I Proteins 0.000 claims 1
- 102000013275 Somatomedins Human genes 0.000 claims 1
- 229940116978 human epidermal growth factor Drugs 0.000 claims 1
- 238000004519 manufacturing process Methods 0.000 claims 1
- GVUGOAYIVIDWIO-UFWWTJHBSA-N nepidermin Chemical compound C([C@@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)NCC(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CS)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O)NC(=O)CNC(=O)[C@@H](NC(=O)[C@@H](NC(=O)[C@H](CS)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CS)NC(=O)[C@H](C)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](C)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@@H](NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CCSC)NC(=O)[C@H](CS)NC(=O)[C@@H](NC(=O)CNC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC=1NC=NC=1)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CS)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)CNC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC=1NC=NC=1)NC(=O)[C@H](CO)NC(=O)[C@H](CC(C)C)NC(=O)[C@H]1N(CCC1)C(=O)[C@H](CS)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CO)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CC(N)=O)C(C)C)[C@@H](C)CC)C(C)C)C(C)C)C1=CC=C(O)C=C1 GVUGOAYIVIDWIO-UFWWTJHBSA-N 0.000 claims 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 abstract description 23
- 230000000694 effects Effects 0.000 abstract description 18
- 230000000259 anti-tumor effect Effects 0.000 abstract description 15
- 231100000331 toxic Toxicity 0.000 abstract description 8
- 230000002588 toxic effect Effects 0.000 abstract description 8
- 210000004881 tumor cell Anatomy 0.000 abstract description 8
- 239000002246 antineoplastic agent Substances 0.000 abstract description 4
- 210000000056 organ Anatomy 0.000 abstract description 3
- 230000009870 specific binding Effects 0.000 abstract description 2
- 229940034982 antineoplastic agent Drugs 0.000 abstract 1
- 239000000562 conjugate Substances 0.000 description 107
- 239000000243 solution Substances 0.000 description 41
- IEDXPSOJFSVCKU-HOKPPMCLSA-N [4-[[(2S)-5-(carbamoylamino)-2-[[(2S)-2-[6-(2,5-dioxopyrrolidin-1-yl)hexanoylamino]-3-methylbutanoyl]amino]pentanoyl]amino]phenyl]methyl N-[(2S)-1-[[(2S)-1-[[(3R,4S,5S)-1-[(2S)-2-[(1R,2R)-3-[[(1S,2R)-1-hydroxy-1-phenylpropan-2-yl]amino]-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl]-3-methoxy-5-methyl-1-oxoheptan-4-yl]-methylamino]-3-methyl-1-oxobutan-2-yl]amino]-3-methyl-1-oxobutan-2-yl]-N-methylcarbamate Chemical compound CC[C@H](C)[C@@H]([C@@H](CC(=O)N1CCC[C@H]1[C@H](OC)[C@@H](C)C(=O)N[C@H](C)[C@@H](O)c1ccccc1)OC)N(C)C(=O)[C@@H](NC(=O)[C@H](C(C)C)N(C)C(=O)OCc1ccc(NC(=O)[C@H](CCCNC(N)=O)NC(=O)[C@@H](NC(=O)CCCCCN2C(=O)CCC2=O)C(C)C)cc1)C(C)C IEDXPSOJFSVCKU-HOKPPMCLSA-N 0.000 description 33
- 230000006870 function Effects 0.000 description 26
- 238000006243 chemical reaction Methods 0.000 description 25
- 210000004027 cell Anatomy 0.000 description 21
- 238000001254 matrix assisted laser desorption--ionisation time-of-flight mass spectrum Methods 0.000 description 17
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 15
- 241000699670 Mus sp. Species 0.000 description 13
- 239000000047 product Substances 0.000 description 13
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 12
- 229940079593 drug Drugs 0.000 description 12
- 238000003786 synthesis reaction Methods 0.000 description 12
- 238000000502 dialysis Methods 0.000 description 11
- 238000003756 stirring Methods 0.000 description 11
- 238000004108 freeze drying Methods 0.000 description 10
- 239000000611 antibody drug conjugate Substances 0.000 description 8
- 229940049595 antibody-drug conjugate Drugs 0.000 description 8
- 238000001727 in vivo Methods 0.000 description 8
- 201000011510 cancer Diseases 0.000 description 7
- 241000699660 Mus musculus Species 0.000 description 6
- 239000008280 blood Substances 0.000 description 6
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 6
- 235000018417 cysteine Nutrition 0.000 description 6
- 238000011580 nude mouse model Methods 0.000 description 6
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 description 5
- 150000001413 amino acids Chemical group 0.000 description 5
- 210000004369 blood Anatomy 0.000 description 5
- 238000002347 injection Methods 0.000 description 5
- 239000007924 injection Substances 0.000 description 5
- ANZJBCHSOXCCRQ-FKUXLPTCSA-N mertansine Chemical compound CO[C@@H]([C@@]1(O)C[C@H](OC(=O)N1)[C@@H](C)[C@@H]1O[C@@]1(C)[C@@H](OC(=O)[C@H](C)N(C)C(=O)CCS)CC(=O)N1C)\C=C\C=C(C)\CC2=CC(OC)=C(Cl)C1=C2 ANZJBCHSOXCCRQ-FKUXLPTCSA-N 0.000 description 5
- 108020003175 receptors Proteins 0.000 description 5
- 102000005962 receptors Human genes 0.000 description 5
- VHYFNPMBLIVWCW-UHFFFAOYSA-N 4-Dimethylaminopyridine Chemical compound CN(C)C1=CC=NC=C1 VHYFNPMBLIVWCW-UHFFFAOYSA-N 0.000 description 4
- 101000851181 Homo sapiens Epidermal growth factor receptor Proteins 0.000 description 4
- PEEHTFAAVSWFBL-UHFFFAOYSA-N Maleimide Chemical compound O=C1NC(=O)C=C1 PEEHTFAAVSWFBL-UHFFFAOYSA-N 0.000 description 4
