KR20230091594A - Salmonella mutant expressing anti-PD-L1 peptide and outer membrane vesicle thereof and pharmaceutical comprising the same - Google Patents
Salmonella mutant expressing anti-PD-L1 peptide and outer membrane vesicle thereof and pharmaceutical comprising the same Download PDFInfo
- Publication number
- KR20230091594A KR20230091594A KR1020210180838A KR20210180838A KR20230091594A KR 20230091594 A KR20230091594 A KR 20230091594A KR 1020210180838 A KR1020210180838 A KR 1020210180838A KR 20210180838 A KR20210180838 A KR 20210180838A KR 20230091594 A KR20230091594 A KR 20230091594A
- Authority
- KR
- South Korea
- Prior art keywords
- cancer
- salmonella
- outer membrane
- peptide
- strain
- Prior art date
Links
- 108090000765 processed proteins & peptides Proteins 0.000 title claims abstract description 50
- 241000607142 Salmonella Species 0.000 title claims abstract description 44
- 239000012528 membrane Substances 0.000 title claims abstract description 25
- 206010028980 Neoplasm Diseases 0.000 claims abstract description 38
- 201000011510 cancer Diseases 0.000 claims abstract description 28
- 210000002472 endoplasmic reticulum Anatomy 0.000 claims abstract description 13
- 239000008194 pharmaceutical composition Substances 0.000 claims abstract description 12
- 108010079246 OMPA outer membrane proteins Proteins 0.000 claims description 25
- 238000000034 method Methods 0.000 claims description 12
- 108090000623 proteins and genes Proteins 0.000 claims description 10
- 239000002246 antineoplastic agent Substances 0.000 claims description 7
- 241000293869 Salmonella enterica subsp. enterica serovar Typhimurium Species 0.000 claims description 6
- 102000004169 proteins and genes Human genes 0.000 claims description 6
- 239000004480 active ingredient Substances 0.000 claims description 5
- 241001354013 Salmonella enterica subsp. enterica serovar Enteritidis Species 0.000 claims description 4
- 206010009944 Colon cancer Diseases 0.000 claims description 3
- 208000017604 Hodgkin disease Diseases 0.000 claims description 3
- 208000021519 Hodgkin lymphoma Diseases 0.000 claims description 3
- 208000010747 Hodgkins lymphoma Diseases 0.000 claims description 3
- 206010058467 Lung neoplasm malignant Diseases 0.000 claims description 3
- 206010061902 Pancreatic neoplasm Diseases 0.000 claims description 3
- 241001355103 Salmonella enterica subsp. enterica serovar Braenderup Species 0.000 claims description 3
- 201000010099 disease Diseases 0.000 claims description 3
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims description 3
- 201000005202 lung cancer Diseases 0.000 claims description 3
- 208000020816 lung neoplasm Diseases 0.000 claims description 3
- 208000015486 malignant pancreatic neoplasm Diseases 0.000 claims description 3
- 208000018795 nasal cavity and paranasal sinus carcinoma Diseases 0.000 claims description 3
- 201000002528 pancreatic cancer Diseases 0.000 claims description 3
- 208000008443 pancreatic carcinoma Diseases 0.000 claims description 3
- 206010061424 Anal cancer Diseases 0.000 claims description 2
- 208000007860 Anus Neoplasms Diseases 0.000 claims description 2
- 206010004593 Bile duct cancer Diseases 0.000 claims description 2
- 206010005003 Bladder cancer Diseases 0.000 claims description 2
- 206010006187 Breast cancer Diseases 0.000 claims description 2
- 208000026310 Breast neoplasm Diseases 0.000 claims description 2
- 208000000461 Esophageal Neoplasms Diseases 0.000 claims description 2
- 208000022072 Gallbladder Neoplasms Diseases 0.000 claims description 2
- 208000008839 Kidney Neoplasms Diseases 0.000 claims description 2
- 206010023825 Laryngeal cancer Diseases 0.000 claims description 2
- 206010062038 Lip neoplasm Diseases 0.000 claims description 2
- 208000032271 Malignant tumor of penis Diseases 0.000 claims description 2
- 102000018697 Membrane Proteins Human genes 0.000 claims description 2
- 108010052285 Membrane Proteins Proteins 0.000 claims description 2
- 208000003445 Mouth Neoplasms Diseases 0.000 claims description 2
- 201000003793 Myelodysplastic syndrome Diseases 0.000 claims description 2
- 208000001894 Nasopharyngeal Neoplasms Diseases 0.000 claims description 2
- 206010061306 Nasopharyngeal cancer Diseases 0.000 claims description 2
- 206010030155 Oesophageal carcinoma Diseases 0.000 claims description 2
- 206010031096 Oropharyngeal cancer Diseases 0.000 claims description 2
- 206010057444 Oropharyngeal neoplasm Diseases 0.000 claims description 2
- 206010033128 Ovarian cancer Diseases 0.000 claims description 2
- 206010061535 Ovarian neoplasm Diseases 0.000 claims description 2
- 208000000821 Parathyroid Neoplasms Diseases 0.000 claims description 2
- 208000002471 Penile Neoplasms Diseases 0.000 claims description 2
- 206010034299 Penile cancer Diseases 0.000 claims description 2
- 206010060862 Prostate cancer Diseases 0.000 claims description 2
- 208000000236 Prostatic Neoplasms Diseases 0.000 claims description 2
- 208000015634 Rectal Neoplasms Diseases 0.000 claims description 2
- 206010038389 Renal cancer Diseases 0.000 claims description 2
- 206010046431 Urethral cancer Diseases 0.000 claims description 2
- 206010046458 Urethral neoplasms Diseases 0.000 claims description 2
- 208000007097 Urinary Bladder Neoplasms Diseases 0.000 claims description 2
- 206010047741 Vulval cancer Diseases 0.000 claims description 2
- 208000004354 Vulvar Neoplasms Diseases 0.000 claims description 2
- 201000011165 anus cancer Diseases 0.000 claims description 2
- 208000026900 bile duct neoplasm Diseases 0.000 claims description 2
- 208000006990 cholangiocarcinoma Diseases 0.000 claims description 2
- 208000029742 colonic neoplasm Diseases 0.000 claims description 2
- 201000004101 esophageal cancer Diseases 0.000 claims description 2
- 201000010175 gallbladder cancer Diseases 0.000 claims description 2
- 201000010982 kidney cancer Diseases 0.000 claims description 2
- 206010023841 laryngeal neoplasm Diseases 0.000 claims description 2
- 208000032839 leukemia Diseases 0.000 claims description 2
- 208000012987 lip and oral cavity carcinoma Diseases 0.000 claims description 2
- 201000006721 lip cancer Diseases 0.000 claims description 2
- 201000007270 liver cancer Diseases 0.000 claims description 2
- 208000014018 liver neoplasm Diseases 0.000 claims description 2
- 208000026045 malignant tumor of parathyroid gland Diseases 0.000 claims description 2
- 201000005962 mycosis fungoides Diseases 0.000 claims description 2
- 208000002154 non-small cell lung carcinoma Diseases 0.000 claims description 2
- 201000006958 oropharynx cancer Diseases 0.000 claims description 2
- 206010038038 rectal cancer Diseases 0.000 claims description 2
- 201000001275 rectum cancer Diseases 0.000 claims description 2
- 201000002314 small intestine cancer Diseases 0.000 claims description 2
- 206010041823 squamous cell carcinoma Diseases 0.000 claims description 2
- 208000013077 thyroid gland carcinoma Diseases 0.000 claims description 2
- 208000029729 tumor suppressor gene on chromosome 11 Diseases 0.000 claims description 2
- 201000005112 urinary bladder cancer Diseases 0.000 claims description 2
- 206010046885 vaginal cancer Diseases 0.000 claims description 2
- 208000013139 vaginal neoplasm Diseases 0.000 claims description 2
- 201000005102 vulva cancer Diseases 0.000 claims description 2
- 125000003275 alpha amino acid group Chemical group 0.000 claims 3
- 208000017572 squamous cell neoplasm Diseases 0.000 claims 1
- 210000004027 cell Anatomy 0.000 abstract description 50
- 210000001744 T-lymphocyte Anatomy 0.000 abstract description 8
- 238000011282 treatment Methods 0.000 abstract description 7
- 230000004913 activation Effects 0.000 abstract description 3
- 230000000259 anti-tumor effect Effects 0.000 abstract description 3
- 230000001939 inductive effect Effects 0.000 abstract description 2
- 230000015572 biosynthetic process Effects 0.000 abstract 1
- 108020004414 DNA Proteins 0.000 description 29
- 239000013598 vector Substances 0.000 description 13
- 241000894006 Bacteria Species 0.000 description 10
- 239000013612 plasmid Substances 0.000 description 10
- 239000012634 fragment Substances 0.000 description 8
- 102100037608 Spectrin alpha chain, erythrocytic 1 Human genes 0.000 description 6
- 150000001413 amino acids Chemical group 0.000 description 6
- 230000002238 attenuated effect Effects 0.000 description 6
- -1 bisulfan Chemical compound 0.000 description 6
- 239000002609 medium Substances 0.000 description 6
- 239000000546 pharmaceutical excipient Substances 0.000 description 6
- 238000002360 preparation method Methods 0.000 description 6
- 101100519207 Mus musculus Pdcd1 gene Proteins 0.000 description 5
- 230000000694 effects Effects 0.000 description 5
- 239000000203 mixture Substances 0.000 description 5
- AOJJSUZBOXZQNB-TZSSRYMLSA-N Doxorubicin Chemical compound O([C@H]1C[C@@](O)(CC=2C(O)=C3C(=O)C=4C=CC=C(C=4C(=O)C3=C(O)C=21)OC)C(=O)CO)[C@H]1C[C@H](N)[C@H](O)[C@H](C)O1 AOJJSUZBOXZQNB-TZSSRYMLSA-N 0.000 description 4
- MXXXVOYFNVJHMA-IUCAKERBSA-N Gly-Arg-Met Chemical compound CSCC[C@@H](C(=O)O)NC(=O)[C@H](CCCN=C(N)N)NC(=O)CN MXXXVOYFNVJHMA-IUCAKERBSA-N 0.000 description 4
- 229940076838 Immune checkpoint inhibitor Drugs 0.000 description 4
- 108091008026 Inhibitory immune checkpoint proteins Proteins 0.000 description 4
- 102000037984 Inhibitory immune checkpoint proteins Human genes 0.000 description 4
- UIUWGMRJTWHIJZ-ULQDDVLXSA-N Pro-Tyr-Lys Chemical compound C1C[C@H](NC1)C(=O)N[C@@H](CC2=CC=C(C=C2)O)C(=O)N[C@@H](CCCCN)C(=O)O UIUWGMRJTWHIJZ-ULQDDVLXSA-N 0.000 description 4
- 239000012274 immune-checkpoint protein inhibitor Substances 0.000 description 4
- 108010064235 lysylglycine Proteins 0.000 description 4
- HQKMJHAJHXVSDF-UHFFFAOYSA-L magnesium stearate Chemical compound [Mg+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O HQKMJHAJHXVSDF-UHFFFAOYSA-L 0.000 description 4
- 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 4
- DNIAPMSPPWPWGF-UHFFFAOYSA-N Propylene glycol Chemical compound CC(O)CO DNIAPMSPPWPWGF-UHFFFAOYSA-N 0.000 description 3
- 108010044940 alanylglutamine Proteins 0.000 description 3
- 230000001093 anti-cancer Effects 0.000 description 3
- WIIZWVCIJKGZOK-RKDXNWHRSA-N chloramphenicol Chemical compound ClC(Cl)C(=O)N[C@H](CO)[C@H](O)C1=CC=C([N+]([O-])=O)C=C1 WIIZWVCIJKGZOK-RKDXNWHRSA-N 0.000 description 3
- 229960005091 chloramphenicol Drugs 0.000 description 3
- 239000003085 diluting agent Substances 0.000 description 3
- 238000004520 electroporation Methods 0.000 description 3
- 238000009472 formulation Methods 0.000 description 3
- 244000005700 microbiome Species 0.000 description 3
- 238000002703 mutagenesis Methods 0.000 description 3
- 231100000350 mutagenesis Toxicity 0.000 description 3
- 108091033319 polynucleotide Proteins 0.000 description 3
- 239000002157 polynucleotide Substances 0.000 description 3
- 102000040430 polynucleotide Human genes 0.000 description 3
- 108091008146 restriction endonucleases Proteins 0.000 description 3
- 239000007787 solid Substances 0.000 description 3
- 239000003826 tablet Substances 0.000 description 3
- 239000013076 target substance Substances 0.000 description 3
- 231100000419 toxicity Toxicity 0.000 description 3
- 230000001988 toxicity Effects 0.000 description 3
- XAXMJQUMRJAFCH-CQDKDKBSSA-N Ala-Tyr-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)[C@@H](N)C)CC1=CC=C(O)C=C1 XAXMJQUMRJAFCH-CQDKDKBSSA-N 0.000 description 2
- GUBGYTABKSRVRQ-XLOQQCSPSA-N Alpha-Lactose Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@H](O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-XLOQQCSPSA-N 0.000 description 2
- CTQIOCMSIJATNX-WHFBIAKZSA-N Asn-Gly-Ala Chemical compound [H]N[C@@H](CC(N)=O)C(=O)NCC(=O)N[C@@H](C)C(O)=O CTQIOCMSIJATNX-WHFBIAKZSA-N 0.000 description 2
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 2
- VTYYLEPIZMXCLO-UHFFFAOYSA-L Calcium carbonate Chemical compound [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 description 2
- 241000588724 Escherichia coli Species 0.000 description 2
- 108010010803 Gelatin Proteins 0.000 description 2
- SUDUYJOBLHQAMI-WHFBIAKZSA-N Gly-Asp-Cys Chemical compound NCC(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CS)C(O)=O SUDUYJOBLHQAMI-WHFBIAKZSA-N 0.000 description 2
- 108010043121 Green Fluorescent Proteins Proteins 0.000 description 2
- 102000004144 Green Fluorescent Proteins Human genes 0.000 description 2
- 101000611936 Homo sapiens Programmed cell death protein 1 Proteins 0.000 description 2
- UAVQIQOOBXFKRC-BYULHYEWSA-N Ile-Asn-Gly Chemical compound CC[C@H](C)[C@H](N)C(=O)N[C@@H](CC(N)=O)C(=O)NCC(O)=O UAVQIQOOBXFKRC-BYULHYEWSA-N 0.000 description 2
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 description 2
- 108010079364 N-glycylalanine Proteins 0.000 description 2
- 108091028043 Nucleic acid sequence Proteins 0.000 description 2
- 101710116435 Outer membrane protein Proteins 0.000 description 2
- 229930040373 Paraformaldehyde Natural products 0.000 description 2
- 229930182555 Penicillin Natural products 0.000 description 2
- 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 2
- 102000018120 Recombinases Human genes 0.000 description 2
- 108010091086 Recombinases Proteins 0.000 description 2
- 229920002472 Starch Polymers 0.000 description 2
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 2
- 229930006000 Sucrose Natural products 0.000 description 2
- NKANXQFJJICGDU-QPLCGJKRSA-N Tamoxifen Chemical compound C=1C=CC=CC=1C(/CC)=C(C=1C=CC(OCCN(C)C)=CC=1)/C1=CC=CC=C1 NKANXQFJJICGDU-QPLCGJKRSA-N 0.000 description 2
- JAGGEZACYAAMIL-CQDKDKBSSA-N Tyr-Lys-Ala Chemical compound C[C@@H](C(=O)O)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CC1=CC=C(C=C1)O)N JAGGEZACYAAMIL-CQDKDKBSSA-N 0.000 description 2
- BUFLLCUFNHESEH-UHFFFAOYSA-N [5-(2-amino-6-oxo-3h-purin-9-yl)-4-hydroxy-2-[[hydroxy(phosphonooxy)phosphoryl]oxymethyl]oxolan-3-yl] phosphono hydrogen phosphate Chemical compound C1=2NC(N)=NC(=O)C=2N=CN1C1OC(COP(O)(=O)OP(O)(O)=O)C(OP(O)(=O)OP(O)(O)=O)C1O BUFLLCUFNHESEH-UHFFFAOYSA-N 0.000 description 2
- RJURFGZVJUQBHK-UHFFFAOYSA-N actinomycin D Natural products CC1OC(=O)C(C(C)C)N(C)C(=O)CN(C)C(=O)C2CCCN2C(=O)C(C(C)C)NC(=O)C1NC(=O)C1=C(N)C(=O)C(C)=C2OC(C(C)=CC=C3C(=O)NC4C(=O)NC(C(N5CCCC5C(=O)N(C)CC(=O)N(C)C(C(C)C)C(=O)OC4C)=O)C(C)C)=C3N=C21 RJURFGZVJUQBHK-UHFFFAOYSA-N 0.000 description 2
- 239000003125 aqueous solvent Substances 0.000 description 2
- 108010018691 arginyl-threonyl-arginine Proteins 0.000 description 2
- 230000001580 bacterial effect Effects 0.000 description 2
- SRBFZHDQGSBBOR-UHFFFAOYSA-N beta-D-Pyranose-Lyxose Natural products OC1COC(O)C(O)C1O SRBFZHDQGSBBOR-UHFFFAOYSA-N 0.000 description 2
- 230000003115 biocidal effect Effects 0.000 description 2
- 229940098773 bovine serum albumin Drugs 0.000 description 2
- 239000002775 capsule Substances 0.000 description 2
- 238000007796 conventional method Methods 0.000 description 2
- 239000006059 cover glass Substances 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 239000000839 emulsion Substances 0.000 description 2
- 210000003527 eukaryotic cell Anatomy 0.000 description 2
- 239000013604 expression vector Substances 0.000 description 2
- 229920000159 gelatin Polymers 0.000 description 2
- 239000008273 gelatin Substances 0.000 description 2
- 235000019322 gelatine Nutrition 0.000 description 2
- 235000011852 gelatine desserts Nutrition 0.000 description 2
- VPZXBVLAVMBEQI-UHFFFAOYSA-N glycyl-DL-alpha-alanine Natural products OC(=O)C(C)NC(=O)CN VPZXBVLAVMBEQI-UHFFFAOYSA-N 0.000 description 2
- 239000008187 granular material Substances 0.000 description 2
- 239000005090 green fluorescent protein Substances 0.000 description 2
- 230000012010 growth Effects 0.000 description 2
- 238000002744 homologous recombination Methods 0.000 description 2
- 230000006801 homologous recombination Effects 0.000 description 2
- 102000048362 human PDCD1 Human genes 0.000 description 2
- 239000008101 lactose Substances 0.000 description 2
- 235000019359 magnesium stearate Nutrition 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 238000007431 microscopic evaluation Methods 0.000 description 2
- 230000004899 motility Effects 0.000 description 2
- 235000015097 nutrients Nutrition 0.000 description 2
- 229920002866 paraformaldehyde Polymers 0.000 description 2
- 230000001717 pathogenic effect Effects 0.000 description 2
- 230000007918 pathogenicity Effects 0.000 description 2
- 229940049954 penicillin Drugs 0.000 description 2
- 239000000843 powder Substances 0.000 description 2
- 230000010076 replication Effects 0.000 description 2
- 239000008107 starch Substances 0.000 description 2
- 235000019698 starch Nutrition 0.000 description 2
- 229960005322 streptomycin Drugs 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 239000005720 sucrose Substances 0.000 description 2
- 239000000829 suppository Substances 0.000 description 2
- 239000000725 suspension Substances 0.000 description 2
- 239000000454 talc Substances 0.000 description 2
- 229910052623 talc Inorganic materials 0.000 description 2
- 235000012222 talc Nutrition 0.000 description 2
- 108091005703 transmembrane proteins Proteins 0.000 description 2
- 102000035160 transmembrane proteins Human genes 0.000 description 2
- 230000007923 virulence factor Effects 0.000 description 2
- 239000000304 virulence factor Substances 0.000 description 2
- 239000000080 wetting agent Substances 0.000 description 2
- FWBHETKCLVMNFS-UHFFFAOYSA-N 4',6-Diamino-2-phenylindol Chemical compound C1=CC(C(=N)N)=CC=C1C1=CC2=CC=C(C(N)=N)C=C2N1 FWBHETKCLVMNFS-UHFFFAOYSA-N 0.000 description 1
- AOJJSUZBOXZQNB-VTZDEGQISA-N 4'-epidoxorubicin Chemical compound O([C@H]1C[C@@](O)(CC=2C(O)=C3C(=O)C=4C=CC=C(C=4C(=O)C3=C(O)C=21)OC)C(=O)CO)[C@H]1C[C@H](N)[C@@H](O)[C@H](C)O1 AOJJSUZBOXZQNB-VTZDEGQISA-N 0.000 description 1
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 description 1
- UPJKSWLLCONYMW-UHFFFAOYSA-N 5'-Adenosine monophosphate Natural products COc1cc(O)c(C(=O)C)c(OC2OC(COC3OC(C)C(O)C(O)C3O)C(O)C(O)C2O)c1 UPJKSWLLCONYMW-UHFFFAOYSA-N 0.000 description 1
- FHVDTGUDJYJELY-UHFFFAOYSA-N 6-{[2-carboxy-4,5-dihydroxy-6-(phosphanyloxy)oxan-3-yl]oxy}-4,5-dihydroxy-3-phosphanyloxane-2-carboxylic acid Chemical compound O1C(C(O)=O)C(P)C(O)C(O)C1OC1C(C(O)=O)OC(OP)C(O)C1O FHVDTGUDJYJELY-UHFFFAOYSA-N 0.000 description 1
- STQGQHZAVUOBTE-UHFFFAOYSA-N 7-Cyan-hept-2t-en-4,6-diinsaeure Natural products C1=2C(O)=C3C(=O)C=4C(OC)=CC=CC=4C(=O)C3=C(O)C=2CC(O)(C(C)=O)CC1OC1CC(N)C(O)C(C)O1 STQGQHZAVUOBTE-UHFFFAOYSA-N 0.000 description 1
- 244000215068 Acacia senegal Species 0.000 description 1
- 235000006491 Acacia senegal Nutrition 0.000 description 1
- JAMAWBXXKFGFGX-KZVJFYERSA-N Ala-Arg-Thr Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H]([C@@H](C)O)C(O)=O JAMAWBXXKFGFGX-KZVJFYERSA-N 0.000 description 1
- VWEWCZSUWOEEFM-WDSKDSINSA-N Ala-Gly-Ala-Gly Chemical compound C[C@H](N)C(=O)NCC(=O)N[C@@H](C)C(=O)NCC(O)=O VWEWCZSUWOEEFM-WDSKDSINSA-N 0.000 description 1
- SYIFFFHSXBNPMC-UWJYBYFXSA-N Ala-Ser-Tyr Chemical compound C[C@@H](C(=O)N[C@@H](CO)C(=O)N[C@@H](CC1=CC=C(C=C1)O)C(=O)O)N SYIFFFHSXBNPMC-UWJYBYFXSA-N 0.000 description 1
- 239000012109 Alexa Fluor 568 Substances 0.000 description 1
- SGYSTDWPNPKJPP-GUBZILKMSA-N Arg-Ala-Arg Chemical compound NC(=N)NCCC[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O SGYSTDWPNPKJPP-GUBZILKMSA-N 0.000 description 1
- MFAMTAVAFBPXDC-LPEHRKFASA-N Arg-Asp-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CC(=O)O)NC(=O)[C@H](CCCN=C(N)N)N)C(=O)O MFAMTAVAFBPXDC-LPEHRKFASA-N 0.000 description 1
- 241000186000 Bifidobacterium Species 0.000 description 1
- 108010006654 Bleomycin Proteins 0.000 description 1
- 108010078791 Carrier Proteins Proteins 0.000 description 1
- PTHCMJGKKRQCBF-UHFFFAOYSA-N Cellulose, microcrystalline Chemical compound OC1C(O)C(OC)OC(CO)C1OC1C(O)C(O)C(OC)C(CO)O1 PTHCMJGKKRQCBF-UHFFFAOYSA-N 0.000 description 1
- 241000193403 Clostridium Species 0.000 description 1
- UDMBCSSLTHHNCD-UHFFFAOYSA-N Coenzym Q(11) Natural products C1=NC=2C(N)=NC=NC=2N1C1OC(COP(O)(O)=O)C(O)C1O UDMBCSSLTHHNCD-UHFFFAOYSA-N 0.000 description 1
- 208000001333 Colorectal Neoplasms Diseases 0.000 description 1
- CMSMOCZEIVJLDB-UHFFFAOYSA-N Cyclophosphamide Chemical compound ClCCN(CCCl)P1(=O)NCCCO1 CMSMOCZEIVJLDB-UHFFFAOYSA-N 0.000 description 1
- DCXGXDGGXVZVMY-GHCJXIJMSA-N Cys-Asn-Ile Chemical compound CC[C@H](C)[C@@H](C(O)=O)NC(=O)[C@H](CC(N)=O)NC(=O)[C@@H](N)CS DCXGXDGGXVZVMY-GHCJXIJMSA-N 0.000 description 1
- HKALUUKHYNEDRS-GUBZILKMSA-N Cys-Leu-Gln Chemical compound SC[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(O)=O HKALUUKHYNEDRS-GUBZILKMSA-N 0.000 description 1
- MHYHLWUGWUBUHF-GUBZILKMSA-N Cys-Val-Arg Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CCCN=C(N)N)C(=O)O)NC(=O)[C@H](CS)N MHYHLWUGWUBUHF-GUBZILKMSA-N 0.000 description 1
- UHDGCWIWMRVCDJ-CCXZUQQUSA-N Cytarabine Chemical compound O=C1N=C(N)C=CN1[C@H]1[C@@H](O)[C@H](O)[C@@H](CO)O1 UHDGCWIWMRVCDJ-CCXZUQQUSA-N 0.000 description 1
- FBPFZTCFMRRESA-FSIIMWSLSA-N D-Glucitol Natural products OC[C@H](O)[C@H](O)[C@@H](O)[C@H](O)CO FBPFZTCFMRRESA-FSIIMWSLSA-N 0.000 description 1
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 description 1
- FBPFZTCFMRRESA-JGWLITMVSA-N D-glucitol Chemical compound OC[C@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-JGWLITMVSA-N 0.000 description 1
- 108700020911 DNA-Binding Proteins Proteins 0.000 description 1
- 102000052510 DNA-Binding Proteins Human genes 0.000 description 1
- 108010092160 Dactinomycin Proteins 0.000 description 1
- 102000000541 Defensins Human genes 0.000 description 1
- 108010002069 Defensins Proteins 0.000 description 1
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 1
- LVGKNOAMLMIIKO-UHFFFAOYSA-N Elaidinsaeure-aethylester Natural products CCCCCCCCC=CCCCCCCCC(=O)OCC LVGKNOAMLMIIKO-UHFFFAOYSA-N 0.000 description 1
- HTIJFSOGRVMCQR-UHFFFAOYSA-N Epirubicin Natural products COc1cccc2C(=O)c3c(O)c4CC(O)(CC(OC5CC(N)C(=O)C(C)O5)c4c(O)c3C(=O)c12)C(=O)CO HTIJFSOGRVMCQR-UHFFFAOYSA-N 0.000 description 1
- 239000004386 Erythritol Substances 0.000 description 1
