CN111606999A - 兼具激活免疫共刺激信号通路和阻断免疫检查点的复制型溶瘤腺病毒及其应用 - Google Patents
兼具激活免疫共刺激信号通路和阻断免疫检查点的复制型溶瘤腺病毒及其应用 Download PDFInfo
- Publication number
- CN111606999A CN111606999A CN201910140526.2A CN201910140526A CN111606999A CN 111606999 A CN111606999 A CN 111606999A CN 201910140526 A CN201910140526 A CN 201910140526A CN 111606999 A CN111606999 A CN 111606999A
- Authority
- CN
- China
- Prior art keywords
- immune
- tumor
- cells
- oncolytic adenovirus
- fusion protein
- 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.)
- Granted
Links
- 108091008036 Immune checkpoint proteins Proteins 0.000 title claims abstract description 69
- 102000037982 Immune checkpoint proteins Human genes 0.000 title claims abstract description 69
- 241000701161 unidentified adenovirus Species 0.000 title claims abstract description 69
- 230000000174 oncolytic effect Effects 0.000 title claims abstract description 67
- 230000003213 activating effect Effects 0.000 title claims abstract description 44
- 230000000903 blocking effect Effects 0.000 title claims abstract description 44
- 230000006870 function Effects 0.000 title claims abstract description 27
- 230000019491 signal transduction Effects 0.000 title claims description 42
- 230000003362 replicative effect Effects 0.000 title claims description 27
- 206010028980 Neoplasm Diseases 0.000 claims abstract description 82
- 241000700605 Viruses Species 0.000 claims abstract description 58
- 108020001507 fusion proteins Proteins 0.000 claims abstract description 41
- 102000037865 fusion proteins Human genes 0.000 claims abstract description 41
- 230000000259 anti-tumor effect Effects 0.000 claims abstract description 36
- 210000004881 tumor cell Anatomy 0.000 claims abstract description 36
- 238000010276 construction Methods 0.000 claims abstract description 17
- 239000003814 drug Substances 0.000 claims abstract description 16
- 210000004027 cell Anatomy 0.000 claims description 98
- 108090000623 proteins and genes Proteins 0.000 claims description 28
- 206010003445 Ascites Diseases 0.000 claims description 26
- 102000004169 proteins and genes Human genes 0.000 claims description 21
- 239000013612 plasmid Substances 0.000 claims description 19
- 238000002360 preparation method Methods 0.000 claims description 17
- 238000000034 method Methods 0.000 claims description 15
- 101000638251 Homo sapiens Tumor necrosis factor ligand superfamily member 9 Proteins 0.000 claims description 14
- 102100032101 Tumor necrosis factor ligand superfamily member 9 Human genes 0.000 claims description 14
- 201000007270 liver cancer Diseases 0.000 claims description 14
- 208000014018 liver neoplasm Diseases 0.000 claims description 14
- 239000006228 supernatant Substances 0.000 claims description 12
- 201000001441 melanoma Diseases 0.000 claims description 10
- 206010006187 Breast cancer Diseases 0.000 claims description 9
- 208000026310 Breast neoplasm Diseases 0.000 claims description 9
- 239000007788 liquid Substances 0.000 claims description 9
- 230000004913 activation Effects 0.000 claims description 8
- 238000012258 culturing Methods 0.000 claims description 6
- 238000012216 screening Methods 0.000 claims description 6
- 230000003321 amplification Effects 0.000 claims description 5
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 5
- 239000013605 shuttle vector Substances 0.000 claims description 5
- 230000004936 stimulating effect Effects 0.000 claims description 5
- 201000009030 Carcinoma Diseases 0.000 claims description 4
- 238000000605 extraction Methods 0.000 claims description 4
- 239000001963 growth medium Substances 0.000 claims description 4
- 238000010257 thawing Methods 0.000 claims description 4
- 238000011053 TCID50 method Methods 0.000 claims description 3
- AIYUHDOJVYHVIT-UHFFFAOYSA-M caesium chloride Chemical compound [Cl-].[Cs+] AIYUHDOJVYHVIT-UHFFFAOYSA-M 0.000 claims description 3
- 238000000432 density-gradient centrifugation Methods 0.000 claims description 3
- 229930027917 kanamycin Natural products 0.000 claims description 3
- 229960000318 kanamycin Drugs 0.000 claims description 3
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 claims description 3
- 229930182823 kanamycin A Natural products 0.000 claims description 3
- 231100000915 pathological change Toxicity 0.000 claims description 3
- 230000036285 pathological change Effects 0.000 claims description 3
- 238000005119 centrifugation Methods 0.000 claims description 2
- 102000008096 B7-H1 Antigen Human genes 0.000 claims 2
- 108010074708 B7-H1 Antigen Proteins 0.000 claims 2
- 125000003275 alpha amino acid group Chemical group 0.000 claims 2
- 230000010076 replication Effects 0.000 abstract description 19
- 230000000694 effects Effects 0.000 abstract description 14
- 239000002246 antineoplastic agent Substances 0.000 abstract description 6
- 229940041181 antineoplastic drug Drugs 0.000 abstract description 6
- 238000013461 design Methods 0.000 abstract description 6
- 238000002474 experimental method Methods 0.000 abstract description 6
- 230000005975 antitumor immune response Effects 0.000 abstract description 5
- 238000009169 immunotherapy Methods 0.000 abstract description 4
- 230000003248 secreting effect Effects 0.000 abstract description 3
- 230000009977 dual effect Effects 0.000 abstract 1
- 210000001723 extracellular space Anatomy 0.000 abstract 1
- 241000699666 Mus <mouse, genus> Species 0.000 description 40
- 238000010790 dilution Methods 0.000 description 15
- 239000012895 dilution Substances 0.000 description 15
- 206010073071 hepatocellular carcinoma Diseases 0.000 description 15
- 230000005934 immune activation Effects 0.000 description 15
- 241000699670 Mus sp. Species 0.000 description 13
- 239000012528 membrane Substances 0.000 description 12
- 230000004083 survival effect Effects 0.000 description 11
- 241000699660 Mus musculus Species 0.000 description 10
- 210000001744 T-lymphocyte Anatomy 0.000 description 10
- 210000002865 immune cell Anatomy 0.000 description 10
- 238000007920 subcutaneous administration Methods 0.000 description 10
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 9
- 239000011780 sodium chloride Substances 0.000 description 9
- 210000001519 tissue Anatomy 0.000 description 9
- 238000011740 C57BL/6 mouse Methods 0.000 description 8
- 102100037850 Interferon gamma Human genes 0.000 description 8
- 108010074328 Interferon-gamma Proteins 0.000 description 8
- 239000007924 injection Substances 0.000 description 8
- 238000002347 injection Methods 0.000 description 8
- 239000000243 solution Substances 0.000 description 8
- 239000002033 PVDF binder Substances 0.000 description 7
- 230000005809 anti-tumor immunity Effects 0.000 description 7
- 210000004698 lymphocyte Anatomy 0.000 description 7
- 229920002981 polyvinylidene fluoride Polymers 0.000 description 7
- 238000005406 washing Methods 0.000 description 7
- 108020004414 DNA Proteins 0.000 description 6
- 108010043121 Green Fluorescent Proteins Proteins 0.000 description 6
- VPZXBVLAVMBEQI-UHFFFAOYSA-N glycyl-DL-alpha-alanine Natural products OC(=O)C(C)NC(=O)CN VPZXBVLAVMBEQI-UHFFFAOYSA-N 0.000 description 6
- 238000002156 mixing Methods 0.000 description 6
- 239000013642 negative control Substances 0.000 description 6
- 210000000952 spleen Anatomy 0.000 description 6
- 230000004614 tumor growth Effects 0.000 description 6
- 102000004144 Green Fluorescent Proteins Human genes 0.000 description 5
- 229910002092 carbon dioxide Inorganic materials 0.000 description 5
- 239000003153 chemical reaction reagent Substances 0.000 description 5
- 238000001514 detection method Methods 0.000 description 5
- 239000012634 fragment Substances 0.000 description 5
- 239000005090 green fluorescent protein Substances 0.000 description 5
- 230000005917 in vivo anti-tumor Effects 0.000 description 5
- 238000011081 inoculation Methods 0.000 description 5
- 244000309459 oncolytic virus Species 0.000 description 5
- 238000001262 western blot Methods 0.000 description 5
- UXHYOWXTJLBEPG-GSSVUCPTSA-N Asn-Thr-Thr Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O UXHYOWXTJLBEPG-GSSVUCPTSA-N 0.000 description 4
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 4
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 4
- SBANPBVRHYIMRR-UHFFFAOYSA-N Leu-Ser-Pro Natural products CC(C)CC(N)C(=O)NC(CO)C(=O)N1CCCC1C(O)=O SBANPBVRHYIMRR-UHFFFAOYSA-N 0.000 description 4
- AZWNCEBQZXELEZ-FXQIFTODSA-N Ser-Pro-Ser Chemical compound OC[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CO)C(O)=O AZWNCEBQZXELEZ-FXQIFTODSA-N 0.000 description 4
- 230000008901 benefit Effects 0.000 description 4
- 238000004113 cell culture Methods 0.000 description 4
- 238000001962 electrophoresis Methods 0.000 description 4
- 238000011156 evaluation Methods 0.000 description 4
- 210000000987 immune system Anatomy 0.000 description 4
- 238000007912 intraperitoneal administration Methods 0.000 description 4
- 230000007246 mechanism Effects 0.000 description 4
- 238000012544 monitoring process Methods 0.000 description 4
- 230000037361 pathway Effects 0.000 description 4
- 108010053725 prolylvaline Proteins 0.000 description 4
- 230000001105 regulatory effect Effects 0.000 description 4
- 108010026333 seryl-proline Proteins 0.000 description 4
- 108010020532 tyrosyl-proline Proteins 0.000 description 4
- HKZAAJSTFUZYTO-LURJTMIESA-N (2s)-2-[[2-[[2-[[2-[(2-aminoacetyl)amino]acetyl]amino]acetyl]amino]acetyl]amino]-3-hydroxypropanoic acid Chemical compound NCC(=O)NCC(=O)NCC(=O)NCC(=O)N[C@@H](CO)C(O)=O HKZAAJSTFUZYTO-LURJTMIESA-N 0.000 description 3
- MEFILNJXAVSUTO-JXUBOQSCSA-N Ala-Leu-Thr Chemical compound C[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)O)C(O)=O MEFILNJXAVSUTO-JXUBOQSCSA-N 0.000 description 3
- 238000002965 ELISA Methods 0.000 description 3
- USTCFDAQCLDPBD-XIRDDKMYSA-N Leu-Asn-Trp Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)N[C@@H](CC1=CNC2=CC=CC=C21)C(=O)O)N USTCFDAQCLDPBD-XIRDDKMYSA-N 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 101001044384 Mus musculus Interferon gamma Proteins 0.000 description 3
- 108010076504 Protein Sorting Signals Proteins 0.000 description 3
