CN107739410B - CD3单链抗体-iRGD融合蛋白、制备及其作为抗肿瘤药物的应用 - Google Patents
CD3单链抗体-iRGD融合蛋白、制备及其作为抗肿瘤药物的应用 Download PDFInfo
- Publication number
- CN107739410B CN107739410B CN201710978345.8A CN201710978345A CN107739410B CN 107739410 B CN107739410 B CN 107739410B CN 201710978345 A CN201710978345 A CN 201710978345A CN 107739410 B CN107739410 B CN 107739410B
- Authority
- CN
- China
- Prior art keywords
- irgd
- fusion protein
- chain antibody
- tumor
- cells
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 229960005540 iRGD Drugs 0.000 title claims abstract description 61
- 102000037865 fusion proteins Human genes 0.000 title claims abstract description 34
- 108020001507 fusion proteins Proteins 0.000 title claims abstract description 34
- 238000002360 preparation method Methods 0.000 title claims abstract description 9
- 239000002246 antineoplastic agent Substances 0.000 title claims abstract description 7
- 229940041181 antineoplastic drug Drugs 0.000 title claims abstract description 7
- 206010028980 Neoplasm Diseases 0.000 claims abstract description 20
- 230000008685 targeting Effects 0.000 claims abstract description 13
- 108090000765 processed proteins & peptides Proteins 0.000 claims abstract description 11
- YHTTWXCDIRTOQX-FQJIPJFPSA-N (6S,9S,15S,18R,23R,26S,29S)-18-amino-6-(4-aminobutyl)-9,26-bis(carboxymethyl)-15-[3-(diaminomethylideneamino)propyl]-2,5,8,11,14,17,25,28-octaoxo-20,21-dithia-1,4,7,10,13,16,24,27-octazabicyclo[27.3.0]dotriacontane-23-carboxylic acid Chemical compound NCCCC[C@@H]1NC(=O)[C@H](CC(O)=O)NC(=O)CNC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@@H](N)CSSC[C@H](NC(=O)[C@H](CC(O)=O)NC(=O)[C@@H]2CCCN2C(=O)CNC1=O)C(O)=O YHTTWXCDIRTOQX-FQJIPJFPSA-N 0.000 claims abstract description 8
- 238000010276 construction Methods 0.000 claims abstract description 5
- 108090000623 proteins and genes Proteins 0.000 claims description 33
- 102000004169 proteins and genes Human genes 0.000 claims description 31
- 230000014509 gene expression Effects 0.000 claims description 22
- 150000001413 amino acids Chemical group 0.000 claims description 14
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 claims description 14
- 239000013604 expression vector Substances 0.000 claims description 11
- 230000001580 bacterial effect Effects 0.000 claims description 9
- PXHVJJICTQNCMI-UHFFFAOYSA-N Nickel Chemical compound [Ni] PXHVJJICTQNCMI-UHFFFAOYSA-N 0.000 claims description 8
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 claims description 8
- 239000004202 carbamide Substances 0.000 claims description 8
- 238000000746 purification Methods 0.000 claims description 8
- 239000002244 precipitate Substances 0.000 claims description 6
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 claims description 5
- 238000001042 affinity chromatography Methods 0.000 claims description 4
- 229910001453 nickel ion Inorganic materials 0.000 claims description 4
- 108010075254 C-Peptide Proteins 0.000 claims description 3
- 238000000527 sonication Methods 0.000 claims description 2
- 206010006187 Breast cancer Diseases 0.000 claims 1
- 208000026310 Breast neoplasm Diseases 0.000 claims 1
- 206010008342 Cervix carcinoma Diseases 0.000 claims 1
- 208000005016 Intestinal Neoplasms Diseases 0.000 claims 1
- 206010058467 Lung neoplasm malignant Diseases 0.000 claims 1
- 206010061902 Pancreatic neoplasm Diseases 0.000 claims 1
- 208000005718 Stomach Neoplasms Diseases 0.000 claims 1
- 208000006105 Uterine Cervical Neoplasms Diseases 0.000 claims 1
- 238000005119 centrifugation Methods 0.000 claims 1
- 201000010881 cervical cancer Diseases 0.000 claims 1
- 206010017758 gastric cancer Diseases 0.000 claims 1
- 201000002313 intestinal cancer Diseases 0.000 claims 1
- 201000007270 liver cancer Diseases 0.000 claims 1
- 208000014018 liver neoplasm Diseases 0.000 claims 1
- 201000005202 lung cancer Diseases 0.000 claims 1
- 208000020816 lung neoplasm Diseases 0.000 claims 1
- 208000015486 malignant pancreatic neoplasm Diseases 0.000 claims 1
- 201000002528 pancreatic cancer Diseases 0.000 claims 1
- 208000008443 pancreatic carcinoma Diseases 0.000 claims 1
- 201000011549 stomach cancer Diseases 0.000 claims 1
- 210000001744 T-lymphocyte Anatomy 0.000 abstract description 27
- 230000000149 penetrating effect Effects 0.000 abstract description 8
- 230000000259 anti-tumor effect Effects 0.000 abstract description 6
