US20230416337A1 - Intestinal expression of programmed death ligand 1 - Google Patents
Intestinal expression of programmed death ligand 1 Download PDFInfo
- Publication number
- US20230416337A1 US20230416337A1 US18/121,557 US202318121557A US2023416337A1 US 20230416337 A1 US20230416337 A1 US 20230416337A1 US 202318121557 A US202318121557 A US 202318121557A US 2023416337 A1 US2023416337 A1 US 2023416337A1
- Authority
- US
- United States
- Prior art keywords
- nucleic acid
- seq
- sequence
- polypeptide
- human
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 102000008096 B7-H1 Antigen Human genes 0.000 title claims abstract description 178
- 108010074708 B7-H1 Antigen Proteins 0.000 title claims abstract description 178
- 230000014509 gene expression Effects 0.000 title claims abstract description 66
- 230000000968 intestinal effect Effects 0.000 title claims abstract description 7
- 238000000034 method Methods 0.000 claims abstract description 17
- 208000027866 inflammatory disease Diseases 0.000 claims abstract description 12
- 150000007523 nucleic acids Chemical class 0.000 claims description 94
- 102000039446 nucleic acids Human genes 0.000 claims description 79
- 108020004707 nucleic acids Proteins 0.000 claims description 79
- 229920001661 Chitosan Polymers 0.000 claims description 71
- 150000001413 amino acids Chemical class 0.000 claims description 43
- 108090000623 proteins and genes Proteins 0.000 claims description 36
- 102000004169 proteins and genes Human genes 0.000 claims description 33
- 101001117317 Homo sapiens Programmed cell death 1 ligand 1 Proteins 0.000 claims description 29
- 239000013604 expression vector Substances 0.000 claims description 25
- 102000048776 human CD274 Human genes 0.000 claims description 23
- 108091028043 Nucleic acid sequence Proteins 0.000 claims description 21
- 239000002105 nanoparticle Substances 0.000 claims description 19
- 230000001225 therapeutic effect Effects 0.000 claims description 14
- 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 claims description 11
- 210000001035 gastrointestinal tract Anatomy 0.000 claims description 10
- 108010076504 Protein Sorting Signals Proteins 0.000 claims description 9
- 239000008103 glucose Substances 0.000 claims description 9
- 239000012528 membrane Substances 0.000 claims description 9
- 208000011231 Crohn disease Diseases 0.000 claims description 7
- 208000022559 Inflammatory bowel disease Diseases 0.000 claims description 6
- 230000010056 antibody-dependent cellular cytotoxicity Effects 0.000 claims description 6
- 230000009827 complement-dependent cellular cytotoxicity Effects 0.000 claims description 6
- 229920005862 polyol Polymers 0.000 claims description 6
- 150000003077 polyols Chemical class 0.000 claims description 6
- 238000006467 substitution reaction Methods 0.000 claims description 6
- 238000001514 detection method Methods 0.000 claims description 5
- 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 claims description 4
- 239000004475 Arginine Substances 0.000 claims description 4
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 claims description 4
- 230000001086 cytosolic effect Effects 0.000 claims description 4
- 238000012217 deletion Methods 0.000 claims description 4
- 230000037430 deletion Effects 0.000 claims description 4
- 206010009900 Colitis ulcerative Diseases 0.000 claims description 2
- 239000000203 mixture Substances 0.000 abstract description 39
- 210000004027 cell Anatomy 0.000 description 105
- 241001465754 Metazoa Species 0.000 description 33
- 239000013612 plasmid Substances 0.000 description 33
- 235000018102 proteins Nutrition 0.000 description 30
- 241000699670 Mus sp. Species 0.000 description 27
- 230000037396 body weight Effects 0.000 description 27
- 208000009329 Graft vs Host Disease Diseases 0.000 description 25
- 208000024908 graft versus host disease Diseases 0.000 description 25
- 108020004414 DNA Proteins 0.000 description 24