- 238000011529 RT qPCR Methods 0.000 description 3
- GLNADSQYFUSGOU-GPTZEZBUSA-J Trypan blue Chemical compound [Na+].[Na+].[Na+].[Na+].C1=C(S([O-])(=O)=O)C=C2C=C(S([O-])(=O)=O)C(/N=N/C3=CC=C(C=C3C)C=3C=C(C(=CC=3)\N=N\C=3C(=CC4=CC(=CC(N)=C4C=3O)S([O-])(=O)=O)S([O-])(=O)=O)C)=C(O)C2=C1N GLNADSQYFUSGOU-GPTZEZBUSA-J 0.000 description 3
- 210000000683 abdominal cavity Anatomy 0.000 description 3
- 230000008827 biological function Effects 0.000 description 3
- 238000004364 calculation method Methods 0.000 description 3
- 201000011510 cancer Diseases 0.000 description 3
- 238000011161 development Methods 0.000 description 3
- 239000003085 diluting agent Substances 0.000 description 3
- 230000003828 downregulation Effects 0.000 description 3
- 229940079593 drug Drugs 0.000 description 3
- 239000012636 effector Substances 0.000 description 3
- 239000012091 fetal bovine serum Substances 0.000 description 3
- 108010078144 glutaminyl-glycine Proteins 0.000 description 3
- XKUKSGPZAADMRA-UHFFFAOYSA-N glycyl-glycyl-glycine Natural products NCC(=O)NCC(=O)NCC(O)=O XKUKSGPZAADMRA-UHFFFAOYSA-N 0.000 description 3
- 230000036039 immunity Effects 0.000 description 3
- 238000001727 in vivo Methods 0.000 description 3
- 230000002401 inhibitory effect Effects 0.000 description 3
- 230000002601 intratumoral effect Effects 0.000 description 3
- 230000001404 mediated effect Effects 0.000 description 3
- 239000002609 medium Substances 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 210000000822 natural killer cell Anatomy 0.000 description 3
- 210000002966 serum Anatomy 0.000 description 3
- 238000002791 soaking Methods 0.000 description 3
- 238000002560 therapeutic procedure Methods 0.000 description 3
- 238000012546 transfer Methods 0.000 description 3
- 208000012260 Accidental injury Diseases 0.000 description 2
- MCKSLROAGSDNFC-ACZMJKKPSA-N Ala-Asp-Gln Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CCC(N)=O)C(O)=O MCKSLROAGSDNFC-ACZMJKKPSA-N 0.000 description 2
- NKJBKNVQHBZUIX-ACZMJKKPSA-N Ala-Gln-Asp Chemical compound C[C@H](N)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(O)=O)C(O)=O NKJBKNVQHBZUIX-ACZMJKKPSA-N 0.000 description 2
- VNYMOTCMNHJGTG-JBDRJPRFSA-N Ala-Ile-Ser Chemical compound [H]N[C@@H](C)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CO)C(O)=O VNYMOTCMNHJGTG-JBDRJPRFSA-N 0.000 description 2
- XHNLCGXYBXNRIS-BJDJZHNGSA-N Ala-Lys-Ile Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O XHNLCGXYBXNRIS-BJDJZHNGSA-N 0.000 description 2
- BDQNLQSWRAPHGU-DLOVCJGASA-N Ala-Phe-Cys Chemical compound C[C@@H](C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CS)C(=O)O)N BDQNLQSWRAPHGU-DLOVCJGASA-N 0.000 description 2
- MTDDMSUUXNQMKK-BPNCWPANSA-N Ala-Tyr-Arg Chemical compound C[C@@H](C(=O)N[C@@H](CC1=CC=C(C=C1)O)C(=O)N[C@@H](CCCN=C(N)N)C(=O)O)N MTDDMSUUXNQMKK-BPNCWPANSA-N 0.000 description 2
- LMPKCSXZJSXBBL-NHCYSSNCSA-N Arg-Gln-Val Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](C(C)C)C(O)=O LMPKCSXZJSXBBL-NHCYSSNCSA-N 0.000 description 2
- BMNVSPMWMICFRV-DCAQKATOSA-N Arg-His-Asp Chemical compound NC(N)=NCCC[C@H](N)C(=O)N[C@H](C(=O)N[C@@H](CC(O)=O)C(O)=O)CC1=CN=CN1 BMNVSPMWMICFRV-DCAQKATOSA-N 0.000 description 2
- VEAIMHJZTIDCIH-KKUMJFAQSA-N Arg-Phe-Gln Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CCC(N)=O)C(O)=O VEAIMHJZTIDCIH-KKUMJFAQSA-N 0.000 description 2
- KMFPQTITXUKJOV-DCAQKATOSA-N Arg-Ser-Leu Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(O)=O KMFPQTITXUKJOV-DCAQKATOSA-N 0.000 description 2
- QCTOLCVIGRLMQS-HRCADAONSA-N Arg-Tyr-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CC2=CC=C(C=C2)O)NC(=O)[C@H](CCCN=C(N)N)N)C(=O)O QCTOLCVIGRLMQS-HRCADAONSA-N 0.000 description 2
- IARGXWMWRFOQPG-GCJQMDKQSA-N Asn-Ala-Thr Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](C)C(=O)N[C@@H]([C@@H](C)O)C(O)=O IARGXWMWRFOQPG-GCJQMDKQSA-N 0.000 description 2
- MFFOYNGMOYFPBD-DCAQKATOSA-N Asn-Arg-Leu Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(O)=O MFFOYNGMOYFPBD-DCAQKATOSA-N 0.000 description 2
- VYLVOMUVLMGCRF-ZLUOBGJFSA-N Asn-Asp-Ser Chemical compound NC(=O)C[C@H](N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CO)C(O)=O VYLVOMUVLMGCRF-ZLUOBGJFSA-N 0.000 description 2
- FAEFJTCTNZTPHX-ACZMJKKPSA-N Asn-Gln-Ala Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](C)C(O)=O FAEFJTCTNZTPHX-ACZMJKKPSA-N 0.000 description 2
- OKZOABJQOMAYEC-NUMRIWBASA-N Asn-Gln-Thr Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O OKZOABJQOMAYEC-NUMRIWBASA-N 0.000 description 2
- HYQYLOSCICEYTR-YUMQZZPRSA-N Asn-Gly-Leu Chemical compound [H]N[C@@H](CC(N)=O)C(=O)NCC(=O)N[C@@H](CC(C)C)C(O)=O HYQYLOSCICEYTR-YUMQZZPRSA-N 0.000 description 2
- GJFYPBDMUGGLFR-NKWVEPMBSA-N Asn-Gly-Pro Chemical compound C1C[C@@H](N(C1)C(=O)CNC(=O)[C@H](CC(=O)N)N)C(=O)O GJFYPBDMUGGLFR-NKWVEPMBSA-N 0.000 description 2
- GQRDIVQPSMPQME-ZPFDUUQYSA-N Asn-Ile-Leu Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC(C)C)C(O)=O GQRDIVQPSMPQME-ZPFDUUQYSA-N 0.000 description 2
- MLJZMGIXXMTEPO-UBHSHLNASA-N Asn-Trp-Ser Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CC1=CNC2=C1C=CC=C2)C(=O)N[C@@H](CO)C(O)=O MLJZMGIXXMTEPO-UBHSHLNASA-N 0.000 description 2
- UGIBTKGQVWFTGX-BIIVOSGPSA-N Asp-Asn-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CC(=O)N)NC(=O)[C@H](CC(=O)O)N)C(=O)O UGIBTKGQVWFTGX-BIIVOSGPSA-N 0.000 description 2
- MRYDJCIIVRXVGG-QEJZJMRPSA-N Asp-Trp-Glu Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CC1=CNC2=C1C=CC=C2)C(=O)N[C@@H](CCC(O)=O)C(O)=O MRYDJCIIVRXVGG-QEJZJMRPSA-N 0.000 description 2
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 2
- 210000001266 CD8-positive T-lymphocyte Anatomy 0.000 description 2
- 102000029816 Collagenase Human genes 0.000 description 2
- 108060005980 Collagenase Proteins 0.000 description 2
- 108010049207 Death Domain Receptors Proteins 0.000 description 2
- 102000009058 Death Domain Receptors Human genes 0.000 description 2
- 238000011510 Elispot assay Methods 0.000 description 2
- NVEASDQHBRZPSU-BQBZGAKWSA-N Gln-Gln-Gly Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCC(N)=O)C(=O)NCC(O)=O NVEASDQHBRZPSU-BQBZGAKWSA-N 0.000 description 2
- QQAPDATZKKTBIY-YUMQZZPRSA-N Gln-Gly-Met Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)NCC(=O)N[C@@H](CCSC)C(O)=O QQAPDATZKKTBIY-YUMQZZPRSA-N 0.000 description 2
- OSCLNNWLKKIQJM-WDSKDSINSA-N Gln-Ser-Gly Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](CO)C(=O)NCC(O)=O OSCLNNWLKKIQJM-WDSKDSINSA-N 0.000 description 2
- JVSBYEDSSRZQGV-GUBZILKMSA-N Glu-Asp-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H](CC(O)=O)NC(=O)[C@@H](N)CCC(O)=O JVSBYEDSSRZQGV-GUBZILKMSA-N 0.000 description 2
- IQACOVZVOMVILH-FXQIFTODSA-N Glu-Glu-Ser Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CO)C(O)=O IQACOVZVOMVILH-FXQIFTODSA-N 0.000 description 2
- GJBUAAAIZSRCDC-GVXVVHGQSA-N Glu-Leu-Val Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C(C)C)C(O)=O GJBUAAAIZSRCDC-GVXVVHGQSA-N 0.000 description 2
- UJMNFCAHLYKWOZ-DCAQKATOSA-N Glu-Lys-Gln Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCC(N)=O)C(O)=O UJMNFCAHLYKWOZ-DCAQKATOSA-N 0.000 description 2
- RMWAOBGCZZSJHE-UMNHJUIQSA-N Glu-Val-Pro Chemical compound CC(C)[C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@H](CCC(=O)O)N RMWAOBGCZZSJHE-UMNHJUIQSA-N 0.000 description 2
- KRRMJKMGWWXWDW-STQMWFEESA-N Gly-Arg-Phe Chemical compound NC(=N)NCCC[C@H](NC(=O)CN)C(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 KRRMJKMGWWXWDW-STQMWFEESA-N 0.000 description 2
- XVYKMNXXJXQKME-XEGUGMAKSA-N Gly-Ile-Tyr Chemical compound NCC(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 XVYKMNXXJXQKME-XEGUGMAKSA-N 0.000 description 2
- PGXZHYYGOPKYKM-IHRRRGAJSA-N His-Pro-Lys Chemical compound C1C[C@H](N(C1)C(=O)[C@H](CC2=CN=CN2)N)C(=O)N[C@@H](CCCCN)C(=O)O PGXZHYYGOPKYKM-IHRRRGAJSA-N 0.000 description 2
- RIVKTKFVWXRNSJ-GRLWGSQLSA-N Ile-Ile-Gln Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CCC(=O)N)C(=O)O)N RIVKTKFVWXRNSJ-GRLWGSQLSA-N 0.000 description 2
- HUORUFRRJHELPD-MNXVOIDGSA-N Ile-Leu-Glu Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(=O)O)C(=O)O)N HUORUFRRJHELPD-MNXVOIDGSA-N 0.000 description 2
- 108010014726 Interferon Type I Proteins 0.000 description 2
- 102000002227 Interferon Type I Human genes 0.000 description 2
- KWTVLKBOQATPHJ-SRVKXCTJSA-N Leu-Ala-Lys Chemical compound C[C@@H](C(=O)N[C@@H](CCCCN)C(=O)O)NC(=O)[C@H](CC(C)C)N KWTVLKBOQATPHJ-SRVKXCTJSA-N 0.000 description 2
- IASQBRJGRVXNJI-YUMQZZPRSA-N Leu-Cys-Gly Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CS)C(=O)NCC(O)=O IASQBRJGRVXNJI-YUMQZZPRSA-N 0.000 description 2
- VQPPIMUZCZCOIL-GUBZILKMSA-N Leu-Gln-Ala Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](C)C(O)=O VQPPIMUZCZCOIL-GUBZILKMSA-N 0.000 description 2
- CFZZDVMBRYFFNU-QWRGUYRKSA-N Leu-His-Gly Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CC1=CNC=N1)C(=O)NCC(O)=O CFZZDVMBRYFFNU-QWRGUYRKSA-N 0.000 description 2
- DRWMRVFCKKXHCH-BZSNNMDCSA-N Leu-Phe-Leu Chemical compound CC(C)C[C@H]([NH3+])C(=O)N[C@H](C(=O)N[C@@H](CC(C)C)C([O-])=O)CC1=CC=CC=C1 DRWMRVFCKKXHCH-BZSNNMDCSA-N 0.000 description 2
- RRVCZCNFXIFGRA-DCAQKATOSA-N Leu-Pro-Asn Chemical compound [H]N[C@@H](CC(C)C)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CC(N)=O)C(O)=O RRVCZCNFXIFGRA-DCAQKATOSA-N 0.000 description 2
- AKVBOOKXVAMKSS-GUBZILKMSA-N Leu-Ser-Gln Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCC(N)=O)C(O)=O AKVBOOKXVAMKSS-GUBZILKMSA-N 0.000 description 2
- SBANPBVRHYIMRR-GARJFASQSA-N Leu-Ser-Pro Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CO)C(=O)N1CCC[C@@H]1C(=O)O)N SBANPBVRHYIMRR-GARJFASQSA-N 0.000 description 2
- HWMQRQIFVGEAPH-XIRDDKMYSA-N Leu-Ser-Trp Chemical compound C1=CC=C2C(C[C@H](NC(=O)[C@H](CO)NC(=O)[C@@H](N)CC(C)C)C(O)=O)=CNC2=C1 HWMQRQIFVGEAPH-XIRDDKMYSA-N 0.000 description 2
- SQUFDMCWMFOEBA-KKUMJFAQSA-N Leu-Ser-Tyr Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 SQUFDMCWMFOEBA-KKUMJFAQSA-N 0.000 description 2
- AIQWYVFNBNNOLU-RHYQMDGZSA-N Leu-Thr-Val Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C(C)C)C(O)=O AIQWYVFNBNNOLU-RHYQMDGZSA-N 0.000 description 2
