CN102596234B - 针对表达天冬酰胺酰-β-羟化酶的肿瘤的树突细胞疫苗 - Google Patents
针对表达天冬酰胺酰-β-羟化酶的肿瘤的树突细胞疫苗 Download PDFInfo
- Publication number
- CN102596234B CN102596234B CN201080033781.4A CN201080033781A CN102596234B CN 102596234 B CN102596234 B CN 102596234B CN 201080033781 A CN201080033781 A CN 201080033781A CN 102596234 B CN102596234 B CN 102596234B
- Authority
- CN
- China
- Prior art keywords
- aah
- tumor
- cell
- dendritic cell
- cancer
- 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
- 206010028980 Neoplasm Diseases 0.000 title claims abstract description 94
- 229940029030 dendritic cell vaccine Drugs 0.000 title description 5
- 210000004443 dendritic cell Anatomy 0.000 claims abstract description 104
- 238000000034 method Methods 0.000 claims abstract description 51
- 239000000427 antigen Substances 0.000 claims abstract description 32
- 102000036639 antigens Human genes 0.000 claims abstract description 32
- 108091007433 antigens Proteins 0.000 claims abstract description 32
- 102000004127 Cytokines Human genes 0.000 claims abstract description 21
- 108090000695 Cytokines Proteins 0.000 claims abstract description 21
- 238000000926 separation method Methods 0.000 claims abstract description 9
- 108010017213 Granulocyte-Macrophage Colony-Stimulating Factor Proteins 0.000 claims abstract description 8
- 229960005486 vaccine Drugs 0.000 claims abstract description 6
- 210000004027 cell Anatomy 0.000 claims description 74
- 201000011510 cancer Diseases 0.000 claims description 27
- 108010074328 Interferon-gamma Proteins 0.000 claims description 16
- 102100037850 Interferon gamma Human genes 0.000 claims description 15
- 108090000978 Interleukin-4 Proteins 0.000 claims description 10
- 230000012010 growth Effects 0.000 claims description 9
- 230000004614 tumor growth Effects 0.000 claims description 9
- 108010029697 CD40 Ligand Proteins 0.000 claims description 7
- 102100032937 CD40 ligand Human genes 0.000 claims description 7
- 102100039620 Granulocyte-macrophage colony-stimulating factor Human genes 0.000 claims description 7
- 206010070834 Sensitisation Diseases 0.000 claims description 7
- 230000008313 sensitization Effects 0.000 claims description 7
- 239000011324 bead Substances 0.000 claims description 6
- 210000002950 fibroblast Anatomy 0.000 claims description 5
- 238000000746 purification Methods 0.000 claims description 5
- 206010006187 Breast cancer Diseases 0.000 claims description 4
- 206010058467 Lung neoplasm malignant Diseases 0.000 claims description 4
- 206010027476 Metastases Diseases 0.000 claims description 4
- 210000000265 leukocyte Anatomy 0.000 claims description 4
- 239000007790 solid phase Substances 0.000 claims description 4
- 206010005003 Bladder cancer Diseases 0.000 claims description 3
- 241000283690 Bos taurus Species 0.000 claims description 3
- 208000003174 Brain Neoplasms Diseases 0.000 claims description 3
- 206010008342 Cervix carcinoma Diseases 0.000 claims description 3
- 241000283073 Equus caballus Species 0.000 claims description 3
- 241000282326 Felis catus Species 0.000 claims description 3
- 206010061902 Pancreatic neoplasm Diseases 0.000 claims description 3
- 241000009328 Perro Species 0.000 claims description 3
- 239000004793 Polystyrene Substances 0.000 claims description 3
- 206010038389 Renal cancer Diseases 0.000 claims description 3
- 208000006265 Renal cell carcinoma Diseases 0.000 claims description 3
- 241000282898 Sus scrofa Species 0.000 claims description 3
- 208000007097 Urinary Bladder Neoplasms Diseases 0.000 claims description 3
- 208000006105 Uterine Cervical Neoplasms Diseases 0.000 claims description 3
- -1 aspartyl Chemical group 0.000 claims description 3
- 201000010881 cervical cancer Diseases 0.000 claims description 3
- 201000001343 fallopian tube carcinoma Diseases 0.000 claims description 3
- 201000005264 laryngeal carcinoma Diseases 0.000 claims description 3
- 208000015486 malignant pancreatic neoplasm Diseases 0.000 claims description 3
- 229920002223 polystyrene Polymers 0.000 claims description 3
- 230000002265 prevention Effects 0.000 claims description 3
- 201000001514 prostate carcinoma Diseases 0.000 claims description 3
- 201000010174 renal carcinoma Diseases 0.000 claims description 3
- 201000005112 urinary bladder cancer Diseases 0.000 claims description 3
- 206010033128 Ovarian cancer Diseases 0.000 claims description 2
- 206010061535 Ovarian neoplasm Diseases 0.000 claims description 2
- 208000033781 Thyroid carcinoma Diseases 0.000 claims description 2
- 208000024770 Thyroid neoplasm Diseases 0.000 claims description 2
- 201000002510 thyroid cancer Diseases 0.000 claims description 2
- 208000013077 thyroid gland carcinoma Diseases 0.000 claims description 2
- 239000003814 drug Substances 0.000 claims 9
- 206010017993 Gastrointestinal neoplasms Diseases 0.000 claims 1
- 201000008274 breast adenocarcinoma Diseases 0.000 claims 1
- 201000010175 gallbladder cancer Diseases 0.000 claims 1
- 201000007487 gallbladder carcinoma Diseases 0.000 claims 1
- 102000004457 Granulocyte-Macrophage Colony-Stimulating Factor Human genes 0.000 abstract 1
- 230000036039 immunity Effects 0.000 description 27
- 102000013529 alpha-Fetoproteins Human genes 0.000 description 22
- 108010026331 alpha-Fetoproteins Proteins 0.000 description 22
- 241000699670 Mus sp. Species 0.000 description 20
- 210000004988 splenocyte Anatomy 0.000 description 19
- 241000699666 Mus <mouse, genus> Species 0.000 description 16
- 238000002649 immunization Methods 0.000 description 15
- 230000003053 immunization Effects 0.000 description 15
- 238000002560 therapeutic procedure Methods 0.000 description 13