- 210000002865 immune cell Anatomy 0.000 abstract description 5
- 210000004881 tumor cell Anatomy 0.000 abstract description 5
- 238000002474 experimental method Methods 0.000 abstract description 3
- 230000001225 therapeutic effect Effects 0.000 abstract description 2
- RAXXELZNTBOGNW-UHFFFAOYSA-N imidazole Natural products C1=CNC=N1 RAXXELZNTBOGNW-UHFFFAOYSA-N 0.000 description 24
- 210000004027 cell Anatomy 0.000 description 13
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 9
- 239000007788 liquid Substances 0.000 description 7
- 210000003819 peripheral blood mononuclear cell Anatomy 0.000 description 7
- 230000006698 induction Effects 0.000 description 6
- 239000006228 supernatant Substances 0.000 description 6
- 230000000694 effects Effects 0.000 description 5
- 238000001962 electrophoresis Methods 0.000 description 5
- 230000002147 killing effect Effects 0.000 description 5
- 239000003550 marker Substances 0.000 description 5
- 230000035515 penetration Effects 0.000 description 5
- 239000013612 plasmid Substances 0.000 description 5
- 241000894006 Bacteria Species 0.000 description 4
- 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 4
- 101001023379 Homo sapiens Lysosome-associated membrane glycoprotein 1 Proteins 0.000 description 4
- 102100035133 Lysosome-associated membrane glycoprotein 1 Human genes 0.000 description 4
- 230000003213 activating effect Effects 0.000 description 4
- 238000001514 detection method Methods 0.000 description 4
- 230000001939 inductive effect Effects 0.000 description 4
- NOIIUHRQUVNIDD-UHFFFAOYSA-N 3-[[oxo(pyridin-4-yl)methyl]hydrazo]-N-(phenylmethyl)propanamide Chemical compound C=1C=CC=CC=1CNC(=O)CCNNC(=O)C1=CC=NC=C1 NOIIUHRQUVNIDD-UHFFFAOYSA-N 0.000 description 3
- 102100027207 CD27 antigen Human genes 0.000 description 3
- 101000914511 Homo sapiens CD27 antigen Proteins 0.000 description 3
- 230000027455 binding Effects 0.000 description 3
- 239000003814 drug Substances 0.000 description 3
- 230000001376 precipitating effect Effects 0.000 description 3
- 108020003175 receptors Proteins 0.000 description 3
- 102000005962 receptors Human genes 0.000 description 3
- 238000004153 renaturation Methods 0.000 description 3
- 230000028327 secretion Effects 0.000 description 3
- 238000012163 sequencing technique Methods 0.000 description 3
- 101710163270 Nuclease Proteins 0.000 description 2
- 108010003723 Single-Domain Antibodies Proteins 0.000 description 2
- ANHVRCNNGJMJNG-BZSNNMDCSA-N Tyr-Tyr-Cys Chemical compound C1=CC(=CC=C1C[C@@H](C(=O)N[C@@H](CC2=CC=C(C=C2)O)C(=O)N[C@@H](CS)C(=O)O)N)O ANHVRCNNGJMJNG-BZSNNMDCSA-N 0.000 description 2
- 238000002659 cell therapy Methods 0.000 description 2
- 239000003398 denaturant Substances 0.000 description 2
- 238000000502 dialysis Methods 0.000 description 2
- 238000003114 enzyme-linked immunosorbent spot assay Methods 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- 230000004927 fusion Effects 0.000 description 2
- 108010063718 gamma-glutamylaspartic acid Proteins 0.000 description 2
- 238000000338 in vitro Methods 0.000 description 2
- 229910052759 nickel Inorganic materials 0.000 description 2
- 102000004196 processed proteins & peptides Human genes 0.000 description 2
- 108010026333 seryl-proline Proteins 0.000 description 2
- 239000013598 vector Substances 0.000 description 2
- 238000012795 verification Methods 0.000 description 2
- 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 1
- JBVSSSZFNTXJDX-YTLHQDLWSA-N Ala-Ala-Thr Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)[C@H](C)NC(=O)[C@H](C)N JBVSSSZFNTXJDX-YTLHQDLWSA-N 0.000 description 1
- WYPUMLRSQMKIJU-BPNCWPANSA-N Ala-Arg-Tyr Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O WYPUMLRSQMKIJU-BPNCWPANSA-N 0.000 description 1
- IOFVWPYSRSCWHI-JXUBOQSCSA-N Ala-Thr-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](C)N IOFVWPYSRSCWHI-JXUBOQSCSA-N 0.000 description 1
- GIVATXIGCXFQQA-FXQIFTODSA-N Arg-Ala-Ser Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@H](C)NC(=O)[C@@H](N)CCCN=C(N)N GIVATXIGCXFQQA-FXQIFTODSA-N 0.000 description 1
- ZZZWQALDSQQBEW-STQMWFEESA-N Arg-Gly-Tyr Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)NCC(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O ZZZWQALDSQQBEW-STQMWFEESA-N 0.000 description 1
- GMUOCGCDOYYWPD-FXQIFTODSA-N Asn-Pro-Ser Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CO)C(O)=O GMUOCGCDOYYWPD-FXQIFTODSA-N 0.000 description 1