- JGFZNNIVVJXRND-UHFFFAOYSA-N N,N-Diisopropylethylamine (DIPEA) Chemical compound CCN(C(C)C)C(C)C JGFZNNIVVJXRND-UHFFFAOYSA-N 0.000 description 4
- 241000700159 Rattus Species 0.000 description 4
- NLMBVBUNULOTNS-HOKPPMCLSA-N [4-[[(2s)-5-(carbamoylamino)-2-[[(2s)-2-[6-(2,5-dioxopyrrol-1-yl)hexanoylamino]-3-methylbutanoyl]amino]pentanoyl]amino]phenyl]methyl n-[(2s)-1-[[(2s)-1-[[(3r,4s,5s)-1-[(2s)-2-[(1r,2r)-3-[[(1s,2r)-1-hydroxy-1-phenylpropan-2-yl]amino]-1-methoxy-2-methyl-3-o Chemical compound C1([C@H](O)[C@@H](C)NC(=O)[C@H](C)[C@@H](OC)[C@@H]2CCCN2C(=O)C[C@H]([C@H]([C@@H](C)CC)N(C)C(=O)[C@@H](NC(=O)[C@H](C(C)C)N(C)C(=O)OCC=2C=CC(NC(=O)[C@H](CCCNC(N)=O)NC(=O)[C@@H](NC(=O)CCCCCN3C(C=CC3=O)=O)C(C)C)=CC=2)C(C)C)OC)=CC=CC=C1 NLMBVBUNULOTNS-HOKPPMCLSA-N 0.000 description 4
- 230000008901 benefit Effects 0.000 description 4
- 239000011230 binding agent Substances 0.000 description 4
- 230000007547 defect Effects 0.000 description 4
- 230000004048 modification Effects 0.000 description 4
- 238000012986 modification Methods 0.000 description 4
- 229940041181 antineoplastic drug Drugs 0.000 description 3
- 230000015572 biosynthetic process Effects 0.000 description 3
- 238000004440 column chromatography Methods 0.000 description 3
- 238000011161 development Methods 0.000 description 3
- 230000018109 developmental process Effects 0.000 description 3
- 239000003480 eluent Substances 0.000 description 3
- 238000011156 evaluation Methods 0.000 description 3
- 238000003384 imaging method Methods 0.000 description 3
- 239000011259 mixed solution Substances 0.000 description 3
- 239000013612 plasmid Substances 0.000 description 3
- 239000000843 powder Substances 0.000 description 3
- 210000002966 serum Anatomy 0.000 description 3
- 230000004083 survival effect Effects 0.000 description 3
- 238000005160 1H NMR spectroscopy Methods 0.000 description 2
- 241000588724 Escherichia coli Species 0.000 description 2
- 101000599951 Homo sapiens Insulin-like growth factor I Proteins 0.000 description 2
- 241000699666 Mus <mouse, genus> Species 0.000 description 2
- 125000000539 amino acid group Chemical group 0.000 description 2
- 230000003698 anagen phase Effects 0.000 description 2
- 210000001099 axilla Anatomy 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 230000027455 binding Effects 0.000 description 2
- 239000006285 cell suspension Substances 0.000 description 2
- 238000002512 chemotherapy Methods 0.000 description 2
- 230000004087 circulation Effects 0.000 description 2
- 239000002254 cytotoxic agent Substances 0.000 description 2
- 229940127089 cytotoxic agent Drugs 0.000 description 2
- 239000008367 deionised water Substances 0.000 description 2
- 229910021641 deionized water Inorganic materials 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 239000000975 dye Substances 0.000 description 2
- 238000002296 dynamic light scattering Methods 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 239000007850 fluorescent dye Substances 0.000 description 2
- 102000044162 human IGF1 Human genes 0.000 description 2
- 230000006698 induction Effects 0.000 description 2
- 230000005764 inhibitory process Effects 0.000 description 2
- 238000011068 loading method Methods 0.000 description 2
- 238000001819 mass spectrum Methods 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 230000001404 mediated effect Effects 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 238000000655 nuclear magnetic resonance spectrum Methods 0.000 description 2
- 239000012074 organic phase Substances 0.000 description 2
- 230000035699 permeability Effects 0.000 description 2
- 229920001184 polypeptide Polymers 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 102000004196 processed proteins & peptides Human genes 0.000 description 2
- 108090000765 processed proteins & peptides Proteins 0.000 description 2
- 235000018102 proteins Nutrition 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 239000000523 sample Substances 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 125000003396 thiol group Chemical group [H]S* 0.000 description 2
- ANRHNWWPFJCPAZ-UHFFFAOYSA-M thionine Chemical compound [Cl-].C1=CC(N)=CC2=[S+]C3=CC(N)=CC=C3N=C21 ANRHNWWPFJCPAZ-UHFFFAOYSA-M 0.000 description 2
- 230000004614 tumor growth Effects 0.000 description 2
- YNHKVOGCDPODMT-UHFFFAOYSA-N 1-(2-aminoethyl)pyrrole-2,5-dione;2,2,2-trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F.NCCN1C(=O)C=CC1=O YNHKVOGCDPODMT-UHFFFAOYSA-N 0.000 description 1
- LMDZBCPBFSXMTL-UHFFFAOYSA-N 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide Substances CCN=C=NCCCN(C)C LMDZBCPBFSXMTL-UHFFFAOYSA-N 0.000 description 1
- HZAXFHJVJLSVMW-UHFFFAOYSA-N 2-Aminoethan-1-ol Chemical group NCCO HZAXFHJVJLSVMW-UHFFFAOYSA-N 0.000 description 1
- FPQQSJJWHUJYPU-UHFFFAOYSA-N 3-(dimethylamino)propyliminomethylidene-ethylazanium;chloride Chemical compound Cl.CCN=C=NCCCN(C)C FPQQSJJWHUJYPU-UHFFFAOYSA-N 0.000 description 1
