TWI496886B - 提升感染性與惡性疾病之治療之免疫反應 - Google Patents
提升感染性與惡性疾病之治療之免疫反應 Download PDFInfo
- Publication number
- TWI496886B TWI496886B TW101122686A TW101122686A TWI496886B TW I496886 B TWI496886 B TW I496886B TW 101122686 A TW101122686 A TW 101122686A TW 101122686 A TW101122686 A TW 101122686A TW I496886 B TWI496886 B TW I496886B
- Authority
- TW
- Taiwan
- Prior art keywords
- il2ss
- dna
- pvac
- mpd
- pharmaceutical composition
- Prior art date
Links
- 201000010099 disease Diseases 0.000 title claims description 18
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 title claims description 18
- 230000003211 malignant effect Effects 0.000 title claims description 16
- 208000015181 infectious disease Diseases 0.000 title claims description 14
- 230000002458 infectious effect Effects 0.000 title claims description 12
- 230000002708 enhancing effect Effects 0.000 title claims description 4
- 238000000034 method Methods 0.000 title description 8
- 108010021064 CTLA-4 Antigen Proteins 0.000 claims description 49
- 102000008203 CTLA-4 Antigen Human genes 0.000 claims description 49
- 239000008194 pharmaceutical composition Substances 0.000 claims description 27
- 102000040430 polynucleotide Human genes 0.000 claims description 20
- 108091033319 polynucleotide Proteins 0.000 claims description 20
- 239000002157 polynucleotide Substances 0.000 claims description 20
- 230000028993 immune response Effects 0.000 claims description 17
- 239000012634 fragment Substances 0.000 claims description 15
- 239000002502 liposome Substances 0.000 claims description 14
- 239000003937 drug carrier Substances 0.000 claims description 13
- 239000003446 ligand Substances 0.000 claims description 8
- 230000030833 cell death Effects 0.000 claims description 7
- 230000027455 binding Effects 0.000 claims description 6
- 206010009944 Colon cancer Diseases 0.000 claims description 5
- 206010027480 Metastatic malignant melanoma Diseases 0.000 claims description 5
- 208000021039 metastatic melanoma Diseases 0.000 claims description 5
- 208000001333 Colorectal Neoplasms Diseases 0.000 claims description 4
- 206010025323 Lymphomas Diseases 0.000 claims description 4
- 206010033128 Ovarian cancer Diseases 0.000 claims description 4
- 206010061535 Ovarian neoplasm Diseases 0.000 claims description 4
- 206010061902 Pancreatic neoplasm Diseases 0.000 claims description 4
- 239000002671 adjuvant Substances 0.000 claims description 4
- 230000002924 anti-infective effect Effects 0.000 claims description 4
- 206010073071 hepatocellular carcinoma Diseases 0.000 claims description 4
- 231100000844 hepatocellular carcinoma Toxicity 0.000 claims description 4
- 208000015486 malignant pancreatic neoplasm Diseases 0.000 claims description 4
- 201000002528 pancreatic cancer Diseases 0.000 claims description 4
- 208000008443 pancreatic carcinoma Diseases 0.000 claims description 4
- 208000031261 Acute myeloid leukaemia Diseases 0.000 claims description 3
- 208000033776 Myeloid Acute Leukemia Diseases 0.000 claims description 3
- 239000002246 antineoplastic agent Substances 0.000 claims description 3
- 229940041181 antineoplastic drug Drugs 0.000 claims description 3
- 208000018821 hormone-resistant prostate carcinoma Diseases 0.000 claims description 3
- 238000004519 manufacturing process Methods 0.000 claims description 3
- 208000002154 non-small cell lung carcinoma Diseases 0.000 claims description 3
- 208000029729 tumor suppressor gene on chromosome 11 Diseases 0.000 claims description 3
- 238000010276 construction Methods 0.000 claims description 2
- 239000003814 drug Substances 0.000 claims description 2
- 238000007918 intramuscular administration Methods 0.000 claims description 2
- 238000007920 subcutaneous administration Methods 0.000 claims description 2
- 230000000699 topical effect Effects 0.000 claims description 2
- 230000001154 acute effect Effects 0.000 claims 1
- 210000001185 bone marrow Anatomy 0.000 claims 1
- 208000032839 leukemia Diseases 0.000 claims 1
- 108020004414 DNA Proteins 0.000 description 67
- 102000053602 DNA Human genes 0.000 description 66
- 241000699670 Mus sp. Species 0.000 description 59
- 108010041986 DNA Vaccines Proteins 0.000 description 38
- 229940021995 DNA vaccine Drugs 0.000 description 38
- 108090000623 proteins and genes Proteins 0.000 description 34
- 102000008096 B7-H1 Antigen Human genes 0.000 description 31
- 108091028043 Nucleic acid sequence Proteins 0.000 description 29
- 125000003275 alpha amino acid group Chemical group 0.000 description 29
- 108010074708 B7-H1 Antigen Proteins 0.000 description 28
- 102000004169 proteins and genes Human genes 0.000 description 27
- 210000004027 cell Anatomy 0.000 description 26
- 239000013598 vector Substances 0.000 description 26
- 241000282414 Homo sapiens Species 0.000 description 17
- 108090000765 processed proteins & peptides Proteins 0.000 description 16
- 208000006265 Renal cell carcinoma Diseases 0.000 description 13
- 150000001413 amino acids Chemical group 0.000 description 13
- 210000002966 serum Anatomy 0.000 description 13
- 206010028980 Neoplasm Diseases 0.000 description 12
- 101000889276 Homo sapiens Cytotoxic T-lymphocyte protein 4 Proteins 0.000 description 11
- 241001529936 Murinae Species 0.000 description 11
- 230000036039 immunity Effects 0.000 description 11
- 239000002773 nucleotide Substances 0.000 description 11
- 125000003729 nucleotide group Chemical group 0.000 description 11
- 102000004196 processed proteins & peptides Human genes 0.000 description 11
- 229960005486 vaccine Drugs 0.000 description 11
- 241000699666 Mus <mouse, genus> Species 0.000 description 10
- 210000004989 spleen cell Anatomy 0.000 description 10
- 201000011510 cancer Diseases 0.000 description 9
- 230000003053 immunization Effects 0.000 description 9
- -1 PD-1 Proteins 0.000 description 8
- 230000037361 pathway Effects 0.000 description 8
- 229920001184 polypeptide Polymers 0.000 description 8
- 230000001105 regulatory effect Effects 0.000 description 8
- 101000914484 Homo sapiens T-lymphocyte activation antigen CD80 Proteins 0.000 description 7
- 102100027222 T-lymphocyte activation antigen CD80 Human genes 0.000 description 7
- 230000000694 effects Effects 0.000 description 7
- 238000002649 immunization Methods 0.000 description 7
- 230000008685 targeting Effects 0.000 description 7