- RTFXPCYMDYBZNQ-SRVKXCTJSA-N Asn-Tyr-Asn Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CC(N)=O)C(O)=O RTFXPCYMDYBZNQ-SRVKXCTJSA-N 0.000 description 1
- HTSSXFASOUSJQG-IHPCNDPISA-N Asp-Tyr-Trp Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CC1=CNC2=C1C=CC=C2)C(O)=O HTSSXFASOUSJQG-IHPCNDPISA-N 0.000 description 1
- 101100361281 Caenorhabditis elegans rpm-1 gene Proteins 0.000 description 1
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 1
- 108010019670 Chimeric Antigen Receptors Proteins 0.000 description 1
- 108090000695 Cytokines Proteins 0.000 description 1
- 102000004127 Cytokines Human genes 0.000 description 1
- 238000001712 DNA sequencing Methods 0.000 description 1
- XOKGKOQWADCLFQ-GARJFASQSA-N Gln-Arg-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CCC(=O)N)N)C(=O)O XOKGKOQWADCLFQ-GARJFASQSA-N 0.000 description 1
- QFJPFPCSXOXMKI-BPUTZDHNSA-N Gln-Gln-Trp Chemical compound C1=CC=C2C(=C1)C(=CN2)C[C@@H](C(=O)O)NC(=O)[C@H](CCC(=O)N)NC(=O)[C@H](CCC(=O)N)N QFJPFPCSXOXMKI-BPUTZDHNSA-N 0.000 description 1
- FGYPOQPQTUNESW-IUCAKERBSA-N Gln-Gly-Leu Chemical compound CC(C)C[C@@H](C(=O)O)NC(=O)CNC(=O)[C@H](CCC(=O)N)N FGYPOQPQTUNESW-IUCAKERBSA-N 0.000 description 1
- ILKYYKRAULNYMS-JYJNAYRXSA-N Gln-Lys-Phe Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O ILKYYKRAULNYMS-JYJNAYRXSA-N 0.000 description 1
- ZEEPYMXTJWIMSN-GUBZILKMSA-N Gln-Lys-Ser Chemical compound NCCCC[C@@H](C(=O)N[C@@H](CO)C(O)=O)NC(=O)[C@@H](N)CCC(N)=O ZEEPYMXTJWIMSN-GUBZILKMSA-N 0.000 description 1
- 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 1
- LKDIBBOKUAASNP-FXQIFTODSA-N Glu-Ala-Glu Chemical compound OC(=O)CC[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@@H](CCC(O)=O)C(O)=O LKDIBBOKUAASNP-FXQIFTODSA-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
- JVZLZVJTIXVIHK-SXNHZJKMSA-N Glu-Trp-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)[C@H](CC1=CNC2=CC=CC=C21)NC(=O)[C@H](CCC(=O)O)N JVZLZVJTIXVIHK-SXNHZJKMSA-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
- MHHUEAIBJZWDBH-YUMQZZPRSA-N Gly-Asp-Lys Chemical compound C(CCN)C[C@@H](C(=O)O)NC(=O)[C@H](CC(=O)O)NC(=O)CN MHHUEAIBJZWDBH-YUMQZZPRSA-N 0.000 description 1
- BYYNJRSNDARRBX-YFKPBYRVSA-N Gly-Gln-Gly Chemical compound NCC(=O)N[C@@H](CCC(N)=O)C(=O)NCC(O)=O BYYNJRSNDARRBX-YFKPBYRVSA-N 0.000 description 1
- WDXLKVQATNEAJQ-BQBZGAKWSA-N Gly-Pro-Asp Chemical compound NCC(=O)N1CCC[C@H]1C(=O)N[C@@H](CC(O)=O)C(O)=O WDXLKVQATNEAJQ-BQBZGAKWSA-N 0.000 description 1
- FFALDIDGPLUDKV-ZDLURKLDSA-N Gly-Thr-Ser Chemical compound [H]NCC(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CO)C(O)=O FFALDIDGPLUDKV-ZDLURKLDSA-N 0.000 description 1
- KOYUSMBPJOVSOO-XEGUGMAKSA-N Gly-Tyr-Ile Chemical compound [H]NCC(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O KOYUSMBPJOVSOO-XEGUGMAKSA-N 0.000 description 1
- DNVDEMWIYLVIQU-RCOVLWMOSA-N Gly-Val-Asp Chemical compound NCC(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC(O)=O)C(O)=O DNVDEMWIYLVIQU-RCOVLWMOSA-N 0.000 description 1
- YGHSQRJSHKYUJY-SCZZXKLOSA-N Gly-Val-Pro Chemical compound CC(C)[C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)CN YGHSQRJSHKYUJY-SCZZXKLOSA-N 0.000 description 1
- ZJSMFRTVYSLKQU-DJFWLOJKSA-N His-Asp-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)[C@H](CC(=O)O)NC(=O)[C@H](CC1=CN=CN1)N ZJSMFRTVYSLKQU-DJFWLOJKSA-N 0.000 description 1
- 108010093488 His-His-His-His-His-His Proteins 0.000 description 1
- YKUAGFAXQRYUQW-KKUMJFAQSA-N His-Tyr-Cys Chemical compound C1=CC(=CC=C1C[C@@H](C(=O)N[C@@H](CS)C(=O)O)NC(=O)[C@H](CC2=CN=CN2)N)O YKUAGFAXQRYUQW-KKUMJFAQSA-N 0.000 description 1
- KUHFPGIVBOCRMV-MNXVOIDGSA-N Ile-Gln-Leu Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)N[C@@H](CC(C)C)C(=O)O)N KUHFPGIVBOCRMV-MNXVOIDGSA-N 0.000 description 1
- 102100037850 Interferon gamma Human genes 0.000 description 1
- 108010074328 Interferon-gamma Proteins 0.000 description 1
- LJHGALIOHLRRQN-DCAQKATOSA-N Leu-Ala-Arg Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@H](C(O)=O)CCCN=C(N)N LJHGALIOHLRRQN-DCAQKATOSA-N 0.000 description 1
- KAFOIVJDVSZUMD-UHFFFAOYSA-N Leu-Gln-Gln Natural products CC(C)CC(N)C(=O)NC(CCC(N)=O)C(=O)NC(CCC(N)=O)C(O)=O KAFOIVJDVSZUMD-UHFFFAOYSA-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
- LMVOVCYVZBBWQB-SRVKXCTJSA-N Lys-Asp-Lys Chemical compound NCCCC[C@H](N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@H](C(O)=O)CCCCN LMVOVCYVZBBWQB-SRVKXCTJSA-N 0.000 description 1