- 229960000549 4-dimethylaminophenol Drugs 0.000 description 1
- 244000063299 Bacillus subtilis Species 0.000 description 1
- 235000014469 Bacillus subtilis Nutrition 0.000 description 1
- 208000031648 Body Weight Changes Diseases 0.000 description 1
- QKQHVOJVOUDJFG-UHFFFAOYSA-N CC(C)(S(=O)(=O)NCCC(=O)O)S(=O)(=O)NCCC(=O)O Chemical compound CC(C)(S(=O)(=O)NCCC(=O)O)S(=O)(=O)NCCC(=O)O QKQHVOJVOUDJFG-UHFFFAOYSA-N 0.000 description 1
- 241001198387 Escherichia coli BL21(DE3) Species 0.000 description 1
- 239000007821 HATU Substances 0.000 description 1
- 101001012157 Homo sapiens Receptor tyrosine-protein kinase erbB-2 Proteins 0.000 description 1
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 1
- XUJNEKJLAYXESH-REOHCLBHSA-N L-Cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 description 1
- 206010058467 Lung neoplasm malignant Diseases 0.000 description 1
- 229930126263 Maytansine Natural products 0.000 description 1
- 206010033128 Ovarian cancer Diseases 0.000 description 1
- 206010061535 Ovarian neoplasm Diseases 0.000 description 1
- 108091008606 PDGF receptors Proteins 0.000 description 1
- 229930012538 Paclitaxel Natural products 0.000 description 1
- 102000011653 Platelet-Derived Growth Factor Receptors Human genes 0.000 description 1
- 102100030086 Receptor tyrosine-protein kinase erbB-2 Human genes 0.000 description 1
- 238000003917 TEM image Methods 0.000 description 1
- 241001052560 Thallis Species 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 238000001042 affinity chromatography Methods 0.000 description 1
- 125000003172 aldehyde group Chemical group 0.000 description 1
- 235000001014 amino acid Nutrition 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 229940125644 antibody drug Drugs 0.000 description 1
- 239000012964 benzotriazole Substances 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 230000005540 biological transmission Effects 0.000 description 1
- 230000017531 blood circulation Effects 0.000 description 1
- 230000037396 body weight Effects 0.000 description 1
- 201000007983 brain glioma Diseases 0.000 description 1
- 230000009702 cancer cell proliferation Effects 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000012650 click reaction Methods 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 125000004122 cyclic group Chemical group 0.000 description 1
- 231100000433 cytotoxic Toxicity 0.000 description 1
- 230000001472 cytotoxic effect Effects 0.000 description 1
- 231100000135 cytotoxicity Toxicity 0.000 description 1
- 230000003013 cytotoxicity Effects 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 239000002552 dosage form Substances 0.000 description 1
- 238000012377 drug delivery Methods 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 230000012202 endocytosis Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000032050 esterification Effects 0.000 description 1
- 238000005886 esterification reaction Methods 0.000 description 1
- 238000000855 fermentation Methods 0.000 description 1
- 230000004151 fermentation Effects 0.000 description 1
- 238000002189 fluorescence spectrum Methods 0.000 description 1
- 229920000669 heparin Polymers 0.000 description 1
- ZFGMDIBRIDKWMY-PASTXAENSA-N heparin Chemical compound CC(O)=N[C@@H]1[C@@H](O)[C@H](O)[C@@H](COS(O)(=O)=O)O[C@@H]1O[C@@H]1[C@@H](C(O)=O)O[C@@H](O[C@H]2[C@@H]([C@@H](OS(O)(=O)=O)[C@@H](O[C@@H]3[C@@H](OC(O)[C@H](OS(O)(=O)=O)[C@H]3O)C(O)=O)O[C@@H]2O)CS(O)(=O)=O)[C@H](O)[C@H]1O ZFGMDIBRIDKWMY-PASTXAENSA-N 0.000 description 1
- 229960001008 heparin sodium Drugs 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 230000005917 in vivo anti-tumor Effects 0.000 description 1
- 239000000411 inducer Substances 0.000 description 1
- 239000003446 ligand Substances 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 201000005202 lung cancer Diseases 0.000 description 1
- 208000020816 lung neoplasm Diseases 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 238000001840 matrix-assisted laser desorption--ionisation time-of-flight mass spectrometry Methods 0.000 description 1
- WKPWGQKGSOKKOO-RSFHAFMBSA-N maytansine Chemical compound CO[C@@H]([C@@]1(O)C[C@](OC(=O)N1)([C@H]([C@@H]1O[C@@]1(C)[C@@H](OC(=O)[C@H](C)N(C)C(C)=O)CC(=O)N1C)C)[H])\C=C\C=C(C)\CC2=CC(OC)=C(Cl)C1=C2 WKPWGQKGSOKKOO-RSFHAFMBSA-N 0.000 description 1
- 230000037353 metabolic pathway Effects 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 238000010172 mouse model Methods 0.000 description 1
- 238000007709 nanocrystallization Methods 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 229960001592 paclitaxel Drugs 0.000 description 1
- 230000000149 penetrating effect Effects 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 239000011541 reaction mixture Substances 0.000 description 1
- 230000009257 reactivity Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000012827 research and development Methods 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