- 239000013598 vector Substances 0.000 description 22
- 210000001744 T-lymphocyte Anatomy 0.000 description 21
- 238000001890 transfection Methods 0.000 description 21
- 235000001014 amino acid Nutrition 0.000 description 20
- 229940024606 amino acid Drugs 0.000 description 20
- 102100040678 Programmed cell death protein 1 Human genes 0.000 description 17
- 101710089372 Programmed cell death protein 1 Proteins 0.000 description 17
- 239000003826 tablet Substances 0.000 description 16
- 239000000872 buffer Substances 0.000 description 15
- 238000009472 formulation Methods 0.000 description 15
- 239000002773 nucleotide Substances 0.000 description 14
- 125000003729 nucleotide group Chemical group 0.000 description 14
- 239000003981 vehicle Substances 0.000 description 12
- 108090000765 processed proteins & peptides Proteins 0.000 description 11
- 238000011282 treatment Methods 0.000 description 11
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 11
- -1 ICOS Proteins 0.000 description 10
- 201000010099 disease Diseases 0.000 description 10
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 10
- 229960001031 glucose Drugs 0.000 description 10
- 229920001184 polypeptide Polymers 0.000 description 10
- 102000004196 processed proteins & peptides Human genes 0.000 description 10
- 241000792859 Enema Species 0.000 description 9
- 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 9
- 239000007920 enema Substances 0.000 description 9
- 229940095399 enema Drugs 0.000 description 9
- 238000000338 in vitro Methods 0.000 description 9
- 230000001965 increasing effect Effects 0.000 description 9
- 230000010354 integration Effects 0.000 description 9
- 239000007951 isotonicity adjuster Substances 0.000 description 9
- 238000004020 luminiscence type Methods 0.000 description 9
- 230000003442 weekly effect Effects 0.000 description 9
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 8
- 239000012097 Lipofectamine 2000 Substances 0.000 description 8
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 8
- 238000003556 assay Methods 0.000 description 8
- 239000012091 fetal bovine serum Substances 0.000 description 8
- 210000004379 membrane Anatomy 0.000 description 8
- 239000006228 supernatant Substances 0.000 description 8
- 206010009887 colitis Diseases 0.000 description 7
- 238000001943 fluorescence-activated cell sorting Methods 0.000 description 7
- 239000012634 fragment Substances 0.000 description 7
- 230000006698 induction Effects 0.000 description 7
- 230000003993 interaction Effects 0.000 description 7
- 239000002609 medium Substances 0.000 description 7
- 108091033319 polynucleotide Proteins 0.000 description 7
- 102000040430 polynucleotide Human genes 0.000 description 7
- 239000002157 polynucleotide Substances 0.000 description 7
- 239000003755 preservative agent Substances 0.000 description 7
- 230000010076 replication Effects 0.000 description 7
- 230000004083 survival effect Effects 0.000 description 7
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 6
- 102100024216 Programmed cell death 1 ligand 1 Human genes 0.000 description 6
- 206010051379 Systemic Inflammatory Response Syndrome Diseases 0.000 description 6
- 108020001507 fusion proteins Proteins 0.000 description 6
- 102000037865 fusion proteins Human genes 0.000 description 6
- 230000002068 genetic effect Effects 0.000 description 6
- HQKMJHAJHXVSDF-UHFFFAOYSA-L magnesium stearate Chemical compound [Mg+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O HQKMJHAJHXVSDF-UHFFFAOYSA-L 0.000 description 6
- 239000000546 pharmaceutical excipient Substances 0.000 description 6
- 229920000642 polymer Polymers 0.000 description 6
- 230000011664 signaling Effects 0.000 description 6
- 238000012546 transfer Methods 0.000 description 6
- 238000002965 ELISA Methods 0.000 description 5
- 102000001708 Protein Isoforms Human genes 0.000 description 5