- ONPDTSFZAIWMDI-AVGNSLFASA-N Lys-Leu-Gln Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(O)=O ONPDTSFZAIWMDI-AVGNSLFASA-N 0.000 description 1
- RMOKGALPSPOYKE-KATARQTJSA-N Lys-Thr-Ser Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CO)C(O)=O RMOKGALPSPOYKE-KATARQTJSA-N 0.000 description 1
- RIPJMCFGQHGHNP-RHYQMDGZSA-N Lys-Val-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](C(C)C)NC(=O)[C@H](CCCCN)N)O RIPJMCFGQHGHNP-RHYQMDGZSA-N 0.000 description 1
- VOOINLQYUZOREH-SRVKXCTJSA-N Met-Gln-Leu Chemical compound CC(C)C[C@@H](C(=O)O)NC(=O)[C@H](CCC(=O)N)NC(=O)[C@H](CCSC)N VOOINLQYUZOREH-SRVKXCTJSA-N 0.000 description 1
- CIDICGYKRUTYLE-FXQIFTODSA-N Met-Ser-Ala Chemical compound CSCC[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@@H](C)C(O)=O CIDICGYKRUTYLE-FXQIFTODSA-N 0.000 description 1
- WRXOPYNEKGZWAZ-FXQIFTODSA-N Met-Ser-Cys Chemical compound CSCC[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@@H](CS)C(O)=O WRXOPYNEKGZWAZ-FXQIFTODSA-N 0.000 description 1
- WSPQHZOMTFFWGH-XGEHTFHBSA-N Met-Thr-Cys Chemical compound CSCC[C@H](N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CS)C(O)=O WSPQHZOMTFFWGH-XGEHTFHBSA-N 0.000 description 1
- 108010079364 N-glycylalanine Proteins 0.000 description 1
- 108010002311 N-glycylglutamic acid Proteins 0.000 description 1
- 235000016496 Panda oleosa Nutrition 0.000 description 1
- 240000000220 Panda oleosa Species 0.000 description 1
- FZHBZMDRDASUHN-NAKRPEOUSA-N Pro-Ala-Ile Chemical compound CC[C@H](C)[C@H](NC(=O)[C@H](C)NC(=O)[C@@H]1CCCN1)C(O)=O FZHBZMDRDASUHN-NAKRPEOUSA-N 0.000 description 1
- 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 1
- VTFXTWDFPTWNJY-RHYQMDGZSA-N Pro-Leu-Thr Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)O)C(O)=O VTFXTWDFPTWNJY-RHYQMDGZSA-N 0.000 description 1
- JUJCUYWRJMFJJF-AVGNSLFASA-N Pro-Lys-Arg Chemical compound NC(N)=NCCC[C@@H](C(O)=O)NC(=O)[C@H](CCCCN)NC(=O)[C@@H]1CCCN1 JUJCUYWRJMFJJF-AVGNSLFASA-N 0.000 description 1
- HBZBPFLJNDXRAY-FXQIFTODSA-N Ser-Ala-Val Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](C)C(=O)N[C@@H](C(C)C)C(O)=O HBZBPFLJNDXRAY-FXQIFTODSA-N 0.000 description 1
- UIGMAMGZOJVTDN-WHFBIAKZSA-N Ser-Gly-Ser Chemical compound OC[C@H](N)C(=O)NCC(=O)N[C@@H](CO)C(O)=O UIGMAMGZOJVTDN-WHFBIAKZSA-N 0.000 description 1
- SFTZWNJFZYOLBD-ZDLURKLDSA-N Ser-Gly-Thr Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)CNC(=O)[C@@H](N)CO SFTZWNJFZYOLBD-ZDLURKLDSA-N 0.000 description 1
- RHAPJNVNWDBFQI-BQBZGAKWSA-N Ser-Pro-Gly Chemical compound OC[C@H](N)C(=O)N1CCC[C@H]1C(=O)NCC(O)=O RHAPJNVNWDBFQI-BQBZGAKWSA-N 0.000 description 1
- WLJPJRGQRNCIQS-ZLUOBGJFSA-N Ser-Ser-Asn Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(N)=O)C(O)=O WLJPJRGQRNCIQS-ZLUOBGJFSA-N 0.000 description 1
- BMKNXTJLHFIAAH-CIUDSAMLSA-N Ser-Ser-Leu Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(O)=O BMKNXTJLHFIAAH-CIUDSAMLSA-N 0.000 description 1
- AABIBDJHSKIMJK-FXQIFTODSA-N Ser-Ser-Met Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCSC)C(O)=O AABIBDJHSKIMJK-FXQIFTODSA-N 0.000 description 1
- XQJCEKXQUJQNNK-ZLUOBGJFSA-N Ser-Ser-Ser Chemical compound OC[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@@H](CO)C(O)=O XQJCEKXQUJQNNK-ZLUOBGJFSA-N 0.000 description 1
- VGQVAVQWKJLIRM-FXQIFTODSA-N Ser-Ser-Val Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CO)C(=O)N[C@@H](C(C)C)C(O)=O VGQVAVQWKJLIRM-FXQIFTODSA-N 0.000 description 1
- YXGCIEUDOHILKR-IHRRRGAJSA-N Ser-Tyr-Met Chemical compound CSCC[C@@H](C(=O)O)NC(=O)[C@H](CC1=CC=C(C=C1)O)NC(=O)[C@H](CO)N YXGCIEUDOHILKR-IHRRRGAJSA-N 0.000 description 1
- VVKVHAOOUGNDPJ-SRVKXCTJSA-N Ser-Tyr-Ser Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CO)C(O)=O VVKVHAOOUGNDPJ-SRVKXCTJSA-N 0.000 description 1
- BEBVVQPDSHHWQL-NRPADANISA-N Ser-Val-Glu Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCC(O)=O)C(O)=O BEBVVQPDSHHWQL-NRPADANISA-N 0.000 description 1
- LGIMRDKGABDMBN-DCAQKATOSA-N Ser-Val-Lys Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CCCCN)C(=O)O)NC(=O)[C@H](CO)N LGIMRDKGABDMBN-DCAQKATOSA-N 0.000 description 1
- 230000006044 T cell activation Effects 0.000 description 1
- DGDCHPCRMWEOJR-FQPOAREZSA-N Thr-Ala-Tyr Chemical compound C[C@@H](O)[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 DGDCHPCRMWEOJR-FQPOAREZSA-N 0.000 description 1