- 238000002390 rotary evaporation Methods 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 230000003595 spectral effect Effects 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- RCINICONZNJXQF-MZXODVADSA-N taxol Chemical compound O([C@@H]1[C@@]2(C[C@@H](C(C)=C(C2(C)C)[C@H](C([C@]2(C)[C@@H](O)C[C@H]3OC[C@]3([C@H]21)OC(C)=O)=O)OC(=O)C)OC(=O)[C@H](O)[C@@H](NC(=O)C=1C=CC=CC=1)C=1C=CC=CC=1)O)C(=O)C1=CC=CC=C1 RCINICONZNJXQF-MZXODVADSA-N 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 238000004627 transmission electron microscopy Methods 0.000 description 1
- 230000004565 tumor cell growth Effects 0.000 description 1
- 239000013598 vector Substances 0.000 description 1
- 210000003462 vein Anatomy 0.000 description 1
Images
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
- A61K47/62—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
-
- 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
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
- A61K31/425—Thiazoles
- A61K31/427—Thiazoles not condensed and containing further heterocyclic rings
-
- 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
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/535—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines
- A61K31/5365—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines ortho- or peri-condensed with heterocyclic ring systems
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
- A61K38/07—Tetrapeptides
-
- 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
- A61K47/545—Heterocyclic compounds
-
- 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
- A61K47/65—Peptidic linkers, binders or spacers, e.g. peptidic enzyme-labile linkers
-
- 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
- A61K47/69—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 conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
- A61K47/6921—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 conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
- A61K47/6927—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 conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores
- A61K47/6929—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 conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
Abstract
本发明用于肿瘤主动靶向治疗的偶联物是由具有肿瘤主动靶向功能的亲水性亲和体和疏水性抗肿瘤细胞毒素通过小分子连接基偶联得到,其中,亲和体对多种肿瘤细胞表面过量表达的受体具有特异性结合活性和高度亲和力;细胞毒素是高效的抗肿瘤药物。还公开了其纳米颗粒、制备方法及其应用。本发明涉及的亲和体‑细胞毒素偶联物在水中可自组装形成亲和体为外壳、细胞毒素为内核的纳米颗粒,因为亲和体能够特异性识别并结合肿瘤细胞表面过量表达的受体,所以该纳米颗粒可在肿瘤部位有效富集,并通过连接基生物降解,释放出细胞毒素并达到有效治疗浓度,抑制肿瘤增长的同时,减少对正常器官和组织的毒副作用,为肿瘤的主动靶向精准治疗提供新方案。
Description
技术领域
本发明涉及生物和纳米医药技术领域,特别涉及一类用于肿瘤主动靶向治疗的亲和体-细胞毒素偶联物及其纳米颗粒的制备方法和应用。
背景技术
恶性肿瘤(癌症)是严重危及人类生存和社会发展的重大疾病,已成为全世界亟待解决的难题及重大挑战。目前,化学疗法依然是癌症治疗的主要手段,小分子细胞毒素具有结构明确、渗透性高、细胞毒性强等优势,但是由于其分子量较小,靶向特异性缺失,给药后会被机体快速代谢,且常引起机体的严重毒副作用,临床应用受到限制。
抗体药物偶联(antibody-drug conjugate,ADC)技术,结合了单克隆抗体的靶向选择性和小分子细胞毒素的高效性,为肿瘤的“精准医疗”提供了新方法。作为一类新颖的肿瘤治疗药物,ADC的研发不仅受到广泛关注,而且已有产品应用于临床。但现有的ADC药物尚存在如下许多不足:(1)抗体分子量过大(1.5×105Da),导致药物在肿瘤内部的渗透率很低;(2)抗体表面与小分子细胞毒素偶联位点数目不确定,导致其批次间载药率不一致,难以得到均一的产品;(3)缺乏优质的靶标及更为理想的抗体,以进一步提升内吞率以及抗体介导的生物效应。因此,如何克服以上不足,研制药物输送比高,细胞毒素携带均一的ADC药物迫在眉睫。基于此,尝试采用其他亲和性更强,对肿瘤组织或细胞选择性更高,且能与细胞毒素均一结合的靶向载体,开发新型主动靶向抗肿瘤药物不仅必要,而且科学意义和临床应用价值巨大。
发明内容
本发明提供了一种用于肿瘤主动靶向治疗的亲和体-细胞毒素偶联物,可以解决现有技术中的上述缺陷。
本发明的技术方案如下:
一种用于肿瘤主动靶向治疗的亲和体-细胞毒素偶联物,主要包括:细胞毒素和具有主动靶向功能的亲和体,其中,所述的偶联物由所述细胞毒素与所述亲和体之间通过小分子连接基共价偶联而得;
较佳地,所述的亲和体选自:靶向人表皮生长因子受体(HER)的亲和体ZHER2:342、ZEGFR:1907,靶向人***-1受体的亲和体ZIGF1R:4551和靶向人血小板源生长因子受体β的亲和体ZPDGFRβ:07591中的至少一种,它们的氨基酸序列依次如式(II~V):
HMCVDNKFNKEMRNAYWEIALLPNLNNQQKRAFIRSLYDDPSQSANLLAEAKKLNDAQAPK (II)
MGHHHHHHHHHHSSGHIDDDDKHM VDNKFNKEMWAAWEEIRNLPNLNGWQMTAFIASLVDDPSQSANLLAEAKKLNDAQAPK C(III)
MGHHHHHHHHHHSSGHIDDDDKHMAENKFNKEGFYAALEILIPNLTQKQRGAFISSLSDDPSQSANLLAEAKKLNDAQGGGC (IV)
MGHHHHHHHHHHSSGHIDDDKHMAENKFNKELIEAAAEIDALPNLNRRQWNAFIKSLVDDPSQSANLLAEAKKLNDAQAPKC (V)
较佳地,所述的细胞毒素选自埃博霉素B及其衍生物、美登素及其衍生物、海兔毒素(MMAE)及其衍生物中的至少一种。
较佳地,所述的小分子连接基为具有马来酰亚胺基封端的连接基,且小分子连接基通过马来酰亚胺基与亲和体的巯基进行连接。
较佳地,所述的小分子连接基选自以下式(II)、式(III)、式(IV)中的至少一种;
其中,R1选自:-烷基-C(O)NH-,-烷基-SO2NH-或C1-8烷氧基;R2选自羧基或酰氯,n、m各自独立地选自2~10;
所述烷基选自C1-8烷基;所述烷基、烷氧基为未取代的或被1、2、3个选自下组的取代基所取代:卤素、氰基、乙酰基、羟基、羟甲基、羟乙基、羧基、C1-8烷基、C1-8烷氧基、卤代C1-8烷基、C3-8环烷基、C3-8环烷氧基、卤代C1-8烷氧基、-C(O)C1-10烷基、-C(O)OC1-10烷基、-OC(O)C1-10烷基、-CONRa0Rb0;Ra0、Rb0各自独立地为氢或C1-8烷基。
一种用于肿瘤主动靶向治疗的亲和体-细胞毒素偶联物纳米颗粒,由如上任一项所述的偶联物在水介质中自组装得到。
较佳地,所述纳米颗粒的壳层由具有主动靶向功能的亲和体构成,所述纳米颗粒的内核由所述的小分子连接基及细胞毒素构成,所述纳米颗粒的粒径小于200nm。
一种用于肿瘤主动靶向治疗的亲和体-细胞毒素偶联物纳米颗粒的制备方法,包括步骤:(1)将含羟基和/或氨基和/或巯基的疏水性细胞毒素与小分子连接基键合起来,如酯化、点击等化学反应,再进一步与主动靶向亲和体进行偶联反应,得到具有主动靶向功能的亲和体-细胞毒素偶联物;
(2)将一定量步骤(1)所述亲和体-细胞毒素偶联物溶于一定体积的有机溶剂中,室温下将其滴入缓慢搅拌的超纯水中,除去有机溶剂,得到具有主动靶向功能的偶联物纳米颗粒水溶液。
较佳的,步骤(2)中的有机溶剂选自二甲基亚砜、N,N’-二甲基甲酰胺、乙醇、异丙醇中的至少一种。
一种如上任一项所述的用于肿瘤主动靶向治疗的亲和体-细胞毒素偶联物、所述的纳米颗粒或所述的的制备方法制得的纳米颗粒在制备用于肿瘤主动靶向治疗的药物中的应用。
与现有技术相比,本发明的有益效果如下:
本发明用于肿瘤主动靶向治疗的偶联物是由具有肿瘤主动靶向功能的亲水性亲和体和疏水性抗肿瘤细胞毒素通过连接基偶联得到,其中,亲和体是含58个氨基酸残基及3个α-螺旋结构,且对多种肿瘤细胞表面过量表达的受体具有特异性结合活性和高度亲和力的单链多肽分子;
本发明的具有主动靶向功能的亲和体-细胞毒素偶联物可在水中自组装形成纳米颗粒,外层为具有主动靶向功能的亲和体,其能与肿瘤细胞表面过量表达的受体进行高特异性结合,增强药物在肿瘤部位的富集,使药物更高效地进入肿瘤细胞,减少对正常细胞及组织的毒副作用;当偶联物纳米颗粒进入肿瘤细胞后,有机小分子连接基中缩硫酮键或酰胺键发生降解,释放出细胞毒素,杀死肿瘤细胞,降低对正常细胞的毒副作用,达到靶向治疗癌症的目的。
当然,实施本发明的任一产品并不一定需要同时达到以上所述的所有优点。
附图说明
图1为实施例1中具有主动靶向功能偶联物ZHER2:342-Epo B的Maldi-Tof-MS谱图;