- UNXHWFMMPAWVPI-UHFFFAOYSA-N Erythritol Natural products OCC(O)C(O)CO UNXHWFMMPAWVPI-UHFFFAOYSA-N 0.000 description 1
- GHASVSINZRGABV-UHFFFAOYSA-N Fluorouracil Chemical compound FC1=CNC(=O)NC1=O GHASVSINZRGABV-UHFFFAOYSA-N 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- GWCRIHNSVMOBEQ-BQBZGAKWSA-N Gly-Arg-Ser Chemical compound [H]NCC(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CO)C(O)=O GWCRIHNSVMOBEQ-BQBZGAKWSA-N 0.000 description 1
- XQHSBNVACKQWAV-WHFBIAKZSA-N Gly-Asp-Asn Chemical compound [H]NCC(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(N)=O)C(O)=O XQHSBNVACKQWAV-WHFBIAKZSA-N 0.000 description 1
- QGZSAHIZRQHCEQ-QWRGUYRKSA-N Gly-Asp-Tyr Chemical compound NCC(=O)N[C@@H](CC(O)=O)C(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 QGZSAHIZRQHCEQ-QWRGUYRKSA-N 0.000 description 1
- 229920000084 Gum arabic Polymers 0.000 description 1
- CVEFOCIRMVGWDS-XIRDDKMYSA-N His-Cys-Trp Chemical compound C([C@H](N)C(=O)N[C@@H](CS)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(O)=O)C1=CN=CN1 CVEFOCIRMVGWDS-XIRDDKMYSA-N 0.000 description 1
- 101000619708 Homo sapiens Peroxiredoxin-6 Proteins 0.000 description 1
- 108091008036 Immune checkpoint proteins Proteins 0.000 description 1
- 102000037982 Immune checkpoint proteins Human genes 0.000 description 1
- SRBFZHDQGSBBOR-HWQSCIPKSA-N L-arabinopyranose Chemical compound O[C@H]1COC(O)[C@H](O)[C@H]1O SRBFZHDQGSBBOR-HWQSCIPKSA-N 0.000 description 1
- FBOZXECLQNJBKD-ZDUSSCGKSA-N L-methotrexate Chemical compound C=1N=C2N=C(N)N=C(N)C2=NC=1CN(C)C1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 FBOZXECLQNJBKD-ZDUSSCGKSA-N 0.000 description 1
- FQZPTCNSNPWHLJ-AVGNSLFASA-N Leu-Gln-Lys Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCCCN)C(O)=O FQZPTCNSNPWHLJ-AVGNSLFASA-N 0.000 description 1
- 239000000232 Lipid Bilayer Substances 0.000 description 1
- 102000004895 Lipoproteins Human genes 0.000 description 1
- 108090001030 Lipoproteins Proteins 0.000 description 1
- 108010047357 Luminescent Proteins Proteins 0.000 description 1
- 102000006830 Luminescent Proteins Human genes 0.000 description 1
- GUYHHBZCBQZLFW-GUBZILKMSA-N Lys-Gln-Cys Chemical compound C(CCN)C[C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)N[C@@H](CS)C(=O)O)N GUYHHBZCBQZLFW-GUBZILKMSA-N 0.000 description 1
- BDFHWFUAQLIMJO-KXNHARMFSA-N Lys-Thr-Pro Chemical compound C[C@H]([C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@H](CCCCN)N)O BDFHWFUAQLIMJO-KXNHARMFSA-N 0.000 description 1
- 229930195725 Mannitol Natural products 0.000 description 1
- 206010027476 Metastases Diseases 0.000 description 1
- 229920000168 Microcrystalline cellulose Polymers 0.000 description 1
- 229930192392 Mitomycin Natural products 0.000 description 1
- 101100476480 Mus musculus S100a8 gene Proteins 0.000 description 1
- 101100107522 Mus musculus Slc1a5 gene Proteins 0.000 description 1
- NWIBSHFKIJFRCO-WUDYKRTCSA-N Mytomycin Chemical compound C1N2C(C(C(C)=C(N)C3=O)=O)=C3[C@@H](COC(N)=O)[C@@]2(OC)[C@@H]2[C@H]1N2 NWIBSHFKIJFRCO-WUDYKRTCSA-N 0.000 description 1
- ZDZOTLJHXYCWBA-VCVYQWHSSA-N N-debenzoyl-N-(tert-butoxycarbonyl)-10-deacetyltaxol Chemical compound O([C@H]1[C@H]2[C@@](C([C@H](O)C3=C(C)[C@@H](OC(=O)[C@H](O)[C@@H](NC(=O)OC(C)(C)C)C=4C=CC=CC=4)C[C@]1(O)C3(C)C)=O)(C)[C@@H](O)C[C@H]1OC[C@]12OC(=O)C)C(=O)C1=CC=CC=C1 ZDZOTLJHXYCWBA-VCVYQWHSSA-N 0.000 description 1
- 101100068676 Neurospora crassa (strain ATCC 24698 / 74-OR23-1A / CBS 708.71 / DSM 1257 / FGSC 987) gln-1 gene Proteins 0.000 description 1
- 108700028353 OmpC Proteins 0.000 description 1
- 108700006385 OmpF Proteins 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 229930012538 Paclitaxel Natural products 0.000 description 1
- 102000004503 Perforin Human genes 0.000 description 1
- 108010056995 Perforin Proteins 0.000 description 1
- 102100022239 Peroxiredoxin-6 Human genes 0.000 description 1
- 239000002202 Polyethylene glycol Substances 0.000 description 1
- DMKWYMWNEKIPFC-IUCAKERBSA-N Pro-Gly-Arg Chemical compound [H]N1CCC[C@H]1C(=O)NCC(=O)N[C@@H](CCCNC(N)=N)C(O)=O DMKWYMWNEKIPFC-IUCAKERBSA-N 0.000 description 1
- 239000012980 RPMI-1640 medium Substances 0.000 description 1
- 108020005091 Replication Origin Proteins 0.000 description 1
- YMEXHZTVKDAKIY-GHCJXIJMSA-N Ser-Asn-Ile Chemical compound CC[C@H](C)[C@H](NC(=O)[C@H](CC(N)=O)NC(=O)[C@@H](N)CO)C(O)=O YMEXHZTVKDAKIY-GHCJXIJMSA-N 0.000 description 1
- 101150088517 TCTA gene Proteins 0.000 description 1
- 244000299461 Theobroma cacao Species 0.000 description 1
- 235000005764 Theobroma cacao ssp. cacao Nutrition 0.000 description 1
- 235000005767 Theobroma cacao ssp. sphaerocarpum Nutrition 0.000 description 1
- JMZKMSTYXHFYAK-VEVYYDQMSA-N Thr-Arg-Asn Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](CC(=O)N)C(=O)O)N)O JMZKMSTYXHFYAK-VEVYYDQMSA-N 0.000 description 1
- GFRIEEKFXOVPIR-RHYQMDGZSA-N Thr-Pro-Lys Chemical compound C[C@@H](O)[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCCCN)C(O)=O GFRIEEKFXOVPIR-RHYQMDGZSA-N 0.000 description 1
- 239000013504 Triton X-100 Substances 0.000 description 1
- 229920004890 Triton X-100 Polymers 0.000 description 1
- TZNNEYFZZAHLBL-BPUTZDHNSA-N Trp-Arg-Asp Chemical compound [H]N[C@@H](CC1=CNC2=C1C=CC=C2)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(O)=O)C(O)=O TZNNEYFZZAHLBL-BPUTZDHNSA-N 0.000 description 1
- NULQKGDFWHIGMD-NYVOZVTQSA-N Trp-Cys-Trp Chemical compound [H]N[C@@H](CC1=CNC2=C1C=CC=C2)C(=O)N[C@@H](CS)C(=O)N[C@@H](CC1=CNC2=C1C=CC=C2)C(O)=O NULQKGDFWHIGMD-NYVOZVTQSA-N 0.000 description 1
- LGEYOIQBBIPHQN-UWJYBYFXSA-N Tyr-Ala-Ser Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@H](C)NC(=O)[C@@H](N)CC1=CC=C(O)C=C1 LGEYOIQBBIPHQN-UWJYBYFXSA-N 0.000 description 1
- OHNXAUCZVWGTLL-KKUMJFAQSA-N Tyr-His-Cys Chemical compound C1=CC(=CC=C1C[C@@H](C(=O)N[C@@H](CC2=CN=CN2)C(=O)N[C@@H](CS)C(=O)O)N)O OHNXAUCZVWGTLL-KKUMJFAQSA-N 0.000 description 1
- TVXBFESIOXBWNM-UHFFFAOYSA-N Xylitol Natural products OCCC(O)C(O)C(O)CCO TVXBFESIOXBWNM-UHFFFAOYSA-N 0.000 description 1
- 235000010489 acacia gum Nutrition 0.000 description 1
- RJURFGZVJUQBHK-IIXSONLDSA-N actinomycin D Chemical compound C[C@H]1OC(=O)[C@H](C(C)C)N(C)C(=O)CN(C)C(=O)[C@@H]2CCCN2C(=O)[C@@H](C(C)C)NC(=O)[C@H]1NC(=O)C1=C(N)C(=O)C(C)=C2OC(C(C)=CC=C3C(=O)N[C@@H]4C(=O)N[C@@H](C(N5CCC[C@H]5C(=O)N(C)CC(=O)N(C)[C@@H](C(C)C)C(=O)O[C@@H]4C)=O)C(C)C)=C3N=C21 RJURFGZVJUQBHK-IIXSONLDSA-N 0.000 description 1
- UDMBCSSLTHHNCD-KQYNXXCUSA-N adenosine 5'-monophosphate Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](COP(O)(O)=O)[C@@H](O)[C@H]1O UDMBCSSLTHHNCD-KQYNXXCUSA-N 0.000 description 1
- 229940009456 adriamycin Drugs 0.000 description 1
- 239000000443 aerosol Substances 0.000 description 1
- 229940072056 alginate Drugs 0.000 description 1
- 235000010443 alginic acid Nutrition 0.000 description 1
- 229920000615 alginic acid Polymers 0.000 description 1
- XCPGHVQEEXUHNC-UHFFFAOYSA-N amsacrine Chemical compound COC1=CC(NS(C)(=O)=O)=CC=C1NC1=C(C=CC=C2)C2=NC2=CC=CC=C12 XCPGHVQEEXUHNC-UHFFFAOYSA-N 0.000 description 1
- 229960001220 amsacrine Drugs 0.000 description 1
- 238000011394 anticancer treatment Methods 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- PYMYPHUHKUWMLA-WDCZJNDASA-N arabinose Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)C=O PYMYPHUHKUWMLA-WDCZJNDASA-N 0.000 description 1
- PYMYPHUHKUWMLA-UHFFFAOYSA-N arabinose Natural products OCC(O)C(O)C(O)C=O PYMYPHUHKUWMLA-UHFFFAOYSA-N 0.000 description 1
- 108010093581 aspartyl-proline Proteins 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
- 230000027455 binding Effects 0.000 description 1
- 239000011230 binding agent Substances 0.000 description 1
- 239000003124 biologic agent Substances 0.000 description 1
- 229960001561 bleomycin Drugs 0.000 description 1
- OYVAGSVQBOHSSS-UAPAGMARSA-O bleomycin A2 Chemical compound N([C@H](C(=O)N[C@H](C)[C@@H](O)[C@H](C)C(=O)N[C@@H]([C@H](O)C)C(=O)NCCC=1SC=C(N=1)C=1SC=C(N=1)C(=O)NCCC[S+](C)C)[C@@H](O[C@H]1[C@H]([C@@H](O)[C@H](O)[C@H](CO)O1)O[C@@H]1[C@H]([C@@H](OC(N)=O)[C@H](O)[C@@H](CO)O1)O)C=1N=CNC=1)C(=O)C1=NC([C@H](CC(N)=O)NC[C@H](N)C(N)=O)=NC(N)=C1C OYVAGSVQBOHSSS-UAPAGMARSA-O 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 210000004204 blood vessel Anatomy 0.000 description 1
- 238000006664 bond formation reaction Methods 0.000 description 1
- 235000014121 butter Nutrition 0.000 description 1
- 235000001046 cacaotero Nutrition 0.000 description 1
- 229910000019 calcium carbonate Inorganic materials 0.000 description 1
- 239000001506 calcium phosphate Substances 0.000 description 1
- 229910000389 calcium phosphate Inorganic materials 0.000 description 1
- 235000011010 calcium phosphates Nutrition 0.000 description 1
- 239000000378 calcium silicate Substances 0.000 description 1
- 229910052918 calcium silicate Inorganic materials 0.000 description 1
- 235000012241 calcium silicate Nutrition 0.000 description 1
- OYACROKNLOSFPA-UHFFFAOYSA-N calcium;dioxido(oxo)silane Chemical compound [Ca+2].[O-][Si]([O-])=O OYACROKNLOSFPA-UHFFFAOYSA-N 0.000 description 1
- 229960004562 carboplatin Drugs 0.000 description 1
- 190000008236 carboplatin Chemical compound 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 230000021164 cell adhesion Effects 0.000 description 1
- 230000005754 cellular signaling Effects 0.000 description 1
- 239000001913 cellulose Substances 0.000 description 1
- 235000010980 cellulose Nutrition 0.000 description 1
- 229920002678 cellulose Polymers 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 230000003399 chemotactic effect Effects 0.000 description 1
- JCKYGMPEJWAADB-UHFFFAOYSA-N chlorambucil Chemical compound OC(=O)CCCC1=CC=C(N(CCCl)CCCl)C=C1 JCKYGMPEJWAADB-UHFFFAOYSA-N 0.000 description 1
- 229960004630 chlorambucil Drugs 0.000 description 1
- DQLATGHUWYMOKM-UHFFFAOYSA-L cisplatin Chemical compound N[Pt](N)(Cl)Cl DQLATGHUWYMOKM-UHFFFAOYSA-L 0.000 description 1
- 229960004316 cisplatin Drugs 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000012790 confirmation Methods 0.000 description 1
- 230000021615 conjugation Effects 0.000 description 1
- 210000002808 connective tissue Anatomy 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 239000013601 cosmid vector Substances 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 229960004397 cyclophosphamide Drugs 0.000 description 1
- 229940127089 cytotoxic agent Drugs 0.000 description 1
- 229960000640 dactinomycin Drugs 0.000 description 1
- STQGQHZAVUOBTE-VGBVRHCVSA-N daunorubicin Chemical compound O([C@H]1C[C@@](O)(CC=2C(O)=C3C(=O)C=4C=CC=C(C=4C(=O)C3=C(O)C=21)OC)C(C)=O)[C@H]1C[C@H](N)[C@H](O)[C@H](C)O1 STQGQHZAVUOBTE-VGBVRHCVSA-N 0.000 description 1
- 229960000975 daunorubicin Drugs 0.000 description 1
- 230000034994 death Effects 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 238000012217 deletion Methods 0.000 description 1
- 230000037430 deletion Effects 0.000 description 1
- 239000008121 dextrose Substances 0.000 description 1
- 239000007884 disintegrant Substances 0.000 description 1
- 229960003668 docetaxel Drugs 0.000 description 1
- 229960004679 doxorubicin Drugs 0.000 description 1
- 239000003937 drug carrier Substances 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 229960001904 epirubicin Drugs 0.000 description 1
- UNXHWFMMPAWVPI-ZXZARUISSA-N erythritol Chemical compound OC[C@H](O)[C@H](O)CO UNXHWFMMPAWVPI-ZXZARUISSA-N 0.000 description 1
- 235000019414 erythritol Nutrition 0.000 description 1
- 229940009714 erythritol Drugs 0.000 description 1
- 150000002148 esters Chemical class 0.000 description 1
- LVGKNOAMLMIIKO-QXMHVHEDSA-N ethyl oleate Chemical compound CCCCCCCC\C=C/CCCCCCCC(=O)OCC LVGKNOAMLMIIKO-QXMHVHEDSA-N 0.000 description 1
- 229940093471 ethyl oleate Drugs 0.000 description 1
- VJJPUSNTGOMMGY-MRVIYFEKSA-N etoposide Chemical compound COC1=C(O)C(OC)=CC([C@@H]2C3=CC=4OCOC=4C=C3[C@@H](O[C@H]3[C@@H]([C@@H](O)[C@@H]4O[C@H](C)OC[C@H]4O3)O)[C@@H]3[C@@H]2C(OC3)=O)=C1 VJJPUSNTGOMMGY-MRVIYFEKSA-N 0.000 description 1
- 229960005420 etoposide Drugs 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000000945 filler Substances 0.000 description 1
- 229960000390 fludarabine Drugs 0.000 description 1
- GIUYCYHIANZCFB-FJFJXFQQSA-N fludarabine phosphate Chemical compound C1=NC=2C(N)=NC(F)=NC=2N1[C@@H]1O[C@H](COP(O)(O)=O)[C@@H](O)[C@@H]1O GIUYCYHIANZCFB-FJFJXFQQSA-N 0.000 description 1
- 229960002949 fluorouracil Drugs 0.000 description 1
- 235000003599 food sweetener Nutrition 0.000 description 1
- 230000009036 growth inhibition Effects 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- HOMGKSMUEGBAAB-UHFFFAOYSA-N ifosfamide Chemical compound ClCCNP1(=O)OCCCN1CCCl HOMGKSMUEGBAAB-UHFFFAOYSA-N 0.000 description 1
- 229960001101 ifosfamide Drugs 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 230000008595 infiltration Effects 0.000 description 1
- 238000001764 infiltration Methods 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- VMPHSYLJUKZBJJ-UHFFFAOYSA-N lauric acid triglyceride Natural products CCCCCCCCCCCC(=O)OCC(OC(=O)CCCCCCCCCCC)COC(=O)CCCCCCCCCCC VMPHSYLJUKZBJJ-UHFFFAOYSA-N 0.000 description 1
- 239000003446 ligand Substances 0.000 description 1
- 230000000670 limiting effect Effects 0.000 description 1
- 239000002502 liposome Substances 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 229940057995 liquid paraffin Drugs 0.000 description 1
- 239000000314 lubricant Substances 0.000 description 1
- 239000000845 maltitol Substances 0.000 description 1
- 235000010449 maltitol Nutrition 0.000 description 1
- VQHSOMBJVWLPSR-WUJBLJFYSA-N maltitol Chemical compound OC[C@H](O)[C@@H](O)[C@@H]([C@H](O)CO)O[C@H]1O[C@H](CO)[C@@H](O)[C@H](O)[C@H]1O VQHSOMBJVWLPSR-WUJBLJFYSA-N 0.000 description 1
- 229940035436 maltitol Drugs 0.000 description 1
- 239000000594 mannitol Substances 0.000 description 1
- 235000010355 mannitol Nutrition 0.000 description 1
- HAWPXGHAZFHHAD-UHFFFAOYSA-N mechlorethamine Chemical compound ClCCN(C)CCCl HAWPXGHAZFHHAD-UHFFFAOYSA-N 0.000 description 1
- 229960004961 mechlorethamine Drugs 0.000 description 1
- SGDBTWWWUNNDEQ-LBPRGKRZSA-N melphalan Chemical compound OC(=O)[C@@H](N)CC1=CC=C(N(CCCl)CCCl)C=C1 SGDBTWWWUNNDEQ-LBPRGKRZSA-N 0.000 description 1
- 229960001924 melphalan Drugs 0.000 description 1
- GLVAUDGFNGKCSF-UHFFFAOYSA-N mercaptopurine Chemical compound S=C1NC=NC2=C1NC=N2 GLVAUDGFNGKCSF-UHFFFAOYSA-N 0.000 description 1
- HEBKCHPVOIAQTA-UHFFFAOYSA-N meso ribitol Natural products OCC(O)C(O)C(O)CO HEBKCHPVOIAQTA-UHFFFAOYSA-N 0.000 description 1
- 230000009401 metastasis Effects 0.000 description 1
- 230000001394 metastastic effect Effects 0.000 description 1
- 206010061289 metastatic neoplasm Diseases 0.000 description 1
- 229960000485 methotrexate Drugs 0.000 description 1
- 229920000609 methyl cellulose Polymers 0.000 description 1
- 239000001923 methylcellulose Substances 0.000 description 1
- 235000010981 methylcellulose Nutrition 0.000 description 1
- LXCFILQKKLGQFO-UHFFFAOYSA-N methylparaben Chemical compound COC(=O)C1=CC=C(O)C=C1 LXCFILQKKLGQFO-UHFFFAOYSA-N 0.000 description 1
- 235000019813 microcrystalline cellulose Nutrition 0.000 description 1
- 239000008108 microcrystalline cellulose Substances 0.000 description 1
- 229940016286 microcrystalline cellulose Drugs 0.000 description 1
- 239000002480 mineral oil Substances 0.000 description 1
- 235000010446 mineral oil Nutrition 0.000 description 1
- 229960004857 mitomycin Drugs 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000001338 necrotic effect Effects 0.000 description 1
- OSTGTTZJOCZWJG-UHFFFAOYSA-N nitrosourea Chemical compound NC(=O)N=NO OSTGTTZJOCZWJG-UHFFFAOYSA-N 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 235000008390 olive oil Nutrition 0.000 description 1
- 239000004006 olive oil Substances 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 229960001592 paclitaxel Drugs 0.000 description 1
- 238000007911 parenteral administration Methods 0.000 description 1
- 230000036961 partial effect Effects 0.000 description 1
- 150000003904 phospholipids Chemical class 0.000 description 1
- 239000006187 pill Substances 0.000 description 1
- 239000013600 plasmid vector Substances 0.000 description 1
- 229920000729 poly(L-lysine) polymer Polymers 0.000 description 1
- 229920001223 polyethylene glycol Polymers 0.000 description 1
- 229920000136 polysorbate Polymers 0.000 description 1
- 239000001267 polyvinylpyrrolidone Substances 0.000 description 1
- 235000013855 polyvinylpyrrolidone Nutrition 0.000 description 1
- 229920000036 polyvinylpyrrolidone Polymers 0.000 description 1
- 239000003755 preservative agent Substances 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- CPTBDICYNRMXFX-UHFFFAOYSA-N procarbazine Chemical compound CNNCC1=CC=C(C(=O)NC(C)C)C=C1 CPTBDICYNRMXFX-UHFFFAOYSA-N 0.000 description 1
- 229960000624 procarbazine Drugs 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 210000001236 prokaryotic cell Anatomy 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- QELSKZZBTMNZEB-UHFFFAOYSA-N propylparaben Chemical compound CCCOC(=O)C1=CC=C(O)C=C1 QELSKZZBTMNZEB-UHFFFAOYSA-N 0.000 description 1
- 229960003415 propylparaben Drugs 0.000 description 1
- 230000005855 radiation Effects 0.000 description 1
- 230000003362 replicative effect Effects 0.000 description 1
- 230000035939 shock Effects 0.000 description 1
- 238000002741 site-directed mutagenesis Methods 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 239000000600 sorbitol Substances 0.000 description 1
- 235000010356 sorbitol Nutrition 0.000 description 1
- 230000009870 specific binding Effects 0.000 description 1
- 239000004094 surface-active agent Substances 0.000 description 1
- 239000000375 suspending agent Substances 0.000 description 1
- 239000003765 sweetening agent Substances 0.000 description 1
- 239000006188 syrup Substances 0.000 description 1
- 235000020357 syrup Nutrition 0.000 description 1
- 229960001603 tamoxifen Drugs 0.000 description 1
- RCINICONZNJXQF-MZXODVADSA-N taxol Chemical compound O([C@@H]1[C@@]2(C[C@@H](C(C)=C(C2(C)C)[C@H](C([C@]2(C)[C@@H](O)C[C@H]3OC[C@]3([C@H]21)OC(C)=O)=O)OC(=O)C)OC(=O)[C@H](O)[C@@H](NC(=O)C=1C=CC=CC=1)C=1C=CC=CC=1)O)C(=O)C1=CC=CC=C1 RCINICONZNJXQF-MZXODVADSA-N 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 230000001131 transforming effect Effects 0.000 description 1
- QORWJWZARLRLPR-UHFFFAOYSA-H tricalcium bis(phosphate) Chemical compound [Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O QORWJWZARLRLPR-UHFFFAOYSA-H 0.000 description 1
- 210000004881 tumor cell Anatomy 0.000 description 1
- 241001148471 unidentified anaerobic bacterium Species 0.000 description 1
- 201000000360 urethra cancer Diseases 0.000 description 1
- 235000015112 vegetable and seed oil Nutrition 0.000 description 1
- 239000008158 vegetable oil Substances 0.000 description 1
- 239000003981 vehicle Substances 0.000 description 1
- JXLYSJRDGCGARV-CFWMRBGOSA-N vinblastine Chemical compound C([C@H](C[C@]1(C(=O)OC)C=2C(=CC3=C([C@]45[C@H]([C@@]([C@H](OC(C)=O)[C@]6(CC)C=CCN([C@H]56)CC4)(O)C(=O)OC)N3C)C=2)OC)C[C@@](C2)(O)CC)N2CCC2=C1NC1=CC=CC=C21 JXLYSJRDGCGARV-CFWMRBGOSA-N 0.000 description 1
- 229960003048 vinblastine Drugs 0.000 description 1
- OGWKCGZFUXNPDA-UHFFFAOYSA-N vincristine Natural products C1C(CC)(O)CC(CC2(C(=O)OC)C=3C(=CC4=C(C56C(C(C(OC(C)=O)C7(CC)C=CCN(C67)CC5)(O)C(=O)OC)N4C=O)C=3)OC)CN1CCC1=C2NC2=CC=CC=C12 OGWKCGZFUXNPDA-UHFFFAOYSA-N 0.000 description 1
- 229960004528 vincristine Drugs 0.000 description 1
- OGWKCGZFUXNPDA-XQKSVPLYSA-N vincristine Chemical compound C([N@]1C[C@@H](C[C@]2(C(=O)OC)C=3C(=CC4=C([C@]56[C@H]([C@@]([C@H](OC(C)=O)[C@]7(CC)C=CCN([C@H]67)CC5)(O)C(=O)OC)N4C=O)C=3)OC)C[C@@](C1)(O)CC)CC1=C2NC2=CC=CC=C12 OGWKCGZFUXNPDA-XQKSVPLYSA-N 0.000 description 1
- 239000013603 viral vector Substances 0.000 description 1
- 230000001018 virulence Effects 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- 239000000811 xylitol Substances 0.000 description 1
- 235000010447 xylitol Nutrition 0.000 description 1
- HEBKCHPVOIAQTA-SCDXWVJYSA-N xylitol Chemical compound OC[C@H](O)[C@@H](O)[C@H](O)CO HEBKCHPVOIAQTA-SCDXWVJYSA-N 0.000 description 1
- 229960002675 xylitol Drugs 0.000 description 1
Images
Classifications
-
- 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
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
-
- 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
- A61K35/74—Bacteria
-
- 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
- C07K14/70503—Immunoglobulin superfamily
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Genetics & Genomics (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Zoology (AREA)
- Medicinal Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Biochemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Microbiology (AREA)
- Wood Science & Technology (AREA)
- Immunology (AREA)
- General Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Biophysics (AREA)
- Biomedical Technology (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Animal Behavior & Ethology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Physics & Mathematics (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Plant Pathology (AREA)
- Cell Biology (AREA)
- Toxicology (AREA)
- Gastroenterology & Hepatology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Mycology (AREA)
- Epidemiology (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
본 발명은 항PD-L1 펩티드를 발현하는 살모넬라 변이주 및 이의 외막 소포체, 및 이를 포함하는 약학 조성물에 관한 것으로, 상기 살모넬라 변이주는 외막 단백질 A의 세포외 루프에서 항PD-L1 펩티드를 발현함으로써 살모넬라 특성으로 인해 항PD-L1 펩티드를 종양 내부까지 안정적 및 효율적으로 전달할 수 있고, 암세포의 PD-1와의 결합 형성을 통해 T세포의 활성화를 유도하여 항종양 효과를 나타내므로, 이러한 살모넬라 변이주 또는 이로부터 분리된 외막 소포체를 암치료에 활용할 수 있다.The present invention relates to a Salmonella mutant expressing an anti-PD-L1 peptide, an outer membrane endoplasmic reticulum thereof, and a pharmaceutical composition comprising the same. As a result, the anti-PD-L1 peptide can be stably and efficiently delivered to the inside of the tumor, and the anti-tumor effect is shown by inducing the activation of T cells through the formation of bonds with PD-1 of cancer cells. Outer membrane endoplasmic reticulum can be used for cancer treatment.