- 230000014509 gene expression Effects 0.000 description 12
- 210000001744 T-lymphocyte Anatomy 0.000 description 11
- 108090000765 processed proteins & peptides Proteins 0.000 description 11
- 230000004044 response Effects 0.000 description 11
- 210000001519 tissue Anatomy 0.000 description 10
- 102000004388 Interleukin-4 Human genes 0.000 description 9
- 230000000694 effects Effects 0.000 description 9
- 229920001184 polypeptide Polymers 0.000 description 9
- 102000004196 processed proteins & peptides Human genes 0.000 description 9
- 235000018102 proteins Nutrition 0.000 description 9
- 102000004169 proteins and genes Human genes 0.000 description 9
- 108090000623 proteins and genes Proteins 0.000 description 9
- 238000010586 diagram Methods 0.000 description 7
- 238000002965 ELISA Methods 0.000 description 6
- 102000013462 Interleukin-12 Human genes 0.000 description 6
- 108010065805 Interleukin-12 Proteins 0.000 description 6
- 230000000259 anti-tumor effect Effects 0.000 description 6
- 230000001225 therapeutic effect Effects 0.000 description 6
- 238000011282 treatment Methods 0.000 description 6
- 240000002853 Nelumbo nucifera Species 0.000 description 5
- 235000006508 Nelumbo nucifera Nutrition 0.000 description 5
- 235000006510 Nelumbo pentapetala Nutrition 0.000 description 5
- 210000000612 antigen-presenting cell Anatomy 0.000 description 5
- 230000006698 induction Effects 0.000 description 5
- 230000008569 process Effects 0.000 description 5
- 230000028327 secretion Effects 0.000 description 5
- 230000004936 stimulating effect Effects 0.000 description 5
- 210000004881 tumor cell Anatomy 0.000 description 5
- 102100022108 Aspartyl/asparaginyl beta-hydroxylase Human genes 0.000 description 4
- 101000901030 Homo sapiens Aspartyl/asparaginyl beta-hydroxylase Proteins 0.000 description 4
- 230000000890 antigenic effect Effects 0.000 description 4
- 230000007541 cellular toxicity Effects 0.000 description 4
- 208000006990 cholangiocarcinoma Diseases 0.000 description 4
- 201000010099 disease Diseases 0.000 description 4
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 4
- 239000002158 endotoxin Substances 0.000 description 4
- 210000000232 gallbladder Anatomy 0.000 description 4
- 238000011081 inoculation Methods 0.000 description 4
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 4
- 229920006008 lipopolysaccharide Polymers 0.000 description 4
- 239000000243 solution Substances 0.000 description 4
- 230000001629 suppression Effects 0.000 description 4
- 231100000820 toxicity test Toxicity 0.000 description 4
- 208000026310 Breast neoplasm Diseases 0.000 description 3
- 201000009030 Carcinoma Diseases 0.000 description 3
- 108010062580 Concanavalin A Proteins 0.000 description 3
- 239000012981 Hank's balanced salt solution Substances 0.000 description 3
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 description 3
- 241001465754 Metazoa Species 0.000 description 3
- 229930182555 Penicillin Natural products 0.000 description 3
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 3
- 201000008275 breast carcinoma Diseases 0.000 description 3
- 230000003197 catalytic effect Effects 0.000 description 3
- 230000007969 cellular immunity Effects 0.000 description 3
- 210000001072 colon Anatomy 0.000 description 3
- 230000003013 cytotoxicity Effects 0.000 description 3
- 231100000135 cytotoxicity Toxicity 0.000 description 3
- 239000000975 dye Substances 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 230000028993 immune response Effects 0.000 description 3
- 230000005847 immunogenicity Effects 0.000 description 3
- 230000001976 improved effect Effects 0.000 description 3
- 238000000338 in vitro Methods 0.000 description 3
- 210000005075 mammary gland Anatomy 0.000 description 3
- 230000035800 maturation Effects 0.000 description 3
- 239000011325 microbead Substances 0.000 description 3
- 230000002018 overexpression Effects 0.000 description 3
- 229940049954 penicillin Drugs 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 229940041022 streptomycins Drugs 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- 238000012546 transfer Methods 0.000 description 3
- 230000003442 weekly effect Effects 0.000 description 3
- GHKCSRZBNZQHKW-UHFFFAOYSA-N 1-sulfanylethanol Chemical class CC(O)S GHKCSRZBNZQHKW-UHFFFAOYSA-N 0.000 description 2
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 description 2
- BTBUEUYNUDRHOZ-UHFFFAOYSA-N Borate Chemical compound [O-]B([O-])[O-] BTBUEUYNUDRHOZ-UHFFFAOYSA-N 0.000 description 2
- 206010009944 Colon cancer Diseases 0.000 description 2
- 101710088194 Dehydrogenase Proteins 0.000 description 2
- 102100020715 Fms-related tyrosine kinase 3 ligand protein Human genes 0.000 description 2
- 101710162577 Fms-related tyrosine kinase 3 ligand protein Proteins 0.000 description 2
- 239000007995 HEPES buffer Substances 0.000 description 2
- 102100022297 Integrin alpha-X Human genes 0.000 description 2
- 241000124008 Mammalia Species 0.000 description 2
- 239000012980 RPMI-1640 medium Substances 0.000 description 2
- 235000018259 Solanum vestissimum Nutrition 0.000 description 2
- 240000002825 Solanum vestissimum Species 0.000 description 2
- 208000009956 adenocarcinoma Diseases 0.000 description 2
- 230000003321 amplification Effects 0.000 description 2
- 239000007853 buffer solution Substances 0.000 description 2
- 230000006037 cell lysis Effects 0.000 description 2
- 230000036755 cellular response Effects 0.000 description 2
- 238000002512 chemotherapy Methods 0.000 description 2
- 230000000875 corresponding effect Effects 0.000 description 2
- 210000001151 cytotoxic T lymphocyte Anatomy 0.000 description 2
- 230000034994 death Effects 0.000 description 2