- 238000012360 testing method Methods 0.000 description 7
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 6
- 230000004927 fusion Effects 0.000 description 6
- 230000004614 tumor growth Effects 0.000 description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 6
- 102100024321 Alkaline phosphatase, placental type Human genes 0.000 description 5
- 102100039498 Cytotoxic T-lymphocyte protein 4 Human genes 0.000 description 5
- 102000008070 Interferon-gamma Human genes 0.000 description 5
- 108010074328 Interferon-gamma Proteins 0.000 description 5
- 108060001084 Luciferase Proteins 0.000 description 5
- 239000005089 Luciferase Substances 0.000 description 5
- 101000859077 Mus musculus Cytotoxic T-lymphocyte protein 4 Proteins 0.000 description 5
- 108010076504 Protein Sorting Signals Proteins 0.000 description 5
- 102000043321 human CTLA4 Human genes 0.000 description 5
- 239000007924 injection Substances 0.000 description 5
- 238000002347 injection Methods 0.000 description 5
- 230000003993 interaction Effects 0.000 description 5
- 229960003130 interferon gamma Drugs 0.000 description 5
- 102000039446 nucleic acids Human genes 0.000 description 5
- 108020004707 nucleic acids Proteins 0.000 description 5
- 150000007523 nucleic acids Chemical class 0.000 description 5
- 108010031345 placental alkaline phosphatase Proteins 0.000 description 5
- 101000611936 Homo sapiens Programmed cell death protein 1 Proteins 0.000 description 4
- 108091061960 Naked DNA Proteins 0.000 description 4
- 239000004480 active ingredient Substances 0.000 description 4
- 230000006870 function Effects 0.000 description 4
- 201000001441 melanoma Diseases 0.000 description 4
- 108020004999 messenger RNA Proteins 0.000 description 4
- 239000000203 mixture Substances 0.000 description 4
- 244000052769 pathogen Species 0.000 description 4
- 239000002953 phosphate buffered saline Substances 0.000 description 4
- 229920002477 rna polymer Polymers 0.000 description 4
- 238000006467 substitution reaction Methods 0.000 description 4
- BTBUEUYNUDRHOZ-UHFFFAOYSA-N Borate Chemical compound [O-]B([O-])[O-] BTBUEUYNUDRHOZ-UHFFFAOYSA-N 0.000 description 3
- 229940045513 CTLA4 antagonist Drugs 0.000 description 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- 101001117317 Homo sapiens Programmed cell death 1 ligand 1 Proteins 0.000 description 3
- 101100519207 Mus musculus Pdcd1 gene Proteins 0.000 description 3
- 229920000362 Polyethylene-block-poly(ethylene glycol) Polymers 0.000 description 3
- 210000001744 T-lymphocyte Anatomy 0.000 description 3
- 230000001093 anti-cancer Effects 0.000 description 3
- 239000000427 antigen Substances 0.000 description 3
- 108091007433 antigens Proteins 0.000 description 3
- 102000036639 antigens Human genes 0.000 description 3
- 125000002091 cationic group Chemical group 0.000 description 3
- 239000002299 complementary DNA Substances 0.000 description 3
- 229940000406 drug candidate Drugs 0.000 description 3
- 239000003777 experimental drug Substances 0.000 description 3
- 230000012010 growth Effects 0.000 description 3
- 102000048362 human PDCD1 Human genes 0.000 description 3
- 210000000987 immune system Anatomy 0.000 description 3
- 229920000642 polymer Polymers 0.000 description 3
- 230000028327 secretion Effects 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 238000002560 therapeutic procedure Methods 0.000 description 3
- 210000004881 tumor cell Anatomy 0.000 description 3
- 239000003981 vehicle Substances 0.000 description 3
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 2
- 108010017213 Granulocyte-Macrophage Colony-Stimulating Factor Proteins 0.000 description 2
- 102100039620 Granulocyte-macrophage colony-stimulating factor Human genes 0.000 description 2
- 241000282412 Homo Species 0.000 description 2
- 101000914514 Homo sapiens T-cell-specific surface glycoprotein CD28 Proteins 0.000 description 2
- 102000003839 Human Proteins Human genes 0.000 description 2
- 108090000144 Human Proteins Proteins 0.000 description 2
- 108060003951 Immunoglobulin Proteins 0.000 description 2
- 102000000588 Interleukin-2 Human genes 0.000 description 2
- 101100407308 Mus musculus Pdcd1lg2 gene Proteins 0.000 description 2
- 101001117316 Mus musculus Programmed cell death 1 ligand 1 Proteins 0.000 description 2
- 229920002873 Polyethylenimine Polymers 0.000 description 2
- 108700030875 Programmed Cell Death 1 Ligand 2 Proteins 0.000 description 2
- 102100024213 Programmed cell death 1 ligand 2 Human genes 0.000 description 2
- 102100027213 T-cell-specific surface glycoprotein CD28 Human genes 0.000 description 2
- 239000013543 active substance Substances 0.000 description 2
- 230000005904 anticancer immunity Effects 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 230000003833 cell viability Effects 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- 210000004978 chinese hamster ovary cell Anatomy 0.000 description 2
- 238000007796 conventional method Methods 0.000 description 2
- 231100000433 cytotoxic Toxicity 0.000 description 2
- 230000001472 cytotoxic effect Effects 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 239000003995 emulsifying agent Substances 0.000 description 2
- 230000002068 genetic effect Effects 0.000 description 2
- 230000002209 hydrophobic effect Effects 0.000 description 2
- 230000001900 immune effect Effects 0.000 description 2
- 102000018358 immunoglobulin Human genes 0.000 description 2
- 230000004957 immunoregulator effect Effects 0.000 description 2
- 230000001506 immunosuppresive effect Effects 0.000 description 2
- 230000002401 inhibitory effect Effects 0.000 description 2
- 230000005764 inhibitory process Effects 0.000 description 2
- 239000007927 intramuscular injection Substances 0.000 description 2
- 238000010255 intramuscular injection Methods 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 239000003550 marker Substances 0.000 description 2
- 230000036961 partial effect Effects 0.000 description 2
- 230000001717 pathogenic effect Effects 0.000 description 2
- 210000002706 plastid Anatomy 0.000 description 2
- 230000001681 protective effect Effects 0.000 description 2
- 108020003175 receptors Proteins 0.000 description 2
- 230000000638 stimulation Effects 0.000 description 2
- 230000001225 therapeutic effect Effects 0.000 description 2