- UNURFMVMXLENAZ-KJEVXHAQSA-N Thr-Arg-Tyr Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O UNURFMVMXLENAZ-KJEVXHAQSA-N 0.000 description 1
- LIXBDERDAGNVAV-XKBZYTNZSA-N Thr-Gln-Ser Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CO)C(O)=O LIXBDERDAGNVAV-XKBZYTNZSA-N 0.000 description 1
- SPVHQURZJCUDQC-VOAKCMCISA-N Thr-Lys-Leu Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(O)=O SPVHQURZJCUDQC-VOAKCMCISA-N 0.000 description 1
- LHNNQVXITHUCAB-QTKMDUPCSA-N Thr-Met-His Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC1=CN=CN1)C(=O)O)N)O LHNNQVXITHUCAB-QTKMDUPCSA-N 0.000 description 1
- XHWCDRUPDNSDAZ-XKBZYTNZSA-N Thr-Ser-Glu Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CO)C(=O)N[C@@H](CCC(=O)O)C(=O)O)N)O XHWCDRUPDNSDAZ-XKBZYTNZSA-N 0.000 description 1
- IQPWNQRRAJHOKV-KATARQTJSA-N Thr-Ser-Lys Chemical compound C[C@@H](O)[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@H](C(O)=O)CCCCN IQPWNQRRAJHOKV-KATARQTJSA-N 0.000 description 1
- YRJOLUDFVAUXLI-GSSVUCPTSA-N Thr-Thr-Asp Chemical compound C[C@@H](O)[C@H](N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@H](C(O)=O)CC(O)=O YRJOLUDFVAUXLI-GSSVUCPTSA-N 0.000 description 1
- BBPCSGKKPJUYRB-UVOCVTCTSA-N Thr-Thr-Leu Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(C)C)C(O)=O BBPCSGKKPJUYRB-UVOCVTCTSA-N 0.000 description 1
- MNYNCKZAEIAONY-XGEHTFHBSA-N Thr-Val-Ser Chemical compound C[C@@H](O)[C@H](N)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CO)C(O)=O MNYNCKZAEIAONY-XGEHTFHBSA-N 0.000 description 1
- WNZRNOGHEONFMS-PXDAIIFMSA-N Trp-Ile-Tyr Chemical compound [H]N[C@@H](CC1=CNC2=C1C=CC=C2)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O WNZRNOGHEONFMS-PXDAIIFMSA-N 0.000 description 1
- PKZIWSHDJYIPRH-JBACZVJFSA-N Trp-Tyr-Gln Chemical compound [H]N[C@@H](CC1=CNC2=C1C=CC=C2)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CCC(N)=O)C(O)=O PKZIWSHDJYIPRH-JBACZVJFSA-N 0.000 description 1
- UOXPLPBMEPLZBW-WDSOQIARSA-N Trp-Val-Lys Chemical compound C1=CC=C2C(C[C@H](N)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCCCN)C(O)=O)=CNC2=C1 UOXPLPBMEPLZBW-WDSOQIARSA-N 0.000 description 1
- XHALUUQSNXSPLP-UFYCRDLUSA-N Tyr-Arg-Phe Chemical compound C([C@H](N)C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](CC=1C=CC=CC=1)C(O)=O)C1=CC=C(O)C=C1 XHALUUQSNXSPLP-UFYCRDLUSA-N 0.000 description 1
- BEIGSKUPTIFYRZ-SRVKXCTJSA-N Tyr-Asp-Asp Chemical compound C1=CC(=CC=C1C[C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)N[C@@H](CC(=O)O)C(=O)O)N)O BEIGSKUPTIFYRZ-SRVKXCTJSA-N 0.000 description 1
- LDKDSFQSEUOCOO-RPTUDFQQSA-N Tyr-Thr-Phe Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O LDKDSFQSEUOCOO-RPTUDFQQSA-N 0.000 description 1
- 108010064997 VPY tripeptide Proteins 0.000 description 1
- AZSHAZJLOZQYAY-FXQIFTODSA-N Val-Ala-Ser Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@@H](CO)C(O)=O AZSHAZJLOZQYAY-FXQIFTODSA-N 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 108010086434 alanyl-seryl-glycine Proteins 0.000 description 1
- 108010060035 arginylproline Proteins 0.000 description 1
- 108010077245 asparaginyl-proline Proteins 0.000 description 1
- 108010040443 aspartyl-aspartic acid Proteins 0.000 description 1
- 108010092854 aspartyllysine Proteins 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000001110 calcium chloride Substances 0.000 description 1
- 229910001628 calcium chloride Inorganic materials 0.000 description 1
- 230000022534 cell killing Effects 0.000 description 1
- 230000015861 cell surface binding Effects 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 238000004440 column chromatography Methods 0.000 description 1
- 238000004624 confocal microscopy Methods 0.000 description 1
- NKLPQNGYXWVELD-UHFFFAOYSA-M coomassie brilliant blue Chemical compound [Na+].C1=CC(OCC)=CC=C1NC1=CC=C(C(=C2C=CC(C=C2)=[N+](CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=2C=CC(=CC=2)N(CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=C1 NKLPQNGYXWVELD-UHFFFAOYSA-M 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 239000012154 double-distilled water Substances 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 238000001976 enzyme digestion Methods 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- VPZXBVLAVMBEQI-UHFFFAOYSA-N glycyl-DL-alpha-alanine Natural products OC(=O)C(C)NC(=O)CN VPZXBVLAVMBEQI-UHFFFAOYSA-N 0.000 description 1
- 108010078326 glycyl-glycyl-valine Proteins 0.000 description 1