- 108010029485 Protein Isoforms Proteins 0.000 description 5
- 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 5
- 229930006000 Sucrose Natural products 0.000 description 5
- 239000011230 binding agent Substances 0.000 description 5
- 239000002775 capsule Substances 0.000 description 5
- 238000004113 cell culture Methods 0.000 description 5
- 239000003153 chemical reaction reagent Substances 0.000 description 5
- 239000003795 chemical substances by application Substances 0.000 description 5
- 239000012228 culture supernatant Substances 0.000 description 5
- 230000006196 deacetylation Effects 0.000 description 5
- 238000003381 deacetylation reaction Methods 0.000 description 5
- 230000007423 decrease Effects 0.000 description 5
- 238000000684 flow cytometry Methods 0.000 description 5
- 230000006870 function Effects 0.000 description 5
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 description 5
- 210000000952 spleen Anatomy 0.000 description 5
- 239000005720 sucrose Substances 0.000 description 5
- 229960004793 sucrose Drugs 0.000 description 5
- 210000001519 tissue Anatomy 0.000 description 5
- 101000611936 Homo sapiens Programmed cell death protein 1 Proteins 0.000 description 4
- 229920000168 Microcrystalline cellulose Polymers 0.000 description 4
- 239000004698 Polyethylene Substances 0.000 description 4
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 4
- 101710120037 Toxin CcdB Proteins 0.000 description 4
- 230000004913 activation Effects 0.000 description 4
- 239000003963 antioxidant agent Substances 0.000 description 4
- 235000006708 antioxidants Nutrition 0.000 description 4
- 239000006143 cell culture medium Substances 0.000 description 4
- 239000002299 complementary DNA Substances 0.000 description 4
- 150000001875 compounds Chemical class 0.000 description 4
- 230000003247 decreasing effect Effects 0.000 description 4
- 210000004443 dendritic cell Anatomy 0.000 description 4
- 238000011033 desalting Methods 0.000 description 4
- 239000002552 dosage form Substances 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- 239000010408 film Substances 0.000 description 4
- 238000007306 functionalization reaction Methods 0.000 description 4
- 102000048362 human PDCD1 Human genes 0.000 description 4
- 230000001976 improved effect Effects 0.000 description 4
- 229930027917 kanamycin Natural products 0.000 description 4
- 229960000318 kanamycin Drugs 0.000 description 4
- 229930182823 kanamycin A Natural products 0.000 description 4
- 235000019813 microcrystalline cellulose Nutrition 0.000 description 4
- 239000008108 microcrystalline cellulose Substances 0.000 description 4
- 229940016286 microcrystalline cellulose Drugs 0.000 description 4
- 210000000056 organ Anatomy 0.000 description 4
- 239000008194 pharmaceutical composition Substances 0.000 description 4
- 108020003175 receptors Proteins 0.000 description 4
- 102000005962 receptors Human genes 0.000 description 4
- 239000011780 sodium chloride Substances 0.000 description 4
- 239000002904 solvent Substances 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
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 3
- VBICKXHEKHSIBG-UHFFFAOYSA-N 1-monostearoylglycerol Chemical compound CCCCCCCCCCCCCCCCCC(=O)OCC(O)CO VBICKXHEKHSIBG-UHFFFAOYSA-N 0.000 description 3
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 3
- 108020004705 Codon Proteins 0.000 description 3
- 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 3
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 3
- 206010061218 Inflammation Diseases 0.000 description 3
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 description 3
- 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 3
- 229930195725 Mannitol Natural products 0.000 description 3
- 239000012124 Opti-MEM Substances 0.000 description 3
- 229920002472 Starch Polymers 0.000 description 3
- 230000006044 T cell activation Effects 0.000 description 3