- CNGOEHJCLVCJHN-SRVKXCTJSA-N Lys-Pro-Glu Chemical compound NCCCC[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCC(O)=O)C(O)=O CNGOEHJCLVCJHN-SRVKXCTJSA-N 0.000 description 2
- UXJHNUBJSQQIOC-SZMVWBNQSA-N Met-Trp-Val Chemical compound [H]N[C@@H](CCSC)C(=O)N[C@@H](CC1=CNC2=C1C=CC=C2)C(=O)N[C@@H](C(C)C)C(O)=O UXJHNUBJSQQIOC-SZMVWBNQSA-N 0.000 description 2
- 241001529936 Murinae Species 0.000 description 2
- YBAFDPFAUTYYRW-UHFFFAOYSA-N N-L-alpha-glutamyl-L-leucine Natural products CC(C)CC(C(O)=O)NC(=O)C(N)CCC(O)=O YBAFDPFAUTYYRW-UHFFFAOYSA-N 0.000 description 2
- AJHCSUXXECOXOY-UHFFFAOYSA-N N-glycyl-L-tryptophan Natural products C1=CC=C2C(CC(NC(=O)CN)C(O)=O)=CNC2=C1 AJHCSUXXECOXOY-UHFFFAOYSA-N 0.000 description 2
- 108010079364 N-glycylalanine Proteins 0.000 description 2
- 108010019160 Pancreatin Proteins 0.000 description 2
- KHGNFPUMBJSZSM-UHFFFAOYSA-N Perforine Natural products COC1=C2CCC(O)C(CCC(C)(C)O)(OC)C2=NC2=C1C=CO2 KHGNFPUMBJSZSM-UHFFFAOYSA-N 0.000 description 2
- PBXYXOAEQQUVMM-ULQDDVLXSA-N Phe-His-Met Chemical compound CSCC[C@@H](C(=O)O)NC(=O)[C@H](CC1=CN=CN1)NC(=O)[C@H](CC2=CC=CC=C2)N PBXYXOAEQQUVMM-ULQDDVLXSA-N 0.000 description 2
- YDUGVDGFKNXFPL-IXOXFDKPSA-N Phe-Thr-Cys Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CS)C(=O)O)NC(=O)[C@H](CC1=CC=CC=C1)N)O YDUGVDGFKNXFPL-IXOXFDKPSA-N 0.000 description 2
- ABEFOXGAIIJDCL-SFJXLCSZSA-N Phe-Thr-Trp Chemical compound C([C@H](N)C(=O)N[C@@H]([C@H](O)C)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(O)=O)C1=CC=CC=C1 ABEFOXGAIIJDCL-SFJXLCSZSA-N 0.000 description 2
- ONPFOYPPPOHMNH-UVBJJODRSA-N Pro-Ala-Trp Chemical compound C[C@@H](C(=O)N[C@@H](CC1=CNC2=CC=CC=C21)C(=O)O)NC(=O)[C@@H]3CCCN3 ONPFOYPPPOHMNH-UVBJJODRSA-N 0.000 description 2
- SWXSLPHTJVAWDF-VEVYYDQMSA-N Pro-Asn-Thr Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O SWXSLPHTJVAWDF-VEVYYDQMSA-N 0.000 description 2
- CLNJSLSHKJECME-BQBZGAKWSA-N Pro-Gly-Ala Chemical compound OC(=O)[C@H](C)NC(=O)CNC(=O)[C@@H]1CCCN1 CLNJSLSHKJECME-BQBZGAKWSA-N 0.000 description 2
- VPBQDHMASPJHGY-JYJNAYRXSA-N Pro-Trp-Ser Chemical compound C1C[C@H](NC1)C(=O)N[C@@H](CC2=CNC3=CC=CC=C32)C(=O)N[C@@H](CO)C(=O)O VPBQDHMASPJHGY-JYJNAYRXSA-N 0.000 description 2
- BQWCDDAISCPDQV-XHNCKOQMSA-N Ser-Gln-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CCC(=O)N)NC(=O)[C@H](CO)N)C(=O)O BQWCDDAISCPDQV-XHNCKOQMSA-N 0.000 description 2
- YRBGKVIWMNEVCZ-WDSKDSINSA-N Ser-Glu-Gly Chemical compound OC[C@H](N)C(=O)N[C@@H](CCC(O)=O)C(=O)NCC(O)=O YRBGKVIWMNEVCZ-WDSKDSINSA-N 0.000 description 2
- IAORETPTUDBBGV-CIUDSAMLSA-N Ser-Leu-Cys Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CS)C(=O)O)NC(=O)[C@H](CO)N IAORETPTUDBBGV-CIUDSAMLSA-N 0.000 description 2
- YUJLIIRMIAGMCQ-CIUDSAMLSA-N Ser-Leu-Ser Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(O)=O YUJLIIRMIAGMCQ-CIUDSAMLSA-N 0.000 description 2
- IXZHZUGGKLRHJD-DCAQKATOSA-N Ser-Leu-Val Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C(C)C)C(O)=O IXZHZUGGKLRHJD-DCAQKATOSA-N 0.000 description 2
- CKDXFSPMIDSMGV-GUBZILKMSA-N Ser-Pro-Val Chemical compound [H]N[C@@H](CO)C(=O)N1CCC[C@H]1C(=O)N[C@@H](C(C)C)C(O)=O CKDXFSPMIDSMGV-GUBZILKMSA-N 0.000 description 2
- OLKICIBQRVSQMA-SRVKXCTJSA-N Ser-Ser-Tyr Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O OLKICIBQRVSQMA-SRVKXCTJSA-N 0.000 description 2
- XYEXCEPTALHNEV-RCWTZXSCSA-N Thr-Arg-Arg Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O XYEXCEPTALHNEV-RCWTZXSCSA-N 0.000 description 2
- FHDLKMFZKRUQCE-HJGDQZAQSA-N Thr-Glu-Arg Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O FHDLKMFZKRUQCE-HJGDQZAQSA-N 0.000 description 2
- MXNAOGFNFNKUPD-JHYOHUSXSA-N Thr-Phe-Thr Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H]([C@@H](C)O)C(O)=O MXNAOGFNFNKUPD-JHYOHUSXSA-N 0.000 description 2
- MEBDIIKMUUNBSB-RPTUDFQQSA-N Thr-Phe-Tyr Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O MEBDIIKMUUNBSB-RPTUDFQQSA-N 0.000 description 2
- RVMNUBQWPVOUKH-HEIBUPTGSA-N Thr-Ser-Thr Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)O)C(O)=O RVMNUBQWPVOUKH-HEIBUPTGSA-N 0.000 description 2
- CCZXBOFIBYQLEV-IHPCNDPISA-N Trp-Leu-Leu Chemical compound CC(C)C[C@H](NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](N)Cc1c[nH]c2ccccc12)C(O)=O CCZXBOFIBYQLEV-IHPCNDPISA-N 0.000 description 2
- 102100031988 Tumor necrosis factor ligand superfamily member 6 Human genes 0.000 description 2
- 108050002568 Tumor necrosis factor ligand superfamily member 6 Proteins 0.000 description 2
- YCMXFKWYJFZFKS-LAEOZQHASA-N Val-Gln-Asp Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)N[C@@H](CC(=O)O)C(=O)O)N YCMXFKWYJFZFKS-LAEOZQHASA-N 0.000 description 2
- ROLGIBMFNMZANA-GVXVVHGQSA-N Val-Glu-Leu Chemical compound CC(C)C[C@@H](C(=O)O)NC(=O)[C@H](CCC(=O)O)NC(=O)[C@H](C(C)C)N ROLGIBMFNMZANA-GVXVVHGQSA-N 0.000 description 2
- HGJRMXOWUWVUOA-GVXVVHGQSA-N Val-Leu-Gln Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)O)NC(=O)[C@H](C(C)C)N HGJRMXOWUWVUOA-GVXVVHGQSA-N 0.000 description 2
- CXWJFWAZIVWBOS-XQQFMLRXSA-N Val-Lys-Pro Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CCCCN)C(=O)N1CCC[C@@H]1C(=O)O)N CXWJFWAZIVWBOS-XQQFMLRXSA-N 0.000 description 2
- DVLWZWNAQUBZBC-ZNSHCXBVSA-N Val-Thr-Pro Chemical compound C[C@H]([C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@H](C(C)C)N)O DVLWZWNAQUBZBC-ZNSHCXBVSA-N 0.000 description 2
- 238000007605 air drying Methods 0.000 description 2
- 108010024078 alanyl-glycyl-serine Proteins 0.000 description 2
- 150000001413 amino acids Chemical group 0.000 description 2
- 238000011398 antitumor immunotherapy Methods 0.000 description 2
- 108010069205 aspartyl-phenylalanine Proteins 0.000 description 2
- 238000007664 blowing Methods 0.000 description 2
- 230000037396 body weight Effects 0.000 description 2
- 239000007853 buffer solution Substances 0.000 description 2
- 230000030833 cell death Effects 0.000 description 2
- 239000013592 cell lysate Substances 0.000 description 2
- 239000006285 cell suspension Substances 0.000 description 2
- 229960002424 collagenase Drugs 0.000 description 2
- 239000002299 complementary DNA Substances 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 230000029087 digestion Effects 0.000 description 2
- 210000003162 effector t lymphocyte Anatomy 0.000 description 2
- 238000003114 enzyme-linked immunosorbent spot assay Methods 0.000 description 2
- 238000000684 flow cytometry Methods 0.000 description 2
- 238000007710 freezing Methods 0.000 description 2
- 230000008014 freezing Effects 0.000 description 2
- 230000004927 fusion Effects 0.000 description 2
- 238000012215 gene cloning Methods 0.000 description 2
- 108010057083 glutamyl-aspartyl-leucine Proteins 0.000 description 2
- 108010067216 glycyl-glycyl-glycine Proteins 0.000 description 2
- 108010066198 glycyl-leucyl-phenylalanine Proteins 0.000 description 2
- 108010050848 glycylleucine Proteins 0.000 description 2
- 108010084389 glycyltryptophan Proteins 0.000 description 2
- 238000000227 grinding Methods 0.000 description 2
- 230000036737 immune function Effects 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 230000008595 infiltration Effects 0.000 description 2
- 238000001764 infiltration Methods 0.000 description 2
- 238000001802 infusion Methods 0.000 description 2
- 208000014674 injury Diseases 0.000 description 2
- 239000007928 intraperitoneal injection Substances 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 229940055695 pancreatin Drugs 0.000 description 2
- 239000000137 peptide hydrolase inhibitor Substances 0.000 description 2
- 229930192851 perforin Natural products 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 108020003175 receptors Proteins 0.000 description 2
- 238000007789 sealing Methods 0.000 description 2
- 230000028327 secretion Effects 0.000 description 2
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 description 2
- 230000009885 systemic effect Effects 0.000 description 2
- 231100000057 systemic toxicity Toxicity 0.000 description 2
- 239000008399 tap water Substances 0.000 description 2
- 235000020679 tap water Nutrition 0.000 description 2
- 230000008685 targeting Effects 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 230000001225 therapeutic effect Effects 0.000 description 2
- 230000009466 transformation Effects 0.000 description 2
- 108010080629 tryptophan-leucine Proteins 0.000 description 2
- 210000003171 tumor-infiltrating lymphocyte Anatomy 0.000 description 2
- 230000003612 virological effect Effects 0.000 description 2
- 239000011534 wash buffer Substances 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 239000012224 working solution Substances 0.000 description 2
- PMQXMXAASGFUDX-SRVKXCTJSA-N Ala-Lys-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)[C@H](C)N)CCCCN PMQXMXAASGFUDX-SRVKXCTJSA-N 0.000 description 1
- KGSJCPBERYUXCN-BPNCWPANSA-N Arg-Ala-Tyr Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O KGSJCPBERYUXCN-BPNCWPANSA-N 0.000 description 1
- RKRSYHCNPFGMTA-CIUDSAMLSA-N Arg-Glu-Asn Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(N)=O)C(O)=O RKRSYHCNPFGMTA-CIUDSAMLSA-N 0.000 description 1
- ORJQQZIXTOYGGH-SRVKXCTJSA-N Asn-Lys-Leu Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(O)=O ORJQQZIXTOYGGH-SRVKXCTJSA-N 0.000 description 1
- FMNBYVSGRCXWEK-FOHZUACHSA-N Asn-Thr-Gly Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)NCC(O)=O FMNBYVSGRCXWEK-FOHZUACHSA-N 0.000 description 1
- JPSODRNUDXONAS-XIRDDKMYSA-N Asn-Trp-His Chemical compound C1=CC=C2C(=C1)C(=CN2)C[C@@H](C(=O)N[C@@H](CC3=CN=CN3)C(=O)O)NC(=O)[C@H](CC(=O)N)N JPSODRNUDXONAS-XIRDDKMYSA-N 0.000 description 1
- FAEIQWHBRBWUBN-FXQIFTODSA-N Asp-Arg-Ser Chemical compound C(C[C@@H](C(=O)N[C@@H](CO)C(=O)O)NC(=O)[C@H](CC(=O)O)N)CN=C(N)N FAEIQWHBRBWUBN-FXQIFTODSA-N 0.000 description 1
- UGKZHCBLMLSANF-CIUDSAMLSA-N Asp-Asn-Leu Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(C)C)C(O)=O UGKZHCBLMLSANF-CIUDSAMLSA-N 0.000 description 1
- SVFOIXMRMLROHO-SRVKXCTJSA-N Asp-Asp-Phe Chemical compound OC(=O)C[C@H](N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 SVFOIXMRMLROHO-SRVKXCTJSA-N 0.000 description 1
- DTNUIAJCPRMNBT-WHFBIAKZSA-N Asp-Gly-Ala Chemical compound [H]N[C@@H](CC(O)=O)C(=O)NCC(=O)N[C@@H](C)C(O)=O DTNUIAJCPRMNBT-WHFBIAKZSA-N 0.000 description 1