Description
본 발명은 항PD-L1 펩티드를 발현하는 살모넬라 변이주 및 이의 외막 소포체, 및 이를 포함하는 약학 조성물에 관한 것이다.The present invention relates to a Salmonella mutant expressing an anti-PD-L1 peptide, outer membrane endoplasmic reticulum thereof, and a pharmaceutical composition containing the same.
PD-L1(Programmed cell death-ligand 1)은 세포 표면에서 발현되는 막관통단백질(transmembrane protein)의 일종으로, 주로 암세포의 표면에서 발현되어 T세포 표면의 PD-1(programmed death-1)과 결합을 형성하는데, 이로 인해 T세포의 활성화가 차단되어 T세포에 의한 암세포의 사멸이 억제된다. 즉, 종양에서는 체내 면역관문(immune checkpoint) 기능의 변화를 나타내어 T세포의 기능을 억제함으로로써 T세포의 공격을 회피하게 된다. 따라서 PD-L1 또는 PD-1에 특이적으로 결합하는 항체나 억제제와 같은 면역관문억제제(immune checkpoint inhibitor)를 이용하여 T세포의 활성을 유도함으로써 항종양 효과를 얻을 수 있다. 이러한 면역관문억제제의 기능 및 효과를 향상시키기 위해서는 면역관문억제제를 종양 내부까지 안정적이고 효율적으로 전달할 수 있는 전달체에 대한 연구가 필요하다.PD-L1 (Programmed cell death-ligand 1) is a type of transmembrane protein expressed on the cell surface. It is mainly expressed on the surface of cancer cells and binds to PD-1 (programmed death-1) on the surface of T cells. , which blocks the activation of T cells and suppresses the death of cancer cells by T cells. In other words, tumors exhibit changes in the immune checkpoint function in the body to suppress the function of T cells, thereby avoiding the attack of T cells. Therefore, an antitumor effect can be obtained by inducing the activity of T cells using PD-L1 or an immune checkpoint inhibitor such as an antibody or inhibitor that specifically binds to PD-1. In order to improve the functions and effects of these immune checkpoint inhibitors, it is necessary to study a delivery system that can stably and efficiently deliver the immune checkpoint inhibitor to the inside of the tumor.
한편, 일부 세균은 종양 내에서 증식하고 축척되는 것을 선호하는 것으로 보고된 바 있다. 종양 중심부에 위치한 네크로틱 영역(necrotic region)은 외부로부터 약물전달이 효율적으로 이루어지지 않으며, 낮은 산소분압과 풍부한 영양분, 면역반응의 결핍 등 혐기성 세균이 선호하는 서식지의 특성을 제공하기 때문에 클로스트리듐(Clostridium), 비피도박테리움(Bifidobacterium), 살모넬라(Salmonella) 등이 종양에서 증식하며, 이 과정에서 종양의 축소 및 성장 억제 등이 관찰되어 이들 균주를 항암치료에 사용하고 있다.On the other hand, it has been reported that some bacteria prefer to proliferate and accumulate in tumors. The necrotic region, located in the center of the tumor, does not efficiently deliver drugs from the outside, and provides characteristics of a habitat favored by anaerobic bacteria, such as low oxygen partial pressure, abundant nutrients, and lack of immune response. (Clostridium), Bifidobacterium, Salmonella, etc. proliferate in tumors, and in this process, tumor shrinkage and growth inhibition are observed, and these strains are used for anticancer treatment.
살모넬라의 경우 운동성을 가진 침습성 그람 음성균으로, 종양세포에서 생산된 주화성 화합물이나 풍부한 영양분에 의해 살모넬라가 종양내로 이동하여 군집화하는 것으로 알려져 있다. 이러한 살모넬라의 특성을 활용하여, 살모넬라의 독성을 약화시키고 항암물질을 탑재한 살모넬라 재조합 균주가 우수한 항암 효과를 나타낸다는 것이 보고되었다 (Nishikawa H, et al., J Clin Invest. 2006, 116(7):1946-54; Clairmont, C. et al. J.Infect. Dis. 2000, 181:1996-2002). 하지만 면역관문억제제의 전달체로서 약독화 살모넬라를 이용한 경우는 아직 확인된 바 없다.Salmonella is an invasive gram-negative bacterium with motility, and it is known that Salmonella migrates into tumors and colonizes by chemotactic compounds produced in tumor cells or abundant nutrients. Using these characteristics of Salmonella, it has been reported that recombinant Salmonella strains that weaken the toxicity of Salmonella and are loaded with anticancer substances exhibit excellent anticancer effects (Nishikawa H, et al., J Clin Invest. 2006, 116(7) :1946-54; Clairmont, C. et al. J. Infect. Dis. 2000, 181:1996-2002). However, the use of attenuated Salmonella as a delivery vehicle for immune checkpoint inhibitors has not yet been confirmed.
본 발명은 PD-L1에 특이적으로 결합하는 항PD-L1 펩티드를 세포 표면에 제시하는 신규한 살모넬라 변이주를 제공하는 것을 목적으로 한다.An object of the present invention is to provide a novel Salmonella mutant strain that presents an anti-PD-L1 peptide that specifically binds to PD-L1 on the cell surface.
또한, 본 발명은 상기 살모넬라 변이주로부터 분리된 외막 소포체를 제공하는 것을 목적을 한다.In addition, an object of the present invention is to provide an outer membrane endoplasmic reticulum isolated from the Salmonella mutant strain.
또한, 본 발명은 상기 살모넬라 변이주 또는 외막 소포체를 유효성분으로 포함하는 암 예방 또는 치료용 약학 조성물을 제공하는 것을 목적으로 한다.In addition, an object of the present invention is to provide a pharmaceutical composition for preventing or treating cancer comprising the Salmonella mutant strain or outer membrane vesicles as an active ingredient.
본 발명의 일 양상은 외막 단백질 A의 세포외 루프에서 항PD-L1 펩티드를 발현하는 살모넬라 변이주를 제공한다.One aspect of the present invention provides a Salmonella mutant expressing an anti-PD-L1 peptide in an extracellular loop of outer membrane protein A.
본 발명에서 사용된 "외막 단백질(outer membrane protein, Omp)”은 그람 음성균의 외막에 존재하는 단백질을 의미하며, 그 종류로는 OmpA, OmpC, OmpF, OmpG, OmpT, OmpX, Omp31 등이 있고, 주로 전형적인 β-barrel 구조를 가지는 막관통단백질로서 하나 이상의 세포외 루프(extracellular loop, EL)를 가지고 있다.As used in the present invention, "outer membrane protein (Omp)" refers to a protein present in the outer membrane of Gram-negative bacteria, and its types include OmpA, OmpC, OmpF, OmpG, OmpT, OmpX, Omp31, etc. It is a transmembrane protein with a typical β-barrel structure and has one or more extracellular loops (EL).
본 발명에서 사용된 OmpA는 4개의 세포외 루프 (EL1~4)를 포함하며, 이러한 세포외 루프가 세균의 독성, 운동성 등과 관련 있는 것으로 알려져 있다.OmpA used in the present invention includes four extracellular loops (EL1 to 4), and it is known that these extracellular loops are related to bacterial toxicity and motility.
본 발명의 일 구체예에 따르면, 상기 OmpA는 살모넬라에서 유래한 것일 수 있다.According to one embodiment of the present invention, the OmpA may be derived from Salmonella.
보다 구체적으로, 상기 OmpA는 살모넬라 티피뮤리움(Salmonella typhimurium), 살모넬라 브렌데럽(Salmonella braenderup) 및 살모넬라 엔테리티디스(Salmonella enteritidis)로 이루어진 군에서 선택된 1종 이상일 수 있으나, 이에 제한되지 않는다.More specifically, the OmpA may be at least one selected from the group consisting of Salmonella typhimurium , Salmonella braenderup and Salmonella enteritidis , but is not limited thereto.
본 발명의 일 구체예에 따르면, 상기 외막 단백질 A의 세포외 루프는 외막 단백질 A의 N 말단에서 2번째에 위치한 것일 수 있다.According to one embodiment of the present invention, the extracellular loop of the outer membrane protein A may be located second from the N-terminus of the outer membrane protein A.
살모넬라 외막 단백질 A의 세포외 루프에 대해서는 명확한 기능이 밝혀지지 않았으나, 세포외 루프의 기능이 명확하게 밝혀진 대장균(E. coli)을 참고하면 EL1 및 2는 병원성에 크게 기여하며, 특히 EL2는 세포 부착 및 침투 기능을 수행하기 때문에 EL2에 목적하는 펩티드 서열을 제시함으로써 항PD-L1 펩티드 서열을 외부로 노출시킬 뿐만 아니라 EL2에 의한 병독성을 약화시킬 수 있다.Although a clear function of the extracellular loop of Salmonella outer membrane protein A has not been identified, referring to Escherichia coli ( E. coli ), in which the function of the extracellular loop has been clearly identified, EL1 and 2 contribute greatly to pathogenicity, and in particular, EL2 is cell adhesion. And since it performs an infiltration function, by presenting a desired peptide sequence to EL2, the anti-PD-L1 peptide sequence can be exposed to the outside and virulence caused by EL2 can be attenuated.
본 발명에서 사용된 “항PD-L1 펩티드(anti-PD-L1 peptide)”는 PD-L1에 특이적으로 결합할 수 있는 펩티드를 의미하며, 공지된 인간 PD-1과 서열 상동성이 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 또는 100%일 수 있다.The “anti-PD-L1 peptide” used in the present invention means a peptide that can specifically bind to PD-L1, and has 15% sequence homology with known human PD-1. , 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or It can be 100%.
본 발명의 일 구체예에 따르면, 상기 항PD-L1 펩티드는 서열번호 1 내지 3 중 하나 이상의 아미노산 서열로 이루어진 것일 수 있다.According to one embodiment of the present invention, the anti-PD-L1 peptide may consist of one or more amino acid sequences of SEQ ID NOs: 1 to 3.
상기 항PD-L1 펩티드의 인간 PD-1에 대한 아미노산 서열 상동성은 CVRARTR (서열번호 1)가 57%, CLQKTPKQC (서열번호 2) 44%, YASYHCWCWRDPGRS (서열번호 3)가 20%이다.The amino acid sequence homology of the anti-PD-L1 peptide to human PD-1 was 57% for CVRARTR (SEQ ID NO: 1), 44% for CLQKTPKQC (SEQ ID NO: 2), and 20% for YASYHCWCWRDPGRS (SEQ ID NO: 3).
또한, 본 발명의 일 구체예에 따르면, 상기 항PD-L1 펩티드는 4 내지 6 중 하나 이상의 염기서열로 암호화된 것일 수 있다. In addition, according to one embodiment of the present invention, the anti-PD-L1 peptide may be encoded by one or more nucleotide sequences from 4 to 6.
이러한 항PD-L1 펩티드는 EL2의 아미노산 서열 GRMPYKGDNINGAYKAQ (서열번호 7) 내부에 위치하여 세포 표면에 발현 또는 제시될 수 있다.This anti-PD-L1 peptide is located inside the amino acid sequence GRMPYKGDNINGAYKAQ (SEQ ID NO: 7) of EL2 and can be expressed or displayed on the cell surface.
보다 구체적으로, 상기 항PD-L1 펩티드는 아미노산 서열 중 N 말단에서 5번째 내지 15번째, 7번째 내지 13번째, 또는 9번째 내지 11번째에 위치하는 것일 수 있다.More specifically, the anti-PD-L1 peptide may be located at positions 5 to 15, 7 to 13, or 9 to 11 from the N-terminus of the amino acid sequence.
이러한 외막 단백질 A의 EL2 내부에 PD-L1 펩티드를 제시하도록 유전자 조적된 살모넬라 변이주는 세포외 루프 서열과 함께 항PD-L1 펩티드 서열을 모두 포함한다.The Salmonella mutant strain genetically engineered to present the PD-L1 peptide inside EL2 of the outer membrane protein A contains both the anti-PD-L1 peptide sequence along with the extracellular loop sequence.
본 발명의 일 구체예에 따르면, 상기 살모넬라 변이주는 서열번호 8 내지 10 중 하나 이상의 아미노산 서열로 이루어진 세포 표면 단백질을 발현하는 것일 수 있다.According to one embodiment of the present invention, the mutant strain of Salmonella may express a cell surface protein consisting of one or more amino acid sequences of SEQ ID NOs: 8 to 10.
본 발명의 일 구체예에 따르면, 상기 살모넬라 변이주는 살모넬라 티피뮤리움(Salmonella typhimurium), 살모넬라 브렌데럽(Salmonella braenderup) 및 살모넬라 엔테리티디스(Salmonella enteritidis)로 이루어진 군에서 선택된 1종 이상에서 유래한 약독화 균주인 것일 수 있다.According to one embodiment of the present invention, the Salmonella mutant is Salmonella typhimurium ( Salmonella typhimurium ), Salmonella blenderup ( Salmonella braenderup ) and Salmonella enteritidis ( Salmonella enteritidis ) Attenuated from at least one selected from the group consisting of It may be a strain.
본 발명에서 사용된 “약독화”는 미생물의 병원성을 감소시키도록 변형시키는 것을 의미하며, 미생물을 개체 (예컨대, 인간)에게 투여한 경우 독성 및 다른 부작용을 감소시킬 목적으로 이루어진다. 약독화 균주는 당업계에 공지된 다양한 방법을 통하여 제조될 수 있다. 예를 들어, 약독화는 개체에 대한 세균의 위험 요소에 해당하는 독성 인자(virulence factor)를 결실시키거나 또는 파괴하여 달성된다. 상기 결실 및 파괴는 당업계에 잘 알려져 있으며, 예컨대, 상동성 재조합, 화학적 변이유발, 조사 변이유발 또는 트랜스포존 변이유발 등과 같은 방법에 의해 실시된다. "Attenuation" used in the present invention means modification to reduce the pathogenicity of microorganisms, and is made for the purpose of reducing toxicity and other side effects when microorganisms are administered to a subject (eg, human). Attenuated strains can be prepared through various methods known in the art. For example, attenuation is achieved by deleting or destroying a virulence factor that represents a bacterial risk factor for the subject. Such deletions and disruptions are well known in the art and are performed by methods such as, for example, homologous recombination, chemical mutagenesis, radiation mutagenesis, or transposon mutagenesis.
살모넬라의 약독화 균주는 독성 인자로 작용하는 5'-아데노신 모노포스페이트, 사이토라이신, 디펜신 내성, DNAK, 핌브리애, 리포단백질, LPS 등이 결실된 것일 수 있다.The attenuated strain of Salmonella may be one in which 5'-adenosine monophosphate, cytolysin, defensin resistance, DNAK, fimbriae, lipoprotein, LPS, etc., which act as virulence factors, are deleted.
본 발명의 일 실시예에서는 살모넬라의 ppGpp 유전자가 결손된 약독화 균주를 사용하였다.In one embodiment of the present invention, an attenuated strain in which the ppGpp gene of Salmonella is missing was used.
이와 같은 본 발명의 외막 단백질 A의 세포외 루프에서 항PD-L1 펩티드를 발현하는 살모넬라 변이주는 살모넬라 특성으로 인해 종양 내부까지 안정적 및 효율적으로 전달되어 암세포의 PD-1에 특이적인 결합을 형성할 수 있다.Such a Salmonella mutant expressing the anti-PD-L1 peptide in the extracellular loop of the outer membrane protein A of the present invention is stably and efficiently delivered to the inside of the tumor due to the characteristics of Salmonella, and can form a specific binding to PD-1 of cancer cells. there is.
이러한 살모넬라 변이주는 외막 단백질 A의 세포외 루프를 암호화하는 유전자 및 항PD-L1 펩티드를 암호하하는 유전자를 포함하는 재조합 벡터를 제조하는 단계; 상기 재조합 벡터를 숙주세포에 형질전환하는 단계; 및 상기 형질전환된 형질전환체를 수득하는 단계를 통해 제조될 수 있다.Preparing a recombinant vector containing the gene encoding the extracellular loop of outer membrane protein A and the gene encoding the anti-PD-L1 peptide; transforming the recombinant vector into a host cell; And it can be prepared through the step of obtaining the transformed transformant.
상기 재조합 벡터는 적합한 숙주세포 내로 형질전환된 후, 숙주세포의 게놈과 무관하게 복제 가능하거나 게놈 그 자체에 봉합될 수 있으며, 발현 벡터로 사용될 수 있다. 여기서 "적합한 숙주세포"란 벡터가 복제 가능한 것으로서 복제가 개시되는 특정 염기서열인 복제 원점을 포함할 수 있다.After the recombinant vector is transformed into a suitable host cell, it can be replicated independently of the genome of the host cell or can be sutured to the genome itself, and can be used as an expression vector. Here, "a suitable host cell" is a vector capable of replicating, and may include a replication origin, which is a specific nucleotide sequence at which replication is initiated.
상기 재조합 벡터는 플라스미드 벡터(plasmid vector), 바이러스 벡터(viral vector) 및 코스미드 벡터(cosmid vector)로 이루어진 군으로부터 선택되는 어느 하나일 수 있다.The recombinant vector may be any one selected from the group consisting of a plasmid vector, a viral vector and a cosmid vector.
상기 플라스미드의 경우 원핵생물 또는 진핵세포에 접합(conjugation)에 의한 전달과 복제가 가능한 플라스미드일 수 있다. 예를 들면, 상기 플라스미드는 pBR계, pUC계, pBluescriptII계, pGEM계, pTZ계, pCL계 또는 pET계 플라스미드일 수 있다. 구체적으로는 pDZ, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, pCC1BAC, pUC18, pBAD, pJK001, pCM, 또는 pAWP 등일 수 있으나, 이에 제한되는 것은 아니다.In the case of the plasmid, it may be a plasmid capable of delivery and replication by conjugation to prokaryotic or eukaryotic cells. For example, the plasmid may be a pBR-based, pUC-based, pBluescriptII-based, pGEM-based, pTZ-based, pCL-based or pET-based plasmid. Specifically, it may be pDZ, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, pCC1BAC, pUC18, pBAD, pJK001, pCM, or pAWP, but is not limited thereto.
본 발명의 일 구체예에 따르면, 상기 재조합 벡터는 서열번호 13 내지 15 중 하나의 염기서열을 포함하는 것일 수 있다.According to one embodiment of the present invention, the recombinant vector may include one of the nucleotide sequences of SEQ ID NOs: 13 to 15.
상기 재조합 세포는 생체내 또는 시험관내에서 전술한 재조합 벡터 또는 폴리뉴클레오티드로 형질감염, 형질전환, 또는 감염된 세포를 포함하며, 재조합 숙주세포, 재조합 미생물을 의미한다. 여기서 “형질전환”은 단백질을 암호화하는 폴리뉴클레오티드를 포함하는 발현 벡터를 숙주세포 내에 도입하여 숙주세포 내에서 상기 폴리뉴클레오티드가 암호화하는 단백질을 발현할 수 있도록 하는 것을 의미하며, 상기 발현 벡터가 도입된 숙주세포를 형질전환체, 돌연변이 균주, 또는 변이주라 한다. 이러한 숙주세포는 당업계에 공지된 것을 제한 없이 사용할 수 있다. The recombinant cell includes cells transfected, transformed, or infected with the above-described recombinant vector or polynucleotide in vivo or in vitro, and refers to a recombinant host cell or a recombinant microorganism. Here, "transformation" means introducing an expression vector containing a polynucleotide encoding a protein into a host cell so that the protein encoded by the polynucleotide can be expressed in the host cell. Host cells are referred to as transformants, mutant strains, or mutant strains. As such a host cell, those known in the art may be used without limitation.
이러한 재조합 벡터를 통해 본 발명에 따른 항PD-L1 펩티드가 삽입된 외막 단백질 A의 세포외 루프를 세포 표면에 발현하는 변이주를 얻을 수 있다.Through such a recombinant vector, a mutant expressing the outer membrane protein A extracellular loop into which the anti-PD-L1 peptide according to the present invention has been inserted can be obtained on the cell surface.
또한, 본 발명의 다른 양상은 상기 살모넬라 변이주에서 분리된 외막 소포체를 제공한다.In addition, another aspect of the present invention provides outer membrane endoplasmic reticulum isolated from the Salmonella mutant strain.
본 발명에서 사용된 “외막 소포체(outer membrane vesicle, omv)”는 그람 음성균이 생산하여 주변 환경으로 배출시키는 구형 지질 이중층의 소포체를 의미하며, 세포막에서 유래되어 LPS(lipopolysaccharide), 세포막간 단백질(periplasmic proteins), 외막 단백질(Omp), 인지질, DNA, DNA-결합 단백질, 병원성 인자 등을 포함한다. As used in the present invention, “outer membrane vesicle (omv)” refers to a spherical lipid bilayer vesicle that is produced by Gram-negative bacteria and released into the surrounding environment. proteins), outer membrane proteins (Omps), phospholipids, DNA, DNA-binding proteins, pathogenic factors, and the like.
이러한 외막 소포체는 표면-발현된 인자들을 통해 진핵세포 및 다른 세균과 상호작용하고 병원성 인자들의 운반을 매개하는 것으로 알려져 있어 (Ketsy and Kuehn, 2004), 목적 물질을 전달하는 시스템으로 활용된다.These outer membrane endoplasmic reticulum are known to interact with eukaryotic cells and other bacteria through surface-expressed factors and mediate the transport of pathogenic factors (Ketsy and Kuehn, 2004), and thus are used as a system for delivering target substances.
본 발명의 일 구체예에 따르면, 상기 외막 소포체는 서열번호 1 내지 3 및 8 내지 10 중 하나 이상의 아미노산 서열로 이루어진 단백질을 포함하는 것일 수 있다.According to one embodiment of the present invention, the outer membrane endoplasmic reticulum may include a protein consisting of one or more amino acid sequences of SEQ ID NOs: 1 to 3 and 8 to 10.
본 발명의 일 구체예에 따르면, 상기 외막 소포체는 20 내지 250 nm의 직경을 가지는 것일 수 있다.According to one embodiment of the present invention, the outer membrane vesicles may have a diameter of 20 to 250 nm.
보다 구체적으로, 상기 외막 소포체의 직경은 목적 물질의 전달 효율이 향상될 수 있도록 20 내지 250 nm, 20 내지 200 nm, 20 내지 150 nm, 또는 20 내지 100 nm일 수 있다.More specifically, the diameter of the outer membrane vesicles may be 20 to 250 nm, 20 to 200 nm, 20 to 150 nm, or 20 to 100 nm so as to improve the delivery efficiency of the target substance.