- 241000700605 Viruses Species 0.000 description 3
- 238000007792 addition Methods 0.000 description 3
- 210000001185 bone marrow Anatomy 0.000 description 3
- 239000000969 carrier Substances 0.000 description 3
- 230000020411 cell activation Effects 0.000 description 3
- 230000001413 cellular effect Effects 0.000 description 3
- 238000005119 centrifugation Methods 0.000 description 3
- 230000002338 cryopreservative effect Effects 0.000 description 3
- 230000004069 differentiation Effects 0.000 description 3
- 239000003085 diluting agent Substances 0.000 description 3
- 239000007884 disintegrant Substances 0.000 description 3
- 231100000673 dose–response relationship Toxicity 0.000 description 3
- 239000003814 drug Substances 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 239000000796 flavoring agent Substances 0.000 description 3
- 230000028993 immune response Effects 0.000 description 3
- 238000001727 in vivo Methods 0.000 description 3
- 230000001939 inductive effect Effects 0.000 description 3
- 230000004054 inflammatory process Effects 0.000 description 3
- 239000004615 ingredient Substances 0.000 description 3
- 239000008101 lactose Substances 0.000 description 3
- 231100000225 lethality Toxicity 0.000 description 3
- 239000000314 lubricant Substances 0.000 description 3
- 235000019359 magnesium stearate Nutrition 0.000 description 3
- 239000000594 mannitol Substances 0.000 description 3
- 235000010355 mannitol Nutrition 0.000 description 3
- 229960001855 mannitol Drugs 0.000 description 3
- 230000035772 mutation Effects 0.000 description 3
- 238000007747 plating Methods 0.000 description 3
- 229920001223 polyethylene glycol Polymers 0.000 description 3
- 238000000746 purification Methods 0.000 description 3
- 210000003289 regulatory T cell Anatomy 0.000 description 3
- 239000012679 serum free medium Substances 0.000 description 3
- 239000004017 serum-free culture medium Substances 0.000 description 3
- 239000007787 solid Substances 0.000 description 3
- 210000004988 splenocyte Anatomy 0.000 description 3
- 238000010186 staining Methods 0.000 description 3
- 235000019698 starch Nutrition 0.000 description 3
- 229940032147 starch Drugs 0.000 description 3
- 239000008107 starch Substances 0.000 description 3
- 239000004094 surface-active agent Substances 0.000 description 3
- 239000000725 suspension Substances 0.000 description 3
- 230000035899 viability Effects 0.000 description 3
- HDTRYLNUVZCQOY-UHFFFAOYSA-N α-D-glucopyranosyl-α-D-glucopyranoside Natural products OC1C(O)C(O)C(CO)OC1OC1C(O)C(O)C(O)C(CO)O1 HDTRYLNUVZCQOY-UHFFFAOYSA-N 0.000 description 2
- TVZRAEYQIKYCPH-UHFFFAOYSA-N 3-(trimethylsilyl)propane-1-sulfonic acid Chemical compound C[Si](C)(C)CCCS(O)(=O)=O TVZRAEYQIKYCPH-UHFFFAOYSA-N 0.000 description 2
- KDCGOANMDULRCW-UHFFFAOYSA-N 7H-purine Chemical compound N1=CNC2=NC=NC2=C1 KDCGOANMDULRCW-UHFFFAOYSA-N 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 2
- 238000011740 C57BL/6 mouse Methods 0.000 description 2
- 108091026890 Coding region Proteins 0.000 description 2
- 208000014997 Crohn colitis Diseases 0.000 description 2
- 108090000695 Cytokines Proteins 0.000 description 2
- 102000004127 Cytokines Human genes 0.000 description 2
- 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 2
- 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 2
- RGHNJXZEOKUKBD-SQOUGZDYSA-N D-gluconic acid Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C(O)=O RGHNJXZEOKUKBD-SQOUGZDYSA-N 0.000 description 2
- 241000702421 Dependoparvovirus Species 0.000 description 2
- 241000620209 Escherichia coli DH5[alpha] Species 0.000 description 2
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 description 2
- 108700024394 Exon Proteins 0.000 description 2
- 108010010803 Gelatin Proteins 0.000 description 2
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 description 2
- 108091093094 Glycol nucleic acid Proteins 0.000 description 2