- NVFSJIXJZCDICF-SRVKXCTJSA-N Asp-Lys-Lys Chemical compound C(CCN)C[C@@H](C(=O)N[C@@H](CCCCN)C(=O)O)NC(=O)[C@H](CC(=O)O)N NVFSJIXJZCDICF-SRVKXCTJSA-N 0.000 description 1
- 108010075254 C-Peptide Proteins 0.000 description 1
- 206010010144 Completed suicide Diseases 0.000 description 1
- 102100039498 Cytotoxic T-lymphocyte protein 4 Human genes 0.000 description 1
- 229920001917 Ficoll Polymers 0.000 description 1
- NXPXQIZKDOXIHH-JSGCOSHPSA-N Gln-Gly-Trp Chemical compound C1=CC=C2C(=C1)C(=CN2)C[C@@H](C(=O)O)NC(=O)CNC(=O)[C@H](CCC(=O)N)N NXPXQIZKDOXIHH-JSGCOSHPSA-N 0.000 description 1
- GLWXKFRTOHKGIT-ACZMJKKPSA-N Glu-Asn-Asn Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(N)=O)C(O)=O GLWXKFRTOHKGIT-ACZMJKKPSA-N 0.000 description 1
- IVGJYOOGJLFKQE-AVGNSLFASA-N Glu-Leu-Lys Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CCCCN)C(=O)O)NC(=O)[C@H](CCC(=O)O)N IVGJYOOGJLFKQE-AVGNSLFASA-N 0.000 description 1
- UGVQELHRNUDMAA-BYPYZUCNSA-N Gly-Ala-Gly Chemical compound [NH3+]CC(=O)N[C@@H](C)C(=O)NCC([O-])=O UGVQELHRNUDMAA-BYPYZUCNSA-N 0.000 description 1
- UEGIPZAXNBYCCP-NKWVEPMBSA-N Gly-Cys-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CS)NC(=O)CN)C(=O)O UEGIPZAXNBYCCP-NKWVEPMBSA-N 0.000 description 1
- NSTUFLGQJCOCDL-UWVGGRQHSA-N Gly-Leu-Arg Chemical compound NCC(=O)N[C@@H](CC(C)C)C(=O)N[C@H](C(O)=O)CCCN=C(N)N NSTUFLGQJCOCDL-UWVGGRQHSA-N 0.000 description 1
- TVUWMSBGMVAHSJ-KBPBESRZSA-N Gly-Leu-Phe Chemical compound NCC(=O)N[C@@H](CC(C)C)C(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 TVUWMSBGMVAHSJ-KBPBESRZSA-N 0.000 description 1
- IRJWAYCXIYUHQE-WHFBIAKZSA-N Gly-Ser-Ala Chemical compound OC(=O)[C@H](C)NC(=O)[C@H](CO)NC(=O)CN IRJWAYCXIYUHQE-WHFBIAKZSA-N 0.000 description 1
- SOEGEPHNZOISMT-BYPYZUCNSA-N Gly-Ser-Gly Chemical compound NCC(=O)N[C@@H](CO)C(=O)NCC(O)=O SOEGEPHNZOISMT-BYPYZUCNSA-N 0.000 description 1
- WNGHUXFWEWTKAO-YUMQZZPRSA-N Gly-Ser-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H](CO)NC(=O)CN WNGHUXFWEWTKAO-YUMQZZPRSA-N 0.000 description 1
- YXTFLTJYLIAZQG-FJXKBIBVSA-N Gly-Thr-Arg Chemical compound NCC(=O)N[C@@H]([C@H](O)C)C(=O)N[C@H](C(O)=O)CCCN=C(N)N YXTFLTJYLIAZQG-FJXKBIBVSA-N 0.000 description 1
- MREVELMMFOLESM-HOCLYGCPSA-N Gly-Trp-Val Chemical compound [H]NCC(=O)N[C@@H](CC1=CNC2=C1C=CC=C2)C(=O)N[C@@H](C(C)C)C(O)=O MREVELMMFOLESM-HOCLYGCPSA-N 0.000 description 1
- TVMNTHXFRSXZGR-IHRRRGAJSA-N His-Lys-Val Chemical compound [H]N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C(C)C)C(O)=O TVMNTHXFRSXZGR-IHRRRGAJSA-N 0.000 description 1
- PLCAEMGSYOYIPP-GUBZILKMSA-N His-Ser-Gln Chemical compound NC(=O)CC[C@@H](C(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CC1=CN=CN1 PLCAEMGSYOYIPP-GUBZILKMSA-N 0.000 description 1
- 101000889276 Homo sapiens Cytotoxic T-lymphocyte protein 4 Proteins 0.000 description 1
- 101000831007 Homo sapiens T-cell immunoreceptor with Ig and ITIM domains Proteins 0.000 description 1
- OEQKGSPBDVKYOC-ZKWXMUAHSA-N Ile-Gly-Cys Chemical compound CC[C@H](C)[C@@H](C(=O)NCC(=O)N[C@@H](CS)C(=O)O)N OEQKGSPBDVKYOC-ZKWXMUAHSA-N 0.000 description 1
- NURNJECQNNCRBK-FLBSBUHZSA-N Ile-Thr-Thr Chemical compound CC[C@H](C)[C@H](N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O NURNJECQNNCRBK-FLBSBUHZSA-N 0.000 description 1
- IIKJNQWOQIWWMR-CIUDSAMLSA-N Leu-Cys-Ala Chemical compound C[C@@H](C(=O)O)NC(=O)[C@H](CS)NC(=O)[C@H](CC(C)C)N IIKJNQWOQIWWMR-CIUDSAMLSA-N 0.000 description 1
- HYMLKESRWLZDBR-WEDXCCLWSA-N Leu-Gly-Thr Chemical compound CC(C)C[C@H](N)C(=O)NCC(=O)N[C@@H]([C@@H](C)O)C(O)=O HYMLKESRWLZDBR-WEDXCCLWSA-N 0.000 description 1
- LJBVRCDPWOJOEK-PPCPHDFISA-N Leu-Thr-Ile Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O LJBVRCDPWOJOEK-PPCPHDFISA-N 0.000 description 1
- YIRIDPUGZKHMHT-ACRUOGEOSA-N Leu-Tyr-Tyr Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O YIRIDPUGZKHMHT-ACRUOGEOSA-N 0.000 description 1
- LJADEBULDNKJNK-IHRRRGAJSA-N Lys-Leu-Val Chemical compound CC(C)C[C@H](NC(=O)[C@@H](N)CCCCN)C(=O)N[C@@H](C(C)C)C(O)=O LJADEBULDNKJNK-IHRRRGAJSA-N 0.000 description 1
- SVSQSPICRKBMSZ-SRVKXCTJSA-N Lys-Pro-Gln Chemical compound [H]N[C@@H](CCCCN)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCC(N)=O)C(O)=O SVSQSPICRKBMSZ-SRVKXCTJSA-N 0.000 description 1
- XABXVVSWUVCZST-GVXVVHGQSA-N Lys-Val-Gln Chemical compound NC(=O)CC[C@@H](C(O)=O)NC(=O)[C@H](C(C)C)NC(=O)[C@@H](N)CCCCN XABXVVSWUVCZST-GVXVVHGQSA-N 0.000 description 1
- 108010052285 Membrane Proteins Proteins 0.000 description 1
- AETNZPKUUYYYEK-CIUDSAMLSA-N Met-Glu-Asn Chemical compound CSCC[C@H](N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(N)=O)C(O)=O AETNZPKUUYYYEK-CIUDSAMLSA-N 0.000 description 1
- OCRSGGIJBDUXHU-WDSOQIARSA-N Met-Leu-Trp Chemical compound C1=CC=C2C(C[C@H](NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](N)CCSC)C(O)=O)=CNC2=C1 OCRSGGIJBDUXHU-WDSOQIARSA-N 0.000 description 1
- 101100519207 Mus musculus Pdcd1 gene Proteins 0.000 description 1
- 208000009525 Myocarditis Diseases 0.000 description 1
- 108010065395 Neuropep-1 Proteins 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 239000012124 Opti-MEM Substances 0.000 description 1
- 239000012807 PCR reagent Substances 0.000 description 1
- 241000190594 Parana mammarenavirus Species 0.000 description 1
- 229930182555 Penicillin Natural products 0.000 description 1
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 1
- ULECEJGNDHWSKD-QEJZJMRPSA-N Phe-Ala-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@H](C)NC(=O)[C@@H](N)CC1=CC=CC=C1 ULECEJGNDHWSKD-QEJZJMRPSA-N 0.000 description 1
- WMGVYPPIMZPWPN-SRVKXCTJSA-N Phe-Asp-Asn Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)N[C@@H](CC(=O)N)C(=O)O)N WMGVYPPIMZPWPN-SRVKXCTJSA-N 0.000 description 1
- YVXPUUOTMVBKDO-IHRRRGAJSA-N Phe-Pro-Cys Chemical compound C1C[C@H](N(C1)C(=O)[C@H](CC2=CC=CC=C2)N)C(=O)N[C@@H](CS)C(=O)O YVXPUUOTMVBKDO-IHRRRGAJSA-N 0.000 description 1
- AMBLXEMWFARNNQ-DCAQKATOSA-N Pro-Asn-Leu Chemical compound CC(C)C[C@@H](C(=O)O)NC(=O)[C@H](CC(=O)N)NC(=O)[C@@H]1CCCN1 AMBLXEMWFARNNQ-DCAQKATOSA-N 0.000 description 1
- HQVPQXMCQKXARZ-FXQIFTODSA-N Pro-Cys-Ser Chemical compound C1C[C@H](NC1)C(=O)N[C@@H](CS)C(=O)N[C@@H](CO)C(=O)O HQVPQXMCQKXARZ-FXQIFTODSA-N 0.000 description 1
- UAYHMOIGIQZLFR-NHCYSSNCSA-N Pro-Gln-Val Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](C(C)C)C(O)=O UAYHMOIGIQZLFR-NHCYSSNCSA-N 0.000 description 1
- MCWHYUWXVNRXFV-RWMBFGLXSA-N Pro-Leu-Pro Chemical compound CC(C)C[C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@@H]2CCCN2 MCWHYUWXVNRXFV-RWMBFGLXSA-N 0.000 description 1
- 229940124158 Protease/peptidase inhibitor Drugs 0.000 description 1
- 238000001190 Q-PCR Methods 0.000 description 1
- ULVMNZOKDBHKKI-ACZMJKKPSA-N Ser-Gln-Asp Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(O)=O)C(O)=O ULVMNZOKDBHKKI-ACZMJKKPSA-N 0.000 description 1
- YMTLKLXDFCSCNX-BYPYZUCNSA-N Ser-Gly-Gly Chemical compound OC[C@H](N)C(=O)NCC(=O)NCC(O)=O YMTLKLXDFCSCNX-BYPYZUCNSA-N 0.000 description 1
- UGHCUDLCCVVIJR-VGDYDELISA-N Ser-His-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)[C@H](CC1=CN=CN1)NC(=O)[C@H](CO)N UGHCUDLCCVVIJR-VGDYDELISA-N 0.000 description 1
- NIOYDASGXWLHEZ-CIUDSAMLSA-N Ser-Met-Glu Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CCC(O)=O)C(O)=O NIOYDASGXWLHEZ-CIUDSAMLSA-N 0.000 description 1
- XKFJENWJGHMDLI-QWRGUYRKSA-N Ser-Phe-Gly Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)NCC(O)=O XKFJENWJGHMDLI-QWRGUYRKSA-N 0.000 description 1
- JLKWJWPDXPKKHI-FXQIFTODSA-N Ser-Pro-Asn Chemical compound C1C[C@H](N(C1)C(=O)[C@H](CO)N)C(=O)N[C@@H](CC(=O)N)C(=O)O JLKWJWPDXPKKHI-FXQIFTODSA-N 0.000 description 1
- JZRYFUGREMECBH-XPUUQOCRSA-N Ser-Val-Gly Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](C(C)C)C(=O)NCC(O)=O JZRYFUGREMECBH-XPUUQOCRSA-N 0.000 description 1
- 230000006044 T cell activation Effects 0.000 description 1
- 102100024834 T-cell immunoreceptor with Ig and ITIM domains Human genes 0.000 description 1
- YZUWGFXVVZQJEI-PMVVWTBXSA-N Thr-Gly-His Chemical compound C[C@H]([C@@H](C(=O)NCC(=O)N[C@@H](CC1=CN=CN1)C(=O)O)N)O YZUWGFXVVZQJEI-PMVVWTBXSA-N 0.000 description 1
- WKGAAMOJPMBBMC-IXOXFDKPSA-N Thr-Ser-Phe Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O WKGAAMOJPMBBMC-IXOXFDKPSA-N 0.000 description 1
- ZMYCLHFLHRVOEA-HEIBUPTGSA-N Thr-Thr-Ser Chemical compound C[C@@H](O)[C@H](N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CO)C(O)=O ZMYCLHFLHRVOEA-HEIBUPTGSA-N 0.000 description 1
- KXFYAQUYJKOQMI-QEJZJMRPSA-N Trp-Ser-Gln Chemical compound C1=CC=C2C(C[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCC(N)=O)C(O)=O)=CNC2=C1 KXFYAQUYJKOQMI-QEJZJMRPSA-N 0.000 description 1
- WVRUKYLYMFGKAN-IHRRRGAJSA-N Tyr-Glu-Glu Chemical compound OC(=O)CC[C@@H](C(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@@H](N)CC1=CC=C(O)C=C1 WVRUKYLYMFGKAN-IHRRRGAJSA-N 0.000 description 1
- HZYOWMGWKKRMBZ-BYULHYEWSA-N Val-Asp-Asp Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)N[C@@H](CC(=O)O)C(=O)O)N HZYOWMGWKKRMBZ-BYULHYEWSA-N 0.000 description 1
- PIFJAFRUVWZRKR-QMMMGPOBSA-N Val-Gly-Gly Chemical compound CC(C)[C@H]([NH3+])C(=O)NCC(=O)NCC([O-])=O PIFJAFRUVWZRKR-QMMMGPOBSA-N 0.000 description 1
- HJSLDXZAZGFPDK-ULQDDVLXSA-N Val-Phe-Leu Chemical compound CC(C)C[C@@H](C(=O)O)NC(=O)[C@H](CC1=CC=CC=C1)NC(=O)[C@H](C(C)C)N HJSLDXZAZGFPDK-ULQDDVLXSA-N 0.000 description 1
- KRAHMIJVUPUOTQ-DCAQKATOSA-N Val-Ser-His Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CO)C(=O)N[C@@H](CC1=CN=CN1)C(=O)O)N KRAHMIJVUPUOTQ-DCAQKATOSA-N 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- QPMSXSBEVQLBIL-CZRHPSIPSA-N ac1mix0p Chemical compound C1=CC=C2N(C[C@H](C)CN(C)C)C3=CC(OC)=CC=C3SC2=C1.O([C@H]1[C@]2(OC)C=CC34C[C@@H]2[C@](C)(O)CCC)C2=C5[C@]41CCN(C)[C@@H]3CC5=CC=C2O QPMSXSBEVQLBIL-CZRHPSIPSA-N 0.000 description 1
- 230000004721 adaptive immunity Effects 0.000 description 1
- 108010047495 alanylglycine Proteins 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 230000006023 anti-tumor response Effects 0.000 description 1
- 239000000427 antigen Substances 0.000 description 1
- 108091007433 antigens Proteins 0.000 description 1