- 238000003745 diagnosis Methods 0.000 description 2
- 238000004043 dyeing Methods 0.000 description 2
- 239000012636 effector Substances 0.000 description 2
- 210000003527 eukaryotic cell Anatomy 0.000 description 2
- 239000013613 expression plasmid Substances 0.000 description 2
- 238000000684 flow cytometry Methods 0.000 description 2
- 230000002496 gastric effect Effects 0.000 description 2
- 208000005017 glioblastoma Diseases 0.000 description 2
- 206010073071 hepatocellular carcinoma Diseases 0.000 description 2
- 230000002163 immunogen Effects 0.000 description 2
- 238000011532 immunohistochemical staining Methods 0.000 description 2
- 229960001438 immunostimulant agent Drugs 0.000 description 2
- 239000003022 immunostimulating agent Substances 0.000 description 2
- 230000003308 immunostimulating effect Effects 0.000 description 2
- 238000009169 immunotherapy Methods 0.000 description 2
- 230000003993 interaction Effects 0.000 description 2
- 235000014655 lactic acid Nutrition 0.000 description 2
- 239000004310 lactic acid Substances 0.000 description 2
- 201000007270 liver cancer Diseases 0.000 description 2
- 208000014018 liver neoplasm Diseases 0.000 description 2
- 210000004072 lung Anatomy 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 239000003550 marker Substances 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 230000009401 metastasis Effects 0.000 description 2
- MYWUZJCMWCOHBA-VIFPVBQESA-N methamphetamine Chemical compound CN[C@@H](C)CC1=CC=CC=C1 MYWUZJCMWCOHBA-VIFPVBQESA-N 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 238000003199 nucleic acid amplification method Methods 0.000 description 2
- 108020004707 nucleic acids Proteins 0.000 description 2
- 102000039446 nucleic acids Human genes 0.000 description 2
- 150000007523 nucleic acids Chemical class 0.000 description 2
- 238000011321 prophylaxis Methods 0.000 description 2
- 239000012679 serum free medium Substances 0.000 description 2
- DAEPDZWVDSPTHF-UHFFFAOYSA-M sodium pyruvate Chemical compound [Na+].CC(=O)C([O-])=O DAEPDZWVDSPTHF-UHFFFAOYSA-M 0.000 description 2
- 210000002784 stomach Anatomy 0.000 description 2
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 description 2
- 238000010254 subcutaneous injection Methods 0.000 description 2
- 239000007929 subcutaneous injection Substances 0.000 description 2
- 230000008685 targeting Effects 0.000 description 2
- 210000003462 vein Anatomy 0.000 description 2
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 description 2
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 1
- KZDCMKVLEYCGQX-UDPGNSCCSA-N 2-(diethylamino)ethyl 4-aminobenzoate;(2s,5r,6r)-3,3-dimethyl-7-oxo-6-[(2-phenylacetyl)amino]-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylic acid;hydrate Chemical compound O.CCN(CC)CCOC(=O)C1=CC=C(N)C=C1.N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 KZDCMKVLEYCGQX-UDPGNSCCSA-N 0.000 description 1
- YXHLJMWYDTXDHS-IRFLANFNSA-N 7-aminoactinomycin D Chemical compound C[C@H]1OC(=O)[C@H](C(C)C)N(C)C(=O)CN(C)C(=O)[C@@H]2CCCN2C(=O)[C@@H](C(C)C)NC(=O)[C@H]1NC(=O)C1=C(N)C(=O)C(C)=C2OC(C(C)=C(N)C=C3C(=O)N[C@@H]4C(=O)N[C@@H](C(N5CCC[C@H]5C(=O)N(C)CC(=O)N(C)[C@@H](C(C)C)C(=O)O[C@@H]4C)=O)C(C)C)=C3N=C21 YXHLJMWYDTXDHS-IRFLANFNSA-N 0.000 description 1
- 108700012813 7-aminoactinomycin D Proteins 0.000 description 1
- HRPVXLWXLXDGHG-UHFFFAOYSA-N Acrylamide Chemical compound NC(=O)C=C HRPVXLWXLXDGHG-UHFFFAOYSA-N 0.000 description 1
- 206010001167 Adenocarcinoma of colon Diseases 0.000 description 1
- 102100034278 Annexin A6 Human genes 0.000 description 1
- 108090000656 Annexin A6 Proteins 0.000 description 1
- 238000011725 BALB/c mouse Methods 0.000 description 1
- 208000003170 Bronchiolo-Alveolar Adenocarcinoma Diseases 0.000 description 1
- 206010058354 Bronchioloalveolar carcinoma Diseases 0.000 description 1
- 101150013553 CD40 gene Proteins 0.000 description 1
- 208000017897 Carcinoma of esophagus Diseases 0.000 description 1
- 108020004705 Codon Proteins 0.000 description 1
- 208000001333 Colorectal Neoplasms Diseases 0.000 description 1
- 241000699800 Cricetinae Species 0.000 description 1
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 1
- 102000002322 Egg Proteins Human genes 0.000 description 1
- 108010000912 Egg Proteins Proteins 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 1
- 101000599852 Homo sapiens Intercellular adhesion molecule 1 Proteins 0.000 description 1
- 101000914484 Homo sapiens T-lymphocyte activation antigen CD80 Proteins 0.000 description 1
- 102100037877 Intercellular adhesion molecule 1 Human genes 0.000 description 1
- 108010002350 Interleukin-2 Proteins 0.000 description 1
- 241000581650 Ivesia Species 0.000 description 1
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 description 1
- 206010029260 Neuroblastoma Diseases 0.000 description 1
- 206010030155 Oesophageal carcinoma Diseases 0.000 description 1
- 102000004022 Protein-Tyrosine Kinases Human genes 0.000 description 1
- 108090000412 Protein-Tyrosine Kinases Proteins 0.000 description 1
- 241000700159 Rattus Species 0.000 description 1
- 208000015634 Rectal Neoplasms Diseases 0.000 description 1
- 208000005718 Stomach Neoplasms Diseases 0.000 description 1
- 230000024932 T cell mediated immunity Effects 0.000 description 1
- 102100027222 T-lymphocyte activation antigen CD80 Human genes 0.000 description 1
- 201000009365 Thymic carcinoma Diseases 0.000 description 1
- 101710120037 Toxin CcdB Proteins 0.000 description 1
- 102100040245 Tumor necrosis factor receptor superfamily member 5 Human genes 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 150000001413 amino acids Chemical group 0.000 description 1