- KSXTUUUQYQYKCR-LQDDAWAPSA-M 2,3-bis[[(z)-octadec-9-enoyl]oxy]propyl-trimethylazanium;chloride Chemical compound [Cl-].CCCCCCCC\C=C/CCCCCCCC(=O)OCC(C[N+](C)(C)C)OC(=O)CCCCCCC\C=C/CCCCCCCC KSXTUUUQYQYKCR-LQDDAWAPSA-M 0.000 description 1
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 description 1
- XCHBHXMUDZRHQJ-UHFFFAOYSA-N 4-ethyl-1,3-benzothiazole-2-sulfonic acid Chemical compound CCC1=CC=CC2=C1N=C(S(O)(=O)=O)S2 XCHBHXMUDZRHQJ-UHFFFAOYSA-N 0.000 description 1
- 102000002260 Alkaline Phosphatase Human genes 0.000 description 1
- 108020004774 Alkaline Phosphatase Proteins 0.000 description 1
- 208000023275 Autoimmune disease Diseases 0.000 description 1
- 208000031648 Body Weight Changes Diseases 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 238000011740 C57BL/6 mouse Methods 0.000 description 1
- 241000282472 Canis lupus familiaris Species 0.000 description 1
- 241000283707 Capra Species 0.000 description 1
- 208000005623 Carcinogenesis Diseases 0.000 description 1
- 208000035473 Communicable disease Diseases 0.000 description 1
- 241000699802 Cricetulus griseus Species 0.000 description 1
- 102000004127 Cytokines Human genes 0.000 description 1
- 108090000695 Cytokines Proteins 0.000 description 1
- 238000002965 ELISA Methods 0.000 description 1
- 238000008157 ELISA kit Methods 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 208000001382 Experimental Melanoma Diseases 0.000 description 1
- 241000282326 Felis catus Species 0.000 description 1
- 238000012413 Fluorescence activated cell sorting analysis Methods 0.000 description 1
- 101000834898 Homo sapiens Alpha-synuclein Proteins 0.000 description 1
- 101000652359 Homo sapiens Spermatogenesis-associated protein 2 Proteins 0.000 description 1
- 108020005350 Initiator Codon Proteins 0.000 description 1
- 102000014150 Interferons Human genes 0.000 description 1
- 108010050904 Interferons Proteins 0.000 description 1
- 108091092195 Intron Proteins 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 102000045595 Phosphoprotein Phosphatases Human genes 0.000 description 1
- 108700019535 Phosphoprotein Phosphatases Proteins 0.000 description 1
- 239000004698 Polyethylene Substances 0.000 description 1
- 229920001213 Polysorbate 20 Polymers 0.000 description 1
- 241000288906 Primates Species 0.000 description 1
- 102100040678 Programmed cell death protein 1 Human genes 0.000 description 1
- 206010060862 Prostate cancer Diseases 0.000 description 1
- 208000000236 Prostatic Neoplasms Diseases 0.000 description 1
- 206010042602 Supraventricular extrasystoles Diseases 0.000 description 1
- 230000006044 T cell activation Effects 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 150000001412 amines Chemical class 0.000 description 1
- 125000000539 amino acid group Chemical group 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000000845 anti-microbial effect Effects 0.000 description 1
- 230000000890 antigenic effect Effects 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 230000005784 autoimmunity Effects 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 230000037396 body weight Effects 0.000 description 1
- 230000004579 body weight change Effects 0.000 description 1
- 229940098773 bovine serum albumin Drugs 0.000 description 1
- 239000007975 buffered saline Substances 0.000 description 1
- 210000004899 c-terminal region Anatomy 0.000 description 1
- 230000036952 cancer formation Effects 0.000 description 1
- 231100000504 carcinogenesis Toxicity 0.000 description 1
- 230000022534 cell killing Effects 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- 206010009887 colitis Diseases 0.000 description 1
- 238000002648 combination therapy Methods 0.000 description 1
- 230000000295 complement effect Effects 0.000 description 1
- 230000001461 cytolytic effect Effects 0.000 description 1
- 230000003013 cytotoxicity Effects 0.000 description 1
- 231100000135 cytotoxicity Toxicity 0.000 description 1
- 230000034994 death Effects 0.000 description 1
- 230000000779 depleting effect Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 239000008121 dextrose Substances 0.000 description 1
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000012636 effector Substances 0.000 description 1
- 238000004520 electroporation Methods 0.000 description 1
- 239000003623 enhancer Substances 0.000 description 1
- 230000001747 exhibiting effect Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000013604 expression vector Substances 0.000 description 1
- 238000000684 flow cytometry Methods 0.000 description 1
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 230000009036 growth inhibition Effects 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 1
- 125000000487 histidyl group Chemical class [H]N([H])C(C(=O)O*)C([H])([H])C1=C([H])N([H])C([H])=N1 0.000 description 1
- 102000048776 human CD274 Human genes 0.000 description 1
- 238000003384 imaging method Methods 0.000 description 1
- 230000002519 immonomodulatory effect Effects 0.000 description 1
- 230000003832 immune regulation Effects 0.000 description 1
- 239000000677 immunologic agent Substances 0.000 description 1
- 229940124541 immunological agent Drugs 0.000 description 1
- 230000003308 immunostimulating effect Effects 0.000 description 1
- 238000013101 initial test Methods 0.000 description 1
- 229940079322 interferon Drugs 0.000 description 1
- 230000014828 interferon-gamma production Effects 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 239000007928 intraperitoneal injection Substances 0.000 description 1
- 238000001990 intravenous administration Methods 0.000 description 1
- 229960005386 ipilimumab Drugs 0.000 description 1
- 229920002521 macromolecule Polymers 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 229940101578 microlipid Drugs 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- 230000017066 negative regulation of growth Effects 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 210000001672 ovary Anatomy 0.000 description 1
- 230000008506 pathogenesis Effects 0.000 description 1
- 238000010647 peptide synthesis reaction Methods 0.000 description 1
- 239000008177 pharmaceutical agent Substances 0.000 description 1
- 239000000546 pharmaceutical excipient Substances 0.000 description 1
- 210000002826 placenta Anatomy 0.000 description 1
- 230000003169 placental effect Effects 0.000 description 1
- 229920000573 polyethylene Polymers 0.000 description 1