- 101000914514 Homo sapiens T-cell-specific surface glycoprotein CD28 Proteins 0.000 description 2
- 101000914484 Homo sapiens T-lymphocyte activation antigen CD80 Proteins 0.000 description 2
- 241000701024 Human betaherpesvirus 5 Species 0.000 description 2
- 229920002153 Hydroxypropyl cellulose Polymers 0.000 description 2
- 108700002232 Immediate-Early Genes Proteins 0.000 description 2
- 102000018071 Immunoglobulin Fc Fragments Human genes 0.000 description 2
- 108010091135 Immunoglobulin Fc Fragments Proteins 0.000 description 2
- 108010002352 Interleukin-1 Proteins 0.000 description 2
- DCXYFEDJOCDNAF-REOHCLBHSA-N L-asparagine Chemical compound OC(=O)[C@@H](N)CC(N)=O DCXYFEDJOCDNAF-REOHCLBHSA-N 0.000 description 2
- 241000713666 Lentivirus Species 0.000 description 2
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 2
- 229920000881 Modified starch Polymers 0.000 description 2
- 101100519207 Mus musculus Pdcd1 gene Proteins 0.000 description 2
- 240000007019 Oxalis corniculata Species 0.000 description 2
- 229910019142 PO4 Inorganic materials 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
- 108091093037 Peptide nucleic acid Proteins 0.000 description 2
- 239000002202 Polyethylene glycol Substances 0.000 description 2
- 239000012979 RPMI medium Substances 0.000 description 2
- 206010063837 Reperfusion injury Diseases 0.000 description 2
- 108020005091 Replication Origin Proteins 0.000 description 2
- 206010053879 Sepsis syndrome Diseases 0.000 description 2
- 206010040070 Septic Shock Diseases 0.000 description 2
- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical compound [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 description 2
- 238000000692 Student's t-test Methods 0.000 description 2
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 2
- 230000005867 T cell response Effects 0.000 description 2
- 102100027213 T-cell-specific surface glycoprotein CD28 Human genes 0.000 description 2
- 102100027222 T-lymphocyte activation antigen CD80 Human genes 0.000 description 2
- 108091046915 Threose nucleic acid Proteins 0.000 description 2
- HDTRYLNUVZCQOY-WSWWMNSNSA-N Trehalose Natural products O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-WSWWMNSNSA-N 0.000 description 2
- 239000002250 absorbent Substances 0.000 description 2
- 230000002745 absorbent Effects 0.000 description 2
- DPXJVFZANSGRMM-UHFFFAOYSA-N acetic acid;2,3,4,5,6-pentahydroxyhexanal;sodium Chemical compound [Na].CC(O)=O.OCC(O)C(O)C(O)C(O)C=O DPXJVFZANSGRMM-UHFFFAOYSA-N 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- 230000001154 acute effect Effects 0.000 description 2
- 230000000735 allogeneic effect Effects 0.000 description 2
- HDTRYLNUVZCQOY-LIZSDCNHSA-N alpha,alpha-trehalose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-LIZSDCNHSA-N 0.000 description 2
- 125000003277 amino group Chemical group 0.000 description 2
- XMQFTWRPUQYINF-UHFFFAOYSA-N bensulfuron-methyl Chemical compound COC(=O)C1=CC=CC=C1CS(=O)(=O)NC(=O)NC1=NC(OC)=CC(OC)=N1 XMQFTWRPUQYINF-UHFFFAOYSA-N 0.000 description 2
- 238000004166 bioassay Methods 0.000 description 2
- 229940098773 bovine serum albumin Drugs 0.000 description 2
- CJZGTCYPCWQAJB-UHFFFAOYSA-L calcium stearate Chemical class [Ca+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O CJZGTCYPCWQAJB-UHFFFAOYSA-L 0.000 description 2
- 239000001768 carboxy methyl cellulose Substances 0.000 description 2
- 230000024245 cell differentiation Effects 0.000 description 2
- 210000003169 central nervous system Anatomy 0.000 description 2
- OSASVXMJTNOKOY-UHFFFAOYSA-N chlorobutanol Chemical compound CC(C)(O)C(Cl)(Cl)Cl OSASVXMJTNOKOY-UHFFFAOYSA-N 0.000 description 2
- 230000001684 chronic effect Effects 0.000 description 2
- 238000000576 coating method Methods 0.000 description 2
- 229940075614 colloidal silicon dioxide Drugs 0.000 description 2
- 239000003086 colorant Substances 0.000 description 2
- 230000037011 constitutive activity Effects 0.000 description 2