- 108010089804 glycyl-threonine Proteins 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 238000009169 immunotherapy Methods 0.000 description 1
- 102000006495 integrins Human genes 0.000 description 1
- 108010044426 integrins Proteins 0.000 description 1
- 229930027917 kanamycin Natural products 0.000 description 1
- 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 description 1
- 229960000318 kanamycin Drugs 0.000 description 1
- 229930182823 kanamycin A Natural products 0.000 description 1
- 238000002843 lactate dehydrogenase assay Methods 0.000 description 1
- 239000006166 lysate Substances 0.000 description 1
- 108010057952 lysyl-phenylalanyl-lysine Proteins 0.000 description 1
- 238000000034 method Methods 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 230000037361 pathway Effects 0.000 description 1
- 239000008188 pellet Substances 0.000 description 1
- 108010051242 phenylalanylserine Proteins 0.000 description 1
- 229920002401 polyacrylamide Polymers 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 230000003248 secreting effect Effects 0.000 description 1
- 239000013049 sediment Substances 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 238000009210 therapy by ultrasound Methods 0.000 description 1
- 108010072986 threonyl-seryl-lysine Proteins 0.000 description 1
- 230000001131 transforming effect Effects 0.000 description 1
- 108010038745 tryptophylglycine Proteins 0.000 description 1
- 108010003137 tyrosyltyrosine Proteins 0.000 description 1
- 238000002525 ultrasonication Methods 0.000 description 1
- 108010009962 valyltyrosine Proteins 0.000 description 1
- 210000005166 vasculature Anatomy 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
- C07K16/2809—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against the T-cell receptor (TcR)-CD3 complex
-
- 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
- A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
- C07K2319/10—Fusion polypeptide containing a localisation/targetting motif containing a tag for extracellular membrane crossing, e.g. TAT or VP22
Landscapes
- Health & Medical Sciences (AREA)
- Immunology (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Biophysics (AREA)
- Biochemistry (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Life Sciences & Earth Sciences (AREA)
- Peptides Or Proteins (AREA)
Abstract
本发明公开了一种CD3单链抗体‑iRGD融合蛋白、制备及其作为抗肿瘤药物的应用。本发明将穿透肽iRGD成分作为实现肿瘤主动靶向性、高穿透性的分子，将CD3的单链抗体与其连接，形成一个可以活化T细胞并能有效靶向杀伤肿瘤细胞的融合蛋白。成功构建后经反复实验验证，本发明具有明确肿瘤靶向性和高穿透性，能够切实地增强免疫细胞的抗肿瘤治疗效果，应用于抗肿瘤药物中。
Description
技术领域
本发明属于生物与新医药技术领域，特别涉及一种CD3单链抗体-iRGD融合蛋白、制备及其作为抗肿瘤药物的应用。
背景技术
在越来越引起人们重视的免疫治疗领域，尽管存在类似于嵌合抗原受体T细胞疗法、BiTE等可以实现肿瘤的靶向性，但却在实体瘤的治疗中进展缓慢。免疫细胞难以有效进入肿瘤组织是其中一个重要的原因。因此，在免疫细胞治疗中，同时改善免疫细胞的主动靶向性与穿透性是进一步提高抗肿瘤治疗效果的关键。
发明内容
本发明的目的就在于克服上述缺陷，提供一种CD3单链抗体-iRGD融合蛋白（命名为anti-CD3-iRGD）同时提供上述融合蛋白的制备方法及其作为抗肿瘤药物的应用。
本发明利用含有靶向穿透肽iRGD成分的物质作为实现肿瘤主动靶向性、高穿透性的分子，将CD3的单链抗体与其连接，形成一个可以活化T细胞并能有效靶向杀伤肿瘤细胞的融合蛋白。成功构建后经反复实验验证，本发明具有明确肿瘤靶向性和高穿透性，能够切实地增强免疫细胞的抗肿瘤治疗效果，应用于抗肿瘤药物中。
本发明提供的技术方案是：
本发明提供一种CD3单链抗体-iRGD融合蛋白，所述融合蛋白由靶向人CD3单链抗体和肿瘤穿透肽iRGD经由连接肽（比如：GGGGSGGGGSGGGGS）连接组成。
进一步地，所述融合蛋白包含但不限于CD3单链抗体、单域抗体、单克隆抗体以及能活化T细胞的CD3功能域或其他能活化T细胞的分子，肿瘤靶向穿透肽包含但不限于iRGD、iNGR，以及含前述靶向穿透肽结构的融合肽、融合蛋白或其它修饰成分。
进一步地，所述融合蛋白具有如下氨基酸序列：
（1）由SEQ ID No.1所示的氨基酸序列组成的蛋白质；或
（2）与序列SEQ ID No.1所示的氨基酸序列同源性在80%-100%编码相同功能蛋白质的氨基酸序列；或
（3）SEQ ID No.1所示的氨基酸序列经增加、缺失或者替换一个或多个氨基酸具有同等活性的由（1）衍生的蛋白。
本发明制备所述CD3单链抗体-iRGD融合蛋白的具体制备方法如下：
（1）表达载体pET28a-anti-CD3-iRGD的构建：
表达载体由南京金斯瑞公司合成，且已完成对该质粒的验证，包括核酸电泳及DNA测序；
（2）将表达载体pET28a-anti-CD3-iRGD转化至表达菌株BL21 DE3：
取已经确认的100ng/μL表达载体pET28a-anti-CD3-iRGD 0.1μL-5μL转化入表达菌株BL21 DE3感受态细胞中，涂布到特定抗性的LB平板上，挑选阳性克隆低温冰箱内甘油保存该菌种；
（3）重组anti-CD3-iRGD的诱导表达,变性和复性，及纯化：
1）重组anti-CD3-iRGD 异丙基-β-D-硫代半乳糖苷诱导表达：
对重组anti-CD3-iRGD用1mM 异丙基-β-D-硫代半乳糖苷诱导表达，在诱导4小时后，聚丙烯酰氨凝胶全蛋白电泳；
2）重组anti-CD3-iRGD进行变复性；
收集1000mL经过异丙基-β-D-硫代半乳糖苷诱导表达后的BL21菌体，用PBS、5mM咪唑重悬菌体，350W功率进行超声破碎，12 000 r·min−1离心后收集沉淀，用8M尿素变性剂溶解沉淀，并依次在6M、4M、2M、0M尿素溶液中透析复性；
3）重组anti-CD3-iRGD经AKTA Purifier 900 FPLC系统，镍离子亲和层
析柱纯化：
（4）对重组anti-CD3-iRGD用1mM 异丙基-β-D-硫代半乳糖苷（IPTG）诱导4小时，收集菌液进行超声破碎，离心后收集沉淀，用8M尿素变性剂溶解沉淀，并依次在6M、4M、2M、0M尿素溶液中透析复性，重组anti-CD3-iRGD经AKTA Purifier 900 FPLC系统，镍离子亲和层析柱纯化：用SDS-PAGE方法检测目的蛋白。
本发明的有益效果是：
融合蛋白CD3单链抗体部分与T细胞结合，促进T细胞的多克隆增殖、活化（包括表型改变和细胞因子分泌增多）和对肿瘤细胞的杀伤作用，融合蛋白iRGD部分则通过整合素ανβ3/β5 受体途径靶向至肿瘤局部，并进一步增加T细胞在肿瘤血管和实质中的穿透性。
附图说明
图1是目的基因表达载体pET28a-anti-CD3-iRGD核酸电泳图。
泳道1：完整质粒电泳结果；泳道2：质粒经NcoⅠ和HindⅢ核酸酶酶切后电泳结果；泳道3：DNA marker。
图2是表达载体pET28a-anti-CD3-iRGD中NcoⅠ和HindⅢ位点之间序列的测序结果示意图。