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 1
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 229940023143 protein vaccine Drugs 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 230000010076 replication Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 239000011347 resin Substances 0.000 description 1
- 229920005989 resin Polymers 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 238000013207 serial dilution Methods 0.000 description 1
- 238000009097 single-agent therapy Methods 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 210000004988 splenocyte Anatomy 0.000 description 1
- 150000003431 steroids Chemical class 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 230000001629 suppression Effects 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 238000001890 transfection Methods 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 230000001960 triggered effect Effects 0.000 description 1
- 239000012646 vaccine adjuvant Substances 0.000 description 1
- 229940124931 vaccine adjuvant Drugs 0.000 description 1
- 239000000080 wetting agent Substances 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
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/177—Receptors; Cell surface antigens; Cell surface determinants
- A61K38/1774—Immunoglobulin superfamily (e.g. CD2, CD4, CD8, ICAM molecules, B7 molecules, Fc-receptors, MHC-molecules)
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
- A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- 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
- A61P35/02—Antineoplastic agents specific for leukemia
-
- 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
- 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
- A61K2039/53—DNA (RNA) vaccination
-
- 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
- A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
- A61K2039/55511—Organic adjuvants
- A61K2039/55522—Cytokines; Lymphokines; Interferons
- A61K2039/55527—Interleukins
- A61K2039/55533—IL-2
-
- 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
- A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
- A61K2039/55511—Organic adjuvants
- A61K2039/55555—Liposomes; Vesicles, e.g. nanoparticles; Spheres, e.g. nanospheres; Polymers
-
- 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
- A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
- A61K2039/572—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2 cytotoxic response
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
- A61K2039/6031—Proteins
Description
本發明係關於一種治療感染性與惡性疾病之新穎方法。
細胞毒T淋巴細胞抗原4(Cytotoxic T-lymphocyte antigen-4,CTLA-4)於1987年被發現為免疫球蛋白超家族中之一新成員,其特徵在於其亦具有變異(V)或恆定(C)免疫球蛋白區之重要結構性特徵(Brunet et al.,Nature 328,267-270)。報導指出CTLA-4對於調節免疫系統亦扮演一重要的角色(Keilholz,U.,J Immunother 31,431-439)。CTLA-4被報導可藉由與CD28競爭CD80/CD86之結合位置以降低T細胞之活化(Rudd et al.,Immunol Rev 229,12-26)。雖然CTLA-4可保護個體免於罹患自體免疫性疾病,但它亦會抑制抗癌的免疫力。為了避免CTLA-4於癌症治療中可能造成之不欲的免疫反應,已有許多方法在探討如何操控T細胞之共刺激路徑,以提升抗癌免疫反應。標靶CTLA-4療法為眾多先進的方法中之一種,且此方法已於後期的臨床試驗中顯示出具潛力之結果(Hodi et al.,N Engl J Med 363,711-723;Hodi,F.S.,Asia Pac J Clin Oncol 6 Suppl 1,S16-23;Weber,J.,Oncologist 13 Suppl 4,16-25;及Ribas,A.,Oncologist 13 Suppl 4,10-15)。抗CTLA-4之一種單株抗體,ipilimumab,已於2011年三月被FDA核准用以治療轉移性黑色素瘤。除了轉移性黑色素瘤外,目前CTLA-4抗體於眾多的臨床試驗中用於治療惡性腫瘤,包括胰臟癌、大腸直腸癌、肝細胞癌、淋巴癌、激素抗拒性***癌、卵巢癌、及急性骨髓性白血病。
計畫性死亡-1(Program death-1,PD-1)為CD28超家族中之一員,當與其之配體,計畫性死亡配體1及2(PD-L1及PD-L2),結合時可觸發負向的訊號路徑(Riley,J.L.,Immunol Rev 229,114-125)。PD-1與其配體間的交互作用會抑制增生、
細胞激素的生成、及T細胞之細胞溶解功能,從而耗盡T細胞,並抑制其免疫反應。PD-1/PD-L路徑對於耐受性及免疫力十分重要。此路徑保護組織及器官免受免疫調控之傷害。然而,此路徑已知會被慢性感染的病原菌及腫瘤所利用,以抑制抗微生物性及抗癌之免疫力。為了調控PD-1/PD-L路徑之免疫調控活性,已有標靶此路徑之療法被發展,以用於治療包含感染、自體免疫、乃至癌症等疾病(Weber,J.,Semin Oncol 37,430-439)。
雖然已有許多方法可藉由標靶CTLA-4、PD-1/PD-L1/PD-L2、及其他免疫調控蛋白,以調控免疫反應來對抗感染性及惡性疾病;然而仍需一種可藉由提升免疫力且同時避免抑制免疫反應的新穎方法,以用於抗癌及抗感染之治療中。
本發明提供一種醫藥組合物,可於治療感染性或惡性疾病時,提升受治療個體的免疫反應,該醫藥組合物包含一與表現載體結合的DNA構築體,及醫藥可接受之載劑,其中該DNA構築體包含一多核苷酸序列,該多核苷酸編碼選自由細胞毒T淋巴細胞抗原4(CTLA-4)、計畫性死亡-1(PD-1)、計畫性細胞死亡1配體1(PD-L1)、其片段或其功能性變異體、及其組合所組成之群組中任一者。
在另一方面中,本發明提供一種醫藥組合物,可於治療感染性或惡性疾病時,提升受治療個體的免疫反應,該醫藥組合物包含一重組多胜肽及一醫藥可接受之載劑,其中該重組多胜肽包含一多胜肽序列,該多胜肽序列係選自由CTLA-4、PD-1、PD-L1、其片段或其功能性變異體、及其組合之多胜肽序列所組成之群組。
除非另有定義,所有於此處所使用之技術性及科學性術語具有之意義,對於屬於本發明之領域中具有通常技藝者係為習知意義。當於此處使用時,以下術語具有其所屬之意義,除非另有特別定義者。
於此處使用到「一」時,是指一個或一個以上(如:至少一者)之文述之物體。例如,「一元件」意指一個元件或一個以上之元件。
「多核苷酸」或「核酸」乙詞係指由核苷酸單元組成之聚合體。多核苷酸包括自然存在之核酸,像是去氧核糖核酸(deoxyribonucleic acid,DNA)、及核糖核酸(ribonucleic acid,RNA),以及核酸類似物,包括該等具有非天然存在之核苷酸,像是重組多核苷酸。多核苷酸可被合成,如:可使用一自動化DNA合成儀合成之。「核酸」乙詞典型地係指大的多核苷酸。當核苷酸序列係以DNA序列(如A、T、G、C)代表時,應可瞭解的是亦包括RNA序列(如A、U、G、C);其中「U」取代「T」。「cDNA」乙詞係指一DNA與mRNA成互補或與mRNA相同,且該cDNA可為單股或雙股形式。
「編碼」乙詞係指於多核苷酸(如:基因、cDNA、或mRNA)中之核苷酸的特定序列之固有功能,該序列可作為模板,用以合成生物反應中之其他聚合物及巨分子,該等聚合物及巨分子具有一定義的核苷酸序列(如rRNA、tRNA及mRNA)、或可為一定義的胺基酸序列,藉此以得到生物活性。因此,若由基因所產生的mRNA經過轉錄及轉譯而於細胞或其他生物系統生成一蛋白,則稱該基因編碼該蛋白。應可為具有技藝者所理解的是,因遺傳密碼的簡併性,多種不同之多核苷酸及核酸可編碼相同之多胜肽。對於具有技藝者亦可理解的是,使用常規技術使核苷酸進行替換,且不會改變所述之多核苷酸所編碼的多胜肽序列,以對應任何特定宿主生物體所需使用之密碼子,以於該宿主生物體中表現該多胜肽。因此,除非另有特別所指,「多核苷酸編碼一胺基酸序列」包括所有多核苷酸,其可
互為簡併形式,且其編碼相同的胺基酸序列。多核苷酸編碼蛋白質及RNA,可能包括內含子。
「重組多核苷酸」乙詞係指一多核苷酸具有非天然連結成之序列。一重組多核苷酸可以一載體形式存在。「載體」可包含特定之感興趣核苷酸序列,及一調控序列;載體可被用以表現該特定之核苷酸序列、或用以維持該核苷酸序列以供複製、操控、或於不同位置間進行轉移(如:不同生物體間)。載體可被引入一適當宿主細胞,以進行前述之目的。
載體之例子包含,但不限於:質體、黏質體、噬菌體、YACs、或PACs。典型地,於載體中,特定之核苷酸序列可與調控序列連結,像是當該載體被引入一宿主細胞內時,該特定之核苷酸序列可經由調控序列之控制,於該宿主細胞內表現。該調控序列可包含,例如但非侷限,啟動子序列、起始密碼子、複製子、增強子、操控子序列、分泌訊號序列(如IL2訊號胜肽)、及其他控制序列。較佳地,載體可進一步含有標記子序列(如:抗生素抗性之標記子序列),供後續篩選步驟所用。
「多胜肽」乙詞係指分子或多分子,係由胺基酸殘基透過胜肽鍵連結所組成。多肽胜可被合成,例如,使用一自動化多肽肽合成儀。「蛋白」乙詞典型地係指大型的多肽胜。「胜肽」乙詞典型地係指短的多胜肽。
胺基酸可藉由三個字母或一個字母所表示。表1所列為標準胺基酸縮寫。
「疫苗」乙詞係指一含有活性成分之藥劑或組合物;該活性成分可有效誘導個體產生治療程度之免疫力,已對抗特定病原菌或疾病。傳統上,疫苗之活性成分為衍生自病原菌之多胜肽,該病原菌即為疫苗之標的。「DNA疫苗」乙詞係指活性成分為DNA的疫苗。「蛋白疫苗」乙詞係指活性成分為多胜肽的疫苗。
「醫藥組合物」乙詞係指適用於個體之醫藥用途的組合物。醫藥組合物包含有效量之活性物質及醫藥可接受之載劑。「有效量」乙詞係指該物質的量可有效產生所欲之結果,像是本發明中之免疫反應。「醫藥可接受之載劑」乙詞係指任何標準的醫藥載劑、緩衝液及賦形劑,像是磷酸緩衝鹽水溶液、5%葡萄糖液態溶液、及乳化劑,像是油包水、或水包油乳化劑,及各種不同形式之潤濕劑、及/或佐劑。較佳的醫藥載劑需視活性物質之欲投予的模式決定。投予的典型模式包括腸內的(如口服)、或非經口的(如皮下的、肌肉、靜脈、或腹腔注射;或局部、經皮的、或經黏膜的投予)。「佐劑」乙詞係指藥學的或免疫學的藥劑,其可修飾其他藥劑(如藥物、疫苗)的作用,當單獨投予佐劑時,仍可具有少量之直接效果。佐劑通常包括於疫苗內,以提升接受對象對於所提供之抗原的免疫反應,同時使所注射之外來物質維持於低量。
「個體」乙詞為人類或非人類之哺乳動物。非人類之哺乳動物包括,但不限於靈長類、有蹄類、犬、及貓。
「裸露的DNA」乙詞係指DNA構築體(用以投予一個體),其未與微脂體結合。
多胜肽(或蛋白)之「片段」乙詞係指一多胜肽之胺基端及/或羧基端被去除,但所剩餘之胺基酸序列係與對應位置之天然存在的序列所演繹者相同,例如,來自全長的DNA序列。片段典型地為至少10個胺基酸長度,較佳地為10至50個胺基酸長度、更佳地為50至100個胺基酸長度、再佳地為超過100個胺基酸長度。