- 229910021529 ammonia Inorganic materials 0.000 description 1
- QGZKDVFQNNGYKY-UHFFFAOYSA-N ammonia Natural products N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 1
- 230000002001 anti-metastasis Effects 0.000 description 1
- 230000000118 anti-neoplastic effect Effects 0.000 description 1
- 230000006907 apoptotic process Effects 0.000 description 1
- 210000003719 b-lymphocyte Anatomy 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 238000010241 blood sampling Methods 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 239000000337 buffer salt Substances 0.000 description 1
- 230000003139 buffering effect Effects 0.000 description 1
- 239000002775 capsule Substances 0.000 description 1
- 239000013592 cell lysate Substances 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 230000009087 cell motility Effects 0.000 description 1
- 239000002458 cell surface marker Substances 0.000 description 1
- 229940030156 cell vaccine Drugs 0.000 description 1
- 210000003679 cervix uteri Anatomy 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- 238000003501 co-culture Methods 0.000 description 1
- 201000010897 colon adenocarcinoma Diseases 0.000 description 1
- 208000029742 colonic neoplasm Diseases 0.000 description 1
- 238000004440 column chromatography Methods 0.000 description 1
- 230000002301 combined effect Effects 0.000 description 1
- 239000002299 complementary DNA Substances 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 108091036078 conserved sequence Proteins 0.000 description 1
- 230000002596 correlated effect 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
- 239000012228 culture supernatant Substances 0.000 description 1
- 238000000432 density-gradient centrifugation Methods 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 235000013399 edible fruits Nutrition 0.000 description 1
- 239000003344 environmental pollutant Substances 0.000 description 1
- 210000003743 erythrocyte Anatomy 0.000 description 1
- 201000005619 esophageal carcinoma Diseases 0.000 description 1
- 210000003238 esophagus Anatomy 0.000 description 1
- 235000020776 essential amino acid Nutrition 0.000 description 1
- 239000003797 essential amino acid Substances 0.000 description 1
- 230000017214 establishment of T cell polarity Effects 0.000 description 1
- 238000001943 fluorescence-activated cell sorting Methods 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 206010017758 gastric cancer Diseases 0.000 description 1
- 238000001502 gel electrophoresis Methods 0.000 description 1
- 239000008187 granular material Substances 0.000 description 1
- 230000035876 healing Effects 0.000 description 1
- 210000003958 hematopoietic stem cell Anatomy 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 238000003364 immunohistochemistry Methods 0.000 description 1
- 239000007943 implant Substances 0.000 description 1
- 238000002513 implantation Methods 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 210000000867 larynx Anatomy 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 208000016992 lung adenocarcinoma in situ Diseases 0.000 description 1
- 210000004698 lymphocyte Anatomy 0.000 description 1
- 230000002934 lysing effect Effects 0.000 description 1
- 210000002540 macrophage Anatomy 0.000 description 1
- 238000002826 magnetic-activated cell sorting Methods 0.000 description 1
- 229930182817 methionine Natural products 0.000 description 1
- 208000024191 minimally invasive lung adenocarcinoma Diseases 0.000 description 1
- 239000007758 minimum essential medium Substances 0.000 description 1
- 230000017074 necrotic cell death Effects 0.000 description 1
- 230000008520 organization Effects 0.000 description 1
- 210000001672 ovary Anatomy 0.000 description 1
- 210000003101 oviduct Anatomy 0.000 description 1
- 210000004681 ovum Anatomy 0.000 description 1
- 210000000496 pancreas Anatomy 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 230000000505 pernicious effect Effects 0.000 description 1
- 210000004332 phalangeal cell Anatomy 0.000 description 1
- 239000012071 phase Substances 0.000 description 1
- 231100000719 pollutant Toxicity 0.000 description 1
- 230000003389 potentiating effect Effects 0.000 description 1
- 230000003449 preventive effect Effects 0.000 description 1
- 210000001236 prokaryotic cell Anatomy 0.000 description 1
- 210000002307 prostate Anatomy 0.000 description 1
- 238000012797 qualification Methods 0.000 description 1
- 206010038038 rectal cancer Diseases 0.000 description 1
- 201000001275 rectum cancer Diseases 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 238000009256 replacement therapy Methods 0.000 description 1
- 230000022120 response to tumor cell Effects 0.000 description 1
- 230000000630 rising effect Effects 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 229940054269 sodium pyruvate Drugs 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 238000012453 sprague-dawley rat model Methods 0.000 description 1
- 206010041823 squamous cell carcinoma Diseases 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 238000010561 standard procedure Methods 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 201000011549 stomach cancer Diseases 0.000 description 1
- 229960005322 streptomycin Drugs 0.000 description 1
- 238000007920 subcutaneous administration Methods 0.000 description 1
- 230000008093 supporting effect Effects 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 208000008732 thymoma Diseases 0.000 description 1
- 210000001685 thyroid gland Anatomy 0.000 description 1
- 238000012549 training Methods 0.000 description 1
- GPRLSGONYQIRFK-MNYXATJNSA-N triton Chemical compound [3H+] GPRLSGONYQIRFK-MNYXATJNSA-N 0.000 description 1