图3是anti-CD3-iRGD蛋白的表达与纯化：（左图）泳道1：Marker；泳道2：未诱导；泳道3：诱导后；泳道4：上清；泳道5：沉淀；泳道6：复性后。（右图）泳道1：Marker；泳道2：蛋白样品流穿；泳道3：1%咪唑洗脱蛋白；泳道4：%咪唑洗脱蛋白；泳道5：25%-100%咪唑线性洗脱蛋白，即纯化后。
图4是镍柱亲和层析纯化目的蛋白图谱（箭头指示目的蛋白）。
图5是融合蛋白anti-CD3-iRGD在不同浓度时能分别与MKN45细胞和T细胞表面的受体结合。（半小时后流式检测细胞表面与蛋白的结合，左图为融合蛋白与MKN45细胞表面iRGD受体的结合，右图为融合蛋白与T细胞表面CD3分子的结合）。
图6是融合蛋白anti-CD3-iRGD对T细胞表面CD107a表型的作用。（24h后流式检测细胞表面与蛋白的结合， CD107a是T细胞杀伤功能的标志）。
图7是融合蛋白anti-CD3-iRGD对T细胞表面CD27表型的作用（CD27是T细胞活化的标志）。
图8是融合蛋白促进T细胞IFN-γ的分泌。（24h后通过ELISPOT检测T细胞IFN-γ的分泌量，右图为定量结果）。
图9是融合蛋白anti-CD3-iRGD促进T细胞对MKN45的杀伤作用。（24h和48h后经LDH释放实验检测T细胞的杀伤作用）。
图10是融合蛋白anti-CD3-iRGD促进T细胞在体外3D肿瘤细胞球内的穿透。（6h和24h后在共聚焦显微镜下观察T细胞在肿瘤球中的穿透作用）。
图11是融合蛋白anti-CD3-iRGD促进T细胞对体外3D肿瘤细胞球的杀伤作用。（24h和48h后于倒置光学显微镜下观察肿瘤球的大小和形态）。
具体实施方式
以下结合附图和具体实施方式详细说明本发明的技术方案，但不限制本发明权利要求的保护范围。
实施例1
一种CD3单链抗体-iRGD融合蛋白，所述融合蛋白由靶向人CD3单链抗体和肿瘤穿透肽iRGD经由连接肽（比如：GGGGSGGGGSGGGGS）连接组成。
进一步地，所述融合蛋白包含但不限于CD3单链抗体、单域抗体、单克隆抗体以及能活化T细胞的CD3功能域或其他能活化T细胞的分子，肿瘤靶向穿透肽包含但不限于iRGD、iNGR，以及含前述靶向穿透肽结构的融合肽、融合蛋白或其它修饰成分。
进一步地，所述融合蛋白具有如下氨基酸序列：
（1）由SEQ ID No.1所示的氨基酸序列组成的蛋白质；或
（2）与序列SEQ ID No.1所示的氨基酸序列同源性在80%-100%编码相同功能蛋白质的氨基酸序列；或
（3）SEQ ID No.1所示的氨基酸序列经增加、缺失或者替换一个或多个氨基酸具有同等活性的由（1）衍生的蛋白。
具有抗肿瘤作用的融合蛋白的表达和纯化过程如下：
1、表达载体pET28a-anti-CD3-iRGD的构建：
该载体由南京金斯瑞公司合成，质粒经NcoⅠ和HindⅢ核酸酶酶切后进行核酸电泳，结果显示目的基因片段大小为800bp左右，如图1所示：可见片段大小与预期一致；并将质粒进行测序验证。测序基因的核苷酸序列以及推导的氨基酸序列与设计的结果进行对比分析。经对比表明载体pET28a-anti-CD3-iRGD构建成功，测序结果如图2所示。
2、将表达载体pET28a-anti-CD3-iRGD转化至表达菌株BL21 DE3：
取已经确认的100ng/μL表达载体pET28a-anti-CD3-iRGD 0.1μL-5μL转化入氯化钙制备的表达菌株BL21 DE3感受态细胞中，涂布到特定卡那抗性的LB平板上。挑选阳性克隆低温冰箱内甘油保存该菌种。
3、重组anti-CD3-iRGD的诱导表达及纯化：
1）重组anti-CD3-iRGD IPTG诱导表达：
对重组anti-CD3-iRGD用1mMIPTG诱导表达，诱导4小时后，SDS-PAGE全蛋白电泳，在30KD左右可以看到清晰特异性蛋白条带，在无IPTG诱导和存在IPTG诱导对比显示，anti-CD3-iRGD蛋白已经表达成功。
全蛋白IPTG诱导表达，实验步骤如下：
a）取3μL菌液（表达菌株甘油保存菌）加入3mL LB（卡那抗性）培养基，220rpm 37℃过夜摇菌；
b）测OD600至0.5时；
c）加入浓度为1mM的IPTG；
d）220rpm 37℃ 摇菌4小时；
e）取出菌液，12000g 10min离心去上清；
2）重组anti-CD3-iRGD用8M尿素变性，透析法复性，并经AKTA Purifier 900 FPLC系统，镍离子亲和层析柱纯化：
收集1000mL经过IPTG诱导表达后的BL21 DE3菌液，经4200 r·min−1 离心10min，收集菌体。沉淀重悬于PBS（PH 7.4）、5mM咪唑80mL中进行超声裂解，超声条件为350W、工作3s、间歇3s、总时间为45min。超声裂解物于4 ℃、 12 000 r·min−1离心 20min。收集沉淀于含8M尿素的蛋白变性液中充分溶解，并依次在6M、4M、2M、0M尿素溶液中透析复性，于AKTA Purifier 900 FPLC系统纯化。 镍柱用5倍柱体积PBS（PH7.4）、5mmol l·L−1咪唑平衡后上样,经 PBS(pH 7.4)、40 mmol·L−1咪唑洗杂后,目的蛋白用 PBS (pH7.4)、500 mmol·L−1咪唑洗脱，如图4。洗脱后的纯化蛋白在PBS (pH 7.4)中透析。
4、用SDS-PAGE方法检测目的蛋白：
选取未诱导表达的表达菌株BL21 DE3 pET28a-anti-CD3-iRGD菌液1mL、加入IPTG诱导表达4小时后的菌液1mL进行12000 r·min−1离心 1 min，弃上清，沉淀用100μLddH2O重悬作为“未诱导”与“诱导”；超声后的菌液100μL进行12000 r·min−1离心 1 min，上清移至新的EP管，沉淀用100μLddH2O重悬分别作为“上清”与“沉淀”；经透析法复性的蛋白作为“复性后”，进行SDS-PAGE电泳，电泳后考马斯亮蓝染色并脱色，拍照，如图3。
5、将MKN45和PBMC分别与不同浓度的anti-CD3-iRGD（0、0.1、1、10ug/ml）
冰上孵育30min，1mlPBS洗1遍，再分别加入3ul anti-His流式抗体，避光孵育30min，1mlPBS洗1遍，300g离心5min，留取100ul体积进行流式检测MKN45、PBMC细胞表面与anti-CD3-iRGD的结合，如图5。
6、将MKN45细胞和PBMC细胞按E:T=20：1共孵育，加入不同浓度的anti-CD3-iRGD（0、0.1、1、10ug/ml），24h后流式检测T细胞表面CD107a、CD27的表达。流式结果显示，当蛋白浓度为0.1ug/ml时，T细胞表面CD107a（如图6）、CD27（如图7）的表达与对照组相比已明显上调。
7、将MKN45细胞和PBMC细胞按E:T=20：1共孵育，加入不同浓度的anti-CD3-iRGD（0、0.1、1、10ug/ml），24h后测ELISPOT结果，当蛋白浓度为5ug/ml和10ug/ml时，分泌IFN-γ的T细胞数明显增多，如图8。
8、LDH释放实验： 将MKN45细胞和PBMC细胞按E:T=20：1或40:1共孵育,24h和48h后取100ul上清，加入100ulLDH反应液，避光作用30min后测A492nm处的吸光度，如图9。
9、将MKN45细胞按1000个/ml的浓度加入超低吸附96孔板中，2天后细胞成球，将CFSE标记的PBMC按E:T=4：1加入96孔板中，实验组同时加入10ug/ml的anti-CD3-iRGD蛋白，6h和24h后在共聚焦显微镜下观察T细胞在肿瘤球中的穿透作用（如图10）。或将未标记的PBMC按E:T=5：1、10：1、20:1加入96孔板中，24h和48h后于倒置光学显微镜下观察肿瘤球的大小和形态（如图11）。结果显示，anti-CD3-iRGD能促进T细胞在3D肿瘤球水平的穿透和杀伤作用。
序列表
<110> 南京鼓楼医院
<120> CD3单链抗体-iRGD融合蛋白、制备及其作为抗肿瘤药物的应用
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 263
<212> PRT
<213> 人工序列()
<400> 1
His His His His His His Asp Ile Lys Leu Gln Gln Ser Gly Ala Glu
1 5 10 15
Leu Ala Arg Pro Gly Ala Ser Val Lys Met Ser Cys Lys Thr Ser Gly
20 25 30
Tyr Thr Phe Thr Arg Tyr Thr Met His Trp Val Lys Gln Arg Pro Gly
35 40 45
Gln Gly Leu Glu Trp Ile Gly Tyr Ile Asn Pro Ser Arg Gly Tyr Thr
50 55 60
Asn Tyr Asn Gln Lys Phe Lys Asp Lys Ala Thr Leu Thr Thr Asp Lys
65 70 75 80
Ser Ser Ser Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu Asp
85 90 95
Ser Ala Val Tyr Tyr Cys Ala Arg Tyr Tyr Asp Asp His Tyr Cys Leu
100 105 110
Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser Val Glu Gly
115 120 125
Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Val Asp Asp
130 135 140
Ile Gln Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly Glu
145 150 155 160
Lys Val Thr Met Thr Cys Arg Ala Ser Ser Ser Val Ser Tyr Met Asn
165 170 175
Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys Arg Trp Ile Tyr Asp