一多胜肽(或蛋白)之「功能性變異體」係指將本發明之多胜肽(或蛋白)的主要胺基酸序列進行一個或多個修飾之多胜肽,其仍可保有於本文所述之免疫促進功效。若本發明之多胜肽(或蛋白)之功能性變異體涉及胺基酸之替換、保守胺基酸替換典型地較佳為,如:替換時仍保有原本胺基酸之一特性,如大小、電荷、疏水性、構形等。保守胺基酸替換的實例包括於下列群組中之胺基酸進行替代:(1)M、I、L、V;(2)F、Y、W;(3)K、R、H;(4)A、G;(5)S、T;(6)Q、N;及(7)E、D。其他適用的替換可由具技藝者所輕易建立,且亦可額外地藉由參考公開文獻決定如Voet,Biochemistry,
Wiley,1990;StryerBiochemistry
4th
Ed.,Freeman N.Y.,1995;Peptide Chemistry.A Practical Textbook,
2nd ed.,Miklos Bodanszky,Springer-Verlag,Berlin,1993;Principles of Peptide Synthesis,
2nd ed.,Miklos Bodanszky,Springer-Verlag,Berlin,1993;Chemical Approaches to the Synthesis of Peptides and Proteins
,P.Lloyd-Williams,F.Albericio,E.Giralt,CRC Press,Boca Raton,1997;Bioorganic Chemistry:Peptides and Proteins
,S.M.Hecht,Ed.,Oxford Press,Oxford,1998,Synthetic Peptides:A User's Guide
,Gregory A.Grant(Editor),Oxford University Press,2002、及其類似文獻,前述所有文獻皆被引用於本文中。
本發明之醫藥組合物可由習知方法及一個或多個醫藥可接受之載劑製備。於此處所使用之「醫藥可接受之載劑」乙詞係包含任何標準的醫藥載劑。該載劑可包括,但不限於:食鹽
水、緩衝食鹽水、葡萄糖、水、甘油、乙醇及其組合。
本發明提供一種醫藥組合物,可於治療感染性或惡性疾病時,提升受治療個體的免疫反應,該醫藥組合物包含一與表現載體結合的DNA構築體,及醫藥可接受之載劑,其中該DNA構築體包含一多核苷酸序列,該多核苷酸編碼選自由胞毒T淋巴細胞抗原4(CTLA-4)、計畫性死亡-1(PD-1)、計畫性細胞死亡1配體1(PD-L1)、其片段或其功能性變異體、及其組合所組成之群組中任一者。
根據本發明之具體實施例,該醫藥組合物包含一DNA構築體,其包含一多核苷酸序列,該多核苷酸序列編碼CTLA-4,並與pVAC-1載體融合,且該組合物於黑色素瘤及腎細胞癌模式中提供保護效果。
可推測的是,同時阻斷CTLA-4及PD-1及/或PD-L1應可進一步提升抗病原菌及癌細胞的免疫力。根據本發明之部分具體實施例,該醫藥組合物有能力可誘發專一抗CTLA-4及PD-1及/或PD-L1之免疫力。於部分具體實施例中,本發明之醫藥組合物包含一DNA構築體,其編碼CTLA-4及PD-1,並與一載體融合,如:pVAC-1-IL2ss-CTLA4-PD-1-PLAP及pVAC-1-IL2ss-CTLA4-PD-1。舉例而言,本發明之DNA疫苗包含DNA構築體,其編碼CTLA-4及PD-L1,如:pVAC-1-IL2ss-CTLA4-PD-L1-PLAP及pVAC-1-IL2ss-CTLA4-PD-L1。於該等具體實施例中,於試驗小鼠之抗兩種抗原之抗體力價皆被提高,以及其內之腫瘤生長皆被大幅度抑制。
含有本發明之DNA構築體的醫藥組合物,即DNA疫苗,具有超越已存在之療法的多種優點,該等已存在之療法係標靶與CTLA-4、PD-1、PD-L1相關之路徑及其他免疫調控路徑,像是使用抗體來對抗CTLA-4、PD-1、PD-L1及其他免疫調控分子。例如,相較於抗體,DNA疫苗之大量生產較為容易且成本較低。再者,本發明之CTLA-4-PD-1、CTLA-4-PD-L1及CTLA-4-PD-1-PD-L1融合DNA疫苗提供一種單一療法,可用
以同時抑制此二或三種重要標的。為達成更有效之免疫力而投予多個CTLA-4、PD-1、或PD-L1之專一性抗體在考量到其所需費用時,將變得較為困難。此外,投予兩種實驗性藥物相關之管制問題將會阻礙此種結合性療法之初期試驗。融合基因DNA疫苗之策略可提供一同時操控多種免疫系統路徑之方式,且不需投予多種試驗性藥物,該等投予多種試驗性藥物既不具經濟效益之可行性,亦不為多數國家之管制單位所允許。
於本發明之其他具體實施例中,多種DNA構築體被選殖為DNA疫苗,其可由一個以上之人類、鼠、人類及鼠之嵌合體、其他生物、或人類及其他生物之嵌合體的DNA序列(如編碼CTLA-4、PD-1、PD-L1、或其他之DNA序列)所組成。該DNA序列可能編碼與免疫系統功能、感染性疾病之致病機轉、或惡性腫瘤之腫瘤生成有關之蛋白,如CTLA-4、PD-1、PD-L1、其片段或其功能性變異體及其組合。
在另一方面中,本發明提供一種醫藥組合物,可於治療感染性或惡性疾病時,提升受治療個體的免疫反應,該醫藥組合物包含一重組多胜肽及一醫藥可接受之載劑,其中該重組多胜肽包含一多胜肽序列,該多胜肽序列係選自由CTLA-4、PD-1、PD-L1、其片段或其功能性變異體、及其組合之多胜肽序列所組成之群組。
本發明之具體實施例中,該個體係以抗感染性或抗癌藥物治療,造成該個體之免疫反應受到刺激。於本發明之一實例中,該個體受惡性疾病之治療。
根據本發明,該惡性疾病可為轉移性黑色素瘤、胰臟癌、大腸直腸癌、肝細胞癌、淋巴癌、激素抗拒性***癌、卵巢癌、急性骨髓性白血病、或非小細胞肺癌。
根據一具體實施例,本發明之醫藥組合物包含重組多胜肽,該重組多胜肽包含CTLA-4及PD-1、CTLA-4及PD-L1或CTLA-4及PD-1及PD-L1之多胜肽序列,且該組合物於黑色素瘤及腎細胞癌模式中提供保護效果。
本發明進一步以下列實施例闡述,其係供佐證之目的,而非用以侷限本發明。
為構築CTLA-4之DNA疫苗,選殖人類或小鼠的CTLA-4序列,並於N端及C端分別與IL2分泌訊號序列(IL2ss)及胎盤鹼性磷酸酶(PLAP)之穿膜區域序列融合,再將之構築入哺乳動物表現質體,pVAC-1。所得之pVAC1-IL2ss-hCTLA-4-PLAP(SEQ ID NO:1)及pVAC1-IL2ss-mCTLA-4-PLAP(SEQ ID NO:2)的DNA序列皆列示於圖1a及圖1b中。該等DNA疫苗,如單獨僅有人類或鼠的PD-1或PD-L1者、由CTLA-4及PD-1及/或PD-L1所組成之人類或鼠的融合基因構築體,以及於C端具有或不具有PLAP之穿膜區域者,皆被構築並列示於下文及圖10中:
(1)pVAC-1-IL2ss-hPD-L1(含有編碼人類PD-L1之DNA序列);
(2)pVAC-1-IL2ss-hPD-1(含有編碼人類PD-1之DNA序列);
(3)pVAC-1-IL2ss-hCTLA4-hPD-L1(含有編碼人類CTLA-4及PD-L1之DNA序列);
(4)pVAC-1-IL2ss-hCTLA4-hPD-1(含有編碼人類CTLA-4及PD-1之DNA序列);
(5)pVAC-1-IL2ss-hCTLA4-hPD-1-hPD-L1(含有編碼人類CTLA-4、PD-1及PD-L1之DNA序列);
(6)pVAC-1-IL2ss-mPD-L1(含有編碼小鼠PD-L1之DNA序列);
(7)pVAC-1-IL2ss-mPD-1(含有編碼小鼠PD-1之DNA序列);
(8)pVAC-1-IL2ss-mCTLA4-mPD-L1(含有編碼小鼠
CTLA-4及PD-L1之DNA序列);
(9)pVAC-1-IL2ss-mCTLA4-mPD-1(含有編碼小鼠CTLA-4及PD-1之DNA序列)。
(10)pAC-1-IL2ss-mCTLA4-mPD-1-mPD-L1(含有編碼小鼠CTLA-4、PD-1及PD-L1之DNA序列)。
為構築CTLA-4-PD-1及CTLA-4-PD-L1之重組多胜肽,以與PD1或PD-L1融合的小鼠CTLA-4構築入E.Coli
表現載體,pET56。以鎳-樹脂親和柱(nickel-resin affinity column)純化所得之mCTLA-4-mPD-1-His6
及mCTLA-4-mPD-L1-His6
之蛋白序列係分別為SEQ ID NO:31及SEQ ID NO:32。
PC-PEG-PE微脂體係如下所製備:將5.9 mg之PC(1,2-二棕櫚醯-sn-甘油-3乙基磷酸膽鹼)與14.6 mg之PEG(1,2-二棕櫚醯-sn-甘油-3磷酸乙醇胺-N-聚乙二醇-5000)(Avanti Polar Labs,Inc.)分別溶解於2 ml之溶劑(90%氯仿、10% MeOH)及1 ml之MeOH中,並置入500 ml圓底燒瓶中。將燒瓶置於旋轉蒸發器上,於55℃、真空下進行旋轉,直到液體消失。將燒瓶進一步於室溫30分鐘及加熱5分鐘之三個循環中進行真空乾燥。將溶於6 ml PBS中的6 mg聚乙烯亞胺(PE)(Sigma Aldrich)加入旋轉中之燒瓶,並進行10分鐘加熱、30分鐘室溫的五個循環。將最終溶液以蒸餾水調整至6 ml,並進行一次冷凍解凍步驟(從-20℃至4℃)。將該溶液以一系列濾膜(1.2、0.8、0.45、及0.22 μm)過濾,且仍維持於60℃之水浴中,以得到PC-PEG-PE微脂體。該微脂體/DNA(如實施例1所得之DNA構築體)複合物需於注射入小鼠前兩小時內新鮮製備。將微脂體與製備好之DNA(1 mg/ml)以等量體積於室溫混合20分鐘。於投予各試驗老鼠前,再將
該微脂體/DNA複合物以PBS調整至100μL。
將不同量(從0、25、50至100 μg)之pVAC-1-IL2ss-mCTLA-4、pVAC-1-IL2ss-hCTLA-4、及pVAC-1對照組載體,以肌肉注射至6-7週齡之C57BL/6及Balb/c小鼠。其中該等DNA分別可為裸露DNA形式、或分別與兩種微脂體中之一者結合,即Nancy-Templeton(Templeton et al.,Nat Biotechnol 15,647-652)所發展之DOTAP:Chol或前述製備之PC-PEG-PE。每週進行一次免疫,長達四週。
以前述DNA疫苗藉由肌肉注射免疫小鼠四週後,採小鼠血清並分析其對於PD-1、PD-L1、以及人類及小鼠CTLA-4的專一性。為偵測抗mCTLA-4、hCTLA-4、mPD-L1及mPD-1之可能的抗體,將1μg/ml(於pH8.4之硼酸鹽(BS)緩衝液中)之該等蛋白(R&D systems,Minneapolis,MN,USA)於室溫下固著於96孔之EIA盤4小時,再以含有1%(w/v)之牛血清白蛋白(BS-BSA)之硼酸鹽水進行封閉。以BS-BSA將小鼠血清進行2倍之連續稀釋(1:100至1:3200)後,將其加入二重複孔洞中,並置於4℃培養隔夜。隨後,以含有0.05%(v/v)Tween-20的PBS沖洗培養盤,並與經BS-BSA稀釋至1:2000且與HRP結合之山羊抗鼠IgG於室溫培養2小時。將HRP受質(2,2’-次偶氮基-雙(乙基苯并噻唑啉磺酸))加入,並於室溫培養20分鐘。吸光值係以405 nm於ELISA分析儀上測量。於圖2中,來自被pVAC-1-IL2ss-hCTLA4-PLAP免疫的小鼠血清表現出對於重組人類CTLA4專一的免疫力,且對於小鼠之CTLA4具有較低程度之免疫力;然而,經以pVAC-1
對照組載體免疫之小鼠的血清,對於人類及小鼠之CTLA4皆無顯著的結合活性。當試驗小鼠以編碼融合基因之DNA疫苗免疫時,該等融合基因包括mCTLA4-PD-L1-PLAP、mCTLA4-PD-L1、mCTLA4-PD-1-PLAP、或mCTLA4-PD-1,其皆可誘發mCTLA4及mPD-1、或mCTLA4及mPD-L1之抗血清,如圖15所示。
本發明之DNA疫苗的效力研究係使用黑色素瘤細胞(B16F10)。為了分析抗CTLA-4免疫力的保護效果,以B16F10黑色素瘤細胞接種經本發明之pVAC-1-IL2ss-hCTLA-4-PLAP(「hCTLA4」)免疫的c57/BL小鼠。如圖3所示,經hCTLA4免疫之小鼠的B16F10腫瘤生長速率顯著地低於投予對照組pVAC-1載體(p
=0.004)之小鼠。雖然經hCTLA4免疫之小鼠獲得了足以抑制腫瘤生長之抗CTLA-4的免疫反應,但該等小鼠並未出現任何結腸炎之症狀,此症狀為投予抗CTLA-4單株抗體試驗中,最常發生於癌症病患的副作用(Di Giacomo et al.,Cancer Immunol Immunother 58(8):1297-306)。經以pVAC-1-IL2ss-hCTLA-4-PLAP DNA疫苗或對照組pVAC-1載體處理五週之小鼠,其於體重變化上未顯示出可區別之差異(參見圖4)。
為了瞭解pVAC-1-IL2ss-CTLA-4-PLAP疫苗之抗癌效果的機制,將免疫後的小鼠犧牲,並收集其脾細胞供分析其對於癌細胞之細胞毒殺效果。脾細胞所調控之細胞毒殺能力係使用先前篩選所得之可穩定表現螢光酵素之B16F10細胞(B16F10-luc細胞)測量,其細胞數目已證實與螢光酵素活性有好的關連性;該螢光酵素活性係以每秒之光子計數來表示(參見圖5a)。將B16F10-luc細胞(2000細胞/孔)置於含有
100倍或30倍(2x105
或6x104
細胞)之脾細胞的96孔中。經培養一天後,將螢光酵素受質,螢光素(luciforin),加入該等細胞中,並以IVIS影像系統以每秒之光子計數來量化細胞存活率。結果證實源自以pVAC-1-IL2ss-CTLA-4-PLAP免疫之小鼠的脾細胞對於B16F10的細胞存活率上有顯著之功效(參見圖5b)。
為了瞭解脾細胞對於B16F10-luc之免疫反應,遂測量干擾素γ產生之量。將免疫小鼠的脾細胞與CTLA4抗原一起培養,並以專用於測量干擾素γ之ELISA套組(R&D system)測量受刺激而分泌之干擾素γ。相似地,源自經對照組pVAC-1或本發明之pVAC-1-IL2ss-CTLA-4-PLAP免疫之帶有腫瘤之小鼠的脾細胞,係以相應的腫瘤細胞刺激,再測量干擾素γ的分泌量。取自以pVAC-1-IL2ss-CTLA-4-PLAP DNA疫苗免疫之小鼠的脾細胞所產生之干擾素γ高於取自以pVAC-1免疫之對照組小鼠者(參見圖6)。
鼠腎細胞癌(RENCA)亦使用於效力試驗。以鼠腎細胞癌模式之balb/c小鼠的動物試驗進行。將該小鼠以pVAC-1-mCTLA-4-PLAP免疫,以於小鼠中產生更專一於自體抗原的抗體。在未與陽離子微脂體結合的情況下,該「裸露的」pVAC-1-mCTLA-4 DNA疫苗無法引起顯著之抗mCTLA-4抗體力價(參見圖7a)。由於如實施例3所製備之陽離子微脂體與DNA疫苗之結合可增加轉染效率及提升免疫反應,遂探討該pVAC-1-mCTLA-4-微脂體複合物於誘發抗小鼠CTLA-4之免疫反應,以提供一較佳形式的DNA疫苗。此一方法大幅度地誘發抗小鼠CTLA-4之抗體力價,並較小程度地誘發之抗人類CTLA-4抗體力價(圖7b及圖7c)。相較於以對照組pVAC-1