图2为实施例1中具有主动靶向功能偶联物ZHER2:342-Epo B纳米颗粒的透射电镜照片;
图3为实施例1中具有主动靶向功能偶联物ZHER2:342-Epo B纳米颗粒的动态光散射数据图;
图4为实施例2中所得中间体B-1的高分辨质谱图;
图5为实施例2中具有主动靶向功能偶联物ZHER2:342-DM1的Maldi-Tof-MS谱图;
图6为实施例3中具有主动靶向功能偶联物ZHER2:342-MMAE的Maldi-Tof-MS谱图;
图7为实施例4中具有主动靶向功能偶联物ZEGFR:1907-DM1的Maldi-Tof-MS谱图;
图8为实施例5中具有主动靶向功能偶联物ZEGFR:1907-MMAE的Maldi-Tof-MS谱图;
图9为实施例6中具有主动靶向功能偶联物ZIGF1R:4551-DM1的Maldi-Tof-MS谱图;
图10为实施例7中具有主动靶向功能偶联物ZIGF1R:4551-MMAE的Maldi-Tof-MS谱图;
图11为实施例8中具有主动靶向功能偶联物ZPDGFRβ:07591-DM1的Maldi-Tof-MS谱图;
图12为实施例9中具有主动靶向功能偶联物ZPDGFRβ:07591-MMAE的Maldi-Tof-MS谱图;
图13为实施例10中具有主动靶向功能偶联物纳米颗粒对肿瘤细胞生长抑制作用示意图。
图14为实施例11中具有主动靶向功能偶联物ZHER2:342-Epo B纳米颗粒在SD大鼠体内的药代动力学示意图。
图15为实施例12中具有主动靶向功能偶联物ZHER2:342-Epo B纳米颗粒经荧光修饰后在SKOV-3荷瘤裸鼠体内长时间循环成像示意图。
图16为实施例13中荷瘤小鼠经由具有主动靶向功能偶联物ZHER2:342-MMAE纳米颗粒治疗后其肿瘤体积变化曲线图。
图17为实施例13中荷瘤小鼠经由具有主动靶向功能偶联物ZHER2:342-MMAE纳米颗粒治疗后荷瘤小鼠的体重变化曲线图。
图18为实施例13中荷瘤小鼠经由具有主动靶向功能偶联物ZHER2:342-MMAE纳米颗粒治疗后荷瘤小鼠治疗效果图。
具体实施方式
本发明提供一种用于肿瘤主动靶向治疗的亲和体-细胞毒素偶联物,主要包括细胞毒素和具有主动靶向功能的亲和体,所述的偶联物由所述细胞毒素与所述亲和体之间通过小分子连接基共价偶联而得。
其中,亲和体(Affibody)是一类新型亲和配体,其分子中介导受体结合区域面积达8nm2,与抗原-抗体反应结合区域的面积相类似,且相对分子质量小,结构稳定性高,有“人工抗体”之称,这些独特的性质使其在生物学和免疫学等领域中有广泛的应用前景。
亲和体由58个氨基酸残基组成,相对分子质量约为6.5×103Da,是含3个α-螺旋结构的单链多肽分子。亲和体不仅功能类似于抗体,而且具有抗体所没有的如下优势:(1)相对分子质量小,仅为抗体分子的二十五分之一,稳定性非常高,易制备。(2)具有高度的选择性和亲和性,其亲和力最高可达22pmol/L。(3)氨基酸序列中不含有半胱氨酸,在制备亲和体分子时可定向引入一个半胱氨酸,再通过巯基和其他活性基团的反应将其连接到有效负载之上。(4)能够很快地定位于作用部位,并且具有良好的组织穿透力,能够到达一些抗体分子所无法到达的靶部位。
目前,亲和体的应用领域日益扩大,但在亲和体-药物偶联方面的研究报道仍相对较少。亲合体的独特优势决定了其可作为高亲和力靶向分子连接到抗肿瘤药物上,实现肿瘤主动靶向治疗。
现有技术中有通过细胞结合剂与细胞毒素耦联的示例,细胞结合剂上含2-羟基乙胺基团的氨基酸数目有多个,由于化学反应活性的限制,每个细胞结合剂分子上氧化得到的醛基最终并不能都和细胞毒性剂反应结合,因此造成每个细胞结合剂分子携带的细胞毒性剂分子的数目是不确定的,这一缺陷不可避免地会造成终产物的批次间差异,也是目前抗体药物偶联(ADC)药物迫切需要改善的地方。
本发明以亲和体为靶向载体,制备了亲和体-细胞毒素偶联物。首先,鉴于亲和体分子本身不含有半胱氨酸,我们在亲和体氨基酸序列中定向引入一个半胱氨酸分子,同时对细胞毒素进行马来酰亚胺化修饰;然后,通过半胱氨酸与马来酰亚胺之间的点击反应,将二者偶联得到亲和体-细胞毒素偶联物。在此类偶联物中,一个亲和体分子只与一个细胞毒素分子结合,避免了ADC药物载药比不稳定,批次间差异大的不足。与此同时,基于偶联物的两亲性分子结构,其可在水中自组装形成纳米颗粒。这一策略不仅赋予细胞毒素优异的主动靶向功能,同时赋予偶联物纳米药物具有的被动靶向在肿瘤部位富集的特征(EPR效应),高效的靶向性有效减少了所携带的细胞毒素对机体的毒副作用,纳米化的特征则有效的提高了偶联物的体内生物半衰期,因此具有良好的临床应用前景和巨大的市场价值。因为亲和体能够特异性识别并结合肿瘤细胞表面过量表达的受体,所以由其制备的纳米颗粒可在肿瘤部位有效富集,并通过连接基生物降解,释放出细胞毒素并达到有效治疗浓度,抑制肿瘤增长的同时,减少对正常器官和组织的毒副作用,为肿瘤的主动靶向精准治疗提供新方案。
其中,靶向人表皮生长因子受体(HER)的亲和体ZHER2:342、ZEGFR:1907,靶向人***-1受体的亲和体ZIGF1R:4551和靶向人血小板源生长因子受体β的亲和体ZPDGFRβ:07591等均通过大肠杆菌生物发酵结合诱导表达的方法制备得到。例如亲和体ZHER2:342是根据其氨基酸序列制备对应的DNA碱基序列,并将其连接到质粒上。然后将重组质粒转化进入大肠杆菌BL21(DE3)菌株之中,经过特定的质粒抗性筛选得到携带目标亲和体ZHER2:342基因的大肠杆菌菌株。随后取适量该菌株置于2L大培养瓶中扩大培养,待其生长到一定浓度,加入诱导剂,诱导表达12h后收集菌体。利用高压均质机将菌体细胞破碎,离心取上清,通过Ni-NTA亲和层析柱进行分离纯化,得到亲和体ZHER2:342洗脱液,将得到的亲和体蛋白洗脱溶液进行透析除盐(透析袋截留分子量为3.5kDa),离心浓缩后冷冻干燥,最后得到棉花状亲和体ZHER2:342蛋白,密封保存。
在本文中,由「一数值至另一数值」表示的范围,是一种避免在说明书中一一列举该范围中的所有数值的概要性表示方式。因此,某一特定数值范围的记载,涵盖该数值范围内的任意数值以及由该数值范围内的任意数值界定出的较小数值范围,如同在说明书中明文写出该任意数值和该较小数值范围一样。
下面结合具体实施例,进一步阐述本发明。应该理解,这些实施例仅用于说明本发明,而不用于限定本发明的保护范围。在实际应用中本领域技术人员根据本发明做出的改进和调整,仍属于本发明的保护范围。
实施例1
1.1中间体A-1的合成
将3,3'-丙烷-2,2-二基双(磺胺二基)二丙酸(TK,252.05mg)、1-乙基-(3-二甲基氨基丙基)碳酰二亚胺盐酸盐(EDCI,287.55mg)、4-二甲氨基吡啶(DMAP,12.2mg)和无水三乙胺(TEA,0.28mL)加入反应瓶中,再加入25mL无水二氯甲烷,室温搅拌反应1小时后,再加入埃博霉素B(Epothilone B,507.27mg),随后室温下继续搅拌反应24小时。反应结束后,加入20mL去离子水萃取,收集有机相,用二氯甲烷与甲醇体积比为(20:1)的混合溶液为洗脱剂,经柱色谱分离得到白色粉末状中间体A-1(345.92mg,产率46.6%)。中间体A-1的1H NMR谱中各质子峰的归属如下:δ(ppm from TMS),1H NMR(CDCl3,400MHz)δ7.04(1H,s),6.82(1H,bs),5.34(1H,dd),5.26(1H,dd),4.21(1H,m),4.19(1H,bs),3.56(1H,dq),2.93(4H,m),2.84(1H,dd),2.75(3H,s),2.69(4H,m),2.51(1H,dd),2.35(1H,dd),2.16(1H,ddd),2.12(3H,d),1.95(1H,ddd),1.74(2H,m),1.63(6H,s),1.35(2H,m),1.32(3H,m),1.30(3H,s),1.27(3H,s),1.16(3H,d),1.13(3H,s),0.97(3H,d).HRMS(Esi-TOF,m/z)m/z[M+H]+calcd:C36H55NO9S3:742.3039,found:742.3122.
1.2中间体A-2的合成
将中间体A-1(74.13mg)、N-(2-氨基乙基)马来酰亚胺三氟乙酸盐(38.12mg)、2-(7-氧化苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸盐(HATU,57.04mg)和N,N-二异丙基乙胺(DIPEA,42μL)加入反应瓶中,再加入20mL无水二氯甲烷,室温搅拌反应6小时。反应结束后,加入20mL去离子水萃取,收集有机相,用二氯甲烷与甲醇体积比为(20:1)的混合溶液为洗脱剂,经柱色谱分离得到白色粉末状中间体A-2(74.07mg,产率85.7%)。
本实施例制得中间体A-2的1H NMR谱中各质子峰的归属如下:δ(ppm from TMS),1HNMR(CDCl3,400MHz)7.01(1H,s),6.74(1H,s),6.64(1H,bs),5.49(1H,dd),5.33(1H,dd),4.28(1H,bs),4.12(1H,m),3.75(1H,m),3.71(2H,t),3.50(2H,t),3.20(1H,dq),2.90(4H,m),2.84(1H,dd),2.72(3H,s),2.67(2H,t),2.58(1H,dd),2.49(1H,dd),2.44(2H,t),2.18(1H,d),2.12(3H,d),1.97(1H,d),1.68(2H,m),1.60(6H,s).1.47(2H,m),1.44(3H,m),1.38(3H,s),1.29(3H,s),1.16(3H,d),1.09(3H,s),0.94(3H,d).
1.3偶联物ZHER2:342-Epo B的合成
将亲和体ZHER2:342(10mg)溶于5mL PBS溶液中,随后将中间体A-2(1.22mg)溶于150μL二甲基亚砜,并缓慢的逐滴加入前述的亲和体溶液中,室温搅拌下反应12小时。将反应液置于水中透析提纯,透析完毕后,冷冻干燥得得到白色棉花状偶联物ZHER2:342-Epo B。
本实施例制得偶联物ZHER2:342-Epo B的Maldi-Tof-MS质谱数据如图1所示,表示产物分子量的单峰出现在7941.47,与理论分子量7942.21基本一致。
将上述制得的偶联物ZHER2:342-Epo B溶解在二甲基亚砜中,在室温下将其滴入水中,除去二甲基亚砜,得到偶联物的纳米颗粒水溶液。本实施例制备的基于偶联物ZHER2:342-Epo B抗肿瘤纳米颗粒的透射电镜照片如图2所示,纳米颗粒粒径的平均尺寸在100nm左右;动态光散射数据如图3所示,纳米颗粒粒径的平均尺寸在100nm左右,与透射电镜照片数据基本一致。
本实施例的ZHER2:342-Epo B抗肿瘤纳米粒子具有良好的均一性,可有效降低纳米粒子制备时的批次间差异,提高其质量稳定性;其次,良好的均一性可保证药物纳米颗粒在体内循环时具有一致的药代动力学行为及体内代谢途径,有助于其通过临床评估。
实施例2
2.1中间体B-1的合成
将N2'-去乙酰基-N2'-(3-巯基-1-氧代丙基)美登素(DM1,737.2mg)、乙基双马来酰亚胺(1.1g)、加入反应瓶中,再加入50mL甲醇,室温搅拌反应12小时。反应结束后,旋蒸浓缩,用二氯甲烷与甲醇体积比为(20:1)的混合溶液为洗脱剂,经柱色谱分离得到淡黄色粉末状中间体B-1(883.4mg,产率92.3%)。
本实施例制得中间体B-1的质谱谱图如图4所示,HRMS(Esi-TOF,m/z)m/z[M+H]+calcd:C45H56ClN5O14S:958.3233,found:958.3330.