- 231100000588 tumorigenic Toxicity 0.000 description 1
- 230000000381 tumorigenic effect Effects 0.000 description 1
- 210000003932 urinary bladder Anatomy 0.000 description 1
- 238000002255 vaccination Methods 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/0005—Vertebrate antigens
- A61K39/0011—Cancer antigens
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/46—Cellular immunotherapy
- A61K39/461—Cellular immunotherapy characterised by the cell type used
- A61K39/4615—Dendritic cells
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/46—Cellular immunotherapy
- A61K39/462—Cellular immunotherapy characterized by the effect or the function of the cells
- A61K39/4622—Antigen presenting cells
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/46—Cellular immunotherapy
- A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
- A61K39/4643—Vertebrate antigens
- A61K39/4644—Cancer antigens
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
- A61P35/04—Antineoplastic agents specific for metastasis
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
- A61P37/04—Immunostimulants
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0634—Cells from the blood or the immune system
- C12N5/0639—Dendritic cells, e.g. Langherhans cells in the epidermis
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
- A61K2039/515—Animal cells
- A61K2039/5154—Antigen presenting cells [APCs], e.g. dendritic cells or macrophages
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
- A61K2039/515—Animal cells
- A61K2039/5158—Antigen-pulsed cells, e.g. T-cells
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2500/00—Specific components of cell culture medium
- C12N2500/90—Serum-free medium, which may still contain naturally-sourced components
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/20—Cytokines; Chemokines
- C12N2501/22—Colony stimulating factors (G-CSF, GM-CSF)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/20—Cytokines; Chemokines
- C12N2501/23—Interleukins [IL]
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/20—Cytokines; Chemokines
- C12N2501/24—Interferons [IFN]
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/50—Cell markers; Cell surface determinants
- C12N2501/52—CD40, CD40-ligand (CD154)
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Immunology (AREA)
- Medicinal Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Pharmacology & Pharmacy (AREA)
- Zoology (AREA)
- Genetics & Genomics (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
- Biomedical Technology (AREA)
- Wood Science & Technology (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- Mycology (AREA)
- Epidemiology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Engineering & Computer Science (AREA)
- Oncology (AREA)
- Molecular Biology (AREA)
- Hematology (AREA)
- Toxicology (AREA)
- Gastroenterology & Hepatology (AREA)
- Biophysics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Peptides Or Proteins (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Enzymes And Modification Thereof (AREA)
Abstract
一种含载AAH的成熟树突细胞的疫苗用于治疗哺乳动物对象中表达AAH的肿瘤。一种生产致敏树突细胞的方法,通过使分离的树突细胞接触抗原如AAH来进行。抗原接触步骤之后,树突细胞与细胞因子组合如GM‑CSF和IFN‑γ接触。
Description
相关申请
本申请请求以下申请的权益:2009年7月24日提交的U.S.S.N 61/228,429、2009年8月4日提交的U.S.S.N 61/231,127、2009年9月2日提交的U.S.S.N 61/239,288和2009年9月9日提交的U.S.S.N 61/240,745,其内容通过引用全文纳入本文。
发明背景
肝癌包括肝细胞癌和胆管细胞性肝癌,是世界上最常见癌症中的一种。在美国,肝癌在男性和女性的癌症致死中分别是第五和第九大死因。尽管对肝癌的诊断和局部控制有显著进展,但在美国的致死率仍在持续上升。死亡率升高的原因之一可能在于,与乳腺癌和结直肠癌在内的其他癌症不同,肝癌耐受全身治疗如化疗。因此,肝癌一旦成为全身疾病便没有有效疗法。
发明内容
本发明为长期以来对诊断为癌症的患者进行化疗替代治疗的问题提供了解决方案。降低对象内表达天冬氨酰基(天冬酰胺酰基)-β-羟化酶(AAH或ASPH)肿瘤的生长的方法包括给予对象分离的成熟载AAH树突细胞或细胞群体。表达AAH的肿瘤的生长在给予此类树突细胞疫苗后有降低。例如,肿瘤生长与肿瘤负荷降低10%、20%、50%、75%、2倍、5倍、10倍或更多。所述方法用于降低和消除哺乳动物对象如人患者中表达AAH的肿瘤。所述组合物和方法也适用于伴侣动物和牲畜,如人、犬、猫、马、牛或猪对象。
表达AAH的肿瘤包括大多数肿瘤类型,例如胃肠组织(如食道、胃、结肠)、胰腺、肝(如胆管细胞性肝癌、肝细胞癌)、乳腺、前列腺、子宫颈、卵巢、输卵管、喉、肺、甲状腺、胆囊、肾、膀胱和脑(如成胶质细胞瘤)的肿瘤以及下文描述的大量其他肿瘤。表达AAH的肿瘤包括相比正常组织表达且提高AAH水平的原发性肿瘤,以及通过所述过量表达AAH的原发肿瘤转移所产生的肿瘤。
所述免疫方法中所用的树突细胞优选在给予对象前用含GM-CSF和IFN-γ的细胞因子的组合离体激活。后一步骤导致树突细胞群体的抗肿瘤活性改善。
产生致敏树突细胞的改进方法通过使分离的树突细胞接触抗原如AAH或肿瘤抗原的组合如AAH和甲胎蛋白(AFP)并处理细胞以得到成熟并活化的抗原呈递细胞群进行。抗原接触步骤之后,树突细胞与细胞因子组合接触。例如,所述组合包括GM-CSF和IFN-γ。在其他示例中,组合还包括IL-4。可选地,组合包括CD40L。树突细胞与细胞因子组合接触至少10小时(例如,12、24、36、40、48小时或更久)。抗原为可溶形式或结合于固相载体。例如,所述固相载体包括聚苯乙烯珠如生物可降解珠或颗粒。树突细胞通过已知方法如白血球单采术或细胞分离法获自对象。
获取细胞的对象患有癌症或有发生癌症的风险。例如,所述患者已诊断有肿瘤如表达AAH的肿瘤。有发生癌症如载AAH肿瘤风险的患者包括已鉴定为有此类癌症病患个体的家族史的对象。
本发明还包括含有如上所述产生的致敏树突细胞的疫苗组合物。所述疫苗有助于抑制肿瘤生长、预防肿瘤生长和抑制或减少转移。抑制哺乳动物中肿瘤生长的方法通过下述步骤进行:鉴定患有载AAH肿瘤的对象,并给予所述对象自体树突细胞,所述细胞已经按上述方法致敏。预防哺乳动物中肿瘤发生的方法包括以下步骤:鉴定有发生载AAH肿瘤风险的对象(例如,有癌症家族史的对象),并给予所述对象经抗原致敏并活化的自体树突细胞。预防载AAH肿瘤转移的方法通过鉴定患有载AAH肿瘤的对象,并给予所述对象如上所述的自体树突细胞进行。
本发明的多肽和其他组合物为纯化的。例如,其中基本纯的AAH多肽变体优选通过表达编码该多肽的重组核酸或通过化学合成所述蛋白来获得。当多肽或蛋白与在天然状态下与其共存的污染物(蛋白质或其他天然产生的有机分子)分离时为基本纯。通常,当多肽在制品的蛋白中至少占60重量%时,所述多肽为基本纯的。制品中的蛋白质优选以重量计至少75%,更优选至少90%,最优选至少99%为AAH。纯度通过任何适当方法如柱层析、聚丙烯酰胺凝胶电泳或HPLC分析测定。因此,基本纯的多肽包括衍生自真核细胞但在大肠杆菌或其他原核细胞内或在不同于所述多肽原始来源的真核细胞内生产的重组多肽。
用于所述方法的树突细胞或其他细胞,例如免疫细胞如巨噬细胞、B细胞、T细胞为纯化或分离的。就细胞而言,术语″分离的″指细胞基本不含在天然条件下与其共存的其他细胞类型或细胞材料。例如,特定组织类型或表型的细胞样品占细胞群体至少60%时为″基本纯的″。制品中优选至少75%,更优选至少90%,最优选至少99%或100%为所述细胞群体。纯度通过任何适当的标准方法测定,例如,通过荧光活化的细胞分选(FACS)。
产生经抗原致敏DC的方法相比此前的方法有多种优势。细胞不但加载抗原,还经过后续的细胞因子孵育产生优异的已活化的经抗原致敏的抗原呈递细胞,具有改善的抗肿瘤和抗转移活性。
在阅读下列附图、具体描述和权利要求书后将更充分理解本发明的这些和其他能力以及本发明本身。本文引用的所有文献通过引用纳入本文。
附图简要说明
图1的柱状图显示分离DC的IL-12生产。
图2显示分离DC上表达多种表面DC标记物的系列线图。
图3的柱状图显示表面DC标记物的平均荧光。
图4显示细胞因子存在下生长的CD8+ DC群体增加的系列流式细胞分选图和柱状图。
图5的柱状图显示IFN-γ分泌。
图6的柱状图显示IL-4分泌。
图7是显示细胞毒性试验结果的线图。
图8为细胞表达AAH的示意图。
图9为加载成熟抗原的DC的产生方法的示意图。
图10为用AAH加载DC进行免疫的方案流程图。
图11显示AAH免疫对小鼠中肿瘤生长的治疗效果的确定方法的示意图。
图12显示AAH免疫对小鼠中肿瘤生长的治疗效果的线图。
具体实施方式