DNA疫苗或裸露的pVAC-1-mCTLA-4 DNA疫苗免疫者,以pVAC-1-mCTLA-4-微脂體複合物免疫之小鼠亦顯示對於腎細胞癌(RENCA)之生長抑制(參見圖8,pVAC-1-mCTLA4-微脂體複合物與對照組DNA疫苗相比較:p<0.01)。
由於CTLA-4係藉由與抗原呈現細胞表面上的CD-80(B7.1)及CD-86(B7.2)結合而發揮其功能,遂以純化之6個組胺酸(His6
)標記的CTLA-4蛋白及可穩定表現B7.1及B7.2之中國倉鼠卵巢(CHO)細胞來探討小鼠血清對此等蛋白間交互作用的抑制效果。可穩定表現B7.1或B7.2之CHO細胞係由台灣、台北之中央研究院的Dr.M.H.所提供。在經pVAC-1-IL2ss-hCTLA-4 DNA疫苗或對照組DNA疫苗免疫小鼠的血清存在或不存在下,將細胞(5x105
)與2 ng/ml之6個組胺酸標記的人類CTLA於4℃培養30分鐘。結合的蛋白可由FITC標記之鼠抗his6抗體於1:200下偵測,並進行FACS分析。結果顯示於圖9,其指出His6
-人類CTLA-4(2ng/ml)與於穩定轉染之CHO細胞(5x105
細胞/試驗)上所表現之B7.1/B7.2之間的交互作用會被經pVAC-1-IL2ss-hCTLA-4-PLAP DNA疫苗免疫小鼠的血清所阻斷,其中於1:25稀釋時,該等對照組之血清未發現此種抑制作用的現象(參見圖9)。
除了具有良好結果之pVAC-1-IL2ss-CTLA-4-PLAP DNA疫苗外,本發明亦構築其他同時標靶多種免疫抑制蛋白之DNA疫苗,包括CTLA-4及PD-1或PD-L1(參見圖11及圖12),並探討其效力;類似於pVAC-1-IL2ss-CTLA-4-PLAP,該等構築體亦基於pVAC-1載體。根據本發明,該等疫苗包括:
pVAC1-IL2ss hPD-1-PLAP(SEQ ID NO:3);pVAC1-IL2ss hPD-1(SEQ ID NO:4);pVAC1-IL2ss hPD-L1-PLAP(SEQ ID NO:5);pVAC1-IL2ss hPD-L1(SEQ ID NO:6);pVAC1-IL2ss hCTLA4-hPD-1-PLAP(SEQ ID NO:7);pVAC1-IL2ss hCTLA4-hPD-1(SEQ ID NO:8);pVAC1-IL2ss hCTLA4-hPD-L1-PLAP(SEQ ID NO:9);pVAC1-IL2ss hCTLA4-hPD-L1(SEQ ID NO:10);pVAC1-IL2ss mPD-1-PLAP(SEQ ID NO:11);pVAC1-IL2ss mPD-1(SEQ ID NO:12);pVAC1-IL2ss mPD-L1-PLAP(SEQ ID NO:13);pVAC1-IL2ss mPD-L1(SEQ ID NO:14);pVAC1-IL2ss mCTLA4-mPD-1-PLAP(SEQ ID NO:15);pVAC1-IL2ss mCTLA4-mPD-1(SEQ ID NO:16);pVAC1-IL2ss mCTLA4-mPD-L1-PLAP(SEQ ID NO:17);及pVAC1-IL2ss mCTLA4-mPD-L1(SEQ ID NO:18)。
其中該IL2ss(SEQ ID NO:19)係作為訊號胜肽,其含有21個胺基酸,且與其他分泌蛋白之訊號胜肽一樣具有常見之特徵,該特徵係關於疏水性胺基酸之豐裕度及位置;IL2ss之細胞內切割會發生在Ser20之後,並導致該抗原蛋白之分泌;限制性切位為BamHI或EcoRI;疏水性COOH-端序列的32個殘基(「PLAP」衍生自胎盤鹼性磷酸酶)係為一穿膜區域,以拴住被轉譯之蛋白質至細胞膜上(SEQ ID NO:20)。標靶hCTLA4之序列為編碼hCTLA4之第37至160個殘基之胺基酸序列的DNA序列(SEQ ID NO:21);標靶mCTLA4之序列為編碼mCTLA4之第36至161個殘基之胺基酸序列的DNA序列(SEQ ID NO:22);標靶hPD-1之序列為編碼人類計畫性細胞死亡蛋白1(hPD-1)之第21至170個殘基之胺基酸序列的DNA序列(SEQ ID NO:23);標靶mPD-1之序列為編碼老
鼠計畫性細胞死亡蛋白1(mPD-1)之第21至170個殘基之胺基酸序列的DNA序列(SEQ ID NO:24)。標靶hPDL-1之序列為編碼人類計畫性細胞死亡1配體1(hPD-L1)之第19至238個殘基之胺基酸序列的DNA序列(SEQ ID NO:25);標靶mPD-L1之序列為編碼老鼠計畫性細胞死亡1配體1(mPD-L1)之第19至127個殘基之胺基酸序列的DNA序列(SEQ ID NO:26);標靶hCTLA4及hPD-1之序列為編碼hCTLA4之第37至160個殘基之胺基酸序列及hPD-1之第21至170個殘基之胺基酸序列的DNA序列(hCTLA4-hPD-1)(SEQ ID NO:27);標靶hCTLA4及hPD-L1之序列為編碼hCTLA4之第37至160個殘基之胺基酸序列及PD-L1之第19至238個殘基之胺基酸序列的DNA序列(hCTLA4-hPD-L1)(SEQ ID NO:28);標靶mCTLA4及mPD-1之序列為編碼mCTLA4之第36至161個殘基之胺基酸序列及mPD-1之第21至170個殘基之胺基酸序列的DNA序列(mCTLA4-mPD-1)(SEQ ID NO:29);標靶mCTLA4及mPD-L1之序列為編碼mCTLA4之第36至161個殘基之胺基酸序列及mPD-L1之第19至127個殘基之胺基酸序列的DNA序列(mCTLA4-mPD-L1)(SEQ ID NO:30)。
根據本發明,亦可構築其他DNA疫苗標靶多於兩種的免疫抑制蛋白,如pVAC1-IL2ss-CTLA4-PD-1-PD-L1。部分具體實施例顯示於圖13並列於下方:(1)pVAC1-IL2ss-hCTLA4-hPD1-hPDL1(SEQ ID NO:33 or SEQ ID NO:34);及(2)pVAC1-IL2ss-mCTLA4-mPD1-mPDL1(SEQ ID NO:35 or SEQ ID NO:36);其中標靶hCTLA4、hPD-1及hPD-L1之序列分別為編碼hCTLA4之第37至160個殘基之胺基酸序列、hPD-1之第31至147個殘基之胺基酸序列的DNA序列及hPD-L1之第19至
127或19至238個殘基之胺基酸序列的DNA序列(hCTLA4-hPD-1-hPD-L1);標靶mCTLA4、mPD-1及mPD-L1之序列分別為編碼mCTLA4之第36至161個殘基之胺基酸序列、mPD-1之第31至147個殘基之胺基酸序列的DNA序列及mPD-L1之第19至127或19至237個殘基之胺基酸序列的DNA序列(mCTLA4-mPD-1-mPD-L1)。
以如同前述實施例中所採用之pVAC-1-IL2ss-CTLA4-PLAP DNA疫苗之相似的免疫步驟來探討該等抗原之免疫力。從該等構築體所得之正面結果,證實藉由以pVAC-1-IL2ss-mCTLA4-mPD-L1-PLAP或pVAC-1-IL2ss-mPD-L1-mCTLA-4-PLAP免疫小鼠,就增加抗鼠CTLA-4及PD-L1之抗體力價而言,可同時提升其免疫力(圖14)。如圖14所示,該虛線代表經注射pVAC-1載體之對照組的血清抗體力價,而來自受前述DNA疫苗注射之小鼠的血清係以對照組小鼠之數據標準化(疫苗組vs.對照組相:*
=p<0.05,**
=p<0.01)。
以70 μg之pVAC-1-IL2ss-mCTLA-4-mPD-L1-PLAP、pVAC-1-IL2ss-mCTLA-4-mPD-L1、pVAC-1-IL2ss-mCTLA-4-mPD-1-PLAP、pVAC-1-IL2ss-mCTLA-4-mPD-1、或pVAC-1 DNA疫苗,肌肉注射至6至7週齡之Balb/c小鼠,每週一次,長達三週。第四次免疫亦是使用相同量之DNA疫苗,但在肌肉注射DNA後立刻將兩支電極針置於注射部位之側面,並以2脈沖之電壓、1,000 V/cm進行電穿孔。誘發之抗CTLA4、PD-1及PD-L1之抗血清的測量方式係如同實施例5中所述者。相較於源自以pVAC-1(載體對照組)免疫小鼠的血清,
源自以pVAC-1-IL2ss-mCTLA-4-mPD-L1-PLAP、pVAC-1-IL2ss-mCTLA-4-mPD-L1、pVAC-1-IL2ss-mCTLA-4-mPD-1-PLAP、及pVAC-1-IL2ss-mCTLA-4-mPD-1免疫小鼠的血清皆顯示增加的抗CTLA4之抗體力價(pVAC-1-IL2ss-mCTLA-4-mPD-L1與pVAC-1相比較:p<0.05,圖15a)。再者,相較於源自以pVAC-1免疫小鼠之血清,源自以pVAC-1-IL2ss-mCTLA-4-mPD-1-PLAP、pVAC-1-IL2ss-mCTLA-4-mPD-1、及該等以pVAC-1-IL2ss-mCTLA-4-mPD-L1-PLAP及pVAC-1-IL2ss-mCTLA-4-mPD-L1免疫之小鼠血清皆分別顯示出具有抗PD-1之抗體力價(圖15b)及PD-L1之抗體力價(圖15c)。
在一併投予30 μg的pORF-GM-CSF(作為疫苗佐劑)或pORF載體(對照組)之情況下,以pVAC-1-IL2ss-mCTLA-4-mPD-L1-PLAP、pVAC-1-IL2ss-mCTLA-4-mPD-L1、pVAC-1-IL2ss-mCTLA-4-mPD-1-PLAP、pVAC-1-IL2ss-mCTLA-4-mPD-1、或pVAC-1 DNA疫苗免疫小鼠。該等小鼠受四週的DNA肌肉注射。於第四週最後一次注射後亦於注射部位施行實施例12所述之立即性電穿孔。最後一次注射後一週,將小鼠以皮下注射方式接種鼠的腎細胞癌,RENCA(2x104
細胞/小鼠)。如圖16及圖17所示,相較於以pVAC-1載體對照組免疫之小鼠,經融合DNA疫苗免疫之小鼠,特別是pVAC-1-IL2ss-mCTLA4-mPD-L1及pVAC-1-IL2ss-mCTLA4-mPD-1,呈現顯著被抑制之腫瘤生長(圖17及圖16分別為共同投予或未共同投予GM-CSF DNA之免疫結果)。同樣地,相較於以pVAC-1載體對照組免疫之小鼠,經pVAC-1-IL2ss-mCTLA4-mPD-L1或pVAC-1-IL2ss-mCTLA4-mPD-1免疫之小鼠亦呈現受抑制之CT26的腫瘤生長(參見圖
18)。
對於該領域具有通常技藝者可明瞭的是,上述具體實施例可於不背離本發明之廣義概念下而修改。因此,應明瞭本發明並非侷限於所揭露之特定具體實施例,但此意欲包含屬於本發明之精神及範疇內之各種變化,即如同所附之申請專利範圍所定義者。
於圖式所示之具體實施例,其目的係用於闡述本發明。然而,吾人應明瞭的是,本發明並不侷限於所示之較佳具體實施例。
於圖式中:圖1a提供pVAC-1-IL2ss-hCTLA-4-PLAP之DNA及胺基酸序列。
圖1b提供pVAC-1-IL2ss-mCTLA4-PLAP之DNA及胺基酸序列。
圖2a顯示經pVAC-1-IL2ss-hCTLA-4-PLAP免疫之小鼠,其產生之抗人類及鼠之CTLA-4抗體的結果。
圖2b提供經pVAC-1-IL2ss-hCTLA-4-PLAP免疫之小鼠,於稀釋1:50、1:100、1:200、1:400、及1:800倍時,所產生之抗體的力價標準曲線。
圖3顯示經以pVAC-1-IL2ss-hCTLA-4-PLAP免疫之小鼠,小鼠之B16黑色素瘤(B16F10腫瘤細胞)生長被抑制的結果。
圖4提供經以pVAC-1-IL2ss-hCTLA-4免疫之小鼠及施以pVAC-1載體之對照組小鼠的體重比較結果,結果顯示兩組之間無差異。
圖5a提供B16F10細胞數目與螢光酵素活性之關連性;其中該B16F10細胞可穩定表現螢光酵素(B16F10-luc)。
圖5b結果顯示源自pVAC-1-IL2ss-hCTLA-4-PLAP免疫之
小鼠的脾細胞,其細胞毒殺能力被提升,使得B16F10-luc細胞存活率減少(於效應因子(脾細胞):腫瘤細胞(B16F10-luc)為100:1及30:1之比例)。
圖6提供經以pVAC-1-IL2ss-hCTLA-4-PLAP免疫之小鼠,經由B16F10-luc的刺激,提升脾細胞之干擾素γ的分泌量。
圖7a顯示以未與陽離子微脂體(即「裸露的DNA」)結合之pVAC-1-mCTLA-4-PLAP免疫小鼠,於1:50稀釋之血清中無顯著之抗鼠CTLA-4的抗體力價被誘發。
圖7b及圖7c提供相較於以裸露DNA對照組所免疫之小鼠,以與微脂體結合之pVAC-1-mCTLA-4-PLAP免疫小鼠,可產生抗人類及鼠之CTLA-4抗體的結果。
圖8提供以pVAC-1-mCTLA-4 DNA-微脂體複合物免疫之小鼠,其抑制RENCA腫瘤生長之結果。
圖9提供純化的hCTLA-4及其受器B7.1及B7.2間之交互作用;該受器B7.1及B7.2分別穩定地於CHO-B7.1及CHO-B7.2上表現。該交互作用會被經以pVAC-1-IL2ss-hCTLA-4-PLAP免疫之小鼠的血清抑制。
圖10提供標靶免疫調控蛋白之疫苗選殖策略,該等蛋白可為膜連結的蛋白(a)、或分泌的蛋白(b),其可分別具有或不具有胎盤鹼性磷酸酶(placental alkaline phosphatase,PLAP)之穿膜區域序列。於圖10所示之A及B區域之DNA序列可編碼選自由:CTLA-4、PD-1、PD-L1、其片段或其功能性變異體、及其組合所組成之群組中任一者。
圖11提供部分具體實施例的DNA及胺基酸序列,分別標靶根據本發明之人類蛋白,包括以下載體:(a)pVAC1-IL2ss-hPD-1(21-170aa)-PLAP(4167 bp)、(b)pVAC1-IL2ss-hPD-1(21-170aa)(4171 bp)、(c)pVAC1-IL2ss-hPD-L1(19-238aa)-PLAP(4377 bp)、(d)pVAC1-IL2ss-hPD-L1(19-238aa)(4381 bp)、(e)
pVAC1-IL2ss-hCTLA4-hPD-1(21-170aa)-PLAP(4545 bp)、(f)pVAC1-IL2ss-hCTLA4-hPD-1(21-170aa)(4443 bp)、(g)pVAC1-IL2ss-hCTLA4-hPD-L1(19-238aa)-PLAP(4752 bp)、及(h)pVAC1-IL2ss-hCTLA4-hPD-L1(19-238aa)(4650 bp)。