여기서 목적 물질이란 항PD-L1 펩티드가 해당된다.Here, the target substance corresponds to an anti-PD-L1 peptide.
이러한 외막 소포체는 작은 크기로 인해 변이주 자체보다 종양 내부까지 침투가 용이하며, 상대적으로 보관 및 유통이 용이할 뿐만 아니라 리포좀과 같은 다른 막구조체에 비해 보다 견고한 막 구조를 형성한다는 이점이 있어, 항PD-L1 펩티드의 전달체로서 보다 효율적이다.These outer membrane vesicles are easier to penetrate into the tumor than the mutant strain itself due to their small size, are relatively easy to store and distribute, and have the advantage of forming a more robust membrane structure than other membrane structures such as liposomes, which is anti-PD. -It is more efficient as a transporter of the L1 peptide.
본 발명에 따른 외막 소포체는 통상의 방법에 따라 상기 살모넬라 변이주로부터 분리될 수 있다.The outer membrane endoplasmic reticulum according to the present invention can be isolated from the Salmonella mutant strain according to a conventional method.
예를 들면, 상기 외막 소포체는 전술한 외막 단백질 A의 세포외 루프에서 항PD-L1 펩티드를 발현하는 살모넬라 변이주를 배양하는 단계; 및 상기 변이주의 배양액으로부터 외막 소포체를 분리하는 단계를 통해 제조될 수 있다.For example, culturing a Salmonella mutant expressing an anti-PD-L1 peptide in the outer membrane endoplasmic reticulum described above in the extracellular loop of outer membrane protein A; And it can be prepared through the step of separating the outer membrane endoplasmic reticulum from the culture medium of the mutant strain.
본 발명의 다른 양상은 상기 살모넬라 변이주 또는 이로부터 분리된 외막 소포체를 유효성분으로 포함하는 암 예방 또는 치료용 약학 조성물을 제공한다.Another aspect of the present invention provides a pharmaceutical composition for preventing or treating cancer comprising the Salmonella mutant or outer membrane vesicles isolated therefrom as an active ingredient.
본 발명에서 사용된 "암”은 세포가 정상적인 성장 한계를 무시하고 분열 및 성장하는 공격적(aggressive) 특성, 주위 조직에 침투하는 침투적(invasive) 특성, 및 체내의 다른 부위로 퍼지는 전이적(metastatic) 특성을 갖는 세포에 의한 질병을 총칭하는 의미이다. 특히, 본 발명에서의 암은 악성 종양(malignant tumor)으로서 혈관과 결합성 조직으로 이루어져 일정한 경도와 형태를 갖는 고형암일 수 있다.As used herein, “cancer” refers to aggressive characteristics in which cells divide and grow beyond normal growth limits, invasive characteristics infiltrating surrounding tissues, and metastatic characteristics in which cells spread to other parts of the body. ) In particular, the cancer in the present invention is a malignant tumor and may be a solid cancer having a certain hardness and shape composed of blood vessels and connective tissue.
본 발명에서 사용된 "예방" 또는 "치료"는 암의 성장, 증식 또는 전이를 예방, 억제 및 경감하는 것을 의미한다.As used herein, "prevention" or "treatment" means preventing, inhibiting, and reducing the growth, proliferation, or metastasis of cancer.
본 발명의 일 구체예에 따르면, 상기 암은 유방암, 구강암, 전립선암, 직장암, 비소세포 폐암, 구순암, 간암, 폐암, 항문암, 신장암, 외음부암, 구인두암(oropharyngeal cancer), 비강/부비동암(nasal cavity and paranasal sinus cancer), 비인두암, 요도암, 소장암, 담도암, 방광암, 난소암, 후두암, 식도암, 담낭암, 대장암, 두경부암, 갑상선암, 부갑상선암, 음경암, 질암, 췌장암, 편평세포암, 호치킨 림프종(Hodgkin's lymphoma), 백혈병 관련 질환, 균상 식육종(mycosis fungoides) 및 골수이형성증후군으로 이루어진 군에선 선택된 1종 이상인 것일 수 있다.According to one embodiment of the present invention, the cancer is breast cancer, oral cancer, prostate cancer, rectal cancer, non-small cell lung cancer, lip cancer, liver cancer, lung cancer, anal cancer, kidney cancer, vulvar cancer, oropharyngeal cancer, nasal / Nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, urethra cancer, small intestine cancer, bile duct cancer, bladder cancer, ovarian cancer, laryngeal cancer, esophageal cancer, gallbladder cancer, colon cancer, head and neck cancer, thyroid cancer, parathyroid cancer, penile cancer, vaginal cancer, It may be at least one selected from the group consisting of pancreatic cancer, squamous cell carcinoma, Hodgkin's lymphoma, leukemia-related diseases, mycosis fungoides, and myelodysplastic syndrome.
이러한 약학 조성물은 종래 항암제를 더 포함함으로써 암 치료 효과를 향상시킬 수 있다.Such a pharmaceutical composition can improve cancer treatment effect by further including a conventional anticancer agent.
본 발명의 일 구체예에 따르면, 상기 약학 조성물은 항암제를 더 포함하는 것일 수 있다.According to one embodiment of the present invention, the pharmaceutical composition may further include an anticancer agent.
상기 항암제는 통상 암치료에 사용되는 다양한 항암제를 포함하며, 예를 들어 화학치료제(chemotherapeutic agent), 항암 작은 분자(anti-cancer small molecule), 항체, 암세포에 특이적인 생물학 제제(biological agent) 등을 포함할수 있으며, 이에 제한되지 않는다.The anticancer agent includes various anticancer agents commonly used in cancer treatment, and includes, for example, chemotherapeutic agents, anti-cancer small molecules, antibodies, biological agents specific to cancer cells, and the like. It may include, but is not limited to.
본 발명의 일 구체예에 따르면, 상기 항암제는 도세탁셀(docetaxel), 5-플루오로우라실(5-fluorouracil), 6-머캅토퓨린(6-mercaptopurin), 사이토신-아라비노시드(cytosine-arabinoside), 플루다라빈(fludarabine), 메토트렉세이트(methotrexate), 독소루비신(doxorubicin), 에피루비신(epirubicin), 아멕린(amekrin), 시스플라틴(cisplatin), 카르보플라틴(carboplatin), 프로카르바진(procarbazine), 메클로레타민(mechlorethamine), 사이클로포스파미드(cyclophosphamide), 이포스파미드(ifosfamide), 멜팔란(melphalan), 클로라부실(chlorambucil), 비술판(bisulfan), 니트로소우레아(nitrosourea), 디악티노마이신(dactinomycin), 다우노루비신(daunorubicin), 블레오마이신(bleomycin), 플리코마이신(plicomycin), 미토마이신(mitomycin), 에토포시드(etoposide), 탁목시펜(tamoxifen), 파클리탁셀(paclitaxel), 트랜스플라티눔(transplatinum), 아드리아마이신(adriamycin), 빈크리스틴(vincristin) 및 빈블라스틴(vinblastin)으로 이루어진 군에서 선택된 1종 이상인 것일 수 있다.According to one embodiment of the present invention, the anticancer agent is docetaxel, 5-fluorouracil, 6-mercaptopurin, cytosine-arabinoside , fludarabine, methotrexate, doxorubicin, epirubicin, amekrin, cisplatin, carboplatin, procarbazine, mechlorethamine, cyclophosphamide, ifosfamide, melphalan, chlorambucil, bisulfan, nitrosourea, diac dactinomycin, daunorubicin, bleomycin, plicomycin, mitomycin, etoposide, tamoxifen, paclitaxel , transplatinum, adriamycin, vincristin, and vinblastin.
본 발명에 따른 약학 조성물은 투여를 위하여, 상기 기재한 유효성분 이외에 약학적으로 허용 가능한 담체, 부형제 또는 희석제를 포함할 수 있다. 상기 담체, 부형제 및 희석제로는 락토오스, 덱스트로오스, 수크로오스, 소르비톨, 만니톨, 자일리톨, 에리스리톨, 말티톨, 전분, 아카시아 고무, 알지네이트, 젤라틴, 칼슘 포스페이트, 칼슘 실리케이트, 셀룰로오스, 메틸 셀룰로오스, 미정질 셀룰로오스, 폴리비닐피롤리돈, 물, 메틸히드록시벤조에이트, 프로필히드록시벤조에이트, 탈크, 마그네슘 스테아레이트, 광물유 등을 들 수 있으나, 이에 한정되는 것은 아니다.For administration, the pharmaceutical composition according to the present invention may include pharmaceutically acceptable carriers, excipients or diluents in addition to the above-described active ingredients. The carriers, excipients and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil, and the like, but are not limited thereto.
또한, 본 발명에 따른 약학 조성물은 각각 통상의 방법에 따라 산제, 과립제, 정제, 캡슐제, 현탁액, 에멀젼, 시럽, 에어로졸 등의 경구형 제형, 외용제, 좌제 또는 멸균 주사용액의 형태로 제형화하여 사용할 수 있다. 상세하게는 제형화할 경우 통상 사용하는 충진제, 중량제, 결합제, 습윤제, 붕해제, 계면활성제 등의 희석제 또는 부형제를 사용하여 조제될 수 있다. 경구투여를 위한 고형 제제로는 정제, 환제, 산제, 과립제, 캡슐제 등을 포함하나, 이에 한정되는 것은 아니다. 이러한 고형 제제는 상기 유효성분 외에 적어도 하나 이상의 부형제, 예를 들면, 전분, 칼슘 카보네이트, 수크로오스, 락토오스, 젤라틴 등을 섞어 조제될 수 있다. 또한, 단순한 부형제 이외에 마그네슘 스테아레이트, 탈크 같은 윤활제들도 사용될 수 있다. 경구를 위한 액상물, 리퀴드 파라핀 이외에 여러 가지 부형제, 예를 들면 습윤제, 감미제, 방향제, 보존제 등을 첨가하여 조제될 수 있다. 비경구 투여를 위한 제제는 멸균된 수용액, 비수성 용제, 현탁제, 유제, 동결건조 제제 및 과제를 포함한다. 비수성 용제 및 현탁제로는 프로필렌글리콜, 폴리에틸렌글리콜, 올리브 오일과 같은 식물성 오일, 에틸올레이트와 같은 주사 가능한 에스테르 등이 사용될 수 있다. 좌제의 기제로는 위텝솔, 마크로솔, 트윈 61, 카카오지, 라우린지, 글리세로 젤라틴 등이 사용될 수 있다.In addition, the pharmaceutical composition according to the present invention is formulated in the form of oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories or sterile injection solutions according to conventional methods, respectively. can be used Specifically, when formulated, it may be prepared using diluents or excipients such as commonly used fillers, weighting agents, binders, wetting agents, disintegrants, and surfactants. Solid preparations for oral administration include, but are not limited to, tablets, pills, powders, granules, capsules, and the like. Such a solid preparation may be prepared by mixing at least one or more excipients, for example, starch, calcium carbonate, sucrose, lactose, gelatin, etc., in addition to the active ingredient. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. It may be prepared by adding various excipients, for example, wetting agents, sweeteners, aromatics, and preservatives, in addition to liquids and liquid paraffin for oral use. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized formulations and tablets. Propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used as non-aqueous solvents and suspending agents. As a base for the suppository, Witepsol, Macrosol, Tween 61, cacao butter, laurin paper, glycerogelatin, and the like may be used.
본 발명의 약학 조성물의 적합한 투여량은 환자의 상태 및 체중, 질병의 정도, 약물형태, 시간에 따라 다르지만, 당 업자에 의해 적절하게 선택될 수 있는 바, 상기 조성물의 일일 투여량은 바람직하게는 0.001 mg/kg 내지 1,000 mg/kg이며, 필요에 따라 일일 1회 내지 수회로 나누어 투여할 수 있다.A suitable dose of the pharmaceutical composition of the present invention varies depending on the condition and weight of the patient, the severity of the disease, the drug type, and the time, but can be appropriately selected by a person skilled in the art, and the daily dose of the composition is preferably 0.001 mg/kg to 1,000 mg/kg, and may be divided and administered once a day to several times as needed.
본 발명에 따른 살모넬라 변이주는 외막 단백질 A의 세포외 루프에서 항PD-L1 펩티드를 발현함으로써 살모넬라 특성으로 인해 항PD-L1 펩티드를 종양 내부까지 안정적 및 효율적으로 전달할 수 있고, 암세포의 PD-1와의 결합 형성을 통해 T세포의 활성화를 유도하여 항종양 효과를 나타내므로, 이러한 살모넬라 변이주 또는 이로부터 분리된 외막 소포체를 암치료에 활용할 수 있다.The Salmonella mutant strain according to the present invention expresses the anti-PD-L1 peptide in the extracellular loop of outer membrane protein A, thereby stably and efficiently delivering the anti-PD-L1 peptide to the inside of the tumor due to Salmonella characteristics, and is able to communicate with PD-1 of cancer cells. Since it induces activation of T cells through bond formation and exhibits an antitumor effect, these mutant Salmonella strains or outer membrane vesicles isolated therefrom can be used for cancer treatment.
도 1은 본 발명의 일 실시예에 따른 항PD-L1 펩티드 발현 균주의 제작 과정을 나타낸 흐름도이다.
도 2는 본 발명의 일 실시예에 따른 항PD-L1 펩티드 발현 균주 3종 (7AA, 9AA 및 15AA 균주)을 PCR 증폭 후 10% SDS-PAGE에서 전기영동으로 확인한 결과이다.
도 3은 본 발명의 일 실시예에 따른 항PD-L1 펩티드 발현 균주 3종의 세포외 루프 구조 및 서열을 나타낸 것이다.
도 4는 본 발명의 일 실시예에 따른 항PD-L1 펩티드 발현 균주 3종의 암세포 CT-26, AT-1 및 AsPC-1에 대한 부착 능력을 확인한 결과이다.
도 5는 본 발명의 일 실시예에 따른 항PD-L1 펩티드 발현 균주 3종과 암세포 CT-26의 결합을 형광 발현을 통해 시각적으로 확인한 결과이다.1 is a flow chart showing a manufacturing process of a strain expressing an anti-PD-L1 peptide according to an embodiment of the present invention.
FIG. 2 shows the result of electrophoresis on 10% SDS-PAGE after PCR amplification of three anti-PD-L1 peptide expression strains (7AA, 9AA and 15AA strains) according to an embodiment of the present invention.
Figure 3 shows the structure and sequence of the extracellular loop of three strains expressing the anti-PD-L1 peptide according to an embodiment of the present invention.
4 is a result of confirming the adhesion ability of three anti-PD-L1 peptide-expressing strains to cancer cells CT-26, AT-1 and AsPC-1 according to an embodiment of the present invention.
5 is a result of visually confirming the binding between three anti-PD-L1 peptide expressing strains and cancer cell CT-26 according to an embodiment of the present invention through fluorescence expression.
이하, 첨부된 도면을 참조하며 본 발명을 보다 상세하게 설명한다. 그러나, 이러한 설명은 본 발명의 이해를 돕기 위하여 예시적으로 제시된 것일 뿐, 본 발명의 범위가 이러한 예시적인 설명에 의하여 제한되는 것은 아니다.Hereinafter, the present invention will be described in more detail with reference to the accompanying drawings. However, these descriptions are merely presented as examples to aid understanding of the present invention, and the scope of the present invention is not limited by these exemplary descriptions.
실시예 1. 항PD-L1 펩티드 7AA 발현 균주 제작Example 1. Preparation of anti-PD-L1 peptide 7AA expressing strain
1-1. 재조합 벡터 제작1-1. Construction of recombinant vectors
살모넬라 외막 단백질 A(OmpA)의 세포외 루프(EL) 서열 내에 항PD-L1 펩티드 서열이 삽입된 돌연변이 균주를 제작하기 위한 재조합 벡터를 제작하였다.A recombinant vector was constructed to construct a mutant strain in which an anti-PD-L1 peptide sequence was inserted into the extracellular loop (EL) sequence of Salmonella outer membrane protein A (OmpA).
도 1을 참고하면, Salmonella typhimurium SHJ2168 균주의 genomic DNA를 주형으로 SOA_F-Gibson 및 SOA_R-Cat 프라이머 쌍을 사용하여 PCR을 통해 살모넬라 OmpA 서열을 포함하는 DNA 단편(이하 'OmpA 단편'이라 함) (서열번호 11)을 준비하였다. pKD3 (서열번호 18)를 주형으로 Cat_F 및 Cat_R-Gibson 프라이머 쌍을 사용하여 PCR을 통해 클로람페니콜 항생제 저항성 유전자를 포함하는 DNA 단편(이하 'CmR 단편'이라 함) (서열번호 12)을 준비하였다. 이후 pUC19 플라스미드를 SmaⅠ 제한효소로 절단한 후 SmaⅠ 처리된 pUC19 플라스미드, OmpA 단편 및 CmR 단편을 혼합하여 Gibson assembly kit (NEB, Gibson Assembly® Master Mix)를 사용하여 클로닝하였다. 클로닝한 플라스미드를 주형으로 Loop2_7AA_F 및 Loop2_R 프라이머 쌍을 사용하여 위치 유도 돌연변이(site directed mutagenesis) 기법을 통해 OmpA의 EL2 서열 내에 항PD-L1 펩티드 7AA이 삽입된 재조합 벡터(이하 'pUC19_SalOmpA_7AA 벡터'라 함) (서열번호 13)를 구축하였다.Referring to Figure 1, a DNA fragment containing the Salmonella OmpA sequence (hereinafter referred to as 'OmpA fragment') through PCR using the genomic DNA of the Salmonella typhimurium SHJ2168 strain as a template and the SOA_F-Gibson and SOA_R-Cat primer pairs (sequence No. 11) was prepared. A DNA fragment containing a chloramphenicol antibiotic resistance gene (hereinafter referred to as 'Cm R fragment') (SEQ ID NO: 12) was prepared by PCR using pKD3 (SEQ ID NO: 18) as a template and a pair of Cat_F and Cat_R-Gibson primers. Then, the pUC19 plasmid was digested with SmaI restriction enzyme, and the SmaI-treated pUC19 plasmid, the OmpA fragment, and the CmR fragment were mixed and cloned using a Gibson assembly kit (NEB, Gibson Assembly® Master Mix). A recombinant vector in which the anti-PD-L1 peptide 7AA was inserted into the EL2 sequence of OmpA through site directed mutagenesis using the cloned plasmid as a template and a pair of Loop2_7AA_F and Loop2_R primers (hereinafter referred to as 'pUC19_SalOmpA_7AA vector') (SEQ ID NO: 13) was constructed.
1-2. 돌연변이 균주 제작1-2. Production of mutant strains
pUC19_SalOmpA_7AA 벡터를 주형으로 하여 OmpA-CmR 영역을 SOA_F 및 CmR 영역과 살모넬라 OmpA의 하위 영역 41 mer를 포함하는 Cat_R_SOA 프라이머 쌍을 사용하여 PCR을 통해 증폭한 후 증폭된 DNA에 제한효소 DpnⅠ를 처리하여 주형 플라스미드를 제거한 DNA 단편을 얻었다. Using the pUC19_SalOmpA_7AA vector as a template, the OmpA- CmR region was amplified by PCR using a Cat_R_SOA primer pair containing the SOA_F and CmR regions and the 41-mer subregion of Salmonella OmpA, and then the amplified DNA was treated with restriction enzyme DpnI. A DNA fragment from which the template plasmid was removed was obtained.
이후 ppGpp 결손 및 발광단백질 lux 발현 살모넬라 균주(Salmonella typhimurium ΔppGpp/lux, SHJ2168)에 pKD46 플라스미드 (서열번호 16)를 전기천공법을 통해 형질전환한 후, 형질전환된 SHJ2168 균주에 0.2% L-아라비노스(arabinose)를 첨가하여 람다 레드 재조합효소(lambda red recombinase)를 유도(induction)시켜 전기천공용 컴피턴트 셀(competent cell)을 만들어 주었다. 그런 다음, 컴피턴트 셀에 상기 DpnⅠ 제한효소 처리하여 얻은 DNA 단편을 전기천공법으로 형질전환하여 형질전환체를 제작하였다. 이후 형질전환체를 클로람페니콜 항생제 배지에서 선별하여 상동 재조합을 통해 클로람페니콜 저항성을 보이는 균주들을 대상으로 SOA_F+459 및 Cat_R_SOA 프라이머 쌍을 사용하여 PCR을 수행하였고 (도 2), 최종적으로 항PD-L1 펩티드 7AA가 도입된 돌연변이 균주(이하 '7AA 균주'라 함)를 수득하였다 (도 3). Then, ppGpp deficiency and luminescent protein lux expression Salmonella strain ( Salmonella typhimurium ΔppGpp / lux, SHJ2168) was transformed with pKD46 plasmid (SEQ ID NO: 16) through electroporation, and then 0.2% L-arabinose was added to the transformed SHJ2168 strain. (arabinose) was added to induce lambda red recombinase (lambda red recombinase) to make a competent cell for electroporation (competent cell). Then, the DNA fragment obtained by treatment with the DpnI restriction enzyme was transformed into competent cells by electroporation to prepare transformants. Subsequently, the transformants were selected in a chloramphenicol antibiotic medium, and PCR was performed using SOA_F+459 and Cat_R_SOA primer pairs for strains showing chloramphenicol resistance through homologous recombination (FIG. 2), and finally anti-PD-L1 peptide 7AA A mutant strain into which was introduced (hereinafter referred to as '7AA strain') was obtained (FIG. 3).
실시예 2. 항PD-L1 펩티드 9AA 발현 균주 제작Example 2. Preparation of anti-PD-L1 peptide 9AA expressing strain
Loop2_7AA_F 및 Loop2_R 프라이머 쌍 대신 Loop2_9AA_F 및 Loop2_R 프라이머 쌍을 사용하여 재조합 벡터(이하 'pUC19_SalOmpA_9AA 벡터'라 함) (서열번호 14)를 제작한 것을 제외하고는, 실시예 1과 동일한 방법을 수행하여 항PD-L1 펩티드 9AA가 도입된 돌연변이 균주(이하 '9AA 균주'라 함)를 수득하였다.Anti-PD- A mutant strain into which the L1 peptide 9AA was introduced (hereinafter referred to as '9AA strain') was obtained.
실시예 3. 항PD-L1 펩티드 15AA 발현 균주 제작Example 3. Preparation of anti-PD-L1 peptide 15AA expressing strain
Loop2_7AA_F 및 Loop2_R 프라이머 쌍 대신 Loop2_15AA_F 및 Loop2_R 프라이머 쌍을 사용하여 재조합 벡터(이하 'pUC19_SalOmpA_15AA 벡터'라 함) (서열번호 15)를 제작한 것을 제외하고는, 실시예 1과 동일한 방법을 수행하여 항PD-L1 펩티드 15AA가 도입된 돌연변이 균주(이하 '15AA 균주'라 함)를 수득하였다. Anti-PD- A mutant strain into which the L1 peptide 15AA was introduced (hereinafter referred to as '15AA strain') was obtained.
실시예 1 내지 3에서 사용된 프라이머 서열은 하기 표 1과 같다.Primer sequences used in Examples 1 to 3 are shown in Table 1 below.
실험예 1. 항PD-L1 펩티드 발현 균주 3종의 암세포에 대한 부착 능력 확인Experimental Example 1. Confirmation of adhesion ability to cancer cells of three anti-PD-L1 peptide expression strains
1-1. 암세포에 대한 부착 실험1-1. Adhesion experiments on cancer cells
암세포 CT-26 (대장암 세포주), AT-1 (폐암 세포주) 및 AsPC-1 (췌장암 세포주)에 대한 SHJ2168 균주 및 돌연변이 균주 3종 (7AA, 9AA 및 15AA 균주)의 부착 능력을 확인하였다. The attachment ability of SHJ2168 strain and three mutant strains (7AA, 9AA and 15AA strains) to cancer cells CT-26 (colorectal cancer cell line), AT-1 (lung cancer cell line) and AsPC-1 (pancreatic cancer cell line) was confirmed.
배지 조건으로, CT-26 세포는 10% FBS 및 1% 페니실린/스트렙토마이신을 포함한 DMEM 배지를, AT-1 및 AsPC-1 세포는 10% FBS 및 1% 페니실린/스트렙토마이신을 포함한 RPMI 1640 배지를 사용하였다. As medium conditions, DMEM medium containing 10% FBS and 1% penicillin/streptomycin was used for CT-26 cells, and RPMI 1640 medium containing 10% FBS and 1% penicillin/streptomycin was used for AT-1 and AsPC-1 cells. used
먼저, 12웰 플레이트(12-well plate)에 각 세포를 2Х105 cell/well로 분주하였다. 24시간 후 각 well을 항생제가 없는 배지로 교체 후 SHJ2168 균주 또는 돌연변이 균주를 MOI 1:100으로 감염시켰다. 10분 후 PBS로 3번 세척하고 각 균수를 계산하였다. First, each cell was dispensed in a 12-well plate at 2Х10 5 cell/well. After 24 hours, each well was replaced with an antibiotic-free medium, and the SHJ2168 strain or mutant strain was infected at an MOI of 1:100. After 10 minutes, the cells were washed 3 times with PBS and the number of bacteria was counted.
그 결과, 도 4에 나타낸 바와 같이, CT-26 세포에서는 SHJ2168 균주 대비 돌연변이 균주를 처리한 경우의 균수가 약 4 ~ 6배 증가하였으며, AT-1 세포에서는 SHJ2168 균주 대비 돌연변이 균주를 처리한 경우의 균수가 약 3배 증가한 것으로 나타났다. 반면, AsPC-1 세포에서는 SHJ2168 균주 대비 돌연변이 균주를 처리한 경우의 균수가 약 1.2배 증가하여, 유의미한 차이를 보이지 않았다. As a result, as shown in FIG. 4, in CT-26 cells, the number of bacteria increased about 4 to 6 times when the mutant strain was treated compared to the SHJ2168 strain, and in the case of AT-1 cells treated with the mutant strain compared to the SHJ2168 strain. It was found that the number of bacteria increased about 3 times. On the other hand, in AsPC-1 cells, the number of bacteria increased by about 1.2 times when the mutant strain was treated compared to the SHJ2168 strain, showing no significant difference.