AAH为蛋白质,其表达与多种肿瘤类型相关(参见如:USPN 6,815,415;USPN 6,812,206;USPN 6,797,696;USPN 6,783,758;USPN 6,835,370和USPN 7,094,556)。HAAH过量表达已通过免疫组化染色(IHC)在多种肺癌(例如,腺癌、细支气管肺泡癌和其他非小细胞肺癌如鳞状细胞癌亚型(Luu等,2009,Hum.Pathol.40:639-644),肝癌(如肝细胞癌和胆管癌和胆上皮细胞癌(Wang等,2010,Hepatology 52:164-173),胃肠组织(如结肠、胃、食道)癌、胰腺癌、前列腺癌、卵巢癌、胆管癌、乳腺癌、肾癌、膀胱癌和脑癌(如成胶质细胞瘤和神经母细胞瘤)中测得。肿瘤组织样品和细胞系的另一调查证实在以下其他癌症中(与非癌组织相比)的AAH表达:喉癌、宫颈癌、输卵管癌、肝癌(如胆管癌)、肾癌、乳腺癌、宫颈癌、卵巢癌、输卵管癌、喉癌、肺癌、甲状腺癌、胰腺癌、胸腺癌、前列腺癌、膀胱癌、食道癌、胃癌、胆囊癌、结肠癌和直肠癌(Song等,2010,Chinese J.of Cell.and Mol.Immunol.26:141-144)。HAAH为癌症高度特异性,其已通过免疫组织化学在>99%的测试肿瘤样品(n>1000)中测得,而在相邻的未受侵袭组织或在正常个体的组织样品中没有。
将使用经AAH致敏的细胞的免疫治疗给予患在细胞表面表达AAH的肿瘤患者。这类肿瘤包括肝癌如肝细胞癌和胆管癌以及乳腺和结肠的腺癌。这类治疗策略不但可用于治疗癌症,也可用于预防癌症。
在本发明以前,尽管肝癌的诊断和局部控制有所发展,但尚无有效的全身治疗法。免疫治疗因其特异性而用于治疗全身和局部肝癌。
发现使用AAH的树突细胞疫苗治愈有免疫力小鼠内已建立的肝细胞癌。AAH树突细胞疫苗降低表达AAH的肿瘤的生长以降低肿瘤负荷并根除人体内肿瘤。
免疫治疗
恶性肿瘤的免疫治疗在对肿瘤细胞的特异性响应方面引人关注。免疫治疗的一个原理是癌细胞充满潜在抗原,当由抗原呈递细胞呈递给辅助与细胞毒性T细胞时这些抗原成为免疫原性。不论是否为全身性,肝癌的免疫治疗是可行的临床方法,因为肝癌细胞表达多种肿瘤相关蛋白,这些蛋白在正常组织内不表达或仅少量表达。例如,在成人组织内不表达的AFP在肝细胞癌(HCC)中广泛表达。多项研究显示AFP特异性免疫反应不单可在小鼠内诱发还可在人体内诱发。但是,靶向AFP的HCC免疫治疗临床试验未显示哪怕是部分的肿瘤应答。一些可能的理由有:因为AFP不参与癌的进展,不表达AFP的癌细胞在APP-靶向的免疫治疗中占优势。为使HCC细胞被AFP响应T细胞识别,AFP应与I型主要组织相容性复合抗体(MHC)分子一起呈递于细胞表面,因为AFP不分布在细胞表面。但是,由于I型MHC分子通常在很多癌细胞中被下调,AFP可能不被T细胞恰当地识别。为克服肝癌免疫治疗的问题,进行研究以鉴定与肝癌相关的另一抗原以用来致敏细胞供基于细胞的治疗。考虑到用AFP作为抗原的结果,AAH的抗肿瘤结果出乎意料。
AAH也称为ASPH,在肝细胞癌和胆管细胞癌中强烈表达,但在这些组织类型的正常细胞对应体中不表达。因此,AAH是肿瘤相关蛋白。因其独特性质,AAH可用于肝癌的免疫治疗。首先,AAH赋予癌细胞移动性,该移动性与癌转移相关;因此,靶向表达AAH的细胞的治疗能有效抑制转移灶以及原发肿瘤。其次,由于AAH是膜蛋白,其大部分暴露于胞外空间,易于免疫细胞接触所述蛋白,即使I型分子被下调。再次,除AAH以外,尚未知胆管细胞癌特异性的抗原蛋白。因此,AAH可独特地作为针对肝癌的免疫治疗的靶分子。
免疫治疗方法包括注射载有感兴趣蛋白的树突细胞(DC)。DC是捕获、处理和呈递抗原给T细胞以诱导和控制T细胞介导免疫的专门细胞。DC广泛用于免疫,不单用于实验动物也用于荷瘤患者。本文所述方法包括基于DC免疫接种以诱发针对AAH的免疫。本方法比此前所述产生载有感兴趣蛋白的免疫原性小鼠DC的方法(Gehring等,2008,J.Immunol.Meth.332:18-30)至少在两方面有所改善。其一为刺激DC时,此前采用脂多糖(LPS),这种强热原可能不适于体内使用。其二为根据DC的IL-12分泌和不同表面DC标记物的表达来判断,DC刺激较弱。因此,对产生免疫原性DC的方法有所改进。
树突细胞疫苗
最有前景的HCC免疫治疗方法是利用DC的方法,因为DC具有诱导T细胞介导肿瘤免疫的强大能力。DC源自造血细胞,并专用于捕获并处理抗原,将蛋白转化成肽呈递在MHC分子上并由T细胞识别。DC强效诱导并控制T细胞免疫。
基于DC的免疫治疗是根据肿瘤充满潜在抗原且这些抗原由DC呈递时成为免疫原性这一事实。DC获自对象,例如通过细胞分离法。这些细胞此时为离体,载有(即,接触过)肿瘤抗原(例如,肿瘤细胞裂解物;凋亡或坏死的肿瘤细胞;重组、合成或纯化的肿瘤抗原肽或蛋白;或编码肿瘤抗原的核酸如RNA),经刺激以成熟,并重注射入患者以诱导强T细胞免疫。
AAH肿瘤抗原
AAH是II型膜蛋白,在C-末端区域具有催化域。该蛋白大部分位于细胞膜外,使免疫细胞能接触此蛋白。AAH蛋白在HCC、胆管癌和乳腺或结肠源的腺癌中强烈表达,而在正常组织对应体中很少测得或完全不可检测。因此,AAH是HCC和其他荷AAH肿瘤的基于DC的免疫治疗的理想靶标分子。
AAH在肿瘤细胞中的过量表达提高细胞移动性,该移动性使肿瘤细胞具有恶性表型。对HCC的临床病理学研究发现AAH过量表达与组织学评级和肝内转移有关。因此,靶向表达AAH的肿瘤细胞群体抑制载AAH肿瘤的发生和发展。
表1:人AAH的氨基酸序列
号S83325;His基序带下划线;催化域内的保守序列由粗体标出;催化域包含SEQID NO:1的650-700残基)。
表2:人AAH cDNA序列
(SEQ ID NO:2;GENBANK登录号S83325;编码起始甲硫氨酸的密码子带下划线)。
临床应用
抗原呈递细胞(APC)如DC采用白血球单采术取自对象,如患有癌症或有发生癌症风险的人患者。此类过程通常在单采血液成分中心进行(第1天)。用已知方法纯化的树突细胞接触抗原如AAH或AAH以及AFP。抗原接触后,细胞在细胞因子混合物中培养(第2-3天)。然后将经抗原致敏并激活的DC给予患者(第3-4天)。治疗的示范性过程包括在4周时间内给予3次DC。
用下列材料和方法产生本文所述数据。
动物
7-8周龄的雌性BALB/c(H-2d)小鼠购自HSD有限公司(Harlan Sprague Dawley,Inc.),并保持在无特定病原条件下。
制备磁性微珠
免疫磁珠(1.3μm;卡巴开公司(Calbiochem))用50mM,pH 8.5的硼酸盐缓冲液清洗3遍,并重悬于pH 9.0,含0.1mg/ml AAH或GFP蛋白的50mM硼酸盐缓冲液中。然后,悬液在不断搅拌条件下室温孵育过夜。离心珠粒,用PBS清洗并以固体含量30mg/ml的浓度重悬于PBS。
DC分离
DC分离采用已知方法进行(例如(Gehring等,2008,J.Immunol.Meth.332:18-30))。可选由白血球单采术获得细胞。在第0天和第6天将10μg编码Fms-样酪氨酸激酶受体-3配体(FLT3L)的表达质粒pUMVC3-hFLex注入小鼠尾静脉。脾细胞由注射了FLT3L的小鼠用NH4Cl红细胞裂解缓冲液制备,5x 107个细胞在含10μl磁性微珠的无血清DMEM中孵育4-6小时。收集细胞,用MACS MS柱(美天旎公司(Miltenyi))通过磁场以富集摄取磁珠的细胞。然后,采用Lympholyte M(塞得兰公司(Cedarlane))对细胞进行密度梯度离心以消除游离珠和死细胞。收集活细胞,用汉克斯(Hank′s)缓冲盐溶液(HBSS)清洗2次,并用于后续实验。分离细胞的活力>90%,且分离细胞中CD11c阳性群体的百分数为70-80%。
细胞培养
DC在HEPES缓冲的补充有10%小鼠血清(艾奎泰克生物公司(Equitech Bio))、2mML-谷氨酰胺、50μM 2-巯基乙醇、100U/ml青霉素、100μg/ml链霉素、20ng/ml GM-CSF、100ng/ml IL-4、20ng/ml IFN-γ和1μg/ml CD40L(所有细胞因子购自派普罗泰克公司(Peprotech))的RPMI1640中,在6孔超低贴附平板(康宁公司(Corning))上培养40小时。就细胞因子释放而言,脾细胞生长于含2mM L-谷氨酰胺、50nM 2-巯基乙醇、100U/ml青霉素和100μg/ml链霉素的无血清X-VIVO 15(龙沙公司(Lonza))中。就细胞毒性试验而言,脾细胞生长于HEPES缓冲的补充有10%胎牛血清(大西洋生物公司(Atlantic Bio))、2mM L-谷氨酰胺、50μM 2-巯基乙醇、100U/ml青霉素100μg/ml链霉素、1x非必需氨基酸(龙沙公司)、0.5x氨基酸溶液(英杰公司(Invitrogen)和1mM丙酮酸钠的完全RPMI1640中。SP2/0细胞培养于补充有20%胎牛血清、2mM L-谷氨酰胺、100U/ml青霉素和100μg/ml链霉素的Dulbecco极限必需培养基中。
酶联免疫吸附实验(ELISA)
DC或脾细胞在含或不含AAH蛋白的无血清培养基中生长48小时。然后收集细胞上清液,离心并进行ELISA实验。IL-12、p70、IFN-γ和IL-4的ELISA采用ELISA Ready-SET-Go!试剂盒(易生物科学公司(eBioscience))按照生产商说明书进行。
流式细胞分析
通过流式细胞术按此前所述(Gehring等,2008)分析细胞表面标记物的表达。用3-6x 105个细胞来染色。细胞用染色缓冲液(含5%FBS的HBSS)清洗,与2.5μg/ml小鼠BD FcBlock(BD生物科学公司(BD Biosciences))一起4℃孵育5分钟,再与染色抗体一起在4℃孵育30分钟。所用抗体为抗-CD11c(HL3;BD生物科学公司)、抗-CD40(3/23;BD生物科学公司)、抗-CD54(3E2;BD生物科学公司)、抗-CD80(16-10A1;BD生物科学公司)、抗-CD86(GLI;BD生物科学公司)、抗-I-Ad(AMS-32.1;BD生物科学公司)和抗-CD8a(53-6.7;易生物科学公司),所有抗体均与荧光团偶联。仓鼠、大鼠和小鼠免疫球蛋白G同种型匹配对照也用于染色对照。清洗2遍后,细胞重悬于染色缓冲液,并用FACSCalibur(BD生物科学公司)进行流式细胞分析。通过用7-氨基-放线菌素D(易生物科学公司)的细胞染色将死细胞从分析中除去。用CellQuest软件(BD生物科学公司)然后用FlowJo软件(三星公司(Tree Star))处理数据。
疫苗接种
与细胞因子培养40小时后,在第0天和第14天将2.5x 105细胞皮下注射入右胁和左胁(每只小鼠5x 105个细胞)。在第28天,经免疫的小鼠用于进一步的实验,包括细胞因子释放和细胞毒性试验。
细胞毒性试验
从经免疫小鼠制得脾细胞,将5x 107个脾细胞在含0.5μg/ml AAH蛋白的完全培养基中生长2天,并在添加10ng/ml IL-2后再生长2天。2x 104个靶SP2/0细胞与6-60x 104个效应脾细胞在无血清培养基中混合,200x g离心5分钟,并在CO2培养箱中孵育4小时。收集培养上清液,上清液中释放的乳酸脱氢酶活性通过按生产商的说明书使用LDH细胞毒性检测试剂盒(罗氏公司(Roche))测定。用以下公式计算%靶细胞裂解:
((A效应细胞+靶细胞-A效应细胞-A靶细胞)除以(A靶细胞+曲通X-A靶细胞))x 100,
其中A表示490nm的吸光度。
由经不同细胞因子体外刺激的分离DC分泌的IL-12
此前所用的方法包括DC的体内扩增,磁性微珠的摄取,所述微珠上涂覆有抗原和DC刺激物包括LPS和抗-CD40抗体,并由通过磁性柱来分离DC。不同于用涂覆于珠粒上的刺激物刺激DC,本改进方法涉及通过在多种细胞因子存在下生长两天来刺激分离的DC,由于刺激物应与细胞表面的相应受体相互作用,而被DC摄取的珠粒上的刺激物不能有此相互作用。包括IL-4、IFN-γ和CD40L的一些细胞因子刺激并促进DC成熟。因此,检查这些细胞因子对DC成熟的组合效应。进行DC的扩增、摄取和分离。分离的细胞在GM-CSF,和/或IL-4,和/或IFN-γ,和/或CD40L的存在下培养40小时。始终添加GM-CSF,因其为DC存活所必需。成熟而非未成熟的DC强烈生产辅助与细胞毒性T细胞极化必需的IL-12。因此,IL-12水平用作DC刺激的指示剂。收集培养的上清液,通过ELISA测定IL-12p70的浓度。结果表明当DC培养中存在所有细胞因子时IL-12p70最高(图1)。各细胞因子单独或组合用于刺激。优选用所有细胞因子刺激DC。但是,CD40L是可选的。
成熟DC标记物在经刺激DC表面的表达
为研究细胞因子刺激对成熟DC标记物表达的影响,测定细胞因子存在下生长40小时的DC与无培养DC的标记物表达。泛DC标记物CD11c的表达在培养后未改变(图2、3)。但是,包括CD40、CD54、CD80、CD86和II型MHC(I-Ad)在内的所有成熟DC标记物在生长40小时的DC中都显著上调(图2、3)。CD8a+ DC为能有效刺激CTL的DC子集。评估了细胞因子刺激对CD8a+DC群体的影响。如图4所述,DC中CD8a+群体的百分数在刺激期间显著上升。这些结果表明该方法不用LPS所产生的细胞具有基于DC免疫所需的充分品质。
AAH免疫小鼠的脾细胞响应AAH的IFN-γ分泌