圖12提供部分具體實施例的DNA及胺基酸序列,分別標靶根據本發明之鼠蛋白,包括以下載體:(a)pVAC1-IL2ss-mPD-1(21-170aa)-PLAP(4173 bp)、(b)pVAC1-IL2ss-mPD-1(21-170aa)(4177 bp)、(c)pVAC1-IL2ss-mPD-L1(19-127aa)-PLAP(4053 bp)、(d)pVAC1-IL2ss-mPD-L1(19-127aa)(4057 bp)、(e)pVAC1-IL2ss-mCTLA4-mPD-1(21-170aa)-PLAP(4560 bp)、(f)pVAC1-IL2ss-mCTLA4-mPD-1(21-170aa)(4458 bp)、(g)pVAC1-IL2ss-mCTLA4-mPD-L1(19-127aa)-PLAP(4440 bp)、及(h)pVAC1-IL2ss-mCTLA4-mPD-L1(19-127aa)(4338 bp)。
圖13提供部分具體實施例的DNA及胺基酸序列,分別標靶根據本發明之人類蛋白,包括以下載體:(a)pVAC1-IL2ss-hCTLA4-hPD1(31-147aa)-hPDL1(19-127aa)、(b)pVAC1-IL2ss-hCTLA4-hPD1(31-147aa)-hPDL1(19-238aa)、(c)pVAC1-IL2ss-mCTLA4-mPD1(31-147aa)-mPDL1(19-127aa)、及(d)pVAC1-IL2ss-mCTLA4-mPD1(31-147aa)-mPDL1(19-237aa)。
圖14提供經以pVAC-1-IL2ss-mPD-L1-PLAP、pVAC-1-IL2ss-GM-CSF-mPD-L1-PLAP、pVAC-1-IL2ss-mCTLA4-mPD-L1-PLAP、pVAC-1-IL2ss-mPD-L1-mCTLA4-PLAP、及pVAC-1對照組載體免疫小鼠的結果,用以呈現相較於對照組之pVAC-1載體,DNA疫苗所誘發之抗mPD-L1(a)及mCTLA-4(b)兩者之抗體力價。
圖15提供經以pVAC-1-IL2ss-mCTLA4-mPD-L1-PLAP、pVAC-1-IL2ss-mCTLA4-mPD-1-PLAP、pVAC-1-IL2ss-mCTLA4-mPD-L1、pVAC-1-IL2ss-mCTLA4-mPD-1、及pVAC-1
載體對照組免疫小鼠的結果,用以呈現相較於對照組之pVAC-1載體,DNA疫苗所誘發之抗mCTLA-4、mPD-1及mPD-L1之抗體力價。
圖16提供經以pVAC-1-IL2ss-mCTLA4-mPD-L1-PLAP、pVAC-1-IL2ss-mCTLA4-mPD-1-PLAP、pVAC-1-IL2ss-mCTLA4-mPD-L1、及pVAC-1-IL2ss-mCTLA4-mPD-1免疫小鼠的結果,其可抑制RENCA腫瘤之生長。
圖17提供經以pVAC-1-IL2ss-mCTLA4-mPD-L1-PLAP、pVAC-1-IL2ss-mCTLA4-mPD-1-PLAP、pVAC-1-IL2ss-mCTLA4-mPD-L1、及pVAC-1-IL2ss-mCTLA4-mPD-1,並與GM-CSF DNA結合免疫小鼠的結果,其可抑制RENCA腫瘤之生長。
圖18提供經以pVAC-1-IL2ss-mCTLA4-mPD-L1及pVAC-1-IL2ss-mCTLA4-mPD-1免疫小鼠的結果,其可抑制CT26腫瘤之生長。
<110> 藍耿立
<120> 提升感染性與惡性疾病之治療之免疫反應
<130> IV0188/LKL0001TW
<150> US 61/500,825
<151> 2011-06-24
<160> 36
<170> PatentIn version 3.5
<210> 1
<211> 549
<212> DNA
<213> 人工序列
<220>
<223> IL2ss-hCTLA-4-PLAP
<400> 1
<210> 2
<211> 564
<212> DNA
<213> 人工序列
<220>
<223> IL2ss-mCTLA-4-PLAP
<400> 2
<210> 3
<211> 627
<212> DNA
<213> 人工序列
<220>
<223> IL2ss-hPD-1-PLAP
<400> 3
<210> 4
<211> 525
<212> DNA
<213> 人工序列
<220>
<223> IL2ss-hPD-1
<400> 4
<210> 5
<211> 837
<212> DNA
<213> 人工序列
<220>
<223> IL2ss hPD-L1-PLAP
<400> 5
<210> 6
<211> 735
<212> DNA
<213> 人工序列
<220>
<223> IL2ss-hPD-L1
<400> 6
<210> 7
<211> 1005
<212> DNA
<213> 人工序列
<220>
<223> IL2ss-hCTLA4-hPD-1-PLAP
<400> 7
<210> 8
<211> 903
<212> DNA
<213> 人工序列
<220>
<223> IL2ss-hCTLA4-hPD-1
<400> 8
<210> 9
<211> 1212
<212> DNA
<213> 人工序列
<220>
<223> IL2ss-hCTLA4-hPD-L1-PLAP
<400> 9
<210> 10
<211> 1110
<212> DNA
<213> 人工序列
<220>
<223> IL2ss-hCTLA4-hPD-L1
<400> 10
<210> 11
<211> 633
<212> DNA
<213> 人工序列
<220>
<223> IL2ss-mPD-1-PLAP
<400> 11
<210> 12
<211> 531
<212> DNA
<213> 人工序列
<220>
<223> IL2ss-mPD-1
<400> 12
<210> 13
<211> 513
<212> DNA
<213> 人工序列
<220>
<223> IL2ss-mPD-L1-PLAP
<400> 13
<210> 14
<211> 411
<212> DNA
<213> 人工序列
<220>
<223> IL2ss-mPD-L1
<400> 14
<210> 15
<211> 1020
<212> DNA
<213> 人工序列
<220>
<223> IL2ss-mCTLA4-mPD-1-PLAP
<400> 15
<210> 16
<211> 918
<212> DNA
<213> 人工序列
<220>
<223> IL2ss-mCTLA4-mPD-1
<400> 16
<210> 17
<211> 900
<212> DNA
<213> 人工序列
<220>
<223> IL2ss-mCTLA4-mPD-L1-PLAP
<400> 17
<210> 18
<211> 798
<212> DNA
<213> 人工序列
<220>
<223> IL2ss-mCTLA4-mPD-L1
<400> 18
<210> 19
<211> 63
<212> DNA
<213> 智人
<400> 19
<210> 20
<211> 96
<212> DNA
<213> 智人
<400> 20
<210> 21
<211> 372
<212> DNA
<213> 智人
<400> 21
<210> 22
<211> 378
<212> DNA
<213> 家鼷鼠
<400> 22
<210> 23
<211> 450
<212> DNA
<213> 智人
<400> 23
<210> 24
<211> 450
<212> DNA
<213> 家鼷鼠
<400> 24
<210> 25
<211> 660
<212> DNA
<213> 智人
<400> 25
<210> 26
<211> 327
<212> DNA
<213> 家鼷鼠
<400> 26
<210> 27
<211> 828
<212> DNA
<213> 人工序列
<220>
<223> hCTLA4-hPD-1
<400> 27
<210> 28
<211> 1035
<212> DNA
<213> 人工序列
<220>
<223> hCTLA4-hPD-L1
<400> 28
<210> 29
<211> 834
<212> DNA
<213> 人工序列
<220>
<223> mCTLA4-mPD-1
<400> 29
<210> 30
<211> 714
<212> DNA
<213> 人工序列
<220>
<223> mCTLA4-mPD-L1
<400> 30
<210> 31
<211> 288
<212> PRT
<213> 人工序列
<220>
<223> mCILA4-mPD1-His6
<400> 31
<210> 32
<211> 248
<212> PRT
<213> 人工序列
<220>
<223> mCTLA4-mPDL1-His6
<400> 32
<210> 33
<211> 1131
<212> DNA
<213> 人工序列
<220>
<223> pVAC1-IL2ss-hCTLA4-hPD1(31-147aa)-hPDL1(19-127aa)
<400> 33
<210> 34
<211> 1464
<212> DNA
<213> 人工序列
<220>
<223> pVAC1-IL2ss-hCTLA4-hPD1(31-147aa)-hPDL1(19-238aa)
<400> 34
<210> 35
<211> 1143
<212> DNA
<213> 人工序列
<220>
<223> pVAC1-IL2ss-mCTLA4-mPD1(31-147aa)-mPDL1(19-127aa)
<400> 35
<210> 36
<211> 1473
<212> DNA
<213> 人工序列
<220>
<223> pVAC1-IL2ss-mCTLA4-mPD1(31-147aa)-mPDL1(19-237aa)
<400> 36
Claims (11)
- 一種醫藥組合物,可於治療感染性或惡性疾病時,提升受治療個體的免疫反應,該醫藥組合物包含一與表現載體結合的DNA構築體,及醫藥可接受之載劑,其中該構築體包含一多核苷酸序列,該多核苷酸編碼包含(i)細胞毒T淋巴細胞抗原4(CTLA-4)或其片段、及(ii)選自由計畫性死亡-1(PD-1)或其片段、計畫性細胞死亡配體1(PD-L1)或其片段、及其組合所組成之群組中任一者。
- 如申請專利範圍第1項之醫藥組合物,其中該DNA構築體包含編碼(i)CTLA-4或其片段,及(ii)PD-1或其片段之結合的多核苷酸序列。
- 如申請專利範圍第1項之醫藥組合物,其中該DNA構築體包含編碼(i)CTLA-4或其片段,及(ii)PD-L1或其片段之結合的多核苷酸序列。
- 如申請專利範圍第1項之醫藥組合物,其中該表現載體為pVAC-1。
- 如申請專利範圍第1項之醫藥組合物,其進一步包含佐劑。
- 如申請專利範圍第1項之醫藥組合物,其中該DNA構築體係與微脂體結合。
- 如申請專利範圍第1項之醫藥組合物,其中該個體係罹患感染性或惡性疾病,且以抗感染性或抗癌藥物治療,造成該個體之免疫反應受到刺激。
- 如申請專利範圍第1項之醫藥組合物,其中該惡性疾病係選自由轉移性黑色素瘤、胰臟癌、大腸直腸癌、肝細胞癌、淋巴癌、激素抗拒性***癌、卵巢癌、急性骨髓性白血病、及非小細胞肺癌所組成之群組。
- 一種如申請專利範圍第1項之醫藥組合物用於製備治療感染性或惡性疾病的藥物之用途,其中該醫藥組合物係以局部、皮下或肌肉之方式投予至個體。
- 如申請專利範圍第9項之用途,其中該個體係罹患感染性或惡性疾病,且以抗感染性或抗癌藥物治療,造成該個體之免疫反應受到刺激。
- 如申請專利範圍第9項之用途,其中該惡性疾病係選自由轉移性黑色素瘤、胰臟癌、大腸直腸癌、肝細胞癌、淋巴癌、激素抗拒性***癌、卵巢癌、急性骨髓性白血病、及非小細胞肺癌所組成之群組。
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US201161500825P | 2011-06-24 | 2011-06-24 |
Publications (2)
Publication Number | Publication Date |
---|---|
TW201300529A TW201300529A (zh) | 2013-01-01 |
TWI496886B true TWI496886B (zh) | 2015-08-21 |
Family
ID=47362060
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
TW101122686A TWI496886B (zh) | 2011-06-24 | 2012-06-25 | 提升感染性與惡性疾病之治療之免疫反應 |
Country Status (3)
Country | Link |
---|---|
US (3) | US8609625B2 (zh) |
CN (2) | CN107519486B (zh) |
TW (1) | TWI496886B (zh) |
Families Citing this family (24)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US8609625B2 (en) * | 2011-06-24 | 2013-12-17 | Taipei Veterans General Hospital | Method for enhancing immune response in the treatment of infectious and malignant diseases |
US9856320B2 (en) | 2012-05-15 | 2018-01-02 | Bristol-Myers Squibb Company | Cancer immunotherapy by disrupting PD-1/PD-L1 signaling |
MA37681B2 (fr) * | 2012-05-25 | 2020-07-29 | Cellectis | Procédés pour modifier des lymphocytes t résistants allogéniques et immunosuppresseurs pour l'immunothérapie |
EP2992017B1 (en) | 2013-05-02 | 2020-11-18 | AnaptysBio, Inc. | Antibodies directed against programmed death-1 (pd-1) |
BR112016026197A2 (pt) | 2014-05-13 | 2018-02-20 | Medimmune Limited | anticorpos anti-b7-h1 e anti-ctla-4 para o tratamento de câncer de pulmão de não pequenas células |