- 102000036639 antigens Human genes 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 108010013835 arginine glutamate Proteins 0.000 description 1
- 108010047857 aspartylglycine Proteins 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 238000003149 assay kit Methods 0.000 description 1
- 230000001363 autoimmune Effects 0.000 description 1
- 210000001099 axilla Anatomy 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004899 c-terminal region Anatomy 0.000 description 1
- 239000001569 carbon dioxide Substances 0.000 description 1
- 230000003833 cell viability Effects 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 238000002512 chemotherapy Methods 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 208000037966 cold tumor Diseases 0.000 description 1
- 230000001276 controlling effect Effects 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 239000012228 culture supernatant Substances 0.000 description 1
- 108010060199 cysteinylproline Proteins 0.000 description 1
- 230000009089 cytolysis Effects 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 230000034994 death Effects 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 229940042399 direct acting antivirals protease inhibitors Drugs 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 210000003743 erythrocyte Anatomy 0.000 description 1
- 239000012894 fetal calf serum Substances 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 239000007850 fluorescent dye Substances 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 239000006481 glucose medium Substances 0.000 description 1
- 108010001064 glycyl-glycyl-glycyl-glycine Proteins 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 230000005931 immune cell recruitment Effects 0.000 description 1
- 229940126533 immune checkpoint blocker Drugs 0.000 description 1
- 230000037451 immune surveillance Effects 0.000 description 1
- 238000003119 immunoblot Methods 0.000 description 1
- 230000002163 immunogen Effects 0.000 description 1
- 210000005008 immunosuppressive cell Anatomy 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 230000015788 innate immune response Effects 0.000 description 1
- 230000009545 invasion Effects 0.000 description 1
- 210000003292 kidney cell Anatomy 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 230000003902 lesion Effects 0.000 description 1
- 239000003446 ligand Substances 0.000 description 1
- 239000012160 loading buffer Substances 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 210000000207 lymphocyte subset Anatomy 0.000 description 1
- 230000002934 lysing effect Effects 0.000 description 1
- 108010009298 lysylglutamic acid Proteins 0.000 description 1
- 229910052751 metal Inorganic materials 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 230000004719 natural immunity Effects 0.000 description 1
- 230000003472 neutralizing effect Effects 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 230000009437 off-target effect Effects 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 229940049954 penicillin Drugs 0.000 description 1
- 210000005259 peripheral blood Anatomy 0.000 description 1
- 239000011886 peripheral blood Substances 0.000 description 1
- 230000000144 pharmacologic effect Effects 0.000 description 1
- 108010074082 phenylalanyl-alanyl-lysine Proteins 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 230000002035 prolonged effect Effects 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 239000003531 protein hydrolysate Substances 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 238000001959 radiotherapy Methods 0.000 description 1
- 230000008844 regulatory mechanism Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 108091008146 restriction endonucleases Proteins 0.000 description 1
- 238000003757 reverse transcription PCR Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 108010071207 serylmethionine Proteins 0.000 description 1
- 230000001568 sexual effect Effects 0.000 description 1
- IZTQOLKUZKXIRV-YRVFCXMDSA-N sincalide Chemical compound C([C@@H](C(=O)N[C@@H](CCSC)C(=O)NCC(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC=1C=CC=CC=1)C(N)=O)NC(=O)[C@@H](N)CC(O)=O)C1=CC=C(OS(O)(=O)=O)C=C1 IZTQOLKUZKXIRV-YRVFCXMDSA-N 0.000 description 1
- 235000020183 skimmed milk Nutrition 0.000 description 1
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
- 210000004989 spleen cell Anatomy 0.000 description 1
- 210000004988 splenocyte Anatomy 0.000 description 1
- 229960005322 streptomycin Drugs 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000001356 surgical procedure Methods 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 108010029384 tryptophyl-histidine Proteins 0.000 description 1
- 108010084932 tryptophyl-proline Proteins 0.000 description 1
- 230000002476 tumorcidal effect Effects 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
- 239000013598 vector Substances 0.000 description 1
- 230000029812 viral genome replication Effects 0.000 description 1
Images
Classifications
-
- 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
- C07K14/70503—Immunoglobulin superfamily
- C07K14/70521—CD28, CD152
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/66—Microorganisms or materials therefrom
- A61K35/76—Viruses; Subviral particles; Bacteriophages
- A61K35/761—Adenovirus
-
- 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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- 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
- C07K14/70578—NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
-
- 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
- C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
-
- 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
- C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
- C12N2710/00011—Details
- C12N2710/10011—Adenoviridae
- C12N2710/10022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
-
- 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
- C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
- C12N2710/00011—Details
- C12N2710/10011—Adenoviridae
- C12N2710/10041—Use of virus, viral particle or viral elements as a vector
- C12N2710/10043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
-
- 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
- C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
- C12N2710/00011—Details
- C12N2710/10011—Adenoviridae
- C12N2710/10051—Methods of production or purification of viral material
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Medicinal Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Genetics & Genomics (AREA)
- Immunology (AREA)
- Zoology (AREA)
- Pharmacology & Pharmacy (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Gastroenterology & Hepatology (AREA)
- Virology (AREA)
- Molecular Biology (AREA)
- Biophysics (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Toxicology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Epidemiology (AREA)
- Cell Biology (AREA)
- Microbiology (AREA)
- General Chemical & Material Sciences (AREA)
- Wood Science & Technology (AREA)
- Biotechnology (AREA)
- Biomedical Technology (AREA)
- Mycology (AREA)
- General Engineering & Computer Science (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
本发明涉及肿瘤免疫治疗领域,具体涉及一种新型兼具激活免疫共刺激信号通路和阻断免疫检查点的复制型溶瘤腺病毒AD5 sPD1CD137L及其在抗肿瘤药物中的应用。本发明公开了AD5 sPD1CD137L的设计和构建方法,成功获得了复制型溶瘤腺病毒AD5 sPD1CD137L,该病毒可以在肿瘤细胞内特异性复制,并且能够高表达分泌型融合蛋白sPD1CD137L,该融合蛋白分子能够分泌到胞外,发挥免疫共刺激和阻断免疫检查点的双重功能。实验表明,本发明的新型复制型溶瘤腺病毒具有显著的免疫共刺激和阻断免疫检查点的作用,显著激活抗肿瘤免疫反应,具有显著的抗肿瘤的活性,有极大的开发抗肿瘤药物的前景和价值。
Description
技术领域
本发明涉及肿瘤免疫治疗领域,具体涉及兼具激活免疫共刺激信号通路和阻断免疫检查点的复制型溶瘤腺病毒及其应用。
背景技术
癌症已经成为危害人类生命健康的疾病之首,在我国每年新增的癌症患者约400万人,每年有近300万人死于癌症。除了常规的手术、放疗和化疗,急需新而有效的治疗手段,因此抗癌药物的研发一直是药学研究的热点。
随着不断出现的令人振奋的临床研究结果,抗肿瘤免疫治疗给肿瘤患者带来了希望。免疫系统具有识别并清除异己的能力,肿瘤在发生发展过程中不仅通过多种途径抑制免疫系统的先天性免疫反应,也通过“隐藏身份”和启动免疫负性调控等诸多方法来逃避机体正常免疫系统的监视,从而不被机体清除。肿瘤细胞通过自身或者促进其他细胞表达免疫负性调控分子PD-L1等,来劫持PD-1/PD-L1通路,从而抑制效应淋巴细胞的抗肿瘤活性,进入失能状态,达到逃避免疫监视和免疫清除的目的。因此,阻断免疫检查点PD-1/PD-L1这个通路受到了广泛的关注。阻断免疫检查点已经被证明为有效的抗肿瘤方法之一,至今已有多个免疫检查点阻断剂被批准为用于临床肿瘤治疗的药物。
随着临床研究的进展,阻断免疫检查点的抗肿瘤治疗也遇到了一些亟需解决的问题。首先,阻断免疫检查点的免疫治疗对肿瘤类型和肿瘤患者的普适性(有效率)有待提高。尤其是对一些“冷肿瘤”,即实体肿瘤内只有少量或缺乏淋巴细胞的浸润,抑制免疫检查点的治疗效果甚微。其次,在一些肿瘤患者中,系统性阻断免疫检查点存在“脱靶”而“误伤”正常组织的情况,由此带来了自身性免疫损伤如心肌炎等副作用。
CD137是淋巴细胞的一个极其重要的免疫共刺激分子,参与T淋巴细胞的活化过程。如果缺乏该分子的激活信号,识别肿瘤的T细胞仍不能有效产生抗肿瘤反应。然而,由于机体正常的平衡调控机制,活化的淋巴细胞往往上调免疫负性调控分子的表达,包括PD1、CTLA4、TIGIT等,以避免免疫系统的过度活化。因此,我们假设将阻断免疫检查点PD-1/PD-L1与活化免疫共刺激分子CD137联合起来,能够更有效地发挥免疫细胞的抗肿瘤作用。
进一步的临床试验数据表明,肿瘤浸润淋巴细胞(TILs)的多少、肿瘤局部的免疫活化状态等,是靶向免疫检查点治疗能否显效的重要预测指标。CD8+T细胞介导肿瘤清除过程中,I型干扰素(IFNα/β)通路的活化是靶向免疫检查点治疗的重要事件。因此,如何在肿瘤局部有效诱导I型干扰素介导的免疫活化、增强肿瘤微环境免疫细胞的浸润,可以使肿瘤对靶向免疫检查点的治疗更加敏感,这也许是解决免疫检查点治疗普适性(有效率)不高的有效手段之一。
病毒作为外来侵入颗粒,能够有效激活机体的天然免疫和适应性免疫。随着溶瘤病毒T-Vec在2015年底被FDA批准上市,溶瘤病毒介导的抗肿瘤免疫治疗受到越来越多的关注。我们设想,溶瘤病毒免疫疗法是否能使肿瘤从“冷”变“热”,对靶向免疫检查点和免疫共刺激的治疗更加敏感,从而解决免疫检查点治疗普适性(有效率)不高这一问题。
另外,得益于溶瘤病毒在肿瘤细胞具有选择性复制的优势,重组溶瘤病毒经肿瘤局部注射后,能够在感染的肿瘤微环境内,高表达具有免疫活化作用的目的基因(蛋白),使得免疫活化能够最大限度地被局限在肿瘤微环境,从而避免阻断免疫检查点“脱靶”或者活化CD137实现免疫共刺激而造成系统性“误伤”(免疫检查点治疗遇到的第2个问题)。
目前,缺乏一种兼具激活免疫共刺激信号通路和阻断免疫检查点的复制型溶瘤腺病毒及其应用。
发明内容
本发明的目的在于提供一种既能提供T细胞活化第二信号又能阻断T细胞免疫检查点抑制信号的复制型溶瘤腺病毒及其应用。
为达到上述目的,本发明采用了下列技术方案:本发明的一种兼具激活免疫共刺激信号通路、阻断免疫检查点和桥联肿瘤细胞与免疫细胞的可溶性融合蛋白,所述的可溶性融合蛋白的两端分别为结合PD-L1(肿瘤细胞)的PD1和结合CD137(免疫细胞)的CD137L,PD1和CD137L之间通过linker序列连接。
进一步地,所述的可溶性融合蛋白为sPD1CD137L,sPD1CD137L的蛋白序列和氨基酸序列分别如序列表SEQ ID NO:1和SEQ ID NO:6所示。
本发明所述的兼具激活免疫共刺激信号通路和阻断免疫检查点的可溶性融合蛋白在制备活化抗肿瘤免疫药物中的应用。
本发明所述的兼具激活免疫共刺激信号通路和阻断免疫检查点的可溶性融合蛋白在制备刺激IFN-γ表达药物中的应用。
本发明所述的兼具激活免疫共刺激信号通路和阻断免疫检查点的可溶性融合蛋白在制备抗肿瘤药物中的应用。
进一步地,所述的肿瘤为肝癌、腹水癌、黑色素瘤或乳腺癌。
本发明的一种兼具激活免疫共刺激信号通路和阻断免疫检查点的复制型溶瘤腺病毒,其特征在于:所述的复制型溶瘤腺病毒在肿瘤细胞内复制,并且表达和分泌可溶性融合蛋白,所述的可溶性融合蛋白的两端分别为结合PD-L1的PD1和结合CD137的CD137L,PD1和CD137L之间通过linker序列连接。
进一步地,所述的可溶性融合蛋白为sPD1CD137L,sPD1CD137L的蛋白序列的氨基酸序列如序列表SEQ ID NO:1所示。
本发明所述的兼具激活免疫共刺激信号通路和阻断免疫检查点的复制型溶瘤腺病毒,其特征在于:所述的复制型溶瘤腺病毒能够溶瘤。
本发明所述的兼具激活免疫共刺激信号通路和阻断免疫检查点的复制型溶瘤腺病毒在制备活化抗肿瘤免疫药物中的应用。
本发明所述的兼具激活免疫共刺激信号通路和阻断免疫检查点的复制型溶瘤腺病毒在制备刺激IFN-γ表达药物中的应用。
本发明所述的兼具激活免疫共刺激信号通路和阻断免疫检查点的复制型溶瘤腺病毒在制备抗肿瘤药物中的应用。
进一步地,所述的肿瘤为肝癌、腹水癌、黑色素瘤或乳腺癌。
本发明的一种兼具激活免疫共刺激信号通路和阻断免疫检查点的复制型溶瘤腺病毒AD5 sPD1CD137L的构建方法,包括如下步骤:(1)AD5 sPD1CD137L全长质粒构建:将构建好的穿梭载体AD5-pShuttle-sPD1-CD137L用PmeI线性化后转入感受态pAdEasy-BJ5183中,使用含50ug/ml卡那霉素LB平板的进行筛选,挑取阳性克隆培养鉴定,鉴定正确的克隆质粒重新转化DH5a感受态进行二次筛选鉴定,鉴定正确后进行质粒大提获得AD5-sPD1-CD137L全长质粒;