- 230000006378 damage Effects 0.000 description 2
- 239000008121 dextrose Substances 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 238000001035 drying Methods 0.000 description 2
- 230000008030 elimination Effects 0.000 description 2
- 238000003379 elimination reaction Methods 0.000 description 2
- 239000000839 emulsion Substances 0.000 description 2
- 239000002158 endotoxin Substances 0.000 description 2
- 239000003623 enhancer Substances 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 235000013355 food flavoring agent Nutrition 0.000 description 2
- 239000000499 gel Substances 0.000 description 2
- 239000008273 gelatin Substances 0.000 description 2
- 229920000159 gelatin Polymers 0.000 description 2
- 235000019322 gelatine Nutrition 0.000 description 2
- 235000011852 gelatine desserts Nutrition 0.000 description 2
- 238000001476 gene delivery Methods 0.000 description 2
- 235000010977 hydroxypropyl cellulose Nutrition 0.000 description 2
- 239000001863 hydroxypropyl cellulose Substances 0.000 description 2
- 210000002865 immune cell Anatomy 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 208000015181 infectious disease Diseases 0.000 description 2
- 238000002347 injection Methods 0.000 description 2
- 239000007924 injection Substances 0.000 description 2
- 230000003834 intracellular effect Effects 0.000 description 2
- 208000012947 ischemia reperfusion injury Diseases 0.000 description 2
- 238000002955 isolation Methods 0.000 description 2
- 239000002502 liposome Substances 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 239000012669 liquid formulation Substances 0.000 description 2
- 210000001165 lymph node Anatomy 0.000 description 2
- 239000012139 lysis buffer Substances 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- OSWPMRLSEDHDFF-UHFFFAOYSA-N methyl salicylate Chemical compound COC(=O)C1=CC=CC=C1O OSWPMRLSEDHDFF-UHFFFAOYSA-N 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 150000004682 monohydrates Chemical class 0.000 description 2
- 210000004897 n-terminal region Anatomy 0.000 description 2
- 230000003204 osmotic effect Effects 0.000 description 2
- 238000012261 overproduction Methods 0.000 description 2
- 239000002245 particle Substances 0.000 description 2
- 230000037361 pathway Effects 0.000 description 2
- 229940049954 penicillin Drugs 0.000 description 2
- 230000002093 peripheral effect Effects 0.000 description 2
- 239000010452 phosphate Substances 0.000 description 2
- 239000001267 polyvinylpyrrolidone Substances 0.000 description 2
- 229920000036 polyvinylpyrrolidone Polymers 0.000 description 2
- 235000013855 polyvinylpyrrolidone Nutrition 0.000 description 2
- 239000000843 powder Substances 0.000 description 2
- 230000002335 preservative effect Effects 0.000 description 2
- 230000035755 proliferation Effects 0.000 description 2
- 230000001681 protective effect Effects 0.000 description 2
- 230000009467 reduction Effects 0.000 description 2
- 230000008439 repair process Effects 0.000 description 2
- 230000004044 response Effects 0.000 description 2
- 230000036303 septic shock Effects 0.000 description 2
- 235000019333 sodium laurylsulphate Nutrition 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 239000000600 sorbitol Substances 0.000 description 2
- 229960002920 sorbitol Drugs 0.000 description 2
- 235000010356 sorbitol Nutrition 0.000 description 2
- 230000000638 stimulation Effects 0.000 description 2
- 229960005322 streptomycin Drugs 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 235000000346 sugar Nutrition 0.000 description 2
- 239000000829 suppository Substances 0.000 description 2
- 208000024891 symptom Diseases 0.000 description 2
- 239000006188 syrup Substances 0.000 description 2
- 235000020357 syrup Nutrition 0.000 description 2
- 239000006068 taste-masking agent Substances 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 241001430294 unidentified retrovirus Species 0.000 description 2