RU2722212C9 (ru) * | 2014-08-05 | 2020-07-23 | СиБи ТЕРЕПЬЮТИКС, ИНК. | Анти-pd-l1 антитела |
WO2016030455A1 (en) * | 2014-08-28 | 2016-03-03 | Medimmune Limited | Anti-b7-h1 and anti-ctla-4 antibodies for treating non-small lung cancer |
KR20240046641A (ko) | 2015-04-17 | 2024-04-09 | 알파인 이뮨 사이언시즈, 인코포레이티드 | 조율가능한 친화성을 갖는 면역조절 단백질 |
US20180256715A1 (en) * | 2015-05-13 | 2018-09-13 | The Board Of Regents Of The University Of Texas System | Anti-ctla-4 blockade |
WO2017076360A1 (en) | 2015-11-04 | 2017-05-11 | Taipei Veterans General Hospital | Title of the invention combination therapy for malignant diseases |
CN105331585A (zh) * | 2015-11-13 | 2016-02-17 | 科济生物医药(上海)有限公司 | 携带pd-l1阻断剂的嵌合抗原受体修饰的免疫效应细胞 |
MA43552A (fr) | 2016-04-15 | 2018-11-07 | Alpine Immune Sciences Inc | Protéines immunomodulatrices à variants de cd80 et leurs utilisations |
US11471488B2 (en) | 2016-07-28 | 2022-10-18 | Alpine Immune Sciences, Inc. | CD155 variant immunomodulatory proteins and uses thereof |
WO2018022945A1 (en) | 2016-07-28 | 2018-02-01 | Alpine Immune Sciences, Inc. | Cd112 variant immunomodulatory proteins and uses thereof |
AU2017354070A1 (en) | 2016-11-01 | 2019-05-16 | Anaptysbio, Inc. | Antibodies directed against programmed death- 1 (PD-1) |
BR112019014187A2 (pt) | 2017-01-09 | 2020-02-11 | Tesaro, Inc. | Métodos de tratamento de câncer com anticorpos anti-pd-1 |
NZ756395A (en) | 2017-03-16 | 2024-01-26 | Alpine Immune Sciences Inc | Cd80 variant immunomodulatory proteins and uses thereof |
JP2020511144A (ja) | 2017-03-16 | 2020-04-16 | アルパイン イミューン サイエンシズ インコーポレイテッド | Pd−l2バリアント免疫調節タンパク質及びその使用 |
MA50360A (fr) | 2017-10-10 | 2020-08-19 | Alpine Immune Sciences Inc | Protéines immunomodulatrices de variants de ctla-4 et leurs utilisations |
KR20200067195A (ko) * | 2017-10-18 | 2020-06-11 | 리젠엑스바이오 인크. | 완전-인간 번역후 변형된 항체 치료제 |
KR20200085777A (ko) | 2017-10-18 | 2020-07-15 | 알파인 이뮨 사이언시즈, 인코포레이티드 | 변이체 icos 리간드 면역조절 단백질 및 관련 조성물 및 방법 |
US20190161547A1 (en) * | 2017-11-29 | 2019-05-30 | Wyvern Pharmaceutical Inc. | Methods for regulating endogenous production of checkpoint molecule antagonists |
WO2019106592A2 (en) | 2017-11-30 | 2019-06-06 | Grifols Diagnostic Solutions Inc. | Immunoassays and engineered proteins for monitoring antibody treatments to the immune checkpoint inhibitors pd1 and pd-l1 |
EP4244247A1 (en) * | 2020-11-13 | 2023-09-20 | Sab, Llc | Ungulate-derived polyclonal immunoglobulin specific for pd-l1 and uses thereof |
Family Cites Families (9)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5397703A (en) * | 1992-07-09 | 1995-03-14 | Cetus Oncology Corporation | Method for generation of antibodies to cell surface molecules |
DE19939653A1 (de) * | 1999-08-13 | 2001-02-22 | Thomas Huenig | Verwendung CD28 spezifischer monoklonaler Antikörper zur Herstellung einer pharmazeutischen Zusammensetzung |
PT1210428E (pt) * | 1999-08-23 | 2015-07-21 | Genetics Inst Llc | Pd-1, um recetor para b7-4 e suas utilizações |
US7829084B2 (en) * | 2001-01-17 | 2010-11-09 | Trubion Pharmaceuticals, Inc. | Binding constructs and methods for use thereof |
DE10212108A1 (de) * | 2002-03-13 | 2003-10-02 | Tegenero Ag | Verwendung einer an CD28 bindenden Wirksubstanz zur Herstellung einer pharmazeutischen Zusammensetzung |
US7494779B2 (en) * | 2004-06-14 | 2009-02-24 | Li-Te Chin | Method for producing human antibodies to human CD152 with properties of agonist, antagonist, or inverse agonist |
PE20120341A1 (es) * | 2008-12-09 | 2012-04-24 | Genentech Inc | Anticuerpos anti-pd-l1 y su uso para mejorar la funcion de celulas t |
CN102030828B (zh) * | 2009-09-25 | 2014-10-29 | 上海抗体药物国家工程研究中心有限公司 | 一种高亲和力的CTLA4-Ig融合蛋白突变体 |
US8609625B2 (en) * | 2011-06-24 | 2013-12-17 | Taipei Veterans General Hospital | Method for enhancing immune response in the treatment of infectious and malignant diseases |
-
2012
- 2012-06-25 US US13/532,460 patent/US8609625B2/en active Active
- 2012-06-25 CN CN201710789419.3A patent/CN107519486B/zh active Active
- 2012-06-25 TW TW101122686A patent/TWI496886B/zh active
- 2012-06-25 CN CN201210210339.5A patent/CN102836441B/zh active Active
-
2013
- 2013-11-13 US US14/079,088 patent/US9289480B2/en active Active
-
2016
- 2016-02-16 US US15/044,613 patent/US10071147B2/en active Active
Non-Patent Citations (1)
Title |
---|
Curran MA. et al. "PD-1 and CTLA-4 combination blockade expands infiltrating T cells and reduces regulatory T and myeloid cells within B16 melanoma tumors." Proc Natl Acad Sci U S A. 2010, 107(9):4275-80 * |
Also Published As
Publication number | Publication date |
---|---|
CN107519486A (zh) | 2017-12-29 |
US20140105934A1 (en) | 2014-04-17 |
US20170028040A1 (en) | 2017-02-02 |
US9289480B2 (en) | 2016-03-22 |
US20120328693A1 (en) | 2012-12-27 |
US10071147B2 (en) | 2018-09-11 |
CN102836441B (zh) | 2019-06-11 |
TW201300529A (zh) | 2013-01-01 |
CN102836441A (zh) | 2012-12-26 |
CN107519486B (zh) | 2021-06-11 |
US8609625B2 (en) | 2013-12-17 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
TWI496886B (zh) | 提升感染性與惡性疾病之治療之免疫反應 | |
EP3351261B1 (en) | Combination medicament comprising il-12 and an agent for blockade of t-cell inhibitory molecules for tumour therapy | |
JP6113883B2 (ja) | 受容体に対する親和性が増大した変異型light分子を使用する癌療法のための方法および組成物 | |
US20030022860A1 (en) | CD40 binding molecules and CTL peptides for treating tumors | |
WO2004093831A2 (en) | Cytokine-expressing cellular vaccine combinations | |
TW200400970A (en) | Immunogenic compositions | |
JP2021531819A (ja) | 低adcc/cdc機能性モノクローナル抗体、及びその調製方法と使用 | |
CA3106980A1 (en) | Major histocompatibility complex class ll-expressing cancer cell vaccine and methods of use for producing integrated immune responses | |
CA2588573C (en) | Immunotherapeutic formulations to generate autoantibodies capable to avoid the binding of interleukin-2 to its receptor their use in the treatment of cancer | |
Danishmalik et al. | Tumor regression is mediated via the induction of HER263-71-specific CD8+ CTL activity in a 4T1. 2/HER2 tumor model: no involvement of CD80 in tumor control | |
CA3151223A1 (en) | Human anti-pd-l1 peptide vaccines and methods of their use | |
TWI705972B (zh) | 惡性病變的組合治療 | |
WO2023232022A1 (zh) | 靶向pd-l1和vegf的重组融合蛋白及其制备和用途 | |
US20230390369A1 (en) | Chimeric antigen comprising the extracellular domain of pd-l1 | |
CN110167584A (zh) | 用于针对莱姆病的dna抗体构建体 | |
KR20230164118A (ko) | 암 치료를 위한 치료 조합 |