2.2偶联物ZHER2:342-DM1的合成
将亲和体ZHER2:342(10mg)溶于5mL PBS溶液中,随后将中间体B-1(1.35mg)溶于150μL二甲基亚砜,并缓慢的逐滴加入前述的亲和体溶液中,室温搅拌下反应12小时。将反应液置于水中透析提纯,透析完毕后,冷冻干燥得得到白色棉花状偶联物ZHER2:342-DM1。
本实施例制得偶联物ZHER2:342-DM1的Maldi-Tof质谱图如图5所示,表示产物分子量的单峰出现在8030.09,与理论分子量8038.25基本一致。
将上述制得的偶联物ZHER2:342-DM1溶解在二甲基亚砜中,在室温下将其滴入水中,除去二甲基亚砜,得到偶联物的纳米颗粒水溶液。本实施例制得的基于偶联物ZHER2:342-DM1抗肿瘤纳米颗粒的平均尺寸在100nm左右。
实施例3
3.1偶联物ZHER2:342-MMAE的合成
将亲和体ZHER2:342(10mg)溶于5mL PBS溶液中,随后将中间体mc-vc-PAB-MMAE(1.85mg,购自南京康满林生物医药科技有限公司)溶于150μL二甲基亚砜,并缓慢的逐滴加入前述的亲和体溶液中,室温搅拌下反应12小时。将反应液置于水中透析提纯,透析完毕后,冷冻干燥得得到白色棉花状的偶联物ZHER2:342-MMAE。
本实施例制得偶联物ZHER2:342-MMAE的Maldi-Tof质谱图如图6所示,表示产物分子量的单峰出现在8370.53,与理论分子量8393.63基本一致。
将上述制得的偶联物ZHER2:342-MMAE溶解在二甲基亚砜中,在室温下将其滴入水中,除去二甲基亚砜,得到偶联物的纳米颗粒水溶液。本实施例制得基于偶联物ZHER2:342-MMAE抗肿瘤纳米颗粒的平均尺寸在100nm左右。
实施例4
4.1偶联物ZEGFR:1907-DM1的合成
将亲和体ZEGFR:1907(12.3mg)溶于5mL PBS溶液中,随后将中间体B-1(1.35mg)溶于150μL二甲基亚砜,并缓慢的逐滴加入前述的亲和体溶液中,室温搅拌下反应12小时。将反应液置于水中透析提纯,透析完毕后,冷冻干燥得得到白色棉花状偶联物ZEGFR:1907-DM1。
本实施例制得偶联物ZEGFR:1907-DM1的Maldi-Tof质谱图如图7所示,表示产物分子量的单峰出现在10382.53,与理论分子量10445.32基本一致。
将上述制得的偶联物ZEGFR:1907-DM1溶解在二甲基亚砜中,在室温下将其滴入水中,除去二甲基亚砜,得到偶联物的纳米颗粒水溶液。本实施例制得的基于偶联物ZEGFR:1907-DM1抗肿瘤纳米颗粒的平均尺寸在100nm左右。
实施例5
5.1偶联物ZEGFR:1907-MMAE的合成
将亲和体ZEGFR:1907(12.3mg)溶于5mL PBS溶液中,随后将中间体mc-vc-PAB-MMAE(1.85mg)溶于150μL二甲基亚砜,并缓慢的逐滴加入前述的亲和体溶液中,室温搅拌下反应12小时。将反应液置于水中透析提纯,透析完毕后,冷冻干燥得得到白色棉花状偶联物ZEGFR:1907-MMAE。
本实施例制得偶联物ZEGFR:1907-MMAE的Maldi-Tof质谱图如图8所示,表示产物分子量的单峰出现在10736.46,与理论分子量10803.53基本一致。
将上述制得的偶联物ZEGFR:1907-MMAE溶解在二甲基亚砜中,在室温下将其滴入水中,除去二甲基亚砜,得到偶联物的纳米颗粒水溶液。本实施例制备的基于偶联物ZEGFR:1907-MMAE的抗肿瘤纳米颗粒的平均尺寸在100nm左右。
实施例6
6.1偶联物ZIGF1R:4551-DM1的合成
将亲和体ZIGF1R:4551(12.8mg)溶于5mL PBS溶液中,随后将中间体B-1(1.35mg)溶于150μL二甲基亚砜,并缓慢的逐滴加入前述的亲和体溶液中,室温搅拌下反应12小时。将反应液置于水中透析提纯,透析完毕后,冷冻干燥得得到白色棉花状偶联物ZIGF1R:4551-DM1。
本实施例制得偶联物ZIGF1R:4551-DM1的Maldi-Tof质谱图如图9所示,表示产物分子量的单峰出现在10083.02,与理论分子量10040.25基本一致。
将上述制得的偶联物ZIGF1R:4551-DM1溶解在二甲基亚砜中,在室温下将其滴入水中,除去二甲基亚砜,得到偶联物的纳米颗粒水溶液。本实施例制得的基于偶联物ZIGF1R:4551-DM1抗肿瘤纳米颗粒的平均尺寸在100nm左右。
实施例7
7.1偶联物ZIGF1R:4551-MMAE的合成
将亲和体ZIGF1R:4551(12.8mg)溶于5mL PBS溶液中,随后将中间体mc-vc-PAB-MMAE(1.85mg)溶于150μL二甲基亚砜,并缓慢的逐滴加入前述的亲和体溶液中,室温搅拌下反应12小时。将反应液置于水中透析提纯,透析完毕后,冷冻干燥得得到白色棉花状偶联物ZIGF1R:4551-MMAE。
本实施例制得偶联物ZIGF1R:4551-MMAE的Maldi-Tof质谱图如图10所示,表示产物分子量的单峰出现在10271.08,与理论分子量10398.56基本一致。
将上述制得的偶联物ZIGF1R:4551-MMAE溶解在二甲基亚砜中,在室温下将其滴入水中,除去二甲基亚砜,得到偶联物的纳米颗粒水溶液。本实施例制备的基于偶联物ZIGF1R:4551-MMAE的抗肿瘤纳米颗粒的平均尺寸在100nm左右。
实施例8
8.1偶联物ZPDGFRβ:07591-DM1的合成
将亲和体ZPDGFRβ:07591(13.4mg)溶于5mL PBS溶液中,随后将中间体B-1(1.35mg)溶于150μL二甲基亚砜,并缓慢的逐滴加入前述的亲和体溶液中,室温搅拌下反应12小时。将反应液置于水中透析提纯,透析完毕后,冷冻干燥得得到白色棉花状偶联物ZPDGFRβ:07591-DM1。
本实施例制得偶联物ZPDGFRβ:07591-DM1的Maldi-Tof质谱图如图11所示,表示产物分子量的单峰出现在10474.61,与理论分子量10456.72基本一致。
将上述制得的偶联物ZPDGFRβ:07591-DM1溶解在二甲基亚砜中,在室温下将其滴入水中,除去二甲基亚砜,得到偶联物的纳米颗粒水溶液。本实施例制得的基于偶联物ZPDGFRβ:07591-DM1抗肿瘤纳米颗粒的平均尺寸在100nm左右。
实施例9
9.1偶联物ZPDGFRβ:07591-MMAE的合成
将亲和体ZPDGFRβ:07591(13.4mg)溶于5mL PBS溶液中,随后将中间体mc-vc-PAB-MMAE(1.85mg)溶于150μL二甲基亚砜,并缓慢的逐滴加入前述的亲和体溶液中,室温搅拌下反应12小时。将反应液置于水中透析提纯,透析完毕后,冷冻干燥得得到白色棉花状偶联物ZPDGFRβ:07591-MMAE。
本实施例制得偶联物ZPDGFRβ:07591-MMAE的Maldi-Tof质谱图如图12所示,表示产物分子量的单峰出现在10737.24,与理论分子量10815.03基本一致。
将上述制得的偶联物ZPDGFRβ:07591-MMAE溶解在二甲基亚砜中,在室温下将其滴入水中,除去二甲基亚砜,得到偶联物的纳米颗粒水溶液。本实施例制备的基于偶联物ZPDGFRβ:07591-MMAE的抗肿瘤纳米颗粒的平均尺寸在100nm左右。
实施例10
本实施例提供了用于肿瘤主动靶向治疗的亲和体-细胞毒素偶联物纳米颗粒对癌细胞的作用实验。
将实施例1-9中制备得到的亲和体-细胞毒素偶联物纳米颗粒分别用细胞培养液配制成浓度为0.1、0.5、1、2.5、5、10、20、40、50nmol/L的溶液,然后分别跟SKOV-3细胞(卵巢癌细胞系)、A431细胞(表皮癌细胞系)、A549细胞(肺癌细胞系)和U87细胞(脑胶质瘤细胞系)培养48小时后,采用MTT方法进行细胞活性测试,结果如图13的A、B、C、D所示。
当亲和体-细胞毒素偶联物纳米颗粒的浓度达到10nmol/L时,癌细胞增殖得到明显抑制,且亲和体-细胞毒素偶联物纳米颗粒对癌细胞的抑制作用跟浓度呈正比。说明实施例1-9中制得的亲和体-细胞毒素偶联物纳米颗粒在治疗恶性肿瘤中具有潜在的应用价值。
实施例11
本实施例提供了用于肿瘤主动靶向治疗的亲和体-细胞毒素偶联物ZHER2:342-Epo B纳米颗粒在SD大鼠体内的药代动力学实验。
采用Cy5.5(花青染料5.5)作为体内的荧光探针分子,将Cy5.5共价修饰在实施例1中得到的具有主动靶向功能偶联物ZHER2:342-Epo B纳米粒子的表面,对纳米粒子的体内药代动力学性质进行评价。
随机取2只SD大鼠(~200g)作为实验组,分别尾静脉注射200μL Cy5.5共价修饰的ZHER2:342-Epo B纳米粒子,在设定的时间点(15min,30min,1h,2h,4h,8h,和12h),从老鼠眼眶取血约0.5mL到含有肝素钠的试管中,然后保存于4℃冰箱。采集完所有数据点后,将所得血液样品3000rpm离心10min得到上层血清样品。每个时间点取200μL血清,采用荧光光谱进行分析其中的Cy5.5含量,换算得到相应的ZHER2:342-Epo B的含量。
结果如图14所示,从图中可以推断出,ZHER2:342-Epo B纳米粒子的血液半衰期约为6h,这一数值大大高于常规小分子抗肿瘤药物的血液半衰期(例如,目前临床化疗的一线抗癌药物紫杉醇,其在大鼠体内的血液半衰期仅为1.1h)。在大鼠尾静脉注射ZHER2:342-Epo B纳米粒子12h后,其血液里ZHER2:342-Epo B纳米粒子仍然保持在5.6μg/mL的高浓度,说明ZHER2:342-Epo B纳米粒子可以有效的在血液循环***中保留,较长的血液停留时间亦有利于ZHER2:342-Epo B纳米粒子在肿瘤部位更多的富集,从而达到有效的抗肿瘤效果。