2-2. 형광 현미경 분석2-2. Fluorescence microscopic analysis
실시예 1 내지 3의 돌연변이 균주가 우수한 부착 능력을 나타낸 CT-26 세포에 대해 형광 형미경 분석을 실시하였다. Fluorescence microscopic analysis was performed on CT-26 cells in which the mutant strains of Examples 1 to 3 showed excellent adhesion ability.
SHJ2168 균주 및 돌연변이 균주 3종은 GFP(green fluorescent protein) 발현을 위하여 열충격법(heat shock method)를 통해 pEGFP 플라스미드 (서열번호 17)가 형질전환되었다. 커버글라스(Cover glass)를 화염멸균하여 6웰 플레이트에 넣고 폴리-L-라이신(poly-L-lysine)으로 15분 동안 코팅한 후 PBS로 2번 세척하여 사용하였다. 이후 커버글라스에 CT-26 세포를 2Х105 cell/well로 분주하였다. 24시간 후 항생제가 없는 배지로 교체한 다음, SHJ2168 균주 또는 돌연변이 균주를 MOI 1:100으로 감염시켰다. 감염 10분 후 PBS로 1번 세척하였고, 4% PFA(paraformaldehyde)를 15분 처리하여 세포를 고정시켜주었다. 이후 0.1% 트리톤(triton) X-100을 10분 처리하여 투과(permeabilization)시켜준 후 1% BSA(bovine serum albumin)로 1시간 동안 블로킹(blocking)하였다. 이후 ZO-1 항체 (Cell Signaling, #13663)를 1시간 처리 후 2차 항체로 Alexa fluor 568 (Invitrogen, A-11011)를 처리하여 CT-26 세포를 빨간색으로 형광 염색하였다. 이후 DAPI counterstaining을 진행 후 형광 현미경으로 관찰하였다. The SHJ2168 strain and three mutant strains were transformed with pEGFP plasmid (SEQ ID NO: 17) through a heat shock method to express green fluorescent protein (GFP). A cover glass was flame-sterilized, put into a 6-well plate, coated with poly-L-lysine for 15 minutes, washed twice with PBS, and used. Thereafter, CT-26 cells were seeded on the cover glass at 2Х10 5 cell/well. After 24 hours, the medium was replaced with an antibiotic-free medium, and then the SHJ2168 strain or the mutant strain was infected at an MOI of 1:100. After 10 minutes of infection, the cells were washed once with PBS and treated with 4% PFA (paraformaldehyde) for 15 minutes to fix the cells. Thereafter, 0.1% triton X-100 was treated for 10 minutes to permeabilize, followed by blocking with 1% bovine serum albumin (BSA) for 1 hour. Then, after treatment with ZO-1 antibody (Cell Signaling, #13663) for 1 hour, Alexa fluor 568 (Invitrogen, A-11011) was treated as a secondary antibody, and CT-26 cells were fluorescently stained in red. Thereafter, DAPI counterstaining was performed and observed under a fluorescence microscope.
그 결과, 도 5에 나타낸 바와 같이, SHJ2168 균주에 비해 돌연변이 균주 3종 모두 균수가 증가함을 시각적으로 확인하였다.As a result, as shown in Figure 5, it was visually confirmed that the number of bacteria increased in all three mutant strains compared to the SHJ2168 strain.
종합하면, 본 발명의 일 실시예에 따른 항PD-L1 펩티드를 발현하는 돌연변이 균주 3종 (7AA, 9AA 및 15AA 균주)은 암세포의 PD-L1과 선택적으로 결합하며, 이는 암세포에 의한 T세포의 활성 억제를 효과적으로 차단할 수 있음을 시사한다.In summary, the three mutant strains (7AA, 9AA and 15AA strains) expressing the anti-PD-L1 peptide according to an embodiment of the present invention selectively bind to PD-L1 of cancer cells, which is the anti-PD-L1 peptide expression. It suggests that it can effectively block activity inhibition.
이제까지 본 발명에 대하여 그 바람직한 실시예들을 중심으로 살펴보았다. 본 발명이 속하는 기술 분야에서 통상의 지식을 가진 자는 본 발명이 본 발명의 본질적인 특성에서 벗어나지 않는 범위에서 변형된 형태로 구현될 수 있음을 이해할 수 있을 것이다. 그러므로 개시된 실시예들은 한정적인 관점이 아니라 설명적인 관점에서 고려되어야 한다. 본 발명의 범위는 전술한 설명이 아니라 특허청구범위에 나타나 있으며, 그와 동등한 범위 내에 있는 모든 차이점은 본 발명에 포함된 것으로 해석되어야 할 것이다.So far, the present invention has been looked at with respect to its preferred embodiments. Those skilled in the art to which the present invention pertains will be able to understand that the present invention can be implemented in a modified form without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments should be considered from an illustrative rather than a limiting point of view. The scope of the present invention is shown in the claims rather than the foregoing description, and all differences within the equivalent scope will be construed as being included in the present invention.
<110> Kyungpook National University Industry-Academic Cooperation Foundation <120> Salmonella mutant expressing anti-PD-L1 peptide and outer membrane vesicle thereof and pharmaceutical comprising the same <130> BPN211055 <160> 29 <170> KoPatentIn 3.0 <210> 1 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> anti-PD-L1 peptide 7AA <400> 1 Cys Val Arg Ala Arg Thr Arg 1 5 <210> 2 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> anti-PD-L1 peptide 9AA <400> 2 Cys Leu Gln Lys Thr Pro Lys Gln Cys 1 5 <210> 3 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> anti-PD-L1 peptide 15AA <400> 3 Tyr Ala Ser Tyr His Cys Trp Cys Trp Arg Asp Pro Gly Arg Ser 1 5 10 15 <210> 4 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> anti-PD-L1 peptide 7AA <400> 4 tgtgtgcgcg ctcgcacccg t 21 <210> 5 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> anti-PD-L1 peptide 9AA <400> 5 tgtctgcaga aaacccctaa acagtgt 27 <210> 6 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> anti-PD-L1 peptide 15AA <400> 6 tacgcttctt accattgttg gtgttggcgt gacccgggcc gttct 45 <210> 7 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> EL2 <400> 7 Gly Arg Met Pro Tyr Lys Gly Asp Asn Ile Asn Gly Ala Tyr Lys Ala 1 5 10 15 Gln <210> 8 <211> 24 <212> PRT <213> Artificial Sequence <220> <223> EL2_7AA <400> 8 Gly Arg Met Pro Tyr Lys Gly Asp Cys Val Arg Ala Arg Thr Arg Asn 1 5 10 15 Ile Asn Gly Ala Tyr Lys Ala Gln 20 <210> 9 <211> 26 <212> PRT <213> Artificial Sequence <220> <223> EL2_9AA <400> 9 Gly Arg Met Pro Tyr Lys Gly Asp Cys Leu Gln Lys Thr Pro Lys Gln 1 5 10 15 Cys Asn Ile Asn Gly Ala Tyr Lys Ala Gln 20 25 <210> 10 <211> 32 <212> PRT <213> Artificial Sequence <220> <223> EL2_15AA <400> 10 Gly Arg Met Pro Tyr Lys Gly Asp Tyr Ala Ser Tyr His Cys Trp Cys 1 5 10 15 Trp Arg Asp Pro Gly Arg Ser Asn Ile Asn Gly Ala Tyr Lys Ala Gln 20 25 30 <210> 11 <211> 1053 <212> DNA <213> Artificial Sequence <220> <223> OmpA <400> 11 atgaaaaaga cagctatcgc gattgcagtg gcactggctg gtttcgctac cgtagcgcag 60 gccgctccga aagataacac ctggtacgct ggtgctaaac tgggctggtc tcagtaccat 120 gacaccggct tcattcacaa tgatggcccg actcatgaaa accaactggg cgcaggtgct 180 tttggtggtt accaggttaa cccgtatgtt ggctttgaaa tgggctacga ctggttaggc 240 cgtatgccgt acaaaggcga caacatcaat ggcgcttata aagctcaggg cgttcagttg 300 accgctaaac tgggttatcc aatcactgac gatctggacg tttatacccg tctgggtggt 360 atggtatggc gtgcagacac caagtctaac gtccctggcg gcccgtctac taaagaccac 420 gacaccggcg tttccccggt attcgcgggc ggtatcgagt atgctatcac ccctgaaatc 480 gcaacccgtc tggaatacca gtggactaac aacatcggtg atgccaacac catcggcacc 540 cgtccggaca acggcctgct gagcgtaggt gtttcctacc gtttcggcca gcaagaagct 600 gctccggtag tagctccggc accagctccg gctccggaag tacagaccaa gcacttcact 660 ctgaagtctg acgtactgtt caacttcaac aaatctaccc tgaagccgga aggccagcag 720 gctctggatc agctgtacag ccagctgagc aacctggatc cgaaagacgg ttccgttgtc 780 gttctgggct tcactgaccg tatcggttct gacgcttaca accagggtct gtccgagaaa 840 cgtgctcagt ctgttgttga ttacctgatc tccaaaggta ttccgtctga caaaatctcc 900 gcacgtggta tgggcgaatc taacccggtt accggcaaca cctgtgacaa cgtgaaacct 960 cgcgctgccc tgatcgattg cctggctccg gatcgtcgcg tagagatcga agttaaaggc 1020 gttaaagacg tggtaactca gccgcaggct taa 1053 <210> 12 <211> 639 <212> DNA <213> Artificial Sequence <220> <223> CmR <400> 12 atggagaaaa aaatcactgg atataccacc gttgatatat cccaatggca tcgtaaagaa 60 cattttgagg catttcagtc agttgctcaa tgtacctata accagaccgt tcagctggat 120 attacggcct ttttaaagac cgtaaagaaa aataagcaca agttttatcc ggcctttatt 180 cacattcttg cccgcctgat gaatgctcat ccggaattac gtatggcaat gaaagacggt 240 gagctggtga tatgggatag tgttcaccct tgttacaccg ttttccatga gcaaactgaa 300 acgttttcat cgctctggag tgaataccac gacgatttcc ggcagtttct acacatatat 360 tcgcaagatg tggcgtgtta cggtgaaaac ctggcctatt tccctaaagg gtttattgag 420 aatatgtttt tcgtctcagc caatccctgg gtgagtttca ccagttttga tttaaacgtg 480 gccaatatgg acaacttctt cgcccccgtt ttcaccatgg gcaaatatta tacgcaaggc 540 gacaaggtgc tgatgccgct ggcgattcag gttcatcatg ccgtttgtga tggcttccat 600 gtcggcagat gcttaatgaa tacaacagta ctgcgatga 639 <210> 13 <211> 5464 <212> DNA <213> Artificial Sequence <220> <223> pUC19_SalOmpA_7AA <400> 13 tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta 60 tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag 120 aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg 180 tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg 240 tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg 300 cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga 360 agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc 420 tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt 480 aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact 540 ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg 600 cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt 660 accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt 720 ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct 780 ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg 840 gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt 900 aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt 960 gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc 1020 gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg 1080 cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc 1140 gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg 1200 gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca 1260 ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga 1320 tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct 1380 ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg 1440 cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca 1500 accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata 1560 cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct 1620 tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact 1680 cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa 1740 acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc 1800 atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga 1860 tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga 1920 aaagtgccac ctgacgtcta agaaaccatt attatcatga cattaaccta taaaaatagg 1980 cgtatcacga ggccctttcg tctcgcgcgt ttcggtgatg acggtgaaaa cctctgacac 2040 atgcagctcc cggagacggt cacagcttgt ctgtaagcgg atgccgggag cagacaagcc 2100 cgtcagggcg cgtcagcggg tgttggcggg tgtcggggct ggcttaacta tgcggcatca 2160 gagcagattg tactgagagt gcaccatatg cggtgtgaaa taccgcacag atgcgtaagg 2220 agaaaatacc gcatcaggcg ccattcgcca ttcaggctgc gcaactgttg ggaagggcga 2280 tcggtgcggg cctcttcgct attacgccag ctggcgaaag ggggatgtgc tgcaaggcga 2340 ttaagttggg taacgccagg gttttcccag tcacgacgtt gtaaaacgac ggccagtgaa 2400 ttcgagctcg gtacccgaca gcattccggg ctaaaaattc actctaattt gtatcattaa 2460 gtaaaattag gatttatcct ggactttttt ttacgcgaac gtatctcctt tgagtgctaa 2520 cgtttttttt gcgagaacgc ttgtcagaag cggtttccgc aatttttgct gtacgattta 2580 tcatctgaaa ctgttaaatg atgtgtatat ccgtcatgtt tttttcacat gtctgacgga 2640 gttcacactt gtaagtttcc aactacgttg tagactttac atcgccaggg gtgctcagca 2700 taagccgtag atatcggtag agtaactatt gagcagatcc cccggtgaag gatttaaccg 2760 tgttatctcg ttggagatat tcatggcgta ttttggatga taacgaggcg caaaaaatga 2820 aaaagacagc tatcgcgatt gcagtggcac tggctggttt cgctaccgta gcgcaggccg 2880 ctccgaaaga taacacctgg tacgctggtg ctaaactggg ctggtctcag taccatgaca 2940 ccggcttcat tcacaatgat ggcccgactc atgaaaacca actgggcgca ggtgcttttg 3000 gtggttacca ggttaacccg tatgttggct ttgaaatggg ctacgactgg ttaggccgta 3060 tgccgtacaa aggcgacaac tgtgtgcgcg ctcgcacccg tatcaatggc gcttataaag 3120 ctcagggcgt tcagttgacc gctaaactgg gttatccaat cactgacgat ctggacgttt 3180 atacccgtct gggtggtatg gtatggcgtg cagacaccaa gtctaacgtc cctggcggcc 3240 cgtctactaa agaccacgac accggcgttt ccccggtatt cgcgggcggt atcgagtatg 3300 ctatcacccc tgaaatcgca acccgtctgg aataccagtg gactaacaac atcggtgatg 3360 ccaacaccat cggcacccgt ccggacaacg gcctgctgag cgtaggtgtt tcctaccgtt 3420 tcggccagca agaagctgct ccggtagtag ctccggcacc agctccggct ccggaagtac 3480 agaccaagca cttcactctg aagtctgacg tactgttcaa cttcaacaaa tctaccctga 3540 agccggaagg ccagcaggct ctggatcagc tgtacagcca gctgagcaac ctggatccga 3600 aagacggttc cgttgtcgtt ctgggcttca ctgaccgtat cggttctgac gcttacaacc 3660 agggtctgtc cgagaaacgt gctcagtctg ttgttgatta cctgatctcc aaaggtattc 3720 cgtctgacaa aatctccgca cgtggtatgg gcgaatctaa cccggttacc ggcaacacct 3780 gtgacaacgt gaaacctcgc gctgccctga tcgattgcct ggctccggat cgtcgcgtag 3840 agatcgaagt taaaggcgtt aaagacgtgg taactcagcc gcaggcttaa gtttccgtct 3900 gataaaaaac cccgcgtcgc ggggtttttt gctctggtct ggatgacaac gcctttcagc 3960 gttacttctt gcctaataac gcctgtaaat cctgctttaa cgtggtcatt tgcgtggcat 4020 atttctcttt atgctccgcg gatagaaaca gaagccactg gagcacctca aaaacaccat 4080 catacactaa atcagtaagt tggcagcatc acccgacgca ctttgcgccg aataaatacc 4140 tgtgacggaa gatcacttcg cagaataaat aaatcctggt gtccctgttg ataccgggaa 4200 gccctgggcc aacttttggc gaaaatgaga cgttgatcgg cacgtaagag gttccaactt 4260 tcaccataat gaaataagat cactaccggg cgtatttttt gagttatcga gattttcagg 4320 agctaagaag ctaaaatgga gaaaaaaatc actggatata ccaccgttga tatatcccaa 4380 tggcatcgta aagaacattt tgaggcattt cagtcagttg ctcaatgtac ctataaccag 4440 accgttcagc tggatattac ggccttttta aagaccgtaa agaaaaataa gcacaagttt 4500 tatccggcct ttattcacat tcttgcccgc ctgatgaatg ctcatccgga attccgtatg 4560 gcaatgaaag acggtgagct ggtgatatgg gatagtgttc acccttgtta caccgttttc 4620 catgagcaaa ctgaaacgtt ttcatcgctc tggagtgaat accacgacga tttccggcag 4680 tttctacaca tatattcgca agatgtggcg tgttacggtg aaaacctggc ctatttccct 4740 aaagggttta ttgagaatat gtttttcgtc tcagccaatc cctgggtgag tttcaccagt 4800 tttgatttaa acgtggccaa tatggacaac ttcttcgccc ccgttttcac catgggcaaa 4860 tattatacgc aaggcgacaa ggtgctgatg ccgctggcga ttcaggttca tcatgccgtt 4920 tgtgatggct tccatgtcgg cagaatgctt aatgaattac aacagtactg cgatgagtgg 4980 cagggcgggg cgtaattttt ttaaggcagt tattggtgcc cttaaacgcc tggttgctac 5040 gcctgaataa gtgataataa gcggatgaat ggcagaaatt cgaaagcaaa ttcgacccgg 5100 tcgtcggttc agggcagggt cgttaaatag ccgcttatgt ctattgctgg tttaccggtt 5160 tattgactac cggaagcagt gtgaccgtgt gcttggggat cctctagagt cgacctgcag 5220 gcatgcaagc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct 5280 cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg 5340 agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct 5400 gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg 5460 gcgc 5464 <210> 14 <211> 5470 <212> DNA <213> Artificial Sequence <220> <223> pUC19_SalOmpA_9AA <400> 14 tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta 60 tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag 120 aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg 180 tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg 240 tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg 300 cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga 360 agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc 420 tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt 480 aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact 540 ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg 600 cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt 660 accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt 720 ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct 780 ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg 840 gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt 900 aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt 960 gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc 1020 gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg 1080 cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc 1140 gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg 1200 gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca 1260 ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga 1320 tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct 1380 ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg 1440 cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca 1500 accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata 1560 cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct 1620 tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact 1680 cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa 1740 acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc 1800 atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga 1860 tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga 1920 aaagtgccac ctgacgtcta agaaaccatt attatcatga cattaaccta taaaaatagg 1980 cgtatcacga ggccctttcg tctcgcgcgt ttcggtgatg acggtgaaaa cctctgacac 2040 atgcagctcc cggagacggt cacagcttgt ctgtaagcgg atgccgggag cagacaagcc 2100 cgtcagggcg cgtcagcggg tgttggcggg tgtcggggct ggcttaacta tgcggcatca 2160 gagcagattg tactgagagt gcaccatatg cggtgtgaaa taccgcacag atgcgtaagg 2220 agaaaatacc gcatcaggcg ccattcgcca ttcaggctgc gcaactgttg ggaagggcga 2280 tcggtgcggg cctcttcgct attacgccag ctggcgaaag ggggatgtgc tgcaaggcga 2340 ttaagttggg taacgccagg gttttcccag tcacgacgtt gtaaaacgac ggccagtgaa 2400 ttcgagctcg gtacccgaca gcattccggg ctaaaaattc actctaattt gtatcattaa 2460 gtaaaattag gatttatcct ggactttttt ttacgcgaac gtatctcctt tgagtgctaa 2520 cgtttttttt gcgagaacgc ttgtcagaag cggtttccgc aatttttgct gtacgattta 2580 tcatctgaaa ctgttaaatg atgtgtatat ccgtcatgtt tttttcacat gtctgacgga 2640 gttcacactt gtaagtttcc aactacgttg tagactttac atcgccaggg gtgctcagca 2700 taagccgtag atatcggtag agtaactatt gagcagatcc cccggtgaag gatttaaccg 2760 tgttatctcg ttggagatat tcatggcgta ttttggatga taacgaggcg caaaaaatga 2820 aaaagacagc tatcgcgatt gcagtggcac tggctggttt cgctaccgta gcgcaggccg 2880 ctccgaaaga taacacctgg tacgctggtg ctaaactggg ctggtctcag taccatgaca 2940 ccggcttcat tcacaatgat ggcccgactc atgaaaacca actgggcgca ggtgcttttg 3000 gtggttacca ggttaacccg tatgttggct ttgaaatggg ctacgactgg ttaggccgta 3060 tgccgtacaa aggcgacaac tgtctgcaga aaacccctaa acagtgtatc aatggcgctt 3120 ataaagctca gggcgttcag ttgaccgcta aactgggtta tccaatcact gacgatctgg 3180 acgtttatac ccgtctgggt ggtatggtat ggcgtgcaga caccaagtct aacgtccctg 3240 gcggcccgtc tactaaagac cacgacaccg gcgtttcccc ggtattcgcg ggcggtatcg 3300 agtatgctat cacccctgaa atcgcaaccc gtctggaata ccagtggact aacaacatcg 3360 gtgatgccaa caccatcggc acccgtccgg acaacggcct gctgagcgta ggtgtttcct 3420 accgtttcgg ccagcaagaa gctgctccgg tagtagctcc ggcaccagct ccggctccgg 3480 aagtacagac caagcacttc actctgaagt ctgacgtact gttcaacttc aacaaatcta 3540 ccctgaagcc ggaaggccag caggctctgg atcagctgta cagccagctg agcaacctgg 3600 atccgaaaga cggttccgtt gtcgttctgg gcttcactga ccgtatcggt tctgacgctt 3660 acaaccaggg tctgtccgag aaacgtgctc agtctgttgt tgattacctg atctccaaag 3720 gtattccgtc tgacaaaatc tccgcacgtg gtatgggcga atctaacccg gttaccggca 3780 acacctgtga caacgtgaaa cctcgcgctg ccctgatcga ttgcctggct ccggatcgtc 3840 gcgtagagat cgaagttaaa ggcgttaaag acgtggtaac tcagccgcag gcttaagttt 3900 ccgtctgata aaaaaccccg cgtcgcgggg ttttttgctc tggtctggat gacaacgcct 3960 ttcagcgtta cttcttgcct aataacgcct gtaaatcctg ctttaacgtg gtcatttgcg 4020 tggcatattt ctctttatgc tccgcggata gaaacagaag ccactggagc acctcaaaaa 4080 caccatcata cactaaatca gtaagttggc agcatcaccc gacgcacttt gcgccgaata 4140 aatacctgtg acggaagatc acttcgcaga ataaataaat cctggtgtcc ctgttgatac 4200 cgggaagccc tgggccaact tttggcgaaa atgagacgtt gatcggcacg taagaggttc 4260 caactttcac cataatgaaa taagatcact accgggcgta ttttttgagt tatcgagatt 4320 ttcaggagct aagaagctaa aatggagaaa aaaatcactg gatataccac cgttgatata 4380 tcccaatggc atcgtaaaga acattttgag gcatttcagt cagttgctca atgtacctat 4440 aaccagaccg ttcagctgga tattacggcc tttttaaaga ccgtaaagaa aaataagcac 4500 aagttttatc cggcctttat tcacattctt gcccgcctga tgaatgctca tccggaattc 4560 cgtatggcaa tgaaagacgg tgagctggtg atatgggata gtgttcaccc ttgttacacc 4620 gttttccatg agcaaactga aacgttttca tcgctctgga gtgaatacca cgacgatttc 4680 cggcagtttc tacacatata ttcgcaagat gtggcgtgtt acggtgaaaa cctggcctat 4740 ttccctaaag ggtttattga gaatatgttt ttcgtctcag ccaatccctg ggtgagtttc 4800 accagttttg atttaaacgt ggccaatatg gacaacttct tcgcccccgt tttcaccatg 4860 ggcaaatatt atacgcaagg cgacaaggtg ctgatgccgc tggcgattca ggttcatcat 4920 gccgtttgtg atggcttcca tgtcggcaga atgcttaatg aattacaaca gtactgcgat 4980 gagtggcagg gcggggcgta atttttttaa ggcagttatt ggtgccctta aacgcctggt 5040 tgctacgcct gaataagtga taataagcgg atgaatggca gaaattcgaa agcaaattcg 5100 acccggtcgt cggttcaggg cagggtcgtt aaatagccgc ttatgtctat tgctggttta 5160 ccggtttatt gactaccgga agcagtgtga ccgtgtgctt ggggatcctc tagagtcgac 5220 ctgcaggcat gcaagcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta 5280 tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc 5340 ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg 5400 aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg 5460 tattgggcgc 5470 <210> 15 <211> 5488 <212> DNA <213> Artificial Sequence <220> <223> pUC19_SalOmpA_15AA <400> 15 tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta 60 tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag 120 aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg 180 tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg 240 tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg 300 cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga 360 agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc 420 tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt 480 aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact 540 ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg 600 cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt 660 accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt 720 ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct 780 ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg 840 gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt 900 aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt 960 gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc 1020 gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg 1080 cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc 1140 gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg 1200 gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca 1260 ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga 1320 tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct 1380 ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg 1440 cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca 1500 accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata 1560 cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct 1620 tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact 1680 cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa 1740 acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc 1800 atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga 1860 tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga 1920 aaagtgccac ctgacgtcta agaaaccatt attatcatga cattaaccta taaaaatagg 1980 cgtatcacga ggccctttcg tctcgcgcgt ttcggtgatg acggtgaaaa cctctgacac 2040 atgcagctcc cggagacggt cacagcttgt ctgtaagcgg atgccgggag cagacaagcc 2100 cgtcagggcg cgtcagcggg tgttggcggg tgtcggggct ggcttaacta tgcggcatca 2160 gagcagattg tactgagagt gcaccatatg cggtgtgaaa taccgcacag atgcgtaagg 2220 agaaaatacc gcatcaggcg ccattcgcca ttcaggctgc gcaactgttg ggaagggcga 2280 tcggtgcggg cctcttcgct attacgccag ctggcgaaag ggggatgtgc tgcaaggcga 2340 ttaagttggg taacgccagg gttttcccag tcacgacgtt gtaaaacgac ggccagtgaa 2400 ttcgagctcg gtacccgaca gcattccggg ctaaaaattc actctaattt gtatcattaa 2460 gtaaaattag gatttatcct ggactttttt ttacgcgaac gtatctcctt tgagtgctaa 2520 cgtttttttt gcgagaacgc ttgtcagaag cggtttccgc aatttttgct gtacgattta 2580 tcatctgaaa ctgttaaatg atgtgtatat ccgtcatgtt tttttcacat gtctgacgga 2640 gttcacactt gtaagtttcc aactacgttg tagactttac atcgccaggg gtgctcagca 2700 taagccgtag atatcggtag agtaactatt gagcagatcc cccggtgaag gatttaaccg 2760 tgttatctcg ttggagatat tcatggcgta ttttggatga taacgaggcg caaaaaatga 2820 aaaagacagc tatcgcgatt gcagtggcac tggctggttt cgctaccgta gcgcaggccg 2880 ctccgaaaga taacacctgg tacgctggtg ctaaactggg ctggtctcag taccatgaca 2940 ccggcttcat tcacaatgat ggcccgactc atgaaaacca actgggcgca ggtgcttttg 3000 gtggttacca ggttaacccg tatgttggct ttgaaatggg ctacgactgg ttaggccgta 3060 tgccgtacaa aggcgacaac tacgcttctt accattgttg gtgttggcgt gacccgggcc 3120 gttctatcaa tggcgcttat aaagctcagg gcgttcagtt gaccgctaaa ctgggttatc 3180 caatcactga cgatctggac gtttataccc gtctgggtgg tatggtatgg cgtgcagaca 3240 ccaagtctaa cgtccctggc ggcccgtcta ctaaagacca cgacaccggc gtttccccgg 3300 tattcgcggg cggtatcgag tatgctatca cccctgaaat cgcaacccgt ctggaatacc 3360 agtggactaa caacatcggt gatgccaaca ccatcggcac ccgtccggac aacggcctgc 3420 tgagcgtagg tgtttcctac cgtttcggcc agcaagaagc tgctccggta gtagctccgg 3480 caccagctcc ggctccggaa gtacagacca agcacttcac tctgaagtct gacgtactgt 3540 tcaacttcaa caaatctacc ctgaagccgg aaggccagca ggctctggat cagctgtaca 3600 gccagctgag caacctggat ccgaaagacg gttccgttgt cgttctgggc ttcactgacc 3660 gtatcggttc tgacgcttac aaccagggtc tgtccgagaa acgtgctcag tctgttgttg 3720 attacctgat ctccaaaggt attccgtctg acaaaatctc cgcacgtggt atgggcgaat 3780 ctaacccggt taccggcaac acctgtgaca acgtgaaacc tcgcgctgcc ctgatcgatt 3840 gcctggctcc ggatcgtcgc gtagagatcg aagttaaagg cgttaaagac gtggtaactc 3900 agccgcaggc ttaagtttcc gtctgataaa aaaccccgcg tcgcggggtt ttttgctctg 3960 gtctggatga caacgccttt cagcgttact tcttgcctaa taacgcctgt aaatcctgct 4020 ttaacgtggt catttgcgtg gcatatttct ctttatgctc cgcggataga aacagaagcc 4080 actggagcac ctcaaaaaca ccatcataca ctaaatcagt aagttggcag catcacccga 4140 cgcactttgc gccgaataaa tacctgtgac ggaagatcac ttcgcagaat aaataaatcc 4200 tggtgtccct gttgataccg ggaagccctg ggccaacttt tggcgaaaat gagacgttga 4260 tcggcacgta agaggttcca actttcacca taatgaaata agatcactac cgggcgtatt 4320 ttttgagtta tcgagatttt caggagctaa gaagctaaaa tggagaaaaa aatcactgga 4380 tataccaccg ttgatatatc ccaatggcat cgtaaagaac attttgaggc atttcagtca 4440 gttgctcaat gtacctataa ccagaccgtt cagctggata ttacggcctt tttaaagacc 4500 gtaaagaaaa ataagcacaa gttttatccg gcctttattc acattcttgc ccgcctgatg 4560 aatgctcatc cggaattccg tatggcaatg aaagacggtg agctggtgat atgggatagt 4620 gttcaccctt gttacaccgt tttccatgag caaactgaaa cgttttcatc gctctggagt 4680 gaataccacg acgatttccg gcagtttcta cacatatatt cgcaagatgt ggcgtgttac 4740 ggtgaaaacc tggcctattt ccctaaaggg tttattgaga atatgttttt cgtctcagcc 4800 aatccctggg tgagtttcac cagttttgat ttaaacgtgg ccaatatgga caacttcttc 4860 gcccccgttt tcaccatggg caaatattat acgcaaggcg acaaggtgct gatgccgctg 4920 gcgattcagg ttcatcatgc cgtttgtgat ggcttccatg tcggcagaat gcttaatgaa 4980 ttacaacagt actgcgatga gtggcagggc ggggcgtaat ttttttaagg cagttattgg 5040 tgcccttaaa cgcctggttg ctacgcctga ataagtgata ataagcggat gaatggcaga 5100 aattcgaaag caaattcgac ccggtcgtcg gttcagggca gggtcgttaa atagccgctt 5160 atgtctattg ctggtttacc ggtttattga ctaccggaag cagtgtgacc gtgtgcttgg 5220 ggatcctcta gagtcgacct gcaggcatgc aagcttggcg taatcatggt catagctgtt 5280 tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa 5340 gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact 5400 gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc 5460 ggggagaggc ggtttgcgta ttgggcgc 5488 <210> 16 <211> 6329 <212> DNA <213> Artificial Sequence <220> <223> pKD46 <400> 16 catcgattta ttatgacaac ttgacggcta catcattcac tttttcttca caaccggcac 60 ggaactcgct cgggctggcc ccggtgcatt ttttaaatac ccgcgagaaa tagagttgat 120 cgtcaaaacc aacattgcga ccgacggtgg cgataggcat ccgggtggtg ctcaaaagca 180 gcttcgcctg gctgatacgt tggtcctcgc gccagcttaa gacgctaatc cctaactgct 240 ggcggaaaag atgtgacaga cgcgacggcg acaagcaaac atgctgtgcg acgctggcga 300 tatcaaaatt gctgtctgcc aggtgatcgc tgatgtactg acaagcctcg cgtacccgat 360 tatccatcgg tggatggagc gactcgttaa tcgcttccat gcgccgcagt aacaattgct 420 caagcagatt tatcgccagc agctccgaat agcgcccttc cccttgcccg gcgttaatga 480 tttgcccaaa caggtcgctg aaatgcggct ggtgcgcttc atccgggcga aagaaccccg 540 tattggcaaa tattgacggc cagttaagcc attcatgcca gtaggcgcgc ggacgaaagt 600 aaacccactg gtgataccat tcgcgagcct ccggatgacg accgtagtga tgaatctctc 660 ctggcgggaa cagcaaaata tcacccggtc ggcaaacaaa ttctcgtccc tgatttttca 720 ccaccccctg accgcgaatg gtgagattga gaatataacc tttcattccc agcggtcggt 780 cgataaaaaa atcgagataa ccgttggcct caatcggcgt taaacccgcc accagatggg 840 cattaaacga gtatcccggc agcaggggat cattttgcgc ttcagccata cttttcatac 900 tcccgccatt cagagaagaa accaattgtc catattgcat cagacattgc cgtcactgcg 960 tcttttactg gctcttctcg ctaaccaaac cggtaacccc gcttattaaa agcattctgt 1020 aacaaagcgg gaccaaagcc atgacaaaaa cgcgtaacaa aagtgtctat aatcacggca 1080 gaaaagtcca cattgattat ttgcacggcg tcacactttg ctatgccata gcatttttat 1140 ccataagatt agcggatcct acctgacgct ttttatcgca actctctact gtttctccat 1200 acccgttttt ttgggaattc gagctctaag gaggttataa aaaatggata ttaatactga 1260 aactgagatc aagcaaaagc attcactaac cccctttcct gttttcctaa tcagcccggc 1320 atttcgcggg cgatattttc acagctattt caggagttca gccatgaacg cttattacat 1380 tcaggatcgt cttgaggctc agagctgggc gcgtcactac cagcagctcg cccgtgaaga 1440 gaaagaggca gaactggcag acgacatgga aaaaggcctg ccccagcacc tgtttgaatc 1500 gctatgcatc gatcatttgc aacgccacgg ggccagcaaa aaatccatta cccgtgcgtt 1560 tgatgacgat gttgagtttc aggagcgcat ggcagaacac atccggtaca tggttgaaac 1620 cattgctcac caccaggttg atattgattc agaggtataa aacgaatgag tactgcactc 1680 gcaacgctgg ctgggaagct ggctgaacgt gtcggcatgg attctgtcga cccacaggaa 1740 ctgatcacca ctcttcgcca gacggcattt aaaggtgatg ccagcgatgc gcagttcatc 1800 gcattactga tcgttgccaa ccagtacggc cttaatccgt ggacgaaaga aatttacgcc 1860 tttcctgata agcagaatgg catcgttccg gtggtgggcg ttgatggctg gtcccgcatc 1920 atcaatgaaa accagcagtt tgatggcatg gactttgagc aggacaatga atcctgtaca 1980 tgccggattt accgcaagga ccgtaatcat ccgatctgcg ttaccgaatg gatggatgaa 2040 tgccgccgcg aaccattcaa aactcgcgaa ggcagagaaa tcacggggcc gtggcagtcg 2100 catcccaaac ggatgttacg tcataaagcc atgattcagt gtgcccgtct ggccttcgga 2160 tttgctggta tctatgacaa ggatgaagcc gagcgcattg tcgaaaatac tgcatacact 2220 gcagaacgtc agccggaacg cgacatcact ccggttaacg atgaaaccat gcaggagatt 2280 aacactctgc tgatcgccct ggataaaaca tgggatgacg acttattgcc gctctgttcc 2340 cagatatttc gccgcgacat tcgtgcatcg tcagaactga cacaggccga agcagtaaaa 2400 gctcttggat tcctgaaaca gaaagccgca gagcagaagg tggcagcatg acaccggaca 2460 ttatcctgca gcgtaccggg atcgatgtga gagctgtcga acagggggat gatgcgtggc 2520 acaaattacg gctcggcgtc atcaccgctt cagaagttca caacgtgata gcaaaacccc 2580 gctccggaaa gaagtggcct gacatgaaaa tgtcctactt ccacaccctg cttgctgagg 2640 tttgcaccgg tgtggctccg gaagttaacg ctaaagcact ggcctgggga aaacagtacg 2700 agaacgacgc cagaaccctg tttgaattca cttccggcgt gaatgttact gaatccccga 2760 tcatctatcg cgacgaaagt atgcgtaccg cctgctctcc cgatggttta tgcagtgacg 2820 gcaacggcct tgaactgaaa tgcccgttta cctcccggga tttcatgaag ttccggctcg 2880 gtggtttcga ggccataaag tcagcttaca tggcccaggt gcagtacagc atgtgggtga 2940 cgcgaaaaaa tgcctggtac tttgccaact atgacccgcg tatgaagcgt gaaggcctgc 3000 attatgtcgt gattgagcgg gatgaaaagt acatggcgag ttttgacgag atcgtgccgg 3060 agttcatcga aaaaatggac gaggcactgg ctgaaattgg ttttgtattt ggggagcaat 3120 ggcgatgacg catcctcacg ataatatccg ggtaggcgca atcactttcg tctactccgt 3180 tacaaagcga ggctgggtat ttcccggcct ttctgttatc cgaaatccac tgaaagcaca 3240 gcggctggct gaggagataa ataataaacg aggggctgta tgcacaaagc atcttctgtt 3300 gagttaagaa cgagtatcga gatggcacat agccttgctc aaattggaat caggtttgtg 3360 ccaataccag tagaaacaga cgaagaatcc atgggtatgg acagttttcc ctttgatatg 3420 taacggtgaa cagttgttct acttttgttt gttagtcttg atgcttcact gatagataca 3480 agagccataa gaacctcaga tccttccgta tttagccagt atgttctcta gtgtggttcg 3540 ttgtttttgc gtgagccatg agaacgaacc attgagatca tacttacttt gcatgtcact 3600 caaaaatttt gcctcaaaac tggtgagctg aatttttgca gttaaagcat cgtgtagtgt 3660 ttttcttagt ccgttacgta ggtaggaatc tgatgtaatg gttgttggta ttttgtcacc 3720 attcattttt atctggttgt tctcaagttc ggttacgaga tccatttgtc tatctagttc 3780 aacttggaaa atcaacgtat cagtcgggcg gcctcgctta tcaaccacca atttcatatt 3840 gctgtaagtg tttaaatctt tacttattgg tttcaaaacc cattggttaa gccttttaaa 3900 ctcatggtag ttattttcaa gcattaacat gaacttaaat tcatcaaggc taatctctat 3960 atttgccttg tgagttttct tttgtgttag ttcttttaat aaccactcat aaatcctcat 4020 agagtatttg ttttcaaaag acttaacatg ttccagatta tattttatga atttttttaa 4080 ctggaaaaga taaggcaata tctcttcact aaaaactaat tctaattttt cgcttgagaa 4140 cttggcatag tttgtccact ggaaaatctc aaagccttta accaaaggat tcctgatttc 4200 cacagttctc gtcatcagct ctctggttgc tttagctaat acaccataag cattttccct 4260 actgatgttc atcatctgag cgtattggtt ataagtgaac gataccgtcc gttctttcct 4320 tgtagggttt tcaatcgtgg ggttgagtag tgccacacag cataaaatta gcttggtttc 4380 atgctccgtt aagtcatagc gactaatcgc tagttcattt gctttgaaaa caactaattc 4440 agacatacat ctcaattggt ctaggtgatt ttaatcacta taccaattga gatgggctag 4500 tcaatgataa ttactagtcc ttttcctttg agttgtgggt atctgtaaat tctgctagac 4560 ctttgctgga aaacttgtaa attctgctag accctctgta aattccgcta gacctttgtg 4620 tgtttttttt gtttatattc aagtggttat aatttataga ataaagaaag aataaaaaaa 4680 gataaaaaga atagatccca gccctgtgta taactcacta ctttagtcag ttccgcagta 4740 ttacaaaagg atgtcgcaaa cgctgtttgc tcctctacaa aacagacctt aaaaccctaa 4800 aggcttaagt agcaccctcg caagctcggt tgcggccgca atcgggcaaa tcgctgaata 4860 ttccttttgt ctccgaccat caggcacctg agtcgctgtc tttttcgtga cattcagttc 4920 gctgcgctca cggctctggc agtgaatggg ggtaaatggc actacaggcg ccttttatgg 4980 attcatgcaa ggaaactacc cataatacaa gaaaagcccg tcacgggctt ctcagggcgt 5040 tttatggcgg gtctgctatg tggtgctatc tgactttttg ctgttcagca gttcctgccc 5100 tctgattttc cagtctgacc acttcggatt atcccgtgac aggtcattca gactggctaa 5160 tgcacccagt aaggcagcgg tatcatcaac ggggtctgac gctcagtgga acgaaaactc 5220 acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa 5280 ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta 5340 ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt 5400 tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag 5460 tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag caataaacca 5520 gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc 5580 tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt 5640 tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag 5700 ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt 5760 tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat 5820 ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt 5880 gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc 5940 ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat 6000 cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag 6060 ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt 6120 ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg 6180 gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta 6240 ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc 6300 gcgcacattt ccccgaaaag tgccacctg 6329 <210> 17 <211> 3355 <212> DNA <213> Artificial Sequence <220> <223> pEGFP <400> 17 agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc 60 acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc 120 tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa 180 ttgtgagcgg ataacaattt cacacaggaa acagctatga ccatgattac gccaagcttg 240 catgcctgca ggtcgactct agaggatccc cgggtaccgg tcgccaccat ggtgagcaag 300 ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg agctggacgg cgacgtaaac 360 ggccacaagt tcagcgtgtc cggcgagggc gagggcgatg ccacctacgg caagctgacc 420 ctgaagttca tctgcaccac cggcaagctg cccgtgccct ggcccaccct cgtgaccacc 480 ctgacctacg gcgtgcagtg cttcagccgc taccccgacc acatgaagca gcacgacttc 540 ttcaagtccg ccatgcccga aggctacgtc caggagcgca ccatcttctt caaggacgac 600 ggcaactaca agacccgcgc cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc 660 gagctgaagg gcatcgactt caaggaggac ggcaacatcc tggggcacaa gctggagtac 720 aactacaaca gccacaacgt ctatatcatg gccgacaagc agaagaacgg catcaaggtg 780 aacttcaaga tccgccacaa catcgaggac ggcagcgtgc agctcgccga ccactaccag 840 cagaacaccc ccatcggcga cggccccgtg ctgctgcccg acaaccacta cctgagcacc 900 cagtccgccc tgagcaaaga ccccaacgag aagcgcgatc acatggtcct gctggagttc 960 gtgaccgccg ccgggatcac tctcggcatg gacgagctgt acaagtaaag cggccgcgac 1020 tctagaattc caactgagcg ccggtcgcta ccattaccaa cttgtctggt gtcaaaaata 1080 ataggcctac tagtcggccg tacgggccct ttcgtctcgc gcgtttcggt gatgacggtg 1140 aaaacctctg acacatgcag ctcccggaga cggtcacagc ttgtctgtaa gcggatgccg 1200 ggagcagaca agcccgtcag ggcgcgtcag cgggtgttgg cgggtgtcgg ggctggctta 1260 actatgcggc atcagagcag attgtactga gagtgcacca tatgcggtgt gaaataccgc 1320 acagatgcgt aaggagaaaa taccgcatca ggcggcctta agggcctcgt gatacgccta 1380 tttttatagg ttaatgtcat gataataatg gtttcttaga cgtcaggtgg cacttttcgg 1440 ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg 1500 ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt 1560 attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt 1620 gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg 1680 ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa 1740 cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt 1800 gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag 1860 tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt 1920 gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga 1980 ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt 2040 tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta 2100 gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg 2160 caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc 2220 cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt 2280 atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg 2340 gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg 2400 attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa 2460 cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa 2520 atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga 2580 tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg 2640 ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact 2700 ggcttcagca gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac 2760 cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg 2820 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg 2880 gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga 2940 acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc 3000 gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg 3060 agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc 3120 tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc 3180 agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt 3240 cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc 3300 gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaag 3355 <210> 18 <211> 2804 <212> DNA <213> Artificial Sequence <220> <223> pKD3 <400> 18 gcatgcaagc ttggcactgg ccacgcaaaa aggccatccg tcaggatggc cttctgctta 60 atttgatgcc tggcagttta tggcgggcgt cctgcccgcc accctccggg ccgttgcttc 120 gcaacgttca aatccgctcc cggcggattt gtcctactca ggagagcgtt caccgacaaa 180 caacagataa aacgaaaggc ccagtctttc gactgagcct ttcgttttat ttgatgcctg 240 gcagttccct actctcgcat ggggagaccc cacactacca tcggggggcc atcgatgcag 300 gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt 360 caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa 420 ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt 480 gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt 540 tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt 600 ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg 660 tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga 720 atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa 780 gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga 840 caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa 900 ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca 960 ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta 1020 ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac 1080 ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc 1140 gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag 1200 ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga 1260 taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca tatatacttt 1320 agattgattt aaaacttcat ttttaatttt tgcggccgca agatccgcag ttcaacctgt 1380 tgatagtacg tactaagctc tcatgtttca cgtactaagc tctcatgttt aacgtactaa 1440 gctctcatgt ttaacgaact aaaccctcat ggctaacgta ctaagctctc atggctaacg 1500 tactaagctc tcatgtttca cgtactaagc tctcatgttt gaacaataaa attaatataa 1560 atcagcaact taaatagcct ctaaggtttt aagttttata agaaaaaaaa gaatatataa 1620 ggcttttaaa gcttttaagg tttaacggtt gtggacaaca agccagggat gtaacgcact 1680 gagaagccct tagagcctct caaagcaatt ttcagtgaca caggaacact taacggctga 1740 catgggaatt agccatggtc catatgaata tcctccttag ttcctattcc gaagttccta 1800 ttctctagaa agtataggaa cttcggcgcg cctacctgtg acggaagatc acttcgcaga 1860 ataaataaat cctggtgtcc ctgttgatac cgggaagccc tgggccaact tttggcgaaa 1920 atgagacgtt gatcggcacg taagaggttc caactttcac cataatgaaa taagatcact 1980 accgggcgta ttttttgagt tgtcgagatt ttcaggagct aaggaagcta aaatggagaa 2040 aaaaatcact ggatatacca ccgttgatat atcccaatgg catcgtaaag aacattttga 2100 ggcatttcag tcagttgctc aatgtaccta taaccagacc gttcagctgg atattacggc 2160 ctttttaaag accgtaaaga aaaataagca caagttttat ccggccttta ttcacattct 2220 tgcccgcctg atgaatgctc atccggaatt acgtatggca atgaaagacg gtgagctggt 2280 gatatgggat agtgttcacc cttgttacac cgttttccat gagcaaactg aaacgttttc 2340 atcgctctgg agtgaatacc acgacgattt ccggcagttt ctacacatat attcgcaaga 2400 tgtggcgtgt tacggtgaaa acctggccta tttccctaaa gggtttattg agaatatgtt 2460 tttcgtctca gccaatccct gggtgagttt caccagtttt gatttaaacg tggccaatat 2520 ggacaacttc ttcgcccccg ttttcaccat gggcaaatat tatacgcaag gcgacaaggt 2580 gctgatgccg ctggcgattc aggttcatca tgccgtttgt gatggcttcc atgtcggcag 2640 atgcttaatg aatacaacag tactgcgatg agtggcaggg cggggcgtaa ggcgcgccat 2700 ttaaatgaag ttcctattcc gaagttccta ttctctagaa agtataggaa cttcgaagca 2760 gctccagcct acacaatcgc tcaagacgtg taatgctgca atct 2804 <210> 19 <211> 49 <212> DNA <213> Artificial Sequence <220> <223> SOA_F-Gibson <400> 19 gccagtgaat tcgagctcgg tacccgacag cattccgggc taaaaattc 49 <210> 20 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> SOA_R-Cat <400> 20 ccagtggctt ctgtttctat ccgcggagca taaagagaaa tatgc 45 <210> 21 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Cat_F <400> 21 gatagaaaca gaagccactg g 21 <210> 22 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> Cat_R-Gibson <400> 22 ggtcgactct agaggatccc caagcacacg gtcacactg 39 <210> 23 <211> 44 <212> DNA <213> Artificial Sequence <220> <223> Loop2_7AA_F <400> 23 tgtgtgcgcg ctcgcacccg atcaatggcg cttataaagc tcag 44 <210> 24 <211> 51 <212> DNA <213> Artificial Sequence <220> <223> Loop2_9AA_F <400> 24 tgtctgcaga aaacccctaa acagtgtatc aatggcgctt ataaagctca g 51 <210> 25 <211> 69 <212> DNA <213> Artificial Sequence <220> <223> Loop2_15AA_F <400> 25 tacgcttctt accattgttg gtgttggcgt gacccgggcc gttctatcaa tggcgcttat 60 aaagctcag 69 <210> 26 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Loop2_R <400> 26 gttgtcgcct ttgtacgg 18 <210> 27 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> SOA_F <400> 27 gacagcattc cgggctaaaa attc 24 <210> 28 <211> 65 <212> DNA <213> Artificial Sequence <220> <223> Cat_R_SOA <400> 28 ggtaaaacat tatccgaaac gattgtgcaa ctgatagaag actaaggagg atattcatat 60 ggacc 65 <210> 29 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> SOA_F+459 <400> 29 ggtaggtaat gcggtaggg 19 <110> Kyungpook National University Industry-Academic Cooperation Foundation <120> Salmonella mutant expressing anti-PD-L1 peptide and outer membrane vesicle thereof and pharmaceutical comprising the same <130> BPN211055 <160> 29 <170> KoPatentIn 3.0 <210> 1 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> anti-PD-L1 peptide 7AA <400> 1 Cys Val Arg Ala Arg Thr Arg 1 5 <210> 2 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> anti-PD-L1 peptide 9AA <400> 2 Cys Leu Gln Lys Thr Pro Lys Gln Cys 1 5 <210> 3 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> anti-PD-L1 peptide 15AA <400> 3 Tyr Ala Ser Tyr His Cys Trp Cys Trp Arg Asp Pro Gly Arg Ser 1 5 10 15 <210> 4 <211> 21 <212> DNA < 213> Artificial Sequence <220> <223> anti-PD-L1 peptide 7AA <400> 4 tgtgtgcgcg ctcgcacccg t 21 <210> 5 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> anti- PD-L1 peptide 9AA <400> 5 tgtctgcaga aaacccctaa acagtgt 27 <210> 6 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> anti-PD-L1 peptide 15AA <400> 6 tacgcttctt accattgttg gtgttggcgt gacccgggcc gttct 45 <210> 7 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> EL2 <400> 7 Gly Arg Met Pro Tyr Lys Gly Asp Asn Ile Asn Gly Ala Tyr Lys Ala 1 5 10 15 Gln <210> 8 <211> 24 <212> PRT <213> Artificial Sequence <220> <223> EL2_7AA <400> 8 Gly Arg Met Pro Tyr Lys Gly Asp Cys Val Arg Ala Arg Thr Arg Asn 1 5 10 15 Ile Asn Gly Ala Tyr Lys Ala Gln 20 <210> 9 <211> 26 <212> PRT <213> Artificial Sequence <220> <223> EL2_9AA <400> 9 Gly Arg Met Pro Tyr Lys Gly Asp Cys Leu Gln Lys Thr Pro Lys Gln 1 5 10 15 Cys Asn Ile Asn Gly Ala Tyr Lys Ala Gln 20 25 <210> 10 <211> 32 <212> PRT <213> Artificial Sequence <220> <223> EL2_15AA <400> 10 Gly Arg Met Pro Tyr Lys Gly Asp Tyr Ala Ser Tyr His Cys Trp Cys 1 5 10 15 Trp Arg Asp Pro Gly Arg Ser Asn Ile Asn Gly Ala Tyr Lys Ala Gln 20 25 30 <210> 11 <211> 1053 <212> DNA <213 > Artificial Sequence <220> <223> OmpA <400> 11 atgaaaaaga cagctatcgc gattgcagtg gcactggctg gtttcgctac cgtagcgcag 60 gccgctccga aagataacac ctggtacgct ggtgctaaac tgggctggtc tcagtaccat 120 gacaccggct tcatt cacaa tgatggcccg actcatgaaa accaactggg cgcaggtgct 180 tttggtggtt accaggttaa cccgtatgtt ggctttgaaa tgggctacga ctggttaggc 240 cgtatgccgt acaaaggcga cacatcaat ggcgcttata aagctcaggg cgttcagttg 300 accgc taaac tgggttatcc aatcactgac gatctggacg tttatacccg tctgggtggt 360 atggtatggc gtgcagacac caagtctaac gtccctggcg gcccgtctac taaagaccac 420 gacaccggcg tttccccggt attcgcgggc ggtatcgagt atgctatcac ccctgaaatc 660 ctgaagtctg acgtactgtt caacttcaac aaatctaccc tgaagccgga aggccagcag 720 gctctggatc agctgtacag ccagctgagc aacctggatc cgaaagacgg ttccgttgtc 780 gttctgggct tcactgaccg tatcggttct gacgcttaca accagggtct gtccgagaaa 840 cgtgctcagt ctgttgttga ttacctgatc tccaaaggta ttccgtctga caaaatctcc 900 gc acgtggta tgggcgaatc taacccggtt accggcaaca cctgtgacaa cgtgaaacct 960 cgcgctgccc tgatcgattg cctggctccg gatcgtcgcg tagagatcga agttaaaggc 1020 gttaaagacg tggtaactca gccgcaggct taa 1053 <210> 12 <211> 639 <212> DNA <213> Artificial Sequence <220> <223> CmR <400> 12 atggagaaaa aaatcactgg atataccacc gttgatatat cccaatggca tcgtaaagaa 60 cattttgagg catttcagtc agttgctcaa tgtacctata accagaccgt tcagctggat 120 attacggcct ttttaa agac cgtaaagaaa aataagcaca agttttatcc ggcctttatcc ggcctttatt 180 cacattcttg cccgcctgat gaatgctcat ccggaattac gtatggcaat gaaagacggt 240 gagctggtga tatgggatag tgttcaccct tgttacaccg ttttccatga gcaaactgaa 300 acgttttcat cgctctggag tgaataccac gacgatttcc ggcagtttct acacatatat 360 tcgcaagatg tggcgtgtta cggtgaaaac ctggcctatt tccctaaagg gtttattgag 420 aatatgtttt tcgtctcagc caatccctgg gtgagtttca ccagt tttga tttaaacgtg 480 gccaatatgg acaacttctt cgcccccgtt ttcaccatgg gcaaatatta tacgcaaggc 540 gacaaggtgc tgatgccgct ggcgattcag gttcatcatg ccgtttgtga tggcttccat 600 gtcggcagat gcttaatgaa tacaacagta ctg cgatga 639 <210> 13 <211> 5464 < 212> DNA <213> Artificial Sequence <220> <223> pUC19_SalOmpA_7AA <400> 13 tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta 60 tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag 120 aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg 180 tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg 240 tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg 300 cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga 360 agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc 420 tcc aagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt 480 aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact 540 ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg 600 cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt 660 accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt 720 ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct 780 ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg 840 gtcatgagat tat caaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt 900 aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt 960 gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc 1020 gtgtag ataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg 1080 cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc 1140 gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg 1200 gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca 1260 ggcatcgtgg tgtcacgctc gtcgtttggt atgg cttcat tcagctccgg ttcccaacga 1320 tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct 1380 ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg 1440 cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca 1500 accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata 1560 cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct 1620 tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact 1680 cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa 1740 acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc 1800 atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga 1860 tacatatttg aatgtattta gaaaaataaa caa atagggg ttccgcgcac atttccccga 1920 aaagtgccac ctgacgtcta agaaaccatt attatcatga cattaaccta taaaaatagg 1980 cgtatcacga ggccctttcg tctcgcgcgt ttcggtgatg acggtgaaaa cctctgacac 2040 atgcagctcc cggagacggt cacagcttgt ctgtaagcgg atgccgggag cagacaagcc 2100 cgtcagggcg cgtcagcggg tgttggcggg tgtcggggct gg cttaacta tgcggcatca 2160 gagcagattg tactgagagt gcaccatatg cggtgtgaaa taccgcacag atgcgtaagg 2220 agaaaatacc gcatcaggcg ccattcgcca ttcaggctgc gcaactgttg ggaagggcga 2280 tcggtgcggg cctcttcgct attacg ccag ctggcgaaag ggggatgtgc tgcaaggcga 2340 ttaagttggg taacgccagg gttttcccag tcacgacgtt gtaaaacgac ggccagtgaa 2400 ttcgagctcg gtacccgaca gcattccggg ctaaaaattc actctaattt gtatcattaa 2460 gtaaaattag gatttatcct ggactttttt ttacgcgaac gtatctcctt tgagtgctaa 2520 cgtttttttt gcgagaacgc ttgtcagaag cggtttccgc aatttttgct gt acgattta 2580 tcatctgaaa ctgttaaatg atgtgtatat ccgtcatgtt ttttcacat gtctgacgga 2640 gttcacactt gtaagtttcc aactacgttg tagactttac atcgccaggg gtgctcagca 2700 taagccgtag atatcggtag agtaactatt gagcagatcc cccggtgaag gatttaaccg 2760 tgttatctcg ttggagatat tcatggcgta ttttggatga taacgaggcg caaaaaatga 2820 aaaagacagc tatcgcgatt gcagtggcac tggctggttt cgctaccgta gcgcaggccg 2880 ctccgaaaga taaccctgg taacgctggtg ctaaactggg ctggtctcag taccatgaca 2940 ccggcttcat tcacaatgat ggcccgactc atgaaaacca actgggcgca ggtgcttttg 3000 gt ggttacca ggttaacccg tatgttggct ttgaaatggg ctacgactgg ttaggccgta 3060 tgccgtacaa aggcgacaac tgtgtgcgcg ctcgcacccg tatcaatggc gcttataaag 3120 ctcagggcgt tcagttgacc gctaaactgg gttatccaat cactgacgat ctgg acgttt 3180 atacccgtct gggtggtatg gtatggcgtg cagacaccaa gtctaacgtc cctggcggcc 3240 cgtctactaa agaccacgac accggcgttt ccccggtatt cgcgggcggt atcgagtatg 3300 ctatcacccc tgaaatcgca acccgtctgg aataccagtg gactaacaac atcggtgatg 3360 ccaacaccat cggcacccgt ccggacaacg gcctgctgag cgtaggtgtt tcctaccgtt 3420 tcggccagca agaagctgct ccggtagtag ctccggcacc agctccggct ccggaagtac 3480 agaccaagca cttcactctg aagtctgacg tactgttcaa cttcaacaaa tctaccctga 3540 agccggaagg ccagcaggct ctggatcagc tgtacagcca gctgagcaac ctggatccga 3600 aagacgg ttc cgttgtcgtt ctgggcttca ctgaccgtat cggttctgac gcttacaacc 3660 agggtctgtc cgagaaacgt gctcagtctg ttgttgatta cctgatctcc aaaggtattc 3720 cgtctgacaa aatctccgca cgtggtatgg gcgaatctaa cccggttacc ggcaacacct 3780 gtgacaacgt gaaacctcgc gctgccctga tcgattgcct ggctccggat cgtcgcgtag 3840 agatcgaagt taaaggcgtt aaagac gtgg taactcagcc gcaggcttaa gtttccgtct 3900 gataaaaaac cccgcgtcgc ggggtttttt gctctggtct ggatgacaac gcctttcagc 3960 gttacttctt gcctaataac gcctgtaaat cctgctttaa cgtggtcatt tgcgtggcat 4020 atttctcttt atgctccgcg gatagaaaca gaagccactg gagcacctca aaaacaccat 4080 catacactaa atcagtaagt tggcagcatc acccgacgca ctttgcgccg aataaatacc 4140 tgtgacggaa gatcacttcg cagaataaat aaatcctggt gtccctgttg ataccgggaa 4200 gccctgggcc aacttttggc gaaaatgaga cgttgatcgg cacgtaagag gttccaactt 4260 tcaccataat gaaataagat cactaccggg cgtattt ttt gagttatcga gattttcagg 4320 agctaagaag ctaaaatgga gaaaaaaatc actggatata ccaccgttga tatatcccaa 4380 tggcatcgta aagaacattt tgaggcattt cagtcagttg ctcaatgtac ctataaccag 4440 accgttcagc tggatattac ggcctt ttta aagaccgtaa agaaaaataa gcacaagttt 4500 tatccggcct ttattcacat tcttgcccgc ctgatgaatg ctcatccgga attccgtatg 4560 gcaatgaaag acggtgagct ggtgatatgg gatagtgttc acccttgtta caccgttttc 4620 catgagcaaa ctgaaacgtt ttcatcgctc tggagtgaat accacgacga tttccggcag 4680 tttctacaca tatattcgca agatgtggcg tgttacggt g aaaacctggc ctatttccct 4740 aaagggttta ttgagaatat gtttttcgtc tcagccaatc cctgggtgag tttcaccagt 4800 tttgatttaa acgtggccaa tatggacaac ttcttcgccc ccgttttcac catgggcaaa 4860 tattatacgc aa ggcgacaa ggtgctgatg ccgctggcga ttcaggttca tcatgccgtt 4920 tgtgatggct tccatgtcgg cagaatgctt aatgaattac aacagtactg cgatgagtgg 4980 cagggcgggg cgtaattttt ttaaggcagt tattggtgcc cttaaacgcc tggttgctac 5040 gcctgaataa gtgataataa gcggatgaat ggcagaaatt cgaaagcaaa ttcgacccgg 5100 tcgtcggttc agggcagggt cgttaaatag ccgcttatgt ctattgct gg tttaccggtt 5160 tattgactac cggaagcagt gtgaccgtgt gcttggggat cctctagagt cgacctgcag 5220 gcatgcaagc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct 5280 cacaattcca cacaacatac gagccggaag cataaagtgt aaagcct ggg gtgcctaatg 5340 agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct 5400 gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg 5460 gcgc 5464 <210> 14 <211> 5470 <212> DNA <213> Artificial Sequence <220> <223> pUC19_SalOmpA_9AA <400> 14 tcttccgctt cct cgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta 60 tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag 120 aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg 180 tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg 240 tggcgaaacc cgacaggact ataaagatac caggcgtttc ccc ctggaag ctccctcgtg 300 cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga 360 agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc 420 tccaagctgg gctgtgtgca c gaacccccc gttcagcccg accgctgcgc cttatccggt 480 aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact 540 ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg 600 cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt 660 accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt 7 20 ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct 780 ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg 840 gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt 900 aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt 960 gaggcaccta tctcagcgat gtgg tcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg 1200 gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca 1260 ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga 1320 tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct 1380 ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg 1440 cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca 1500 accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata 1560 cgggataata ccgcgccaca tagca a aaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc 1800 atactcttcc tttttcaata ttattgaagc atttatcagg gttatgtct catgagcgga 1860 tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga 1920 aaagtgccac ctgacgtcta agaaaccatt attatcatga cattaaccta taaaaatagg 1980 cgtatcacga ggccctttcg tctcgc gcgt ttcggtgatg acggtgaaaa cctctgacac 2040 atgcagctcc cggagacggt cacagcttgt ctgtaagcgg atgccgggag cagacaagcc 2100 cgtcagggcg cgtcagcggg tgttggcggg tgtcggggct ggcttaacta tgcggcatca 2160 gagcagatt g tactgagagt gcaccatatg cggtgtgaaa taccgcacag atgcgtaagg 2220 agaaaatacc gcatcaggcg ccattcgcca ttcaggctgc gcaactgttg ggaagggcga 2280 tcggtgcggg cctcttcgct attacgccag ctggcgaaag ggggatgtgc tgcaaggcga 2340 ttaagttggg taacgccagg gttttcccag tcacgacgtt gtaaaacgac ggccagtgaa 2400 ttcgagctcg gtacccga ca gcattccggg ctaaaaattc actctaattt gtatcattaa 2460 gtaaaattag gatttatcct ggactttttt ttacgcgaac gtatctcctt tgagtgctaa 2520 cgtttttttt gcgagaacgc ttgtcagaag cggtttccgc aatttttgct gtacgattta 2 580 tcatctgaaa ctgttaaatg atgtgtatat ccgtcatgtt tttttcacat gtctgacgga 2640 gttcacactt gtaagtttcc aactacgttg tagactttac atcgccaggg gtgctcagca cg ctaccgta gcgcaggccg 2880 ctccgaaaga taaccctgg tacgctggtg ctaaactggg ctggtctcag taccatgaca 2940 ccggcttcat tcacaatgat ggcccgactc atgaaaacca actgggcgca ggtgcttttg 3000 gtggttacca ggttaacccg tatgttggct ttgaaatggg ctacgactgg ttaggccgta 3060 tgccgtacaa aggcgacaac tgtctgcaga aaacccctaa acagtgtatc aatggcgctt 3120 ataaagctca gggcgttcag ttgaccgcta aactgggtta tccaatcact gacgatctgg 3180 acgtttatac ccgtctgggt ggtatggtat ggcgtgcaga caccaagtct aacgtccctg 3240 gcggcccgtc tactaaagac cacgacaccg gcgtttcccc ggtattcgcg ggcggtatcg 3300 agtatgctat cacccctgaa atcgcaaccc gtctggaata ccagtggact aacaacatcg 3360 gtgatgccaa caccatcggc acccgtccgg acaacggcct gctgagcgta ggtgtttcct 3420 accgtttcgg ccagcaagaa gctgctccgg tagtagctcc ggcacc agct ccggctccgg 3480 aagtacagac caagcacttc actctgaagt ctgacgtact gttcaacttc aacaaatcta 3540 ccctgaagcc ggaaggccag caggctctgg atcagctgta cagccagctg agcaacctgg 3600 atccgaaaga cggttccgtt gtcgttctgg gcttcactga ccgtatcggt tctgacgctt 3660 acaaccaggg tctgtccgag aaacgtgctc agtctgttgt tgattacctg atctccaaag 37 20 gtattccgtc tgacaaaatc tccgcacgtg gtatgggcga atctaacccg gttaccggca 3780 acacctgtga caacgtgaaa cctcgcgctg ccctgatcga ttgcctggct ccggatcgtc 3840 gcgtagagat cgaagttaaa ggcgttaaag acgtggtaac tcag ccgcag gcttaagttt 3900 ccgtctgata aaaaaccccg cgtcgcgggg ttttttgctc tggtctggat gacaacgcct 3960 ttcagcgtta cttcttgcct aataacgcct gtaaatcctg ctttaacgtg gtcatttgcg 4020 tggcatattt ctctttatgc tccgcggata gaaacagaag ccactggagc acctcaaaaaa 4080 caccatcata cactaaatca gtaagttggc agcatcaccc gacgcacttt gcgccgaata 4140 atatacctgt g acggaagatc acttcgcaga ataaataaat cctggtgtcc ctgttgatac 4200 cgggaagccc tgggccaact tttggcgaaa atgagacgtt gatcggcacg taagaggttc 4260 caactttcac cataatgaaa taagatcact accgggcgta ttttttgagt tatcgagatt 4320 ttcaggagct aagaagctaa aatggagaaa aaaatcactg gatataccac cgttgatata 4380 tcccaatggc atcgtaaaga acattttgag gcatttcagt cagttgctca atgtacctat 4440 aaccagaccg ttcagctgga tattacggcc tttttaaaga ccgtaaagaa aaataagcac 4500 aagttttatc cggcctttat tcacattctt gcccgcctga tgaatgctca tccggaattc 4560 cgtatggcaa t gaaagacgg tgagctggtg atatgggata gtgttcaccc ttgttacacc 4620 gttttccatg agcaaactga aacgttttca tcgctctgga gtgaatacca cgacgatttc 4680 cggcagtttc tacacatata ttcgcaagat gtggcgtgtt acggtgaaaa cctggcctat 4740 ttccctaaag ggttattga gaatatgttt ttcgtctcag ccaatccctg ggtgagtttc 4800 accagttttg atttaaacgt ggccaatatg gacaacttct tcgcc cccgt tttcaccatg 4860 ggcaaatatt atacgcaagg cgacaaggtg ctgatgccgc tggcgattca ggttcatcat 4920 gccgtttgg atggcttcca tgtcggcaga atgcttaatg aattacaaca gtactgcgat 4980 gagtggcagg gcggggcgta atttttttaa ggcagttatt ggtgccctta aacgcctggt 5040 tgctacgcct gaataagtga taataagcgg atgaatggca gaaattcgaa agcaaattcg 5100 acccggtcgt cggttcaggg cagggtcgtt aaatagccgc ttatgtctat tgctggttta 5160 ccggtttatt gactaccgga agcagtgtga ccgtgtgctt ggggatcctc tagagtcgac 5220 ctgcaggcat gcaagcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta 5280 tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc 5340 ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg 5400 aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcgggg agag gcggtttgcg 5460 tattgggcgc 5470 <210> 15 <211> 5488 <212> DNA <213> Artificial Sequence <220> <223> pUC19_SalOmpA_15AA <400> 15 tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta 60 tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag 120 aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg 180 tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg 240 tggcgaaa cc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg 300 cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga 360 agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgt tcgc 420 tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt 480 aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact 540 ggtaacagga ttagcagagc gaggtatgta ggcggtgcta caga gttctt gaagtggtgg 600 cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt 660 accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt 720 ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct 780 ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg 840 gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt 900 aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt 960 gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg actcc ccgtc 1020 gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg 1080 cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc 1140 gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg 1200 gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca 1260 ggcatcgtgg tgtcacg ctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga 1320 tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct 1380 ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcact g 1440 cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca 1500 accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata 1560 cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct 1620 tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact 1680 cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa 1740 acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc 1800 atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga 1860 tacatatttg aatgt attta gaaaaataaa caaatagggg ttccgcgcac atttccccga 1920 aaagtgccac ctgacgtcta agaaaccatt attatcatga cattaaccta taaaaatagg 1980 cgtatcacga ggccctttcg tctcgcgcgt ttcggtgatg acggtgaaaa cctctgacac 2040 atgcagctcc cggagacggt cacagcttgt ctgtaagcgg atgccgggag cagacaagcc 2100 cgtcagggcg cgtcagcggg tgttggc ggg tgtcggggct ggcttaacta tgcggcatca 2160 gagcagattg tactgagagt gcaccatatg cggtgtgaaa taccgcacag atgcgtaagg 2220 agaaaatacc gcatcaggcg ccattcgcca ttcaggctgc gcaactgttg ggaagggcga 2280 tcggtgcggg cctcttcgct attacgccag ctggcgaaag ggggatgtgc tgcaaggcga 2340 ttaagttggg taacgccagg gttttcccag tcacgacgtt gtaaaacgac ggccagtgaa 2400 ttcgagctcg gtacccgaca gcattccggg ctaaaaattc actctaattt gtatcattaa 2460 gtaaaattag gatttatcct ggactttttt ttacgcgaac gtatctcctt tgagtgctaa 2520 cgtttttttt gcgagaacgc ttgtcagaag cggt ttccgc aatttttgct gtacgattta 2580 tcatctgaaa ctgttaaatg atgtgtatat ccgtcatgtt tttttcacat gtctgacgga 2640 gttcacactt gtaagtttcc aactacgttg tagactttac atcgccaggg gtgctcagca 2700 taagccgtag atatcggtag agta actatt gagcagatcc cccggtgaag gatttaaccg 2760 tgttatctcg ttggagatat tcatggcgta ttttggatga taacgaggcg caaaaaatga 2820 aaaagacagc tatcgcgatt gcagtggcac tggctggttt cgctaccgta gcgcaggccg 2880 ctccgaaaga taacacctgg tacgctggtg ctaaactggg ctggtctcag taccatgaca 2940 ccggcttcat tcacaatgat ggcccgactc atgaaaacca actgggcgca gg tgcttttg 3000 gtggttacca ggttaacccg tatgttggct ttgaaatggg ctacgactgg ttaggccgta 3060 tgccgtacaa aggcgacaac tacgcttctt accattgttg gtgttggcgt gacccgggcc 3120 gttctatcaa tggcgcttat aaagctcagg gcgtt cagtt gaccgctaaa ctgggttatc 3180 caatcactga cgatctggac gtttataccc gtctgggtgg tatggtatgg cgtgcagaca 3240 ccaagtctaa cgtccctggc ggcccgtcta ctaaagacca cgacaccggc gtttccccgg 3300 tattcgcggg cggtatcgag tatgctatca cccctgaaat cgcaacccgt ctggaatacc 3360 agtggactaa cacatcggt gatgccaaca ccatcggcac ccgtccggac aacggcctgc 34 20 tgagcgtagg tgtttcctac cgtttcggcc agcaagaagc tgctccggta gtagctccgg 3480 caccagctcc ggctccggaa gtacagacca agcacttcac tctgaagtct gacgtactgt 3540 tcaacttcaa caatctacc ctgaagccgg aaggccagca ggctctggat cagctgtaca 3600 gccagctgag caacctggat ccgaaagacg gttccgttgt cgttctgggc ttcactgacc 3660 gtatcggttc tgacgcttac aaccagggtc tgtccgagaa acgtgctcag tctgttgttg 3720 attacctgat ctccaaaggt attccgtctg acaaaatctc cgcacgtggt atgggcgaat 3780 ctaacccggt taccggcaac acctgtgaca acgtgaaacc tcgcgctgcc ctgatcgatt 3840 gcct ggctcc ggatcgtcgc gtagagatcg aagttaaagg cgttaaagac gtggtaactc 3900 agccgcaggc ttaagtttcc gtctgataaa aaaccccgcg tcgcggggtt ttttgctctg 3960 gtctggatga caacgccttt cagcgttact tcttgcctaa taacg cctgt aaatcctgct 4020 ttaacgtggt catttgcgtg gcatatttct ctttatgctc cgcggataga aacagaagcc 4080 actggagcac ctcaaaaaca ccatcataca ctaaatcagt aagttggcag catcacccga 4140 cgcactttgc gccgaataaa tacctgtgac ggaagatcac ttcgcagaat aaataaatcc 4200 tggtgtccct gttgataccg ggaagccctg ggccaacttt tggcgaaaat gagacgttga 4260 tcggcacgta agaggttcca actttcacca taatgaaata agatcactac cgggcgtatt 4320 ttttgagtta tcgagatttt caggagctaa gaagctaaaa tggagaaaaa aatcactgga 4380 tataccaccg ttgatatc ccaatggcat cgtaaagaac attttgaggc atttcagtca 4440 gttgctcaat gtacctataa ccagaccgtt cagctggata ttacggcctt tttaaagacc 4500 gtaaagaaaa ataagcacaa gttttatccg gcctttattc acattcttgc ccgcctgatg 4560 aatgctcatc cggaattccg tatggcaatg aaagacggtg agctggtgat atgggatagt 4620 gttcaccctt gttacaccgt tttccatgag caactgaaa cgttttcatc gctctggagt 4680 gaataccacg acgatttccg gcagtt tcta cacatatatt cgcaagatgt ggcgtgttac 4740 ggtgaaaacc tggcctattt ccctaaaggg tttattgaga atatgttttt cgtctcagcc 4800 aatccctggg tgagtttcac cagttttgat ttaaacgtgg ccaatatgga caacttcttc 4860 gcccccgt tt tcaccatggg caaatattat acgcaaggcg acaaggtgct gatgccgctg 4920 gcgattcagg ttcatcatgc cgtttgtgat ggcttccatg tcggcagaat gcttaatgaa 4980 ttacaacagt actgcgatga gtggcagggc ggggcgtaat ttttttaagg cagttatgg 5040 tgcccttaaa cgcctggttg ctacgcctga ataagtgata ataagcggat gaatggcaga 5100 aattcgaaag caaattcgac ccggtcgtcg gttcagggca gggtcgttaa atagccgctt 5160 atgtctattg ctggtttacc ggttattga ctaccggaag cagtgtgacc gttgcttgg 5220 ggatcctcta gagtcgacct gcaggcatgc aagcttggcg taatcatggt catagctgtt 5280 tcctgtgtga aattgttatc c gctcacaat tccacacaac atacgagccg gaagcataaa 5340 gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact 5400 gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc 5460 ggggagaggc ggtttgcgta ttgggcgc 5488 <210> 16 <211> 6329 <212> DNA <213> Artificial Sequence <220> <223> pKD46 <400> 16 catcgattta ttatgacaac ttgacggcta catcattcac tttttcttca caaccggcac 60 ggaactcgct cgggctggcc ccggtgcatt ttttaaatac ccgcgagaaa tagagttgat 120 cgtcaaaacc aacattgcga ccgacggtgg cgataggcat ccgggtggtg ctcaaaagca 180 gcttcgcctg gctgatacgt tggtcctcgc gccagcttaa gacgctaatc cctaactgct 240 ggcggaaaag atgtgacaga cgcgacggcg acaagcaaac atgctgtgcg acgctggcga 300 tatcaaaatt gctgtctgcc aggtgatcgc tgatgtactg acaagcctcg cgtacccgat 360 tatccatcgg tggatggagc gactcgttaa tcgcttccat gcgccgcagt aacaattgct 420 caagcagatt tatcgccagc agctccgaat agcgcccttc cccttgcccg gcgttaatga 480 tttgcccaaa caggtcgctg aaatgcggct ggtgcgcttc atccgggcga aagaaccccg 540 tattggcaaa tattgacggc cagttaagcc attcatgcca gtaggcgcgc ggacgaaagt 600 aaacccactg gtgataccat tcgcgagcct ccggatgacg accgtagtga tgaatctctc 660 ctggcgggaa cagcaaaata tcaccc ggtc ggcaaacaaa ttctcgtccc tgatttttca 720 ccaccccctg accgcgaatg gtgagattga gaatataacc tttcattccc agcggtcggt 780 cgataaaaaa atcgagataa ccgttggcct caatcggcgt taaacccgcc accagatggg 840 cattaaacga gtatcccggc agcaggggat cattttgcgc ttcagccata cttttcatac 900 tcccgccatt cagagaagaa accaattgtc catattgcat cagacattgc cgtcactgcg 960 tcttttactg gctcttctcg ctaaccaaac cggtaacccc gcttattaaa agcattctgt 1020 aacaaagcgg gaccaaagcc atgacaaaaa cgcgtaacaa aagtgtctat aatcacggca 1080 