为检查TH1应答是否被AAH免疫诱发,测定响应AAH蛋白的TH1细胞因子IFN-γ的生产。小鼠以2周间隔用DC免疫两次,所用DC摄取了AAH或GFP-涂覆珠粒或无任何抗原的珠粒。后一次免疫2周后,从免疫小鼠制备脾细胞,在AAH存在下培养48小时,并测定培养上清液中IFN-γ的浓度以检查经AAH免疫小鼠的脾细胞是否响应AAH刺激生产IFN-γ。经AAH免疫小鼠的脾细胞以剂量依赖性方式强烈生产IFN-γ(图5)。尽管在伴刀豆球蛋白A(Con A)存在下,来自三组的脾细胞生产了可观量的IFN-γ,但对照脾细胞在AAH刺激时仅生产微量IFN-γ(图5)。这一结果表明对在免疫小鼠中产生对AAH的TH1细胞响应。
AAH免疫小鼠的脾细胞响应AAH的IL-4分泌
为研究AAH负荷DC是否诱发针对AAH的体液免疫,测定在IFN-γ测定的相同样品中TH2细胞因子IL-4的浓度。不论免疫与否,Con A刺激的脾细胞都产生大量IL-4(图6)。相反,用三种DC免疫的小鼠制得的脾细胞仅生产痕量IL-4(图6),表明针对AAH的TH2细胞响应未被AAH免疫有效诱发。
经AAH免疫小鼠中针对AAH表达细胞的CTL活性
为确定在AAH免疫小鼠中是否诱发响应AAH的CTL,通过用表达AAH的SP2/0细胞为靶细胞评估细胞毒性。SP2/0细胞与AAH蛋白和IL-2活化后的来自免疫小鼠的脾细胞共培养4小时。通过测定濒死细胞释放的乳酸脱氢酶活性评估靶细胞裂解效率。如图7所述,经AAH免疫小鼠的脾细胞的CTL活性高于其他对照脾细胞,表明AAH免疫诱发AAH响应性CTL。
经AAH免疫小鼠中的AAH响应性T细胞
当小鼠用载AAH的DC免疫时,诱发AAH响应性TH1细胞和CTL。抗原特异性T细胞扩增是表明产生抗原响应性T细胞的另一重要指标。
AAH体内免疫的抗肿瘤效应
用AAH致敏DC进行AAH免疫诱发抗肿瘤免疫。小鼠用载AAH的DC以2周间隔免疫两次,后一次免疫2周后,SP2/0细胞植入皮下,测定植入肿瘤尺寸的生长。这一荷瘤模型所得数据反映免疫接种的预防效果。为评估AAH免疫的治疗效果,SP2/0先行植入,然后当肿瘤生长至直径5mm时,用载AAH的DC免疫小鼠,并测定对肿瘤生长的效果。
AAH靶向的免疫治疗还抑制转移。小鼠用载AAH的DC免疫,然后注射SP2/0细胞入尾静脉。两周后,切除肺,对形成的结节计数。免疫小鼠内结节数量相对未免疫小鼠中数量的减少表明载AAH的DC免疫方案抑制荷AAH肿瘤的转移。
用载AAH的成熟树突细胞免疫接种显著降低表达AAH肿瘤的肿瘤负荷(肿瘤体积)。图11和12显示小鼠中AAH免疫接种对肿瘤生长的治疗效果。5×105个BNL 1ME A.7R.1小鼠肝癌细胞皮下植入6周龄BALB/c小鼠胁部。在第10天和第15天,将5×105个载有AAH或GFP的成熟DC或HBSS皮下注射入荷瘤小鼠。肿瘤尺寸每周测定。用如下公式确定肿瘤体积:0.52×(长)×(宽)2。各数据点代表平均肿瘤体积±标准误差(n=6)。发现肿瘤体积在用载AAH的DC免疫后显著减小。使用本领域已知肿瘤模型的这些数据表明用载AAH成熟DC的治疗方法能在体内有效减少和消除载有AAH的肿瘤。此类树突细胞疫苗显示针对任何表达AAH的肿瘤的有效抗肿瘤效果。
通过AAH和AFP同时免疫增强抗肿瘤效果
AFP是对小鼠和人为免疫原性的仅有的HCC相关蛋白。但是,也已显示其抗肿瘤效果不足以用于人HCC患者。但是,用AAH和AFP同时免疫增强对同时表达AAH和AFP的肿瘤如HCC的免疫应答。
表达AFP的SP2/0细胞的稳定系通过导入AFP表达质粒DNA产生。通过使用所述细胞,测定在经载AFP和载AAH的DC免疫小鼠中的体外CTL活性。免疫后的细胞毒性增强表明免疫引起AAH和AFP联合免疫对荷有AAH和/或AFP的肿瘤生长的抗肿瘤效果。
Claims (20)
1.分离的载天冬氨酰基(天冬酰胺酰基)-β-羟化酶(AAH)的成熟树突细胞在制备药物中的用途,所述药物用于降低对象内表达AAH的肿瘤的生长,其中由所述树突细胞降低所述表达AAH的肿瘤的生长并且所述树突细胞用包含GM-CSF和IFN-γ的细胞因子组合离体培养然后给予所述药物,所述成熟树突细胞载有纯化AAH抗原。
2.如权利要求1所述的用途,其特征在于,所述肿瘤选自下组:肝癌、胃肠癌、胰腺癌、乳腺癌、前列腺癌、宫颈癌、卵巢癌、输卵管癌、喉癌、肺癌、甲状腺癌、胆囊癌、肾癌、膀胱癌和脑癌。
3.如权利要求1所述的用途,其特征在于,所述细胞因子组合还包含CD40L。
4.如权利要求1所述的用途,其特征在于,所述树突细胞通过白血球单采术从对象获得。
5.如权利要求4所述的用途,其特征在于,所述对象患有癌症或有发生癌症的风险。
6.如权利要求1、4或5所述的用途,其特征在于,所述对象是人、犬、猫、马、牛或猪对象。
7.一种产生AAH-致敏树突细胞的方法,其包括使分离的树突细胞接触抗原,所述抗原包含纯化AAH,在所述抗原接触步骤后,使所述树突细胞接触细胞因子的组合,所述组合包含GM-CSF、IFN-γ和CD40L。
8.如权利要求7所述的方法,其特征在于,所述组合还包括IL-4。
9.如权利要求7所述的方法,其特征在于,所述树突细胞与所述细胞因子组合接触至少10小时。
10.如权利要求7所述的方法,其特征在于,所述树突细胞与所述细胞因子组合接触至少40小时。
11.如权利要求7所述的方法,其特征在于,所述抗原还包括AFP。
12.如权利要求7所述的方法,其特征在于,所述抗原是可溶形式或所述抗原连接于固相载体。
13.如权利要求12所述的方法,其特征在于,所述固相载体包括聚苯乙烯珠。
14.如权利要求13所述的方法,其特征在于,所述聚苯乙烯珠为生物可降解的。
15.一种疫苗组合物,其包含由权利要求7的方法产生的AAH-致敏树突细胞。
16.一种含载AAH的成熟树突细胞的疫苗,其用于治疗哺乳动物对象中表达AAH的肿瘤,所述树突细胞已按权利要求7所述方法致敏。
17.按权利要求7所述方法致敏的自体树突细胞在药物制备中的用途,所述药物用于抑制哺乳动物对象中的肿瘤生长,所述对象患有载AAH的肿瘤。
18.按权利要求7所述方法致敏的自体树突细胞在药物制备中的用途,所述药物用于预防哺乳动物中的肿瘤发生,所述对象有发生载AAH肿瘤的风险。
19.一种按权利要求7所述方法致敏的自体树突细胞在药物制备中的用途,所述药物用于预防患有载AAH肿瘤的对象中的载AAH肿瘤转移。
20.如权利要求17、18或19所述的用途,其特征在于,所述对象是人、犬、猫、马、牛或猪对象。
Applications Claiming Priority (9)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US22842909P | 2009-07-24 | 2009-07-24 | |
US61/228,429 | 2009-07-24 | ||
US23112709P | 2009-08-04 | 2009-08-04 | |
US61/231,127 | 2009-08-04 | ||
US23928809P | 2009-09-02 | 2009-09-02 | |
US61/239,288 | 2009-09-02 | ||
US24074509P | 2009-09-09 | 2009-09-09 | |
US61/240,745 | 2009-09-09 | ||
PCT/US2010/043056 WO2011011688A2 (en) | 2009-07-24 | 2010-07-23 | DENDRITIC CELL VACCINES FOR ASPARAGINYL- β - HYDROXYLASE EXPRESSING TUMORS |
Publications (2)
Publication Number | Publication Date |
---|---|
CN102596234A CN102596234A (zh) | 2012-07-18 |
CN102596234B true CN102596234B (zh) | 2016-11-16 |
Family
ID=42830734
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN201080033781.4A Active CN102596234B (zh) | 2009-07-24 | 2010-07-23 | 针对表达天冬酰胺酰-β-羟化酶的肿瘤的树突细胞疫苗 |
Country Status (10)
Country | Link |
---|---|
US (1) | US9687537B2 (zh) |
EP (1) | EP2456458B1 (zh) |
JP (2) | JP5872468B2 (zh) |
KR (1) | KR101911380B1 (zh) |
CN (1) | CN102596234B (zh) |
AU (1) | AU2010275443B2 (zh) |
CA (1) | CA2768398C (zh) |
ES (1) | ES2711504T3 (zh) |
NZ (1) | NZ597724A (zh) |
WO (1) | WO2011011688A2 (zh) |
Families Citing this family (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US9687537B2 (en) | 2009-07-24 | 2017-06-27 | Rhode Island Hospital | Dendritic cell vaccines for asparaginyl-β-hydroxylase expressing tumors |
KR20150139855A (ko) * | 2013-03-07 | 2015-12-14 | 네오스템 온콜로지, 엘엘씨 | 개별화된 고순도 간세포 암종 줄기세포, 방법 및 그의 용도 |
US9744223B2 (en) * | 2013-03-15 | 2017-08-29 | Panacea Pharmaceuticals, Inc. | Therapeutic cancer vaccine targeted to HAAH (aspartyl-[asparaginyl]-β-hydroxylase) |
CN103405759B (zh) * | 2013-07-23 | 2014-12-10 | 蔡颖 | 一种应用脐血cd34+细胞制备肿瘤特异性dc疫苗方法 |
WO2015164826A2 (en) * | 2014-04-24 | 2015-10-29 | Rhode Island Hospital | ASPARTATE-β-HYDROXYLASE INDUCES EPITOPE-SPECIFIC T CELL RESPONSES IN TUMORS |
US20220054614A1 (en) * | 2018-12-13 | 2022-02-24 | Rhode Island Hospital | Inhibition of asph expressing tumor growth and progression |
Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN1688602A (zh) * | 2002-10-02 | 2005-10-26 | 霍夫曼-拉罗奇有限公司 | 新mhcⅱ相关肽 |
CN101754768A (zh) * | 2007-07-19 | 2010-06-23 | 健康研究股份有限公司 | 用作癌症疫苗的存活蛋白肽 |
CN101755045A (zh) * | 2007-05-17 | 2010-06-23 | 生物载体株式会社 | 树突状细胞的制备方法 |
Family Cites Families (14)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
AU731937B2 (en) * | 1996-12-10 | 2001-04-05 | Hadasit Medical Research Services & Development Company Ltd | Serum-derived factor inducing cell differentiation and medical uses thereof |
US20030031670A1 (en) | 1999-11-08 | 2003-02-13 | Jack R. Wands | Diagnosis and treatment of malignant neoplasms |
US6835370B2 (en) | 1999-11-08 | 2004-12-28 | Rhode Island Hospital | Diagnosis and treatment of malignant neoplasms |
US20050123545A1 (en) * | 1999-11-08 | 2005-06-09 | Wands Jack R. | Diagnosis and treatment of malignant neoplasms |