实施例12
本实施例提供了用于肿瘤主动靶向治疗的亲和体-细胞毒素偶联物ZHER2:342-Epo B纳米颗粒在荷瘤裸鼠体内长时间循环成像示意图。
采用Cy5.5(花青染料5.5)作为体内的荧光探针分子,将Cy5.5共价修饰在实施例1中得到的具有主动靶向功能偶联物ZHER2:342-Epo B纳米粒子的表面,对纳米粒子的体内循环时间及靶向效果进行评价。制备浓度为4.0×106个/mL处于对数生长期的SKOV-3细胞的细胞悬液,接种到裸鼠右前腋窝皮下,每只接种200μL,当肿瘤体积约500mm3时,分别随机取2只小鼠作为对照组和实验组。实验组注射200μL Cy5.5共价修饰的ZHER2:342-Epo B纳米粒子,对照组注射200μL Cy5.5溶液(实验组溶液与对照组溶液中的Cy5.5含量相同)。在注射4h,12h后,采用ZKKS-Mulaurora成像***拍照并分析小鼠全身的荧光强度。
结果如图15所示,小分子的Cy5.5在注射4小时后,在小鼠体内已无明显存在,12h后已基本代谢完全,与此同时,ZHER2:342-Epo B纳米粒子在注射4小时后,在肿瘤部位有显著富集,并且在其他脏器无明显存在,在小鼠体内循环12h后,依旧能在肿瘤部位观察到明显的荧光信号。这一结果说明了该亲和体-细胞毒素偶联物纳米颗粒具有优异的肿瘤靶向性能并且能在体内长时间循环,在肿瘤治疗中具有潜在的应用价值。
实施例13
本实施例提供了用于肿瘤主动靶向治疗的亲和体-细胞毒素偶联物ZHER2:342-MMAE纳米颗粒对荷瘤裸鼠的肿瘤治疗效果图。
制备浓度为4.0×106个/mL处于对数生长期的SKOV-3细胞的细胞悬液,接种到裸鼠右前腋窝皮下,每只接种200μL,当肿瘤体积约100mm3时,随机分4组,每组5只,分别为PBS对照组、MMAE 0.6mg/kg组,ZHER2:342-MMAE纳米颗粒(MMAE含量0.4mg/kg)组,ZHER2:342-MMAE纳米颗粒(MMAE含量0.6mg/kg)组,其中,PBS组注射200μl PBS,MMAE 0.6mg/kg组注射MMAE0.6mg/kg,ZHER2:342-MMAE(MMAE含量0.4mg/kg)组注射ZHER2:342-MMAE(含MMAE 0.4mg/kg),ZHER2:342-MMAE(MMAE含量0.6mg/kg)组注射ZHER2:342-MMAE(含MMAE 0.6mg/kg),每3天注射一次。每次给药后观察荷瘤裸鼠的生存情况,测量小鼠体重及肿瘤大小并拍照留存。
实验结果如图16,17,18所示,PBS对照组小鼠的肿瘤增长迅速;MMAE 0.6mg/kg组小鼠在给药后体重急剧下降,最终在3次给药以后全部死亡,这一结果说明了MMAE原料药具有强烈的毒副作用;与此同时,荷瘤小鼠经由ZHER2:342-MMAE纳米颗粒(MMAE含量0.4mg/kg)治疗后,肿瘤体积明显减小,剂量增加至0.6mg/kg后,荷瘤小鼠的肿瘤体积明显减小直至基本消失,并且生存状况良好。
这一结果说明了亲和体-细胞毒素偶联物ZHER2:342-MMAE纳米颗粒的靶向性不仅带来了优异的体内抗肿瘤效果同时还有效的减小了MMAE的毒副作用。优异的抗肿瘤效果和良好的生物安全性证明了亲和体-细胞毒素偶联物是极具市场前景的抗体-药物偶联的新剂型。
以上公开的仅为本发明优选实施例。优选实施例并没有详尽叙述所有的细节,也不限制该发明仅为所述的具体实施方式。显然,根据本说明书的内容,可作很多的修改和变化。本说明书选取并具体描述这些实施例,是为了更好地解释本发明的原理和实际应用,从而使所属领域技术人员能很好地利用本发明。本发明仅受权利要求书及其全部范围和等效物的限制。
在本发明及上述实施例的教导下,本领域技术人员很容易预见到,本发明所列举或例举的各原料或其等同替换物、各加工方法或其等同替换物都能实现本发明,以及各原料和加工方法的参数上下限取值、区间值都能实现本发明,在此不一一列举实施例。
Claims (7)
1.用于肿瘤主动靶向治疗的亲和体-细胞毒素偶联物,其特征在于,主要包括细胞毒素和具有主动靶向功能的亲和体,所述的偶联物由所述细胞毒素与所述亲和体之间通过小分子连接基共价偶联而得;
所述的亲和体选自靶向人表皮生长因子受体的亲和体ZHER2:342或ZEGFR:1907、靶向人***受体的亲和体ZIGF1R:4551中的至少一种;
所述的细胞毒素选自埃博霉素B及其衍生物、海兔毒素及其衍生物中的至少一种;
所述的小分子连接基选自以下式(II)、式(III)、式(IV)中的至少一种;
其中,R1选自:-烷基-C(O)NH-,-烷基-SO2NH-或C1-8烷氧基;R2选自羧基或酰氯,n、m各自独立地选自2~10;
所述烷基选自C1-8烷基;所述烷基、烷氧基为未取代的或被1、2、3个选自下组的取代基所取代:卤素、氰基、乙酰基、羟基、羟甲基、羟乙基、羧基、C1-8烷基、C1-8烷氧基、卤代C1-8烷基、C3-8环烷基、C3-8环烷氧基、卤代C1-8烷氧基、-C(O)C1-10烷基、-C(O)OC1-10烷基、-OC(O)C1-10烷基、-CONRa0Rb0;Ra0、Rb0各自独立地为氢或C1-8烷基。
3.一种用于肿瘤主动靶向治疗的亲和体-细胞毒素偶联物纳米颗粒,其特征在于,由如权利要求1或2任一项所述的偶联物在水介质中自组装得到。
4.如权利要求3所述的纳米颗粒,其特征在于,所述纳米颗粒的壳层由具有主动靶向功能的亲和体构成,所述纳米颗粒的内核由所述的小分子连接基及细胞毒素构成,所述纳米颗粒的粒径小于200nm。
5.一种如权利要求3或4所述的用于肿瘤主动靶向治疗的亲和体-细胞毒素偶联物纳米颗粒的制备方法,其特征在于,包括以下步骤:
(1)将含羟基、氨基或巯基中任意一种或几种的疏水性细胞毒素与小分子连接基键合起来,再进一步与主动靶向亲和体进行偶联反应,得到具有主动靶向功能的亲和体-细胞毒素偶联物;
(2)将一定量步骤(1)所述亲和体-细胞毒素偶联物溶于一定体积的有机溶剂中,室温下将其滴入缓慢搅拌的超纯水中,除去有机溶剂,得到具有主动靶向功能的偶联物纳米颗粒水溶液。
6.根据权利要求5所述的制备方法,其特征在于,步骤(2)中的有机溶剂选自二甲基亚砜、N,N’-二甲基甲酰胺、乙醇、异丙醇中的至少一种。
7.一种如权利要求1或2任一项所述的用于肿瘤主动靶向治疗的亲和体-细胞毒素偶联物、或如权利要求3或4所述的纳米颗粒或如权利要求5或6所述的制备方法制得的所述纳米颗粒在制备用于肿瘤主动靶向治疗的药物中的应用。
Priority Applications (5)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202011114498.6A CN112220931B (zh) | 2020-10-16 | 2020-10-16 | 用于肿瘤主动靶向治疗的亲和体-细胞毒素偶联物及其纳米颗粒、制备方法、应用 |
JP2023509447A JP2023537524A (ja) | 2020-10-16 | 2021-02-05 | 腫瘍能動標的治療用のアフィボディ-細胞毒素複合体及びそのナノ粒子、調製方法、使用 |
US18/020,577 US20230310624A1 (en) | 2020-10-16 | 2021-02-05 | Affibody-cytotoxin conjugate for active targeted therapy of tumors, nanoparticle thereof, preparation method thereof and application thereof |
PCT/CN2021/075469 WO2022077815A1 (zh) | 2020-10-16 | 2021-02-05 | 用于肿瘤主动靶向治疗的亲和体-细胞毒素偶联物及其纳米颗粒、制备方法、应用 |
EP21878872.7A EP4230224A1 (en) | 2020-10-16 | 2021-02-05 | Affibody-cytotoxin conjugate used for active tumor targeting therapy, and nanoparticles, preparation method, and application thereof |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202011114498.6A CN112220931B (zh) | 2020-10-16 | 2020-10-16 | 用于肿瘤主动靶向治疗的亲和体-细胞毒素偶联物及其纳米颗粒、制备方法、应用 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN112220931A CN112220931A (zh) | 2021-01-15 |
CN112220931B true CN112220931B (zh) | 2022-03-08 |
Family
ID=74118623
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202011114498.6A Active CN112220931B (zh) | 2020-10-16 | 2020-10-16 | 用于肿瘤主动靶向治疗的亲和体-细胞毒素偶联物及其纳米颗粒、制备方法、应用 |