- 239000013603 viral vector Substances 0.000 description 2
- JCIIKRHCWVHVFF-UHFFFAOYSA-N 1,2,4-thiadiazol-5-amine;hydrochloride Chemical compound Cl.NC1=NC=NS1 JCIIKRHCWVHVFF-UHFFFAOYSA-N 0.000 description 1
- IXPNQXFRVYWDDI-UHFFFAOYSA-N 1-methyl-2,4-dioxo-1,3-diazinane-5-carboximidamide Chemical compound CN1CC(C(N)=N)C(=O)NC1=O IXPNQXFRVYWDDI-UHFFFAOYSA-N 0.000 description 1
- NHBKXEKEPDILRR-UHFFFAOYSA-N 2,3-bis(butanoylsulfanyl)propyl butanoate Chemical compound CCCC(=O)OCC(SC(=O)CCC)CSC(=O)CCC NHBKXEKEPDILRR-UHFFFAOYSA-N 0.000 description 1
- MSWZFWKMSRAUBD-IVMDWMLBSA-N 2-amino-2-deoxy-D-glucopyranose Chemical compound N[C@H]1C(O)O[C@H](CO)[C@@H](O)[C@@H]1O MSWZFWKMSRAUBD-IVMDWMLBSA-N 0.000 description 1
- YRNWIFYIFSBPAU-UHFFFAOYSA-N 4-[4-(dimethylamino)phenyl]-n,n-dimethylaniline Chemical compound C1=CC(N(C)C)=CC=C1C1=CC=C(N(C)C)C=C1 YRNWIFYIFSBPAU-UHFFFAOYSA-N 0.000 description 1
- 208000004998 Abdominal Pain Diseases 0.000 description 1
- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 description 1
- 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 1
- DCXYFEDJOCDNAF-UHFFFAOYSA-N Asparagine Natural products OC(=O)C(N)CC(N)=O DCXYFEDJOCDNAF-UHFFFAOYSA-N 0.000 description 1
- 241000416162 Astragalus gummifer Species 0.000 description 1
- 208000032116 Autoimmune Experimental Encephalomyelitis Diseases 0.000 description 1
- 238000011725 BALB/c mouse Methods 0.000 description 1
- 102000004506 Blood Proteins Human genes 0.000 description 1
- 108010017384 Blood Proteins Proteins 0.000 description 1
- BTBUEUYNUDRHOZ-UHFFFAOYSA-N Borate Chemical compound [O-]B([O-])[O-] BTBUEUYNUDRHOZ-UHFFFAOYSA-N 0.000 description 1
- 241000283690 Bos taurus Species 0.000 description 1
- 108010021064 CTLA-4 Antigen Proteins 0.000 description 1
- 102000008203 CTLA-4 Antigen Human genes 0.000 description 1
- 229940045513 CTLA4 antagonist Drugs 0.000 description 1
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 1
- 241001631457 Cannula Species 0.000 description 1
- 241000283707 Capra Species 0.000 description 1
- BVKZGUZCCUSVTD-UHFFFAOYSA-L Carbonate Chemical compound [O-]C([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-L 0.000 description 1
- 229920002134 Carboxymethyl cellulose Polymers 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
- 241000282693 Cercopithecidae Species 0.000 description 1
- GHXZTYHSJHQHIJ-UHFFFAOYSA-N Chlorhexidine Chemical compound C=1C=C(Cl)C=CC=1NC(N)=NC(N)=NCCCCCCN=C(N)N=C(N)NC1=CC=C(Cl)C=C1 GHXZTYHSJHQHIJ-UHFFFAOYSA-N 0.000 description 1
- KRKNYBCHXYNGOX-UHFFFAOYSA-K Citrate Chemical compound [O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O KRKNYBCHXYNGOX-UHFFFAOYSA-K 0.000 description 1
- 229920002261 Corn starch Polymers 0.000 description 1
- 229920002785 Croscarmellose sodium Polymers 0.000 description 1
- RGHNJXZEOKUKBD-UHFFFAOYSA-N D-gluconic acid Natural products OCC(O)C(O)C(O)C(O)C(O)=O RGHNJXZEOKUKBD-UHFFFAOYSA-N 0.000 description 1
- 229920002307 Dextran Polymers 0.000 description 1
- 206010061818 Disease progression Diseases 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 229920003134 Eudragit® polymer Polymers 0.000 description 1
- 241000287828 Gallus gallus Species 0.000 description 1
- 108700028146 Genetic Enhancer Elements Proteins 0.000 description 1
- 239000004471 Glycine Substances 0.000 description 1
- 206010019233 Headaches Diseases 0.000 description 1
- 101001057504 Homo sapiens Interferon-stimulated gene 20 kDa protein Proteins 0.000 description 1
- 101001055144 Homo sapiens Interleukin-2 receptor subunit alpha Proteins 0.000 description 1
- 102100026878 Interleukin-2 receptor subunit alpha Human genes 0.000 description 1
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 description 1
- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 description 1
- 229930182816 L-glutamine Natural products 0.000 description 1