(2)AD5 sPD1CD137L病毒拯救:AD5 sPD1CD137L全长质粒使用PacI线性化,纯化后6孔板中1ug/well转染293T细胞,5%CO2、37℃培养,2天后将细胞消化后转入10cm平皿,2-3天换液,至80%细胞出现病变,使用10ml培养基将细胞吹下收集至15ml离心管,反复冻融2次,3000rpm/min离心15min,收集病毒上清-80℃保存做为毒种;
(3)病毒扩增:取病毒种液50ul加入60%293T细胞10cm平皿中,5%CO237℃培养,细胞密度至90%以上,按照1传3比例传代,直至80%细胞出现病变,大约有10个平皿细胞,按上述方法收病毒,使用氯化铯密度梯度离心纯化病毒;使用TCID50方法进行滴度测定。
有益效果:本发明提供了一种可以兼具激活免疫共刺激信号通路和阻断免疫检查点的复制型溶瘤腺病毒AD5 sPD1CD137L的设计和构建方法,成功获得了一株新型兼具激活免疫共刺激信号通路和阻断免疫检查点的复制型溶瘤腺病毒AD5 sPD1CD137L,该病毒可以选择性地在肿瘤细胞内和肿瘤部位复制、具有肿瘤靶向性,能够有效溶瘤,并诱导免疫原性细胞死亡。
与现有技术相比,本发明具有如下优点:(1)与此同时,该病毒能够高表达可溶性融合蛋白sPD1CD137L,该蛋白能够分泌到细胞外,在肿瘤微环境中发挥阻断免疫检查点、激活免疫共刺激信号通路,同时还能在肿瘤细胞和免疫细胞间起到桥联,增强抗肿瘤免疫作用。
(2)本发明的兼具激活免疫共刺激信号通路和阻断免疫检查点的复制型溶瘤腺病毒AD5 sPD1CD137L具有显著的活化抗肿瘤免疫作用,能够显著刺激IFN-γ在肿瘤局部高表达且没有明显的全身毒性,显著抑制肿瘤生长、延长生存期,具有显著的抗肿瘤作用。一个病毒,同时整合多种独特的抗肿瘤机制于一身,具有预料不到的抗肿瘤效果。可以用来制备抗肿瘤药物。
(3)本发明设计了一个构建一种新型兼具激活免疫共刺激信号通路和阻断免疫检查点的复制型溶瘤腺病毒AD5 sPD1CD137L的方法,获得了一株新型兼具激活免疫共刺激信号通路和阻断免疫检查点的复制型溶瘤腺病毒AD5 sPD1CD137L,该病毒可以在肿瘤细胞内和肿瘤部位特异性复制,并且能够高表达可溶性融合蛋白sPD1CD137L。可溶性融合蛋白sPD1CD137L的N端是PD1的胞外区,可以特异性地结合PD-L1,从而封闭了PD-L1与效应淋巴细胞上的特异性受体PD1点结合,最终能够阻断PD1/PD-L1的负性调控通路;该融合蛋白的C端是CD137L的胞外区,能够特异性地结合其受体CD137,激活CD137的下游免疫活化通路,起到免疫活化作用。融合蛋白sPD1CD137L能够被大量分泌到细胞外,在肿瘤微环境内同时发挥阻断免疫检查点和免疫共刺激、显著活化免疫的生物学与免疫学功能,同时还能在肿瘤细胞和免疫细胞间起到桥联,增强抗肿瘤免疫作用。
(4)药理学实验表明,本发明的新型复制型溶瘤腺病毒AD5 sPD1CD137L具有显著的活化抗肿瘤免疫作用、刺激刺激IFN-γ高表达,有显著的抗肿瘤的活性,具有非常高的开发抗肿瘤药物的前景和价值。
(5)本发明提供了一种兼具激活免疫共刺激信号通路和阻断免疫检查点的复制型溶瘤腺病毒AD5 sPD1CD137L的设计和构建方法,成功获得了一株兼具激活免疫共刺激信号通路和阻断免疫检查点的复制型溶瘤腺病毒AD5 sPD1CD137L,该病毒可以在肿瘤细胞内和肿瘤部位复制,并且能够高表达可溶性融合蛋白sPD1CD137L,该蛋白能够分泌到细胞外,在肿瘤局部发挥调控免疫检查点和免疫共刺激的生物学和免疫学功能。
(6)可溶性融合蛋白sPD1CD137L的N端是PD1的胞外区,可以特异性地结合PD-L1,从而封闭了PD-L1与效应淋巴细胞上的特异性受体PD1点结合,最终能够阻断PD1/PD-L1的负性调控通路;该融合蛋白的C端是CD137L的胞外区,能够特异性地结合其受体CD137,激活CD137的下游免疫活化通路,起到免疫活化作用,同时还能在肿瘤细胞和免疫细胞间起到桥联,增强抗肿瘤免疫作用。
(7)进一步的本发明的兼具激活免疫共刺激信号通路和阻断免疫检查点的复制型溶瘤腺病毒AD5 sPD1CD137L具有显著的活化抗肿瘤免疫作用,能够刺激IFN-γ高表达。在肿瘤动物模型中,兼具激活免疫共刺激信号通路和阻断免疫检查点的复制型溶瘤腺病毒AD5 sPD1CD137L具有显著的抗肿瘤作用,可以用来制备抗肿瘤药物。
附图说明
图1为本发明的结果显示我们已经成功构建兼具激活免疫共刺激信号通路和阻断免疫检查点的复制型溶瘤腺病毒AD5 sPD1CD137L,图中对照组对应位置没有条带、是空白的,而兼具激活免疫共刺激信号通路和阻断免疫检查点的复制型溶瘤腺病毒AD5sPD1CD137L对应的条带深黑,证明兼具激活免疫共刺激信号通路和阻断免疫检查点的复制型溶瘤腺病毒AD5 sPD1CD137L能够在肿瘤细胞表达并且分泌到细胞外,而且从蛋白大小判断,是我们拟表达的目标蛋白:可溶性的SPD1CD137L。
图2为本发明的结果显示我们所构建的兼具激活免疫共刺激信号通路和阻断免疫检查点的复制型溶瘤腺病毒AD5 sPD1CD137L在肿瘤细胞内具有与对照病毒相同的复制和溶瘤能力。
图3为本发明的肿瘤体积测量数据的结果显示兼具激活免疫共刺激信号通路和阻断免疫检查点的复制型溶瘤腺病毒AD5 sPD1CD137L在体内具有显著的抗肿瘤作用(B16/F10实体瘤模型),能够抑制肿瘤的生长。
图4为本发明结果显示复制型溶瘤腺病毒AD5 sPD1CD137L在小鼠4T1乳腺癌皮下瘤模型中能够抑制肿瘤的生长,显著延长小鼠的生存时间。
图5为本发明结果显示复制型溶瘤腺病毒AD5 sPD1CD137L在小鼠Hepa1-6肝癌细胞皮下瘤模型中能够显著抑制肿瘤的生长,约有60%小鼠肿瘤完全治愈,小鼠达到长期存活。
图6为本发明的生存结果数据显示重组溶瘤腺病毒AD5 sPD1CD137L在体内具有抗肿瘤作用(H22肝癌腹水瘤模型),能显著延长荷瘤小鼠的生存期,70%小鼠完全治愈,长期生存。
图7为本发明通过酶联免疫斑点法ELISpot和ELISA法检测分泌IFN-γ的细胞数和表达水平,数据结果显示兼具激活免疫共刺激信号通路和阻断免疫检查点的复制型溶瘤腺病毒AD5 sPD1CD137L显著增强免疫活化而提高抗肿瘤作用(H22肝癌腹水瘤模型),刺激IFN-γ的高表达,并维持T细胞的活性。
图8为本发明证实复制型溶瘤腺病毒AD5 sPD1CD137L促进抗肿瘤免疫应答依赖于CD8+T细胞,而不依赖于NK细胞。而且CD8+T是腹水中IFN-γ的主要来源。
图9为本发明融合蛋白sPD1CD137L小鼠Hepa1-6肝癌细胞皮下瘤模型中能够显著抑制肿瘤的生长。
图10为本发明复制型溶瘤腺病毒AD5 sPD1CD137L通过裂解肿瘤细胞,使肿瘤相关抗原被释放;同时招募免疫细胞,并提供维持免疫细胞活化的信号,最终产生有效抗肿瘤免疫应答。
具体实施方式
下面结合具体实施例对本发明进行进一步的解释和说明,但应理解,所给出的实施例只作为举例说明,不以任何方式对本发明构成任何限制。
实施例1
新型复制型溶瘤腺病毒AD5 sPD1CD137L的构建、制备、抗肿瘤免疫活化评估和抗肿瘤作用评价
1实验材料和方法
1.1实验材料和仪器
1.1.1实验细胞系
人胚肾细胞株293T、人肝癌细胞株LM3、小鼠黑色素瘤细胞株B16/F10小鼠肝癌细胞株H22和Hepa1-6、小鼠乳腺癌细胞株4T1,使用含10%胎牛血清,100U/I青霉素和1mg/ml链霉素的高糖DMEM培养基培养于37℃、5%CO2的培养箱中。
1.1.2实验仪器
生物安全柜( III advance,Class II Biological SafetyCabinet,The Baker Company),二氧化碳培养箱(FORMA SERIES II WATER JACKET CO2incubator,Thermo),低温离心机(HERAEUS MEGAFUGE 1.0R,Thermo),垂直电泳槽(BIO-RAD),电泳仪(BIO-RAD),半干转-转膜仪(BIO-RAD),免疫印迹曝光系统(Alpha Innotech),PCR仪(PCR Thermal Cycler Dice,TaKaRa),实时定量PCR仪及分析软件(ABI384,SequenceDetection Software,Version 1.3.1),酶标仪(VERSA max microplate reader),整套移液器(eppendorf和RAININ),细胞计数仪(Countstar Automated cell counter,Inno-Alliance Biotech Inc.,Wilmington,USA),流式细胞仪(FACSCalibur,Becton,Dickinsonand Company,USA),FlowJo software(Version 7.6.5,Tree Star Inc,Ashland,Oregon),微孔板振荡器(QiLinBeiEr),核酸纯度浓度检测仪(Biophotometer plus,eppendorf),数显恒温水浴锅(国华电器)。
1.1.3主要实验试剂及耗材
引物均由金斯瑞公司合成。肿瘤细胞培养所需的DMEM高糖培养基,双抗,血清均购自Invitrogen(上海)公司。定量RT-PCR试剂Faststart Universal SYBR Green Master(Roche,04913914001)。Western Blot所需试剂耗材:蛋白酶抑制剂(Roche,11873580001),细胞裂解液(碧云天:P0013),PVDF膜(Roche,03010040001),WB Immobilon ECL发光液(Millipore,WBKLS0500),一抗稀释液(碧云天,P0023A),HRP标记的二抗(Multisciences,GAR007and GAM007,1:5000稀释),其余所需试剂均为国产分析纯,购自南京大学化学化工学院。台盼蓝(碧云天,C0011),Opti-MEM购自Invitrogen(上海)公司。Western Blot抗体:anti-His(金斯瑞,MB001,1:5000稀释)。
1.1.4实验方法
AD5 sPD1CD137L病毒构建
可溶性蛋白sPD1CD137L的基因克隆以及携载sPD1CD137L基因的腺病毒穿梭质粒的构建
小鼠PD1和CD137L都属于膜蛋白,其结构依次是:N端信号肽-胞外区-跨膜区-胞内区C端。PD1与CD137L分别与PD-L1和CD137结合的功能单位是胞外区,sPD1CD137L仅将PD1和CD137L的胞外区进行融合表达,中间使用连接肽(Linker)GGGSGGGSGGGS连接,保留PD1的N端信号肽区域(见图1);
可溶性蛋白sPD1CD137L的基因克隆:分别设计合成引物PD1-F、PD1-R、CD137L-F、CD137L-R,使用PD1-F和PD1-R引物。以小鼠脾脏cDNA为模板扩增得到片段EXO-PD1;使用CD137L-F和CD137L-R引物,以小鼠肝癌细胞Hep1-6的cDNA为模板扩增得到片段EXO-CD137L;体外合成Linker DNA;引物PD1-R与CD137L-F分别有16bp左右与linker序列5’和3’完全一致。使用PCR技术以PD1-F和CD137L-R为引物,将EXO-PD1、linker、EXO-CD137L片段按设计拼接,完成sPD1CD137L基因克隆。sPD1CD137L、EXO-PD1、linker、EXO-CD137L和信号肽的蛋白序列分别如序列表SEQ ID NO:1-SEQ ID NO:5所示;EXO-PD1、EXO-CD137L、linker、sPD1CD137L和信号肽的DNA序列分别如序列表SEQ ID NO:6-SEQ ID NO:10所示。基因模板构建相关引物如表1所示:
表1
携载可溶性蛋白基因的腺病毒穿梭质粒AD5-pShuttle-sPD1CD137L载体的构建:
使用Infusion技术将sPD1片段与AD5-pShuttle(pZD55)连接。具体步骤:首先使用限制性内切酶BglII对AD5-pShuttle(pZD55)线性化,纯化后片段按照sPD1-CD137L:AD5-pShuttle的2:1比例使用Infusion试剂盒(clontech lab.Inc.)进行连接,后经转化扩增验证获得携载sPD1-CD137L基因的腺病毒穿梭质粒AD5-pShuttle-sPD1-CD137L。
1.1.5 AD5 sPD1CD137L病毒构建(质粒构建、病毒拯救与扩增)
A.AD5 sPD1CD137L全长质粒构建:
将构建好的穿梭载体AD5-pShuttle-sPD1-CD137L用PmeI线性化后转入感受态pAdEasy-BJ5183中,使用含50ug/ml卡那霉素LB平板的进行筛选,挑取阳性克隆培养鉴定,鉴定正确的克隆质粒重新转化DH5a感受态进行二次筛选鉴定,鉴定正确后进行质粒大提获得AD5-sPD1-CD137L全长质粒。
B.AD5 sPD1CD137L病毒拯救:
AD5 sPD1CD137L全长质粒使用PacI线性化,纯化后6孔板中1ug/well转染293T细胞,5%CO2、37℃培养,2天后将细胞消化后转入10cm平皿,2-3天换液,至80%细胞出现病变,使用10ml培养基将细胞吹下收集至15ml离心管,反复冻融2次,3000rpm/min离心15min,收集病毒上清-80℃保存做为毒种。
C.病毒扩增:
取病毒种液50ul加入60%293T细胞10cm平皿中,5%CO2 37℃培养,细胞密度至90%以上,按照1传3比例传代,直至80%细胞出现病变,大约有10个平皿细胞,按上述方法收病毒,使用氯化铯密度梯度离心纯化病毒;使用TCID50方法进行滴度测定。
AD5 sPD1CD137L病毒功能评价
A.sPD1CD137L的表达和分泌功能:
AD5 sPD1CD137L病毒感染肿瘤细胞72小时后,收细胞和上清,使用westernBlot检测sPD1CD137L的表达和分泌功能。
B.病毒复制能力:
AD5 sPD1CD137L和AD5con病毒相同MOI感染肿瘤细胞,72小时后收细胞,反复冻融离心后得到等量病毒悬液,使用293T细胞进行病毒滴度测定;分析病毒复制能力变化。
C.溶瘤功能:
分别使用AD5 sPD1CD137L和AD5con病毒按照MOI 1到100病毒量感染肿瘤细胞,48小时后使用MTT检测细胞活性,评价AD5 sPD1CD137L的杀瘤作用。
1.1.6体内研究AD5 sPD1CD137L抗肿瘤效应与机制
A.选用6-8周龄C57BL/6小鼠在右侧腋窝建立皮下瘤模型,每只小鼠一侧接种B16/F10细胞5×105个细胞,4-6天后测量肿瘤大小至200mm3,将小鼠随机分成3组,分别是:无处理组、对照AD5病毒治疗组、AD5 sPD1CD137L病毒治疗组;a.按照分组使用相应病毒瘤内注射,每只注射病毒量5×108pfu,跟踪测量肿瘤体积,体重,至肿瘤体积大于2500mm3判定小鼠死亡,记录小鼠生存期。b.按照分组瘤内注射病毒,每只注射病毒量5×108pfu,注射两次,elispot检测免疫活化。
B.选用6-8周龄C57BL/6小鼠在腹腔建立腹水瘤模型,每只小鼠腹腔接种H22细胞1×107个细胞,第7-8天左右看到小鼠有腹水,将小鼠随机分成3组,分别是:无处理组、对照AD5病毒治疗组、AD5 sPD1CD137L病毒治疗组;a.按照分组使用相应病毒腹腔注射,每只注射病毒量5×108pfu,动态监测体重,直至小鼠死亡,记录小鼠生存期。b.按照分组腹腔注射病毒,每只注射病毒量5×108pfu,注射两次,ELISpot检测免疫活化。
1.1.7 AD5 sPD1CD137L病毒的滴度测定
1.293T细胞种于96孔板,每孔约1×103个细胞,待细胞贴壁后进行滴度测定。
2.病毒梯度的稀释:准备EP管,每个EP管加入1170μl含胎牛血清的DMEM;往第一个EP管中加入130μl病毒溶液,混匀,标记为10-1;从第一个EP管中吸取50μl于第二个EP管中,混匀,标记为10-2;依次类推,直至稀释到所需梯度为止。
3.每孔加入100μl相应梯度的病毒稀释液,每个梯度重复10个孔,37℃培养过夜。
4.5天后,将96孔板放于显微镜下观察GFP,记下每个梯度有GFP的孔数,用于病毒滴度的计算。
5.病毒滴度TCID50的计算公式:
Log10(TCID50)=L+d(s-0.5)+log10(1/v)
L=Log10最高稀释度(如最高稀释度为10倍稀释,L=1)
V=最初每孔细胞培养液的体积(ml/well)
d=Log10稀释度(如为10倍稀释,d=1)
s=各个梯度GFP比率之和
1.2.3实时定量PCR
实时定量PCR的10μl体系组成:2.6μl PCR water,上下游引物各0.2μl,2μl的模板和5μl的SYBR Green荧光染料。样品混合后,于ABI 384PCR仪上进行扩增。
1.2.4细胞总蛋白的提取及浓度测定
1)以六孔板为例,去掉细胞培养上清,用PBS洗涤2遍,去掉PBS,每孔加入200μl的胰酶,消化吹打细胞,并将细胞收入至EP管中,1500rpm离心5min。
2)去掉上清,加入PBS重悬细胞,1500rpm离心5min。
3)去掉PBS,每孔根据细胞量加入相应的含蛋白酶抑制剂的细胞裂解液,涡旋30s,置于冰上10min,重复操作三次。4℃,12000g离心15min。收集上清于另一干净的EP管中。
4)蛋白浓度的测定:根据BCA蛋白浓度测定盒说明书进行检测。取2μl蛋白样品于96孔板中,加入18μl的PBS稀释样品,最后在加入200μl的测定工作液(工作液由试剂A:试剂B=50:1),放置于60℃的烘箱中,30min后,用酶标仪在562nm测定吸光度,根据标准曲线,计算出蛋白样品的浓度。
5)每管加入1/4蛋白裂解液体积的5×loading buffer,混匀后,100℃金属浴5min,冷却后,-20℃保存备用。
1.2.5 Western blot实验
1)配胶和电泳:按照不同要求配制不同浓度的SDS-PAGE分离胶和浓缩胶根据蛋白定量的计算结果,每个样品上样量调为30μg。电泳条件:浓缩胶80V30min,分离胶120V,约80min,前提是将条带分开且不会跑出去。
2)转膜:准备滤纸和PVDF膜,先用甲醇浸泡PVDF膜,再和滤纸一同浸泡在转膜缓冲液中备用。从玻璃板中小心将胶取下,浸泡在转膜缓冲液中,按照负极-滤纸-PVDF膜-胶-滤纸-正极的三明治顺序放置,赶走气泡,根据所需条带大小不同,恒流110mA转膜60-70min。
3)封闭:转膜结束后,立即取出PVDF膜,放入5%脱脂奶粉中室温封闭1h。
4)一抗孵育:4℃孵育一抗过夜。
5)二抗孵育:用washing buffer洗涤条带,每次10min,共三次;再用相应的HPR标记的二抗室温孵育1h。
6)曝光:用washing buffer洗涤条带,每次10min,共三次;用化学发光液在WB曝光仪上曝光,并获取条带图像。
1.2.6台盼兰计数
以六孔板为例,去除细胞上清,用PBS洗涤2遍,去掉PBS,每孔加入200μl胰酶消化,轻轻吹打细胞并收集进入干净的EP管中,1500rpm,离心5min。去掉上清,加入PBS重悬细胞,1500rpm,离心5min。去掉PBS,根据细胞数量加入一定量的PBS重悬细胞,从中取出10μl细胞重悬液,加入10μl 0.2%台盼蓝溶液混合,取混合液20μl于细胞计数板中,用细胞计数仪计数。
1.2.7流式细胞仪检测细胞表面分子
1)取实体瘤细胞(腹水)105cells,加PBS清洗一遍。
2)去掉PBS,每管加入100μl含相应量的流式抗体的PBS,重悬细胞,置于冰上30min,避光。期间拿出样品,轻轻吹打,防止其因沉淀而影响抗体结合效果。
3)30min后,每管加入1ml PBS混合细胞,1500rpm,离心5min。去掉上清,再加入PBS重悬细胞,1500rpm,离心5min。去掉PBS,每管加入300μl PBS重悬,避光。
4)将准备好的样品,用流式细胞仪检测。用FlowJo软件分析实验结果。
1.2.8 Mouse IFN-γELISpot检测
1)ELISpot板子在使用前每孔加入200μl含10%血清的培养基孵育30min以上,放入细胞培养箱中。
2)去掉培养基,每孔加入200μl含细胞的培养体系。细胞体系组成:100μl的肿瘤细胞和100μl的脾脏细胞。混合均匀之后,加入孔板中,放入细胞培养箱中。且在实验结束取出之前,不要随意挪动板子。12h后,取出板子检测。
3)去掉培养基,每孔加入200μl的PBS清洗,需要清洗五遍以上。
4)去掉PBS,每孔加入100μl含一抗的稀释液。一抗稀释液:含0.5%FBS的PBS;一抗稀释比例1:1000。室温放置2h。
5)去掉一抗稀释液,用PBS洗五遍以上;每孔加入100μl二抗稀释液。二抗稀释液:含0.5%FBS的PBS;二抗稀释比例1:1000。室温放置1h。
6)去掉二抗稀释液,用PBS洗五遍以上。每孔加入200μl显色剂显色。待有蓝色斑点出现,且又不显色过头的情况下,甩掉显色液,用自来水洗多遍。
7)将自来水甩掉,室温晾干。注意:不要在室温晾的过久,且晾干过程保持避光。最后用避光保存于封口袋中。扫描读板。
1.2.9肿瘤组织及脾细胞的mouse IFN-γ的ELISpot检测
1)肿瘤组织单细胞悬液制备及mouse IFN-γ的ELISpot检测:处死小鼠,取下一小块的肿瘤组织,用PBS清洗,再放入培养皿中,加入1ml的胶原酶溶液,用剪刀剪碎,将肿瘤组织浑浊液吸入干净的离心管中,再加入1ml胶原酶溶液,放入37℃培养箱2小时,使得肿瘤组织得以消化完全。期间,每隔15min要取出,用枪头吹打混匀。2h后,取出装有肿瘤组织浑浊液的离心管,确认肿瘤组织消化完全。将浑浊液离心,取沉淀即肿瘤组织细胞。用含血清的DMEM重悬,细胞计数,将细胞浓度调为2×106个/ml,取100μl细胞混合液做鼠IFN-γ的ELISpot检测,方法同1.2.8。
2)脾单细胞悬液的制备及鼠IFN-γ的ELISpot检测:处死小鼠,取出脾脏,用PBS清洗,剪下一小块脾脏组织,放于70μl的细胞够滤网中,用5ml的注射器研磨,边研磨边加入适量的PBS冲洗。用Ficoll法去掉红细胞,离心重悬获得脾脏单细胞混合液,细胞计数,将细胞浓度调为2×106个/ml,取100μl与肿瘤细胞混合做鼠IFN-γ的ELISpot检测,方法同1.2.8。
2.实验结果与结论
图1为本发明的表达可溶性sPD1CD137L的重组溶瘤腺病毒的构建(A)重组溶瘤腺病毒AD5con和AD5 sPD1CD137L的基因结构原理图。(B)B16/F10小鼠黑色素瘤细胞分别感染AD5con和AD5 sPD1CD137L,感染复数(MOI)为10,48h后,被感染细胞的上清被收集起来,通过western blot的方法检测融合蛋白sPD1CD137L的表达与分泌。数据代表三次独立性重复实验。GFP,绿色荧光蛋白;E1A,病毒早期区域1复制元件(early region 1);sPD1CD137L,游离融合蛋白PD1CD137L。
图2为本发明的重组溶瘤腺病毒AD5 sPD1CD137L的复制与溶瘤(A)B16/F10小鼠黑色素瘤细胞株、H22小鼠肝癌腹水瘤细胞株、Hepa1-6小鼠肝癌细胞株、LM3人肝癌细胞株分别感染AD5con和AD5 sPD1CD137L,MOI=10,分别在12,24,36,48,60和72h收取细胞,提取病毒基因组DNA,通过Q-PCR检测AD5的拷贝数。(B)B16/F10小鼠黑色素瘤细胞株、H22小鼠肝癌腹水瘤细胞株、Hepa1-6小鼠肝癌细胞株、LM3人肝癌细胞株分别感染AD5con和AD5sPD1CD137后,CCK8检测细胞活率。数据代表三次独立性重复实验.