Country Status (5)
Country | Link |
---|---|
US (1) | US20230310624A1 (zh) |
EP (1) | EP4230224A1 (zh) |
JP (1) | JP2023537524A (zh) |
CN (1) | CN112220931B (zh) |
WO (1) | WO2022077815A1 (zh) |
Families Citing this family (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN111097052B (zh) * | 2020-01-17 | 2022-04-01 | 上海交通大学 | 用于肿瘤主动靶向治疗的两亲性前药及其纳米颗粒的制备方法、应用 |
CN112220931B (zh) * | 2020-10-16 | 2022-03-08 | 上海交通大学 | 用于肿瘤主动靶向治疗的亲和体-细胞毒素偶联物及其纳米颗粒、制备方法、应用 |
CN114732915B (zh) * | 2022-04-12 | 2023-08-29 | 上海交通大学 | 用于结肠癌的主动靶向治疗的纳米颗粒及制备方法、应用 |
Family Cites Families (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2016145536A1 (en) * | 2015-03-18 | 2016-09-22 | Immunobiochem Corporation | Conjugates for the treatment of cancer targeted at intracellular tumor-associated antigens |
CN106822036B (zh) * | 2016-12-15 | 2022-08-09 | 国家纳米科学中心 | 特异靶向多肽自组装纳米载体、载药纳米颗粒及制备方法 |
CN108948152A (zh) * | 2017-05-18 | 2018-12-07 | 中国科学院上海药物研究所 | 一种两亲性穿膜肽键合物、其制备方法及用途 |
CN111097052B (zh) * | 2020-01-17 | 2022-04-01 | 上海交通大学 | 用于肿瘤主动靶向治疗的两亲性前药及其纳米颗粒的制备方法、应用 |
CN112220931B (zh) * | 2020-10-16 | 2022-03-08 | 上海交通大学 | 用于肿瘤主动靶向治疗的亲和体-细胞毒素偶联物及其纳米颗粒、制备方法、应用 |
-
2020
- 2020-10-16 CN CN202011114498.6A patent/CN112220931B/zh active Active
-
2021
- 2021-02-05 US US18/020,577 patent/US20230310624A1/en active Pending
- 2021-02-05 JP JP2023509447A patent/JP2023537524A/ja active Pending
- 2021-02-05 WO PCT/CN2021/075469 patent/WO2022077815A1/zh unknown
- 2021-02-05 EP EP21878872.7A patent/EP4230224A1/en active Pending
Also Published As
Publication number | Publication date |
---|---|
WO2022077815A1 (zh) | 2022-04-21 |
EP4230224A1 (en) | 2023-08-23 |
CN112220931A (zh) | 2021-01-15 |
JP2023537524A (ja) | 2023-09-01 |
US20230310624A1 (en) | 2023-10-05 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN112220931B (zh) | 用于肿瘤主动靶向治疗的亲和体-细胞毒素偶联物及其纳米颗粒、制备方法、应用 | |
US9757342B2 (en) | Method for preparing protein cage, and in situ method for preparing hydrophobic additive-supported core-shell structured polymer-protein particles | |
US20100209353A1 (en) | Tumor targeting protein conjugate and a method for preparing the same | |
CN111097052B (zh) | 用于肿瘤主动靶向治疗的两亲性前药及其纳米颗粒的制备方法、应用 | |
Meng et al. | Preparation of drug-loaded albumin nanoparticles and its application in cancer therapy | |
CN114806546A (zh) | 基于荧光分子的有机框架材料及其制备方法和应用 | |
CN107216362A (zh) | 一种阿糖胞苷两亲性小分子前药及其制备方法和应用 | |
Mu et al. | Zwitterionic rhodamine-CPT prodrug nanoparticles with GSH/H2O2 responsiveness for cancer theranostics | |
CN108309938B (zh) | 主动定制白蛋白冕的药物传递载体及其在药学中的应用 | |
Wan et al. | Robust Strategy for Antibody–Polymer–Drug Conjugation: Significance of Conjugating Orientation and Linker Charge on Targeting Ability | |
CN111643678B (zh) | 基于含巯基的两性离子多肽修饰的阿霉素衍生物、纳米胶束及其制备方法 | |
Dong et al. | Targeted MRI and chemotherapy of ovarian cancer with clinic available nano-drug based nanoprobe | |
KR101973535B1 (ko) | 그래핀 양자점 복합체 | |
CN113004536A (zh) | 一种金属-氨基酸/肽配位聚合物及其应用 | |
CN110003086B (zh) | 一种两亲性小分子ir820-1mt及其制剂以及其制备方法和应用 | |
CN109939081B (zh) | F3多肽靶向的纳米有机金属框架材料(nMOFs)及其制备方法 | |
Bizeau et al. | Protein sustained release from isobutyramide-grafted stellate mesoporous silica nanoparticles | |
CN112237636B (zh) | 一种合成两性离子化的双亲性树状大分子并用其包载抗癌药物的方法 | |
CN113384551B (zh) | 一种减少肿瘤内过量钙离子的仿生纳米载体的制备方法和应用 | |
CN108690119A (zh) | 一种伊文思蓝修饰的多肽类前药及其制备和应用 | |
CN114159560A (zh) | Gsh与ph双响应、化疗与光热联合治疗药物递送载体的制备方法及应用 | |
KR20120126356A (ko) | 양친성 저분자량 히알루론산 복합체를 포함하는 나노 입자 및 그의 제조 방법 | |
CN104338151A (zh) | 新型主被动双靶向金纳米棒制备方法及其肿瘤诊断治疗一体化的新途径 | |
Komedchikova et al. | Targosomes: Anti-HER2 PLGA nanocarriers for bioimaging, chemotherapy and local photothermal treatment of tumors and remote metastases | |
CN109568599A (zh) | 一种脂质体修饰的阿霉素及含阿霉素的纳米粒子 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PB01 | Publication | ||
PB01 | Publication | ||
SE01 | Entry into force of request for substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
GR01 | Patent grant | ||
GR01 | Patent grant |