180 185 190
Thr Ser Lys Val Ala Ser Gly Val Pro Tyr Arg Phe Ser Gly Ser Gly
195 200 205
Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu Ala Glu Asp
210 215 220
Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Leu Thr Phe
225 230 235 240
Gly Ala Gly Thr Lys Leu Glu Leu Lys Gly Gly Gly Gly Ser Cys Arg
245 250 255
Gly Asp Lys Gly Pro Asp Cys
260
Claims (3)
1.一种CD3单链抗体-iRGD融合蛋白，其特征在于：所述融合蛋白由靶向人CD3单链抗体和肿瘤穿透肽iRGD经由连接肽连接组成，所述融合蛋白具有SEQ ID No.1所示的氨基酸序列。
2.一种权利要求1所述CD3单链抗体-iRGD融合蛋白的制备方法，其特征在于，包括以下步骤：
表达载体pET28a-anti-CD3-iRGD的构建；
将表达载体pET28a-anti-CD3-iRGD转化至表达菌株BL21 DE3；
重组anti-CD3-iRGD的诱导表达及纯化；
对重组anti-CD3-iRGD用1mM 异丙基-β-D-硫代半乳糖苷（IPTG）诱导4小时， 收集菌液进行超声破碎，离心后收集沉淀，用8M尿素变性剂溶解沉淀，并依次在6M、4M、2M、0M尿素溶液中透析复性，重组anti-CD3-iRGD经AKTA Purifier 900 FPLC系统，镍离子亲和层析柱纯化；用SDS-PAGE方法检测目的蛋白。
3.权利要求1所述的CD3单链抗体-iRGD融合蛋白在制备抗肿瘤药物中的应用，其特征在于，所述肿瘤包含乳腺癌、胃癌、肝癌、肠癌、胰腺癌、肺癌、宫颈癌。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710978345.8A CN107739410B (zh) | 2017-10-18 | 2017-10-18 | CD3单链抗体-iRGD融合蛋白、制备及其作为抗肿瘤药物的应用 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710978345.8A CN107739410B (zh) | 2017-10-18 | 2017-10-18 | CD3单链抗体-iRGD融合蛋白、制备及其作为抗肿瘤药物的应用 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN107739410A CN107739410A (zh) | 2018-02-27 |
| CN107739410B true CN107739410B (zh) | 2021-07-30 |
Family
ID=61237829
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710978345.8A Active CN107739410B (zh) | 2017-10-18 | 2017-10-18 | CD3单链抗体-iRGD融合蛋白、制备及其作为抗肿瘤药物的应用 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN107739410B (zh) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113795271A (zh) * | 2019-03-08 | 2021-12-14 | 森德治疗公司 | 与irgd共施用以治疗癌症的低剂量细胞因子 |
| EP4431527A1 (en) * | 2023-03-17 | 2024-09-18 | Medizinische Hochschule Hannover | Fusion protein for localized activation of t-cells |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| IL138857A0 (en) * | 1998-04-21 | 2001-10-31 | Micromet Ges For Biomedizinisc | Cd19xcd3 specific polypeptides and uses thereof |
| KR20240033145A (ko) * | 2012-11-13 | 2024-03-12 | 비온테크 에스이 | 클라우딘 발현 암 질환의 치료제 |
| SG11201504602VA (en) * | 2012-12-10 | 2015-07-30 | Hutchinson Fred Cancer Res | Lipocalin fusion partners |
| CN104892766A (zh) * | 2014-03-06 | 2015-09-09 | 南京大学医学院附属鼓楼医院 | 高穿透性纳米抗体融合蛋白制备及其在抗肿瘤中的应用 |
-
2017
- 2017-10-18 CN CN201710978345.8A patent/CN107739410B/zh active Active
Non-Patent Citations (1)
| Title |
|---|
| 抗 C D3单克隆抗体激活的杀伤细胞抗瘤活性研究;曹建平;《沈阳部队医药》;19941231;第7卷(第2期);第110-112页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN107739410A (zh) | 2018-02-27 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| WO2014146350A1 (zh) | 一类突变的具有高耐碱特性的蛋白a及其应用 | |
| CN109529046A (zh) | 一种靶向线粒体的自组装蛋白质纳米颗粒的制备与应用 | |
| CN104829729B (zh) | 一种携带抗Her2/CD3双特异性功能蛋白的人T细胞制备 | |
| CN103319595B (zh) | 抗人afp单链抗体以及融合抗原肽的制备方法和应用 | |
| CN107739410B (zh) | CD3单链抗体-iRGD融合蛋白、制备及其作为抗肿瘤药物的应用 | |
| CN102020717B (zh) | 一种增强淋巴细胞靶向杀伤肿瘤的活性因子及其制备方法 | |
| CN114349840A (zh) | 一种融合蛋白及其制备方法与应用 | |
| CN107226846B (zh) | 新型透明质酸结合肽及透皮吸收与皮下靶向释放制剂 | |
| CN110592057B (zh) | 嵌合裂解酶ILTphg和编码此酶的多核苷酸 | |
| CN108341854A (zh) | 一种肿瘤靶向的新型多肽及其用途 | |
| CN105624082B (zh) | 表达单链抗体scFV或多肽适配体的功能化细菌磁颗粒及其应用 | |
| CN103360497A (zh) | 一种新型抗肿瘤融合蛋白疫苗及其制备方法和应用 | |
| CN109053875B (zh) | 突变型igf-1、重组质粒、重组蛋白及应用 | |
| CN104628863A (zh) | 一种双靶点双弹头抗肿瘤融合蛋白、其编码基因和用途 | |
| CN110903402A (zh) | 一种双特异性融合蛋白及其构建方法与应用 | |
| CN107746432A (zh) | 一种Aβ42修饰蛋白及其表达与纯化方法 | |
| CN107344970B (zh) | 一种重组融合蛋白v12‑cd137l的制备方法及应用 | |
| CN106946984A (zh) | 一种具生物活性的霍乱毒素b亚基蛋白的制备方法 | |
| CN108753751B (zh) | 一种人端粒酶逆转录酶抗原肽及其制备方法 | |
| CN108300725A (zh) | 可溶性单链抗体超抗原融合基因及蛋白和其制备与应用 | |
| CN114478793A (zh) | CPP-scFv融合蛋白及相应的核酸分子、载体、细胞和药物 | |
| CN113563474B (zh) | EpCAM-CD16-NKG2D三特异性抗体及其应用 | |
| CN111116757A (zh) | 带有半乳糖结合凝集素ew29标签的铁蛋白融合蛋白、蛋白笼纳米颗粒及其制备方法 | |
| CN116790616B (zh) | 编码sCXCL16的基因及表达载体、制备方法和应用 | |
| CN104491870B (zh) | 来自lt亚基的细胞穿膜肽lta2作为细胞内药物运输载体的应用 |
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 | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| TR01 | Transfer of patent right | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20240102 Address after: Floor 16, No. 192 Yuzhou Road, Shiyou Road Street, Yuzhong District, Chongqing, 400042 Patentee after: Zhongheng Guoxin (Chongqing) Big Health Research Institute Co.,Ltd. Address before: 210008 No. 321, Gulou District, Jiangsu, Zhongshan Road, Nanjing Patentee before: NANJING DRUM TOWER Hospital |