图3为本发明的重组溶瘤腺病毒AD5 sPD1CD137L的体内抗肿瘤作用(B16/F10黑色素瘤实体瘤模型)(A)在B16/F10皮下瘤模型中评估AD5sPD1CD137L的抗肿瘤效果,实验方案图如图所示。(B)C57BL/6右侧皮下接种5×106B16/F10小鼠黑色素瘤细胞,瘤内注射5×108pfu AD5con和AD5sPD1CD137L,实时监测肿瘤大小。数据代表三次独立性重复试验。Pfu,空斑形成单位;Mock,生理盐水处理作为阴性对照;n.s.无统计学差异;**p<0.01。
图4为本发明的重组溶瘤腺病毒AD5 sPD1CD137L的体内抗肿瘤作用(4T1乳腺癌实体瘤模型)(A)在乳腺癌4T1皮下瘤模型中评估AD5 sPD1CD137L的抗肿瘤效果,实验方案图如图所示。(B)Balb/c右侧皮下接种5×104 4T1小鼠乳腺癌细胞,瘤内注射5×108pfuAD5con和AD5 sPD1CD137L,实时监测肿瘤大小。(C)以肿瘤体积大于2cm3认为小鼠死亡,统计生存曲线。数据代表三次独立性重复试验。Pfu,空斑形成单位;Saline,生理盐水处理作为阴性对照;n.s.无统计学差异;**p<0.01。
图5为本发明的重组溶瘤腺病毒AD5 sPD1CD137L的体内抗肿瘤作用(Hepa1-6肝癌实体瘤模型)(A)在Hepa1-6皮下瘤模型中评估AD5sPD1CD137L的抗肿瘤效果,实验方案图如图所示。(B)C57BL/6小鼠右侧皮下接种5×106Hepa1-6肝癌细胞,瘤内注射5×108pfuAD5con和AD5 sPD1CD137L,肿瘤大小被实时监测。(C)以肿瘤体积大于2cm3认为小鼠死亡,统计生存曲线。数据代表三次独立性重复试验。Pfu,空斑形成单位;Saline,生理盐水处理作为阴性对照;n.s.,无统计学差异;**,p<0.01。
图6为本发明的重组溶瘤腺病毒AD5 sPD1CD137L的体内抗肿瘤作用(H22肝癌腹水瘤模型)(A)在H22肝癌腹水瘤模型中评估AD5 sPD1CD137L的抗肿瘤效果,实验方案图如图所示。(B)C57BL/6腹腔接种5×106H22小鼠肝癌腹水瘤细胞,小鼠出现腹水后,腹腔注射5×108pfu AD5con和AD5 sPD1CD137L,实时监测小鼠生存时间。(C)被治愈的小鼠在90天后,腹腔再次接种5×106H22小鼠肝癌腹水瘤细胞,以未经任何处理的小鼠腹腔接种同样数量的H22细胞作为对照,监测小鼠生存情况。数据代表三次独立性重复实验。Pfu,空斑形成单位;Mock,生理盐水处理作为阴性对照;之前未接种过肿瘤的小鼠;n.s.无统计学差异;***p<0.001。
图7为本发明的重组溶瘤腺病毒AD5 sPD1CD137L增强免疫活化通过H22肝癌腹水瘤模型中评估AD5 sPD1CD137L的免疫活化作用,实验方案图如图Fig.6A所示。(A)C57BL/6腹腔接种5×106H22小鼠肝癌腹水瘤细胞,小鼠出现腹水后经腹腔注射5×108pfu AD5con和AD5 sPD1CD137L,第14天用ELISA方法检测腹水中游离sPD1CD137L水平。(B)ELISpot检测活化的免疫细胞水平。(C)ELISA检测腹水中的IFN-γ水平。数据代表三次独立性重复实验。Mock,生理盐水处理作为阴性对照;*p<0.05。
图8为本发明的重组溶瘤腺病毒AD5 sPD1CD137L清除H22肝癌细胞依赖于CD8+T细胞,而不依赖于NK细胞。(A)通过H22肝癌腹水瘤模型研究AD5sPD1CD137L诱导的抗肿瘤免疫应答的机制,实验方案图如图所示。(B)C57BL/6腹腔接种5×106H22小鼠肝癌腹水瘤细胞,在第10和18天分别注射anti-CD8a或Anti-NK1.1抗体。注射抗体5天后流式检测小鼠外周血中淋巴细胞亚群清除情况。(C)小鼠出现腹水后,经腹腔注射上述中和抗体清除CD8+T细胞或NK细胞后,再腹腔注射5×108pfu AD5 sPD1CD137L,监测小鼠生存率。(D)抗体封闭后检测腹水中IFN-γ的水平。数据代表三次独立性重复实验。Pfu,空斑形成单位;Saline,生理盐水处理组作为阴性对照;#无统计学差异;*p<0.05;***p<0.001。
图9为本发明的融合蛋白sPD1CD137L的体内抗肿瘤作用(Hepa1-6肝癌实体瘤模型)(A)在肝癌Hepa1-6皮下瘤模型中评估融合蛋白sPD1CD137L的抗肿瘤效果,实验方案图如图所示。(B)C57BL/6小鼠右侧皮下接种5×106Hepa1-6肿瘤细胞,待肿瘤出现后,腹腔注射300μl含融合蛋白sPD1CD137L的上清液,实时监测肿瘤大小。n.s.无统计学差异;**p<0.01。
图10为本发明的重组溶瘤腺病毒AD5 sPD1CD137L的工作原理。重组溶瘤腺病毒AD5 sPD1CD137L感染肿瘤细胞,一方面促进肿瘤细胞的裂解,引起肿瘤细胞免疫原性细胞死亡,活化并招募免疫细胞;另一方面感染的肿瘤细胞分泌融合蛋白sPD1CD137L,进入肿瘤微环境。该蛋白sPD1CD137L:1)PD1端能够特异性结合肿瘤细胞及其它表达PD-L1免疫抑制细胞的PD-L1分子,阻止抗肿瘤效应T细胞因PD-L1/PD1信号导致细胞失能耗竭;2)CD137L端能够特异性结合T细胞表面共刺激分子受体CD137,诱导持续的T细胞活化,并招募更多淋巴细胞浸润到肿瘤局部,产生更有效抗肿瘤免疫应答;3)因肿瘤微环境免疫活化而上调的PD-L1,使sPD1CD137L能够被捕获在肿瘤组织局部,不易扩散到血液中。因而降低了脱靶效应;4)融合蛋白是肿瘤细胞和效应T细胞双亲分子,因此可以作为桥梁,促进肿瘤细胞与T细胞的接触,增加T细胞对肿瘤细胞的杀伤。
PD1,细胞死亡受体;PD-L1,细胞死亡受体配体;Adenovirus,腺病毒;Ad5-PC,表达PD1CD137L融合蛋白的腺病毒;Perforin,穿孔素;FAS,自杀相关因子;FASL,FAS配体。
由以上结果可知,本发明提供了一种可以兼具激活免疫共刺激信号通路和阻断免疫检查点的复制型溶瘤腺病毒AD5 sPD1CD137L的设计和构建方法,成功获得了一株新型兼具激活免疫共刺激信号通路和阻断免疫检查点的复制型溶瘤腺病毒AD5 sPD1CD137L,该病毒可以选择性地在肿瘤细胞内和肿瘤部位复制、具有肿瘤靶向性,能够有效溶瘤,并诱导免疫原性细胞死亡。与此同时,该病毒能够高表达可溶性融合蛋白sPD1CD137L,该蛋白能够分泌到细胞外,在肿瘤微环境中发挥阻断免疫检查点、激活免疫共刺激信号通路,进而活化免疫的生物学功能。本发明的兼具激活免疫共刺激信号通路和阻断免疫检查点的复制型溶瘤腺病毒AD5 sPD1CD137L具有显著的活化抗肿瘤免疫作用,能够显著刺激IFN-γ在肿瘤局部高表达且没有明显的全身毒性,显著抑制肿瘤生长、延长生存期,具有显著的抗肿瘤作用。一个病毒,同时整合多种独特的抗肿瘤机制于一身,具有预料不到的抗肿瘤效果。可以用来制备抗肿瘤药物。
以上显示和描述了本发明的基本原理、主要特征和本发明的优点。本行业的技术人员应该了解,本发明不受上述实施例的限制,上述实施例和说明书中描述的只是说明本发明的原理,在不脱离本发明精神和范围的前提下,本发明还会有各种变化和改进,本发明要求保护范围由所附的权利要求书、说明书及其等效物界定。
序列表
<110> 南京大学
<120> 兼具激活免疫共刺激信号通路和阻断免疫检查点的复制型溶瘤腺病毒及其应用
<130> 2019
<160> 10
<170> SIPOSequenceListing 1.0
<210> 1
<211> 385
<212> PRT
<213> mouse(Mus musculus)
<400> 1
Met Trp Val Arg Gln Val Pro Trp Ser Phe Thr Trp Ala Val Leu Gln
1 5 10 15
Leu Ser Trp Gln Ser Gly Trp Leu Leu Glu Val Pro Asn Gly Pro Trp
20 25 30
Arg Ser Leu Thr Phe Tyr Pro Ala Trp Leu Thr Val Ser Glu Gly Ala
35 40 45
Asn Ala Thr Phe Thr Cys Ser Leu Ser Asn Trp Ser Glu Asp Leu Met
50 55 60
Leu Asn Trp Asn Arg Leu Ser Pro Ser Asn Gln Thr Glu Lys Gln Ala
65 70 75 80
Ala Phe Cys Asn Gly Leu Ser Gln Pro Val Gln Asp Ala Arg Phe Gln
85 90 95
Ile Ile Gln Leu Pro Asn Arg His Asp Phe His Met Asn Ile Leu Asp
100 105 110
Thr Arg Arg Asn Asp Ser Gly Ile Tyr Leu Cys Gly Ala Ile Ser Leu
115 120 125
His Pro Lys Ala Lys Ile Glu Glu Ser Pro Gly Ala Glu Leu Val Val
130 135 140
Thr Glu Arg Ile Leu Glu Thr Ser Thr Arg Tyr Pro Ser Pro Ser Pro
145 150 155 160
Lys Pro Glu Gly Arg Phe Gln Gly Met Val Gly Gly Gly Gly Ser Gly
165 170 175
Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Leu Thr Ile Thr Thr Ser
180 185 190
Pro Asn Leu Gly Thr Arg Glu Asn Asn Ala Asp Gln Val Thr Pro Val
195 200 205
Ser His Ile Gly Cys Pro Asn Thr Thr Gln Gln Gly Ser Pro Val Phe
210 215 220
Ala Lys Leu Leu Ala Lys Asn Gln Ala Ser Leu Cys Asn Thr Thr Leu
225 230 235 240
Asn Trp His Ser Gln Asp Gly Ala Gly Ser Ser Tyr Leu Ser Gln Gly
245 250 255
Leu Arg Tyr Glu Glu Asp Lys Lys Glu Leu Val Val Asp Ser Pro Gly
260 265 270
Leu Tyr Tyr Val Phe Leu Glu Leu Lys Leu Ser Pro Thr Phe Thr Asn
275 280 285
Thr Gly His Lys Val Gln Gly Trp Val Ser Leu Val Leu Gln Ala Lys
290 295 300
Pro Gln Val Asp Asp Phe Asp Asn Leu Ala Leu Thr Val Glu Leu Phe
305 310 315 320
Pro Cys Ser Met Glu Asn Lys Leu Val Asp Arg Ser Trp Ser Gln Leu
325 330 335
Leu Leu Leu Lys Ala Gly His Arg Leu Ser Val Gly Leu Arg Ala Tyr
340 345 350
Leu His Gly Ala Gln Asp Ala Tyr Arg Asp Trp Glu Leu Ser Tyr Pro
355 360 365
Asn Thr Thr Ser Phe Gly Leu Phe Leu Val Lys Pro Asp Asn Pro Trp
370 375 380
Glu
385
<210> 2
<211> 170
<212> PRT
<213> mouse(Mus musculus)
<400> 2
Met Trp Val Arg Gln Val Pro Trp Ser Phe Thr Trp Ala Val Leu Gln
1 5 10 15
Leu Ser Trp Gln Ser Gly Trp Leu Leu Glu Val Pro Asn Gly Pro Trp
20 25 30
Arg Ser Leu Thr Phe Tyr Pro Ala Trp Leu Thr Val Ser Glu Gly Ala
35 40 45
Asn Ala Thr Phe Thr Cys Ser Leu Ser Asn Trp Ser Glu Asp Leu Met
50 55 60
Leu Asn Trp Asn Arg Leu Ser Pro Ser Asn Gln Thr Glu Lys Gln Ala
65 70 75 80
Ala Phe Cys Asn Gly Leu Ser Gln Pro Val Gln Asp Ala Arg Phe Gln
85 90 95
Ile Ile Gln Leu Pro Asn Arg His Asp Phe His Met Asn Ile Leu Asp
100 105 110
Thr Arg Arg Asn Asp Ser Gly Ile Tyr Leu Cys Gly Ala Ile Ser Leu
115 120 125
His Pro Lys Ala Lys Ile Glu Glu Ser Pro Gly Ala Glu Leu Val Val
130 135 140
Thr Glu Arg Ile Leu Glu Thr Ser Thr Arg Tyr Pro Ser Pro Ser Pro
145 150 155 160
Lys Pro Glu Gly Arg Phe Gln Gly Met Val
165 170
<210> 3
<211> 200
<212> PRT
<213> mouse(Mus musculus)
<400> 3
Ala Leu Thr Ile Thr Thr Ser Pro Asn Leu Gly Thr Arg Glu Asn Asn
1 5 10 15
Ala Asp Gln Val Thr Pro Val Ser His Ile Gly Cys Pro Asn Thr Thr
20 25 30
Gln Gln Gly Ser Pro Val Phe Ala Lys Leu Leu Ala Lys Asn Gln Ala
35 40 45
Ser Leu Cys Asn Thr Thr Leu Asn Trp His Ser Gln Asp Gly Ala Gly
50 55 60
Ser Ser Tyr Leu Ser Gln Gly Leu Arg Tyr Glu Glu Asp Lys Lys Glu
65 70 75 80
Leu Val Val Asp Ser Pro Gly Leu Tyr Tyr Val Phe Leu Glu Leu Lys
85 90 95
Leu Ser Pro Thr Phe Thr Asn Thr Gly His Lys Val Gln Gly Trp Val
100 105 110
Ser Leu Val Leu Gln Ala Lys Pro Gln Val Asp Asp Phe Asp Asn Leu
115 120 125
Ala Leu Thr Val Glu Leu Phe Pro Cys Ser Met Glu Asn Lys Leu Val
130 135 140
Asp Arg Ser Trp Ser Gln Leu Leu Leu Leu Lys Ala Gly His Arg Leu
145 150 155 160
Ser Val Gly Leu Arg Ala Tyr Leu His Gly Ala Gln Asp Ala Tyr Arg
165 170 175
Asp Trp Glu Leu Ser Tyr Pro Asn Thr Thr Ser Phe Gly Leu Phe Leu
180 185 190
Val Lys Pro Asp Asn Pro Trp Glu
195 200
<210> 4
<211> 15
<212> PRT
<213> 人工序列(Mus musculus)
<400> 4
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
1 5 10 15
<210> 5
<211> 16
<212> PRT
<213> mouse(Mus musculus)
<400> 5
Met Leu Trp Pro Leu Pro Leu Phe Leu Leu Cys Ala Gly Ser Leu Ala
1 5 10 15
<210> 6
<211> 510
<212> DNA
<213> mouse(Mus musculus)
<400> 6
atgtgggtcc ggcaggtacc ctggtcattc acttgggctg tgctgcagtt gagctggcaa 60
tcagggtggc ttctagaggt ccccaatggg ccctggaggt ccctcacctt ctacccagcc 120
tggctcacag tgtcagaggg agcaaatgcc accttcacct gcagcttgtc caactggtcg 180
gaggatctta tgctgaactg gaaccgcctg agtcccagca accagactga aaaacaggcc 240
gccttctgta atggtttgag ccaacccgtc caggatgccc gcttccagat catacagctg 300
cccaacaggc atgacttcca catgaacatc cttgacacac ggcgcaatga cagtggcatc 360
tacctctgtg gggccatctc cctgcacccc aaggcaaaaa tcgaggagag ccctggagca 420
gagctcgtgg taacagagag aatcctggag acctcaacaa gatatcccag cccctcgccc 480
aaaccagaag gccggtttca aggcatggtc 510
<210> 7
<211> 600
<212> DNA
<213> mouse(Mus musculus)
<400> 7
gcgctcacaa tcaccacctc gcccaacctg ggtacccgag agaataatgc agaccaggtc 60
acccctgttt cccacattgg ctgccccaac actacacaac agggctctcc tgtgttcgcc 120
aagctactgg ctaaaaacca agcatcgttg tgcaatacaa ctctgaactg gcacagccaa 180
gatggagctg ggagctcata cctatctcaa ggtctgaggt acgaagaaga caaaaaggag 240
ttggtggtag acagtcccgg gctctactac gtatttttgg aactgaagct cagtccaaca 300
ttcacaaaca caggccacaa ggtgcagggc tgggtctctc ttgttttgca agcaaagcct 360
caggtagatg actttgacaa cttggccctg acagtggaac tgttcccttg ctccatggag 420
aacaagttag tggaccgttc ctggagtcaa ctgttgctcc tgaaggctgg ccaccgcctc 480
agtgtgggtc tgagggctta tctgcatgga gcccaggatg catacagaga ctgggagctg 540
tcttatccca acaccaccag ctttggactc tttcttgtga aacccgacaa cccatgggaa 600
<210> 8
<211> 45
<212> DNA
<213> 人工序列(Mus musculus)
<400> 8
ggtggaggcg gttcaggcgg aggtggctct ggcggtggcg gatcg 45
<210> 9
<211> 1155
<212> DNA
<213> mouse(Mus musculus)
<400> 9
atgtgggtcc ggcaggtacc ctggtcattc acttgggctg tgctgcagtt gagctggcaa 60
tcagggtggc ttctagaggt ccccaatggg ccctggaggt ccctcacctt ctacccagcc 120
tggctcacag tgtcagaggg agcaaatgcc accttcacct gcagcttgtc caactggtcg 180
gaggatctta tgctgaactg gaaccgcctg agtcccagca accagactga aaaacaggcc 240
gccttctgta atggtttgag ccaacccgtc caggatgccc gcttccagat catacagctg 300