gaaaagtcca cattgattat ttgcacggcg tc acactttg ctatgccata gcatttttat 1140 ccataagatt agcggatcct acctgacgct ttttatcgca actctctact gtttctccat 1200 acccgttttt ttgggaattc gagctctaag gaggttataa aaaatggata ttaatactga 1260 aactgagatc aagcaaaagc attcacta ac cccctttcct gttttcctaa tcagcccggc 1320 atttcgcggg cgatattttc acagctattt caggagttca gccatgaacg cttattacat 1380 tcaggatcgt cttgaggctc agagctgggc gcgtcactac cagcagctcg cccgtgaaga 1440 gaaagaggca gaactggcag acgacatgga aaaaggcctg ccccagcacc tgtttgaatc 1500 gctatgcatc gatcatttgc aacgccacgg ggccagcaaa aaat ccatta cccgtgcgtt 1560 tgatgacgat gttgagtttc aggagcgcat ggcagaacac atccggtaca tggttgaaac 1620 cattgctcac caccaggttg atattgattc agaggtataa aacgaatgag tactgcactc 1680 gcaacgctgg ctgggaagct ggctgaacgt gt cggcatgg attctgtcga cccacaggaa 1740 ctgatcacca ctcttcgcca gacggcattt aaaggtgatg ccagcgatgc gcagttcatc 1800 gcattactga tcgttgccaa ccagtacggc cttaatccgt ggacgaaaga aatttacgcc 1860 tttcctgata agcagaatgg catcgttccg gtggtgggcg ttgatggctg gtcccgcatc 1920 atcaatgaaa accagcagtt tgatggcatg gactttgagc aggacaatga atcc tgtaca 1980 tgccggattt accgcaagga ccgtaatcat ccgatctgcg ttaccgaatg gatggatgaa 2040 tgccgccgcg aaccattcaa aactcgcgaa ggcagagaaa tcacggggcc gtggcagtcg 2100 catcccaaac ggatgttacg tcataaagcc atgattcagt gtgcccgtct ggccttcgga 2160 tttgctggta tctatgacaa ggatgaagcc gagcgcattg tcgaaaatac tgcatacact 2220 gcagaacgtc 2400 g ctcttggat tcctgaaaca gaaagccgca gagcagaagg tggcagcatg acaccggaca 2460 ttatcctgca gcgtaccggg atcgatgtga gagctgtcga acagggggat gatgcgtggc 2520 acaaattacg gctcggcgtc atcaccgctt cagaagttca caacgtgata gcaaaacc cc 2580 gctccggaaa gaagtggcct gacatgaaaa tgtcctactt ccacaccctg cttgctgagg 2640 tttgcaccgg tgtggctccg gaagttaacg ctaaagcact ggcctgggga aaacagtacg 2700 agaacgacgc cagaaccctg tttgaattca cttccggcgt gaatgttact gaatccccga 2760 tcatctatcg cgacgaaagt atgcgtaccg cctgctctcc cgatggttta tgcagtgacg 2820 gca acggcct tgaactgaaa tgcccgttta cctcccggga tttcatgaag ttccggctcg 2880 gtggtttcga ggccataaaag tcagcttaca tggcccaggt gcagtacagc atgtgggtga 2940 cgcgaaaaaa tgcctggtac tttgccaact atgacccgcg tatga agcgt gaaggcctgc 3000 attatgtcgt gattgagcgg gatgaaaagt acatggcgag ttttgacgag atcgtgccgg 3060 agttcatcga aaaaatggac gaggcactgg ctgaaattgg ttttgtattt ggggagcaat 3120 ggcgatgacg catcctcacg ataatatccg ggtaggcgca atcactttcg tctactccgt 3180 tacaaagcga ggctgggtat ttcccggcct ttctgttatc cgaaatccac tgaaagcaca 3240 gcggctggct gaggagataa ataataaacg aggggctgta tgcacaaagc atcttctgtt 3300 gagttaagaa cgagtatcga gatggcacat agccttgctc aaattggaat caggtttgtg 3360 ccaataccag tagaaacaga cgaagaatcc atgggtatgg acagttttcc ctttgatatg 3420 taacgg tgaa cagttgttct acttttgttt gttagtcttg atgcttcact gatagataca 3480 agagccataa gaacctcaga tccttccgta tttagccagt atgttctcta gtgtggttcg 3540 ttgtttttgc gtgagccatg agaacgaacc attgagatca tacttacttt gcatgtcact 3600 caaaaatttt gcctcaaaac tggtgagctg aatttttgca gttaaagcat cgtgtagtgt 3660 ttttcttagt ccgt tacgta ggtaggaatc tgatgtaatg gttgttggta ttttgtcacc 3720 attcattttt atctggttgt tctcaagttc ggttacgaga tccatttgtc tatctagttc 3780 aacttggaaa atcaacgtat cagtcgggcg gcctcgctta tcaacacca atttcatatt 3840 gctgtaagtg tttaaatctt tacttattgg tttcaaaacc cattggttaa gccttttaaa 3900 ctcatggtag ttattttcaa gcattaacat gaacttaaat tcatcaaggc taatctctat 3960 atttgccttg tgagttttct tttgtgttag ttcttttaat aaccactcat aaatcctcat 4020 agagtatttg ttttcaaaag acttaacatg ttccagatta tattttatga attttttaa 4080 ctgggaaaaga taaggcaata tctcttcact aaa c gtattggtt ataagtgaac gataccgtcc gttctttcct 4320 tgtagggttt tcaatcgtgg ggttgagtag tgccacacag cataaaatta gcttggtttc 4380 atgctccgtt aagtcatagc gactaatcgc tagttcattt gctttgaaaa caactaattc 4440 agacatacat ctcaattggt ctaggtgatt ttaatcacta taccaattga gatgggctag 4500 tcaatgataa ttactagtcc ttttcctttg agttgtgggt atctgtaaat tctgctagac 4560 ctttgctgga aaacttgtaa attctgctag accctctgta aattccgcta gacctttgg 4620 tgtttttttt gtttatattc aagtggttat aatttataga ataaagaaag aataaaaaaa 4680 gataaaaaga atagatccca gccctgtgta taactcacta ctttagtcag ttccgcagta 4740 ttacaaaagg atgtcgcaaa cgctgtttgc tcctctacaa aacagacctt aaaaccctaa 4800 aggcttaagt agcaccctcg caagctcggt tgcggccgca atcgggcaaa tcgctgaata 4860 ttccttttgt ctccgaccat caggcacctg agtcgctgtc tttttcgtga cattcagttc 4920 gctgcgctca cggctctggc agtgaatggg ggtaaatggc actacaggcg ccttttatgg 4980 attcatgcaa ggaaactacc cataatacaa gaaaagcccg tcacgggctt ctcagggcgt 5040 tttatggcgg gtctgctatg tggtgctatc tgactttttg ctgttcagca gttcctgccc 5100 tctgattttc cagt ctgacc acttcggatt atcccgtgac aggtcattca gactggctaa 5160 tgcacccagt aaggcagcgg tatcatcaac ggggtctgac gctcagtgga acgaaaactc 5220 acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa 5280 ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta 5340 ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt 5 400 tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag 5460 tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag caataaacca 5520 gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccat ccagtc 5580 tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt 5640 tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag 5700 ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt 5760 tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat 5 820 ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt 5880 gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc 5940 ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa aagt gctcat 6000 cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag 6060 ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt 6120 ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg 6180 gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta 6240 ttgtct catg agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc 6300 gcgcacattt ccccgaaaag tgccacctg 6329 <210> 17 <211> 3355 <212> DNA <213> Artificial Sequence <220> < 223> pEGFP <400> 17 agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc 60 acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc 120 tcactcatta ggcaccccag gctttacact tta tgcttcc ggctcgtatg ttgtgtggaa 180 ttgtgagcgg ataacaattt cacacagga acagctatga ccatgattac gccaagcttg 240 catgcctgca ggtcgactct agaggatccc cgggtaccgg tcgccaccat ggtgagcaag 300 ggcgaggagc tgttcaccgg gg tggtgccc atcctggtcg agctggacgg cgacgtaaac 360 ggccacaagt tcagcgtgtc cggcgagggc gagggcgatg ccacctacgg caagctgacc 420 ctgaagttca tctgcaccac cggcaagctg cccgtgccct ggcccaccct cgtgaccacc 480 ctgacctacg gcgtgcagtg cttcagccgc taccccgacc acatgaagca gcacgacttc 54 0 ttcaagtccg ccatgcccga aggctacgtc caggagcgca ccatcttctt caaggacgac 600 ggcaactaca agacccgcgc cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc 660 gagctgaagg gcatcgactt caaggaggac ggcaacatcc tggggcacaa gct ggaggtac 720 aactacaaca gccacaacgt ctatatcatg gccgacaagc agaagaacgg catcaaggtg 780 aacttcaaga tccgccacaa catcgaggac ggcagcgtgc agctcgccga ccactaccag 840 cagaacaccc ccatcggcga cggccccgtg ctgctgcccg acaaccacta cctgagcacc 900 cagtccgccc tgagcaaaga ccccaacgag aagcgcgatc acatggtcct gctggagttc 960 gtgaccgccg ccgggatcac tctcggcatg gacgagctgt acaagtaaag cggccgcgac 1020 tctagaattc caactgagcg ccggtcgcta ccattaccaa cttgtctggt gtcaaaaata 1080 ataggcctac tagtcggccg tacgggccct ttcgtctcgc gcgtt tcggt gatgacggtg 1140 aaaacctctg acacatgcag ctcccggaga cggtcacagc ttgtctgtaa gcggatgccg 1200 ggagcagaca agcccgtcag ggcgcgtcag cgggtgttgg cgggtgtcgg ggctggctta 1260 actatgcggc atcagagcag attgtactga gagtgcacca tatgcggtgt gaaataccgc 1320 acagatgcgt aaggagaaaa taccgcatca ggcggcctta agggcctcgt gatacgccta 1380 tttttatagg ttaatgtcat gataataatg gtttcttaga cgtcaggtgg cacttttcgg 1440 ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg 1500 ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt 1 560 attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt 1620 gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg 1680 ggttacatcg aactggatct caacagcggt aagatcctt g agagttttcg ccccgaagaa 1740 cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt 1800 gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag 1860 tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt 1920 gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga 1980 ccgaaggagc taa ccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt 2040 tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta 2100 gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg 216 0 caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc 2220 cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt 2280 atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg 2340 gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg 2400 attaagcatt gg taactgtc agaccaagtt tactcatata tactttagat tgatttaaaa 2460 cttcattttt aatttaaaag gatctaggg aagatccttt ttgataatct catgaccaaa 2520 atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga 2580 tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg 2640 ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact 2700 ggcttcagca gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac 2760 cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg 2820 gctgctgcca gtggcgataa gtcgtgt ctt accgggttgg actcaagacg atagttaccg 2880 gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga 2940 acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc 3000 gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg 3060 agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc 3120 tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc 3180 agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt 3240 cctgcgttat ccc ctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc 3300 gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaag 3355 <210> 18 <211> 2804 <212> DNA <213> Artificial Sequence <220> <223> pKD3 <400> 18 gcatgcaagc ttggcactgg ccacgcaaaa aggccatccg tcaggatggc cttctgctta 60 atttgatgcc tggcagttta tggcgggcgt cctgcccgcc accctccggg ccgttgcttc 120 gcaacgttca aatccgctcc cggcgg attt gtcctactca ggagagcgtt caccgacaaa 180 caacagataa aacgaaaggc ccagtctttc gactgagcct ttcgttttat ttgatgcctg 240 gcagttccct actctcgcat ggggagaccc cacactacca tcggggggcc atcgatgcag 300 gtggcacttt tc ggggaaat gtgcgcggaa cccctatttg tttattttc taaatacatt 360 caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa 420 ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt 480 gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaa agatgct gaagatcagt 540 tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt 600 ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg 660 tattatcccg tattgacgcc gggcaagagc aactc ggtcg ccgcatacac tattctcaga 720 atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa 780 gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga 840 caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa 900 ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca 9 60 ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta 1020 ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac 1080 ttctgcgctc ggcccttccg gctggctggt ttaattgctga taaatctgga g ccggtgagc 1140 gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag 1200 ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga 1260 taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca tatatacttt 1320 agattgattt aaaacttcat ttttaatttt tgcggccgca agatccgcag ttcaacctgt 1380 tgatagtacg tactaagctc tcatgtttca cgtactaagc tctcatgttt aacgtactaa 1440 gctctcatgt ttaacgaact aaaccctcat ggctaacgta ctaagctctc atggctaacg 1500 tactaagctc tcatgtttca cgtactaagc tctcatgttt gaacaataaa attaatataa 1560 atcag caact taaatagcct ctaaggtttt aagttttata agaaaaaaaa gaatatataa 1620 ggcttttaaa gcttttaagg tttaacggtt gtggacaaca agccagggat gtaacgcact 1680 gagaagccct tagagcctct caaagcaatt ttcagtgaca caggaacact taacggctga 1740 catgggaatt agccatggtc catatgaata tcctccttag ttcctattcc gaagttccta 1800 ttctctagaa agtataggaa cttcggcgc g cctacctgg acggaagatc acttcgcaga 1860 ataaataaat cctggtgtcc ctgttgatac cgggaagccc tgggccaact tttggcgaaa 1920 atgagacgtt gatcggcacg taagaggttc caactttcac cataatgaaa taagatcact 1980 accgggcgta tttt ttgagt tgtcgagatt ttcaggagct aaggaagcta aaatggagaa 2040 aaaaatcact ggatatacca ccgttgatat atcccaatgg catcgtaaag aacattttga 2100 ggcatttcag tcagttgctc aatgtaccta taaccagacc gttcagctgg atattacggc 2160 ctttttaaag accgtaaaga aaaataagca caagttttat ccggccttta ttcacattct 2220 tgcccgcctg atgaatgctc atccgga att acgtatggca atgaaagacg gtgagctggt 2280 gatatgggat agtgttcacc cttgttacac cgttttccat gagcaaactg aaacgttttc 2340 atcgctctgg agtgaatacc acgacgattt ccggcagttt ctacacatat attcgcaaga 2400 tgtggcgtgt tacggtgaaa acctggccta tttccctaaa gggtttattg agaatatgtt 2460 tttcgtctca gccaatccct gggtgagttt caccagtttt gatttaaacg tggccaatat 2520 ggacaacttc ttcgcccccg ttttcaccat gggcaaatat tatacgcaag gcgacaaggt 2580 gctgatgccg ctggcgattc aggttcatca tgccgtttgt gatggcttcc atgtcggcag 2640 atgcttaatg aatacaacag tactgcgatg agtggcaggg cggggcgtaa ggcgcgccat 2700 ttaaatgaag ttcctattcc gaagttccta ttctctagaa agtataggaa cttcgaagca 2760 gctccagcct acacaatcgc tcaagacgtg taatgctgca atct 2804 <210> 19 <211> 49 <212 > DNA <213 > Artificial Sequence <220> <223> SOA_F-Gibson <400> 19 gccagtgaat tcgagctcgg tacccgacag cattccgggc taaaaattc 49 <210> 20 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> SOA_R-Cat <400 > 20 ccagtggctt ctgtttctat ccgcggagca taaagagaaa tatgc 45 <210> 21 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Cat_F <400> 21 gatagaaaca gaagccactg g 21 <210> 22 <211> 39 <2 12 > DNA <213> Artificial Sequence <220> <223> Cat_R-Gibson <400> 22 ggtcgactct agaggatccc caagcacacg gtcacactg 39 <210> 23 <211> 44 <212> DNA <213> Artificial Sequence <220> <223> Loop2_7AA_F < 400> 23 tgtgtgcgcg ctcgcacccg atcaatggcg cttataaagc tcag 44 <210> 24 <211> 51 <212> DNA <213> Artificial Sequence <220> <223> Loop2_9AA_F <400> 24 tgtctgcaga aaacccctaa acagtgtatc aat ggcgctt ataaagctca g 51 <210> 25 <211 > 69 <212> DNA <213> Artificial Sequence <220> <223> Loop2_15AA_F <400> 25 tacgcttctt accattgttg gtgttggcgt gacccgggcc gttctatcaa tggcgcttat 60 aaagctcag 69 <210> 26 <211> 18 <212> DNA <21 3> Artificial Sequence <220 > <223> Loop2_R <400> 26 gttgtcgcct ttgtacgg 18 <210> 27 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> SOA_F <400> 27 gacagcattc cgggctaaaa attc 24 <210> 28 <211 > 65 <212> DNA <213> Artificial Sequence <220> <223> Cat_R_SOA <400> 28 ggtaaaacat tatccgaaac gattgtgcaa ctgatagaag actaaggagg atattcatat 60 ggacc 65 <210> 29 <211> 19 <212> DNA <213> Artificial Sequence <220 > <223> SOA_F+459<400> 29 ggtaggtaat gcggtaggg 19
Claims (10)
상기 외막 단백질 A의 세포외 루프는 외막 단백질 A의 N 말단에서 2번째에 위치한 것인 살모넬라 변이주.The method of claim 1,
The extracellular loop of the outer membrane protein A is a Salmonella mutant strain located second from the N-terminus of the outer membrane protein A.
상기 항PD-L1 펩티드는 서열번호 1 내지 3 중 하나 이상의 아미노산 서열로 이루어진 것인 살모넬라 변이주.The method of claim 1,
The anti-PD-L1 peptide is a Salmonella mutant strain consisting of one or more amino acid sequences of SEQ ID NOs: 1 to 3.
상기 변이주는 서열번호 8 내지 10 중 하나 이상의 아미노산 서열로 이루어진 세포 표면 단백질을 발현하는 것인 살모넬라 변이주.The method of claim 1,
The mutant strain is a Salmonella mutant expressing a cell surface protein consisting of one or more amino acid sequences of SEQ ID NOs: 8 to 10.
상기 살모넬라 변이주는 살모넬라 티피뮤리움(Salmonella typhimurium), 살모넬라 브렌데럽(Salmonella braenderup) 및 살모넬라 엔테리티디스(Salmonella enteritidis)로 이루어진 군에서 선택된 1종 이상의 균주에서 유래한 약독화 균주인 것인 살모넬라 변이주.The method of claim 1,
The Salmonella mutant is a Salmonella strain derived from at least one strain selected from the group consisting of Salmonella typhimurium , Salmonella braenderup and Salmonella enteritidis .
청구항 1 내지 5 중 어느 한 항의 살모넬라 변이주에서 분리된 외막 소포체.The method of claim 1,
Outer membrane endoplasmic reticulum isolated from the Salmonella mutant strain of any one of claims 1 to 5.
상기 외막 소포체는 서열번호 1 내지 3 및 8 내지 10 중 하나 이상의 아미노산 서열로 이루어진 단백질을 포함하는 것인 외막 소포체.The method of claim 6,
The outer membrane endoplasmic reticulum comprises a protein consisting of one or more amino acid sequences of SEQ ID NOs: 1 to 3 and 8 to 10.
상기 암은 유방암, 구강암, 전립선암, 직장암, 비소세포 폐암, 구순암, 간암, 폐암, 항문암, 신장암, 외음부암, 구인두암(oropharyngeal cancer), 비강/부비동암(nasal cavity and paranasal sinus cancer), 비인두암, 요도암, 소장암, 담도암, 방광암, 난소암, 후두암, 식도암, 담낭암, 대장암, 두경부암, 갑상선암, 부갑상선암, 음경암, 질암, 췌장암, 편평세포암, 호치킨 림프종(Hodgkin's lymphoma), 백혈병 관련 질환, 균상 식육종(mycosis fungoides) 및 골수이형성증후군으로 이루어진 군에선 선택된 1종 이상인 것인 약학 조성물.The method of claim 8,
The cancer is breast cancer, oral cancer, prostate cancer, rectal cancer, non-small cell lung cancer, lip cancer, liver cancer, lung cancer, anal cancer, kidney cancer, vulvar cancer, oropharyngeal cancer, nasal cavity and paranasal sinus cancer ), nasopharyngeal cancer, urethral cancer, small intestine cancer, bile duct cancer, bladder cancer, ovarian cancer, laryngeal cancer, esophageal cancer, gallbladder cancer, colon cancer, head and neck cancer, thyroid cancer, parathyroid cancer, penile cancer, vaginal cancer, pancreatic cancer, squamous cell cancer, Hodgkin's lymphoma (Hodgkin's lymphoma), leukemia-related diseases, mycosis fungoides, and myelodysplastic syndrome, one or more selected from the group consisting of a pharmaceutical composition.
상기 약학 조성물은 항암제를 더 포함하는 것인 약학 조성물.The method of claim 8,
The pharmaceutical composition further comprises an anti-cancer agent.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| KR1020210180838A KR20230091594A (en) | 2021-12-16 | 2021-12-16 | Salmonella mutant expressing anti-PD-L1 peptide and outer membrane vesicle thereof and pharmaceutical comprising the same |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| KR1020210180838A KR20230091594A (en) | 2021-12-16 | 2021-12-16 | Salmonella mutant expressing anti-PD-L1 peptide and outer membrane vesicle thereof and pharmaceutical comprising the same |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| KR20230091594A true KR20230091594A (en) | 2023-06-23 |
Family
ID=86993716
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| KR1020210180838A KR20230091594A (en) | 2021-12-16 | 2021-12-16 | Salmonella mutant expressing anti-PD-L1 peptide and outer membrane vesicle thereof and pharmaceutical comprising the same |
Country Status (1)
| Country | Link |
|---|---|
| KR (1) | KR20230091594A (en) |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR101743442B1 (en) | 2016-01-04 | 2017-06-05 | 전북대학교산학협력단 | Vaccine composition for preventing or treating porcine edema disease comprising ghost Salmonella mutant expressing Enterotoxigenic Escherichia coli antigen as effective component |
| KR20200133193A (en) | 2017-02-28 | 2020-11-26 | 진메디신 주식회사 | Anti-tumor composition comprising oncologic adenovirus and immune checkpoint inhibitor |
-
2021
- 2021-12-16 KR KR1020210180838A patent/KR20230091594A/en not_active Application Discontinuation
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR101743442B1 (en) | 2016-01-04 | 2017-06-05 | 전북대학교산학협력단 | Vaccine composition for preventing or treating porcine edema disease comprising ghost Salmonella mutant expressing Enterotoxigenic Escherichia coli antigen as effective component |
| KR20200133193A (en) | 2017-02-28 | 2020-11-26 | 진메디신 주식회사 | Anti-tumor composition comprising oncologic adenovirus and immune checkpoint inhibitor |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| KR101169468B1 (en) | Multi plasmid system for the production of influenza virus | |
| CN110437317B (en) | Adeno-associated virus with variant capsid proteins and uses thereof | |
| KR20080034334A (en) | A microorganism producing glutamic acid in high yield and a process of producing glutamic acid using the same | |
| KR20130116858A (en) | Extraction solvents derived from oil for alcohol removal in extractive fermentation | |
| KR20130105649A (en) | Integration of a polynucleotide encoding a polypeptide that catalyzes pyruvate to acetolactate conversion | |
| KR20130087013A (en) | Extraction solvents derived from oil for alcohol removal in extractive fermentation | |
| DK1748790T3 (en) | Multiplasmidsystem for the production of influenza | |
| CN107849583B (en) | Means and methods for controlling cell proliferation using cell division loci | |
| CN113227365A (en) | Novel acetyltransferases | |
| US20210299275A1 (en) | Gene therapy for the treatment of galactosemia | |
| CN106659805B (en) | Method for inhibiting Ebola virus through miRNA | |
| KR20230091594A (en) | Salmonella mutant expressing anti-PD-L1 peptide and outer membrane vesicle thereof and pharmaceutical comprising the same | |
| CN112592900B (en) | Method for constructing oncolytic adeno-associated virus oAAVs expressing pyroprotein and application thereof | |
| KR20100084689A (en) | Hcv ns3 protease replicon shuttle vectors | |
| CN115707778B (en) | Recombinant coxsackievirus A10 virus-like particles and uses thereof | |
| CN110484517A (en) | A kind of composition and preparation method of the Rift Valley fever virus being used to prepare weak poison, RVFV attenuated vaccine | |
| CN112154209A (en) | AAV-compatible laminin-linker polyprotein | |
| CN108060175B (en) | Construction and application of inducible yeast transformation recombination system | |
| CA2516727C (en) | Hypoxia inducible vegf plasmid for ischemic disease | |
| KR102208879B1 (en) | Nucleic acid construct and use of the same | |
| CN114181318B (en) | Recombinant adeno-associated virus for tissue-specific expression of IDUA fusion protein penetrating blood brain barrier and application thereof | |
| KR102543504B1 (en) | Fluorescent protein variant for detection of cell damage and evaluation method for drug toxicity using the same | |
| KR101146335B1 (en) | TRANSGENIC MOUSE EXPRESSING REPORTER PROTEIN UNDER REGULATION OF α-FETOPROTEIN ENHANCER AND PROMOTER, METHOD FOR PREPARATION THEREOF AND METHOD FOR SCREENING COMPOUNDS INDUCING INCREASE OR DECREASE OF Α-FETOPROTEIN EXPRESSION USING THE SAME | |
| CN114634915A (en) | Mutation library for constructing AAV serotype random insertion, kit and method thereof | |
| CN116554278A (en) | Mutant adeno-associated viruses and their use in the treatment of disease |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| E902 | Notification of reason for refusal |