- AGPKZVBTJJNPAG-WHFBIAKZSA-N L-isoleucine Chemical compound CC[C@H](C)[C@H](N)C(O)=O AGPKZVBTJJNPAG-WHFBIAKZSA-N 0.000 description 1
- ROHFNLRQFUQHCH-YFKPBYRVSA-N L-leucine Chemical compound CC(C)C[C@H](N)C(O)=O ROHFNLRQFUQHCH-YFKPBYRVSA-N 0.000 description 1
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 description 1
- COLNVLDHVKWLRT-QMMMGPOBSA-N L-phenylalanine Chemical compound OC(=O)[C@@H](N)CC1=CC=CC=C1 COLNVLDHVKWLRT-QMMMGPOBSA-N 0.000 description 1
- QIVBCDIJIAJPQS-VIFPVBQESA-N L-tryptophane Chemical compound C1=CC=C2C(C[C@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-VIFPVBQESA-N 0.000 description 1
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 1
- ROHFNLRQFUQHCH-UHFFFAOYSA-N Leucine Natural products CC(C)CC(N)C(O)=O ROHFNLRQFUQHCH-UHFFFAOYSA-N 0.000 description 1
- 238000003231 Lowry assay Methods 0.000 description 1
- 238000009013 Lowry's assay Methods 0.000 description 1
- 239000006142 Luria-Bertani Agar Substances 0.000 description 1
- 239000004472 Lysine Substances 0.000 description 1
- 244000246386 Mentha pulegium Species 0.000 description 1
- 235000016257 Mentha pulegium Nutrition 0.000 description 1
- 235000004357 Mentha x piperita Nutrition 0.000 description 1
- 101001117316 Mus musculus Programmed cell death 1 ligand 1 Proteins 0.000 description 1
- WHNWPMSKXPGLAX-UHFFFAOYSA-N N-Vinyl-2-pyrrolidone Chemical compound C=CN1CCCC1=O WHNWPMSKXPGLAX-UHFFFAOYSA-N 0.000 description 1
- OVRNDRQMDRJTHS-FMDGEEDCSA-N N-acetyl-beta-D-glucosamine Chemical class CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O OVRNDRQMDRJTHS-FMDGEEDCSA-N 0.000 description 1
- 206010028813 Nausea Diseases 0.000 description 1
- 101710163270 Nuclease Proteins 0.000 description 1
- 241000282577 Pan troglodytes Species 0.000 description 1
- 206010034133 Pathogen resistance Diseases 0.000 description 1
- ONIBWKKTOPOVIA-UHFFFAOYSA-N Proline Natural products OC(=O)C1CCCN1 ONIBWKKTOPOVIA-UHFFFAOYSA-N 0.000 description 1
- 229940124158 Protease/peptidase inhibitor Drugs 0.000 description 1
- CZPWVGJYEJSRLH-UHFFFAOYSA-N Pyrimidine Chemical compound C1=CN=CN=C1 CZPWVGJYEJSRLH-UHFFFAOYSA-N 0.000 description 1
- 108020004511 Recombinant DNA Proteins 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 description 1
- 239000004141 Sodium laurylsulphate Substances 0.000 description 1
- 108091081024 Start codon Proteins 0.000 description 1
- 230000033540 T cell apoptotic process Effects 0.000 description 1
- 230000006052 T cell proliferation Effects 0.000 description 1
- AYFVYJQAPQTCCC-UHFFFAOYSA-N Threonine Natural products CC(O)C(N)C(O)=O AYFVYJQAPQTCCC-UHFFFAOYSA-N 0.000 description 1
- 239000004473 Threonine Substances 0.000 description 1
- 229920001615 Tragacanth Polymers 0.000 description 1
- 239000007983 Tris buffer Substances 0.000 description 1
- QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Natural products C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 description 1
- 102100022153 Tumor necrosis factor receptor superfamily member 4 Human genes 0.000 description 1
- 101710165473 Tumor necrosis factor receptor superfamily member 4 Proteins 0.000 description 1
- 201000006704 Ulcerative Colitis Diseases 0.000 description 1
- TVXBFESIOXBWNM-UHFFFAOYSA-N Xylitol Natural products OCCC(O)C(O)C(O)CCO TVXBFESIOXBWNM-UHFFFAOYSA-N 0.000 description 1
- 239000003070 absorption delaying agent Substances 0.000 description 1
- 208000024340 acute graft versus host disease Diseases 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 239000002671 adjuvant Substances 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- 239000011543 agarose gel Substances 0.000 description 1
- 235000004279 alanine Nutrition 0.000 description 1
- 235000010443 alginic acid Nutrition 0.000 description 1
- 229920000615 alginic acid Polymers 0.000 description 1
- 239000000783 alginic acid Substances 0.000 description 1
- 229960001126 alginic acid Drugs 0.000 description 1
- 150000004781 alginic acids Chemical class 0.000 