cccaacaggc atgacttcca catgaacatc cttgacacac ggcgcaatga cagtggcatc 360
tacctctgtg gggccatctc cctgcacccc aaggcaaaaa tcgaggagag ccctggagca 420
gagctcgtgg taacagagag aatcctggag acctcaacaa gatatcccag cccctcgccc 480
aaaccagaag gccggtttca aggcatggtc ggtggaggcg gttcaggcgg aggtggctct 540
ggcggtggcg gatcggcgct cacaatcacc acctcgccca acctgggtac ccgagagaat 600
aatgcagacc aggtcacccc tgtttcccac attggctgcc ccaacactac acaacagggc 660
tctcctgtgt tcgccaagct actggctaaa aaccaagcat cgttgtgcaa tacaactctg 720
aactggcaca gccaagatgg agctgggagc tcatacctat ctcaaggtct gaggtacgaa 780
gaagacaaaa aggagttggt ggtagacagt cccgggctct actacgtatt tttggaactg 840
aagctcagtc caacattcac aaacacaggc cacaaggtgc agggctgggt ctctcttgtt 900
ttgcaagcaa agcctcaggt agatgacttt gacaacttgg ccctgacagt ggaactgttc 960
ccttgctcca tggagaacaa gttagtggac cgttcctgga gtcaactgtt gctcctgaag 1020
gctggccacc gcctcagtgt gggtctgagg gcttatctgc atggagccca ggatgcatac 1080
agagactggg agctgtctta tcccaacacc accagctttg gactctttct tgtgaaaccc 1140
gacaacccat gggaa 1155
<210> 10
<211> 48
<212> DNA
<213> mouse(Mus musculus)
<400> 10
atgctgtggc cactgccgct gttcttgctg tgtgcaggct ccctggct 48
Claims (14)
1.一种兼具激活免疫共刺激信号通路和阻断免疫检查点的可溶性融合蛋白,其特征在于:所述的兼具激活免疫共刺激信号通路和阻断免疫检查点的可溶性融合蛋白的两端分别为结合PD-L1的PD1和结合CD137的CD137L,PD1和CD137L之间通过linker序列连接。
2.如权利要求1所述的兼具激活免疫共刺激信号通路和阻断免疫检查点的可溶性融合蛋白,其特征在于:所述的可溶性融合蛋白为sPD1CD137L,sPD1CD137L的蛋白序列和氨基酸序列分别如序列表SEQ ID NO:1和SEQ ID NO:6所示。
3.权利要求1或2所述的兼具激活免疫共刺激信号通路和阻断免疫检查点的可溶性融合蛋白在制备活化抗肿瘤免疫药物中的应用。
4.权利要求1或2所述的兼具激活免疫共刺激信号通路和阻断免疫检查点的可溶性融合蛋白在制备刺激IFN-γ表达药物中的应用。
5.权利要求1或2所述的兼具激活免疫共刺激信号通路和阻断免疫检查点的可溶性融合蛋白在制备抗肿瘤药物中的应用。
6.如权利要求5所述的兼具激活免疫共刺激信号通路和阻断免疫检查点的可溶性融合蛋白在制备抗肿瘤药物中的应用,其特征在于:所述的肿瘤为肝癌、腹水癌、黑色素瘤或乳腺癌。
7.一种兼具激活免疫共刺激信号通路和阻断免疫检查点的复制型溶瘤腺病毒,其特征在于:所述的复制型溶瘤腺病毒在肿瘤细胞内复制,并且表达和分泌可溶性融合蛋白,所述的可溶性融合蛋白的两端分别为结合PD-L1的PD1和结合CD137的CD137L,PD1和CD137L之间通过linker序列连接。
8.如权利要求7所述的兼具激活免疫共刺激信号通路和阻断免疫检查点的复制型溶瘤腺病毒,其特征在于:所述的可溶性融合蛋白为sPD1CD137L,sPD1CD137L的蛋白序列的氨基酸序列如序列表SEQ ID NO:1所示。
9.权利要求7或8所述的兼具激活免疫共刺激信号通路和阻断免疫检查点的复制型溶瘤腺病毒,其特征在于:所述的复制型溶瘤腺病毒能够溶瘤。
10.权利要求7或8所述的兼具激活免疫共刺激信号通路和阻断免疫检查点的复制型溶瘤腺病毒在制备活化抗肿瘤免疫药物中的应用。
11.权利要求7或8所述的兼具激活免疫共刺激信号通路和阻断免疫检查点的复制型溶瘤腺病毒在制备刺激IFN-γ表达药物中的应用。
12.权利要求7或8所述的兼具激活免疫共刺激信号通路和阻断免疫检查点的复制型溶瘤腺病毒在制备抗肿瘤药物中的应用。
13.如权利要求12所述的兼具激活免疫共刺激信号通路和阻断免疫检查点的复制型溶瘤腺病毒在制备抗肿瘤药物中的应用,其特征在于:所述的肿瘤为肝癌、腹水癌、黑色素瘤或乳腺癌。
14.一种兼具激活免疫共刺激信号通路和阻断免疫检查点的复制型溶瘤腺病毒AD5sPD1CD137L的构建方法,其特征在于
包括如下步骤:(1)AD5sPD1CD137L全长质粒构建:将构建好的穿梭载体AD5-pShuttle-sPD1-CD137L用PmeI线性化后转入感受态pAdEasy-BJ5183中,使用含50ug/ml卡那霉素LB平板的进行筛选,挑取阳性克隆培养鉴定,鉴定正确的克隆质粒重新转化DH5a感受态进行二次筛选鉴定,鉴定正确后进行质粒大提获得AD5-sPD1-CD137L全长质粒;
(2)AD5sPD1CD137L病毒拯救:AD5sPD1CD137L全长质粒使用PacI线性化,纯化后6孔板中1ug/well转染293T细胞,5%CO2、37℃培养,2天后将细胞消化后转入10cm平皿,2-3天换液,至80%细胞出现病变,使用10ml培养基将细胞吹下收集至15ml离心管,反复冻融2次,3000rpm/min离心15min,收集病毒上清-80℃保存做为毒种;
(3)病毒扩增:取病毒种液50ul加入60%293T细胞10cm平皿中,5%CO237℃培养,细胞密度至90%以上,按照1传3比例传代,直至80%细胞出现病变,大约有10个平皿细胞,按上述方法收病毒,使用氯化铯密度梯度离心纯化病毒;使用TCID50方法进行滴度测定。
Priority Applications (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201910140526.2A CN111606999B (zh) | 2019-02-26 | 2019-02-26 | 兼具激活免疫共刺激信号通路和阻断免疫检查点的复制型溶瘤腺病毒及其应用 |
PCT/CN2019/115627 WO2020173123A1 (zh) | 2019-02-26 | 2019-11-05 | 兼具激活免疫共刺激信号通路和阻断免疫检查点的复制型溶瘤腺病毒及其应用 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201910140526.2A CN111606999B (zh) | 2019-02-26 | 2019-02-26 | 兼具激活免疫共刺激信号通路和阻断免疫检查点的复制型溶瘤腺病毒及其应用 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN111606999A true CN111606999A (zh) | 2020-09-01 |
CN111606999B CN111606999B (zh) | 2022-09-06 |
Family
ID=72198025
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN201910140526.2A Active CN111606999B (zh) | 2019-02-26 | 2019-02-26 | 兼具激活免疫共刺激信号通路和阻断免疫检查点的复制型溶瘤腺病毒及其应用 |
Country Status (2)
Country | Link |
---|---|
CN (1) | CN111606999B (zh) |
WO (1) | WO2020173123A1 (zh) |
Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN112852757A (zh) * | 2021-02-01 | 2021-05-28 | 南京大学 | 一种制备新型溶瘤病毒EM/VSV-G Ad5sPVRCD137L的方法 |
CN112941039A (zh) * | 2021-02-01 | 2021-06-11 | 南京大学 | 一种新型类囊泡溶瘤病毒及其在制备抗肿瘤药物上的应用 |
CN113832111A (zh) * | 2020-06-23 | 2021-12-24 | 南京大学 | 一种类外泌体技术用于制备新型溶瘤病毒的方法 |
CN114129711A (zh) * | 2020-09-03 | 2022-03-04 | 南京大学 | 度拉糖肽在制备抗肿瘤药物中的应用 |
Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN102174522A (zh) * | 2011-02-24 | 2011-09-07 | 杭州师范大学 | 一种4-1bbl蛋白的制备方法 |
CN105407902A (zh) * | 2013-03-05 | 2016-03-16 | 贝勒医学院 | 溶瘤病毒 |
CN108026151A (zh) * | 2015-06-19 | 2018-05-11 | 塞巴斯蒂安·科博尔德 | Pd-1-cd28融合蛋白及其在医学中的用途 |
WO2018127917A1 (en) * | 2017-01-05 | 2018-07-12 | Kahr Medical Ltd. | A pd1-41bbl fusion protein and methods of use thereof |
CN108350055A (zh) * | 2015-10-01 | 2018-07-31 | 热生物制品有限公司 | 作为异源嵌合蛋白邻接i型和ii型胞外结构域的组合物和方法 |
Family Cites Families (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP3778881A1 (en) * | 2016-01-08 | 2021-02-17 | Replimune Limited | Modified oncolytic virus |
US20190022203A1 (en) * | 2016-01-11 | 2019-01-24 | Turnstone Limited Partnership | Oncolytic virus and checkpoint inhibitor combination therapy |
CN108728488A (zh) * | 2017-04-19 | 2018-11-02 | 生命序有限公司 | 溶瘤病毒构建体、溶瘤病毒及其应用 |
CN108165536A (zh) * | 2017-12-11 | 2018-06-15 | 浙江大学 | 一种重组溶瘤痘苗病毒及其制备方法与应用 |
-
2019
- 2019-02-26 CN CN201910140526.2A patent/CN111606999B/zh active Active
- 2019-11-05 WO PCT/CN2019/115627 patent/WO2020173123A1/zh active Application Filing
Patent Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN102174522A (zh) * | 2011-02-24 | 2011-09-07 | 杭州师范大学 | 一种4-1bbl蛋白的制备方法 |
CN105407902A (zh) * | 2013-03-05 | 2016-03-16 | 贝勒医学院 | 溶瘤病毒 |
CN108026151A (zh) * | 2015-06-19 | 2018-05-11 | 塞巴斯蒂安·科博尔德 | Pd-1-cd28融合蛋白及其在医学中的用途 |
CN108350055A (zh) * | 2015-10-01 | 2018-07-31 | 热生物制品有限公司 | 作为异源嵌合蛋白邻接i型和ii型胞外结构域的组合物和方法 |
WO2018127917A1 (en) * | 2017-01-05 | 2018-07-12 | Kahr Medical Ltd. | A pd1-41bbl fusion protein and methods of use thereof |
Non-Patent Citations (5)
Title |
---|
D-P XU等: "The systemic administration of Ig-4-1BB ligand in combination with IL-12 gene transfer eradicates hepatic colon carcinoma", 《GENE THERAPY》 * |
PETER M. BLACK等: "《神经系统肿瘤学》", 31 January 2008, 人民卫生出版社 * |
余元勋等: "《中国分子胃癌学》", 30 April 2016, 安徽科学技术出版社 * |
苏春霞: "《肺癌的免疫治疗新进展》", 30 June 2016, 上海科学普及出版社 * |
魏继武: "溶瘤免疫治疗的机遇与挑战", 《医学研究生学报》 * |
Cited By (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN113832111A (zh) * | 2020-06-23 | 2021-12-24 | 南京大学 | 一种类外泌体技术用于制备新型溶瘤病毒的方法 |
CN114129711A (zh) * | 2020-09-03 | 2022-03-04 | 南京大学 | 度拉糖肽在制备抗肿瘤药物中的应用 |
CN114129711B (zh) * | 2020-09-03 | 2024-02-09 | 南京大学 | 度拉糖肽在制备抗肿瘤药物中的应用 |
CN112852757A (zh) * | 2021-02-01 | 2021-05-28 | 南京大学 | 一种制备新型溶瘤病毒EM/VSV-G Ad5sPVRCD137L的方法 |
CN112941039A (zh) * | 2021-02-01 | 2021-06-11 | 南京大学 | 一种新型类囊泡溶瘤病毒及其在制备抗肿瘤药物上的应用 |
Also Published As
Publication number | Publication date |
---|---|
CN111606999B (zh) | 2022-09-06 |
WO2020173123A1 (zh) | 2020-09-03 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN110128550B (zh) | 一种新型的同时阻断免疫检查点pd-l1和tigit的复制型溶瘤腺病毒和应用 | |
CN111606999B (zh) | 兼具激活免疫共刺激信号通路和阻断免疫检查点的复制型溶瘤腺病毒及其应用 | |
US11806374B2 (en) | Isolated recombinant oncolytic adenoviruses, pharmaceutical compositions, and uses thereof for drugs for treatment of tumors and/or cancers | |
CN109554353B (zh) | 分离的重组溶瘤痘病毒、药物组合物及其在治疗肿瘤和/或癌症的药物中的用途 | |
JP7378840B2 (ja) | インターフェロンを発現する腫瘍溶解性ウイルス及びその応用 | |
CN110157686B (zh) | 一种免疫检查点激活免疫共刺激的复制型溶瘤腺病毒及其构建方法和应用 | |
JP7420751B2 (ja) | I型インターフェロン及びcd40-配位子を用いる腫瘍溶解性ウイルス又は抗原提示細胞媒介性癌治療 | |
CN105368859A (zh) | 一种嵌合抗原受体hCD87-CAR及载有hCD87-CAR基因结构的慢病毒及质粒及其应用 | |
CN111607571B (zh) | 一种特异性激活免疫共刺激通路的复制型溶瘤腺病毒及其制备方法和应用 | |
CN110325200A (zh) | 治疗剂及其在治疗肿瘤和/或癌症的药物中的应用 | |
CN110996980B (zh) | 一种用于治疗肿瘤的病毒 | |
CN115212299A (zh) | Car-t和car-m联用在制备抗肿瘤药物中的应用 | |
CN104001185A (zh) | 一种cea阳性肿瘤特异性树突状细胞疫苗的制备方法 | |
Bian et al. | In vivo efficacy of systemic tumor targeting of a viral RNA vector with oncolytic properties using a bispecific adapter protein | |
WO2023124973A1 (zh) | 一种利用外源性抗原和治疗剂联合治疗肿瘤的方法 | |
CN113248577B (zh) | 一种以腺病毒为载体的冠状病毒疫苗及其制备方法 | |
CN111500632B (zh) | 表达st13和trail的溶瘤腺病毒构建及其应用 | |
CN110564700B (zh) | 携带鲎凝集素基因的溶瘤痘苗病毒、构建方法及应用 | |
Yang et al. | An Engineered Influenza a Virus Expressing the Co-Stimulator OX40L as an Oncolytic Agent Against Hepatocellular Carcinoma | |
NL2031110B1 (en) | Recombinant adeno-associated virus vector, recombinant adeno-associated virus aav8-pd1 and use thereof | |
CN112646839A (zh) | 一种经修饰的腺相关病毒 | |
CN114908064A (zh) | 一种能抑制肿瘤进展并延长荷瘤个体生存时间的复制型溶瘤腺病毒及其应用 | |
CN111575287B (zh) | 一种抑制NK细胞KIR 2DL3受体表达的siRNA及其应用 | |
WO2021036244A1 (zh) | 携带安全开关并靶向Her2的嵌合抗原受体T细胞及其制备方法和应用 | |
CN117024605A (zh) | 嵌合抗原受体、表达嵌合抗原受体的小胶质细胞及其应用 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PB01 | Publication | ||
PB01 | Publication | ||
SE01 | Entry into force of request for substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
TA01 | Transfer of patent application right |
Effective date of registration: 20210813 Address after: Room 1611, building a, phase I, Zhongdan Ecological Life Science Industrial Park, No. 3-1, xinjinhu Road, Jiangbei new area, Nanjing, Jiangsu 210000 Applicant after: Nanjing weiyade biomedical Co.,Ltd. Address before: 163 Xianlin Avenue, Qixia District, Nanjing City, Jiangsu Province Applicant before: NANJING University |
|
TA01 | Transfer of patent application right | ||
GR01 | Patent grant | ||
GR01 | Patent grant |