EP1450814B1 (en) | 2001-10-01 | 2016-11-30 | Geistlich Pharma AG | Methods of inhibiting metastases |
TWI259206B (en) * | 2002-09-24 | 2006-08-01 | Univ Nat Cheng Kung | A DNA vaccine containing a tumor associated gene and a cytokine gene and the method producing thereof |
MXPA05009178A (es) * | 2003-02-27 | 2006-03-08 | Northwest Biotherapeutics Inc | Generacion de celulas dendriticas a partir de celulas precursoras dendriticas monociticas con gm-csf en ausencia de citocinas adicionales. |
JP2007535907A (ja) | 2003-11-14 | 2007-12-13 | マサチューセッツ・インスティテュート・オブ・テクノロジー | 抗ヒドロキシラーゼ抗体およびその使用 |
CA2504451A1 (en) * | 2004-08-10 | 2006-02-10 | Geron Corporation | Dendritic cell vaccines for treating cancer made from embryonic stem cells |
EP1800693B1 (en) | 2004-08-24 | 2013-07-17 | Chugai Seiyaku Kabushiki Kaisha | Adjuvant therapy with the use of anti-glypican 3 antibody |
GB2426581A (en) * | 2005-05-27 | 2006-11-29 | Univ Nottingham | Immunoassay methods |
KR100675761B1 (ko) | 2005-09-01 | 2007-01-30 | 주식회사 바이넥스 | 1,2-디옥타노일-sn-글리세롤3-포스페이트를 이용한 급성골수성 백혈병 세포의 수지상 세포로의 전이분화 유도 방법 |
KR20150082688A (ko) * | 2005-12-08 | 2015-07-15 | 노쓰웨스트 바이오써라퓨틱스, 인크. | 미성숙 단핵구성 수지상 세포의 활성화를 유도하기 위한 조성물 및 방법 |
US9687537B2 (en) | 2009-07-24 | 2017-06-27 | Rhode Island Hospital | Dendritic cell vaccines for asparaginyl-β-hydroxylase expressing tumors |
-
2010
- 2010-07-23 US US12/842,494 patent/US9687537B2/en active Active
- 2010-07-23 KR KR1020127004697A patent/KR101911380B1/ko active IP Right Grant
- 2010-07-23 WO PCT/US2010/043056 patent/WO2011011688A2/en active Application Filing
- 2010-07-23 JP JP2012521829A patent/JP5872468B2/ja active Active
- 2010-07-23 ES ES10735417T patent/ES2711504T3/es active Active
- 2010-07-23 CN CN201080033781.4A patent/CN102596234B/zh active Active
- 2010-07-23 NZ NZ597724A patent/NZ597724A/xx not_active IP Right Cessation
- 2010-07-23 EP EP10735417.7A patent/EP2456458B1/en active Active
- 2010-07-23 AU AU2010275443A patent/AU2010275443B2/en not_active Ceased
- 2010-07-23 CA CA2768398A patent/CA2768398C/en active Active
-
2015
- 2015-10-08 JP JP2015199935A patent/JP2016053036A/ja active Pending
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN1688602A (zh) * | 2002-10-02 | 2005-10-26 | 霍夫曼-拉罗奇有限公司 | 新mhcⅱ相关肽 |
CN101755045A (zh) * | 2007-05-17 | 2010-06-23 | 生物载体株式会社 | 树突状细胞的制备方法 |
CN101754768A (zh) * | 2007-07-19 | 2010-06-23 | 健康研究股份有限公司 | 用作癌症疫苗的存活蛋白肽 |
Non-Patent Citations (3)
Title |
---|
Generation of Human Dendritic Cells That Simultaneously Secrete IL-12 and Have Migratory Capacity by Adenoviral Gene Transfer of hCD40L in Combination With IFN-g;Ilka knipperz,et al.;《J.Immunother》;20090630;第32卷(第5期);第524页摘要,第525页左栏第2-4段 * |
High-avidity antitumor T-cell generation by toll receptor 8–primed,myeloid- derived dendritic cells is mediated by IL-12 production;shuwen xu,et al.;《Surgery》;20060831;第140卷(第2期);全文 * |
Proinflammatory Cytokines and CD40 Ligand Enhance Cross-Presentation and Cross-Priming Capability of Human Dendritic Cells Internalizing Apoptotic Cancer Cells;Thomas K.Hoffmann,et al.;《journal of immunotherapy》;20011231;第24卷(第2期);第163页右栏第2段,第164页左栏倒数第1段及右栏第1段,第166页图2,第169页右栏第1-3行,第10-23行 * |
Also Published As
Publication number | Publication date |
---|---|
EP2456458B1 (en) | 2018-12-26 |
JP2013500261A (ja) | 2013-01-07 |
US20110076290A1 (en) | 2011-03-31 |
CN102596234A (zh) | 2012-07-18 |
AU2010275443A1 (en) | 2012-02-02 |
US9687537B2 (en) | 2017-06-27 |
CA2768398A1 (en) | 2011-01-27 |
KR101911380B1 (ko) | 2018-10-25 |
WO2011011688A2 (en) | 2011-01-27 |
ES2711504T3 (es) | 2019-05-06 |
CA2768398C (en) | 2020-01-07 |
AU2010275443B2 (en) | 2016-05-12 |
KR20120052346A (ko) | 2012-05-23 |
NZ597724A (en) | 2013-10-25 |
JP5872468B2 (ja) | 2016-03-01 |
WO2011011688A3 (en) | 2011-03-17 |
JP2016053036A (ja) | 2016-04-14 |
EP2456458A2 (en) | 2012-05-30 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN102597222B (zh) | 用于增殖抗原特异性t细胞的方法 | |
CN103502439B (zh) | 用于抗原特异性t细胞增殖的方法 | |
CN102596234B (zh) | 针对表达天冬酰胺酰-β-羟化酶的肿瘤的树突细胞疫苗 | |
KR100458403B1 (ko) | 생체외 조혈세포의 자극방법 | |
JP6010136B2 (ja) | ナチュラルキラー細胞の製造方法、その方法により製造されたナチュラルキラー細胞、並びにそれを含む腫瘍及び感染性疾患治療用組成物 | |
EA016168B1 (ru) | Способ получения т-клеточной популяции и ее применение | |
CN108251378B (zh) | 一种过表达ptgds基因的间质干细胞外泌体及其制备方法和应用 | |
CN107354133A (zh) | 磁珠偶联多种刺激蛋白的体外扩增nk细胞的方法及应用 | |
CN102719400B (zh) | Hla-a0201限制性抗cea抗原特异性ctl的制备方法 | |
AU2019237832A1 (en) | Method for producing natural killer cells | |
CN105031631A (zh) | 一种HLA-A0201限制性抗Sox2特异性CTL的制备方法及其应用 | |
TW201537172A (zh) | 加強腫瘤生長的方法 | |
CN110628717B (zh) | 一种浸润性t细胞的培养方法 | |
CN1887296B (zh) | 一种诱导抗肿瘤免疫的方法及其在制药中的应用 | |
CN112010960A (zh) | 一种肿瘤抗原复合物体外致敏dc、t细胞获得肿瘤特异性杀伤细胞ctl及其制备方法 | |
CN107557338A (zh) | 特异性识别ny‑eso‑1的t细胞及其与细胞因子的联合的应用 | |
CN102813916A (zh) | 一种快速获取树突状细胞疫苗的制备方法及其应用 | |
CN111996166B (zh) | 一种负载的dc细胞、dc-cik细胞及在肿瘤细胞治疗方面的应用 | |
CN112010944B (zh) | 一种肽及体外扩增cik细胞的方法 | |
CN106676068A (zh) | 一种生物活性肽和体外扩增cik细胞的方法 | |
WO2004039968A1 (ja) | 骨髄由来の不死化樹状細胞株 | |
CN105219715A (zh) | 一种用于激活食管癌特异性免疫反应的试剂盒 | |
CN113151166A (zh) | 一种个体化肿瘤新生抗原特异性cd8细胞的获取方法及应用 | |
CN102336821B (zh) | Melan-A表位肽及其在预防和/或治疗肿瘤中的用途 | |
CN105219721A (zh) | 一种用于激活胰腺癌特异性免疫反应的试剂盒 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
C06 | Publication | ||
PB01 | Publication | ||
C10 | Entry into substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
REG | Reference to a national code |
Ref country code: HK Ref legal event code: DE Ref document number: 1171386 Country of ref document: HK |
|
C14 | Grant of patent or utility model | ||
GR01 | Patent grant | ||
REG | Reference to a national code |
Ref country code: HK Ref legal event code: GR Ref document number: 1171386 Country of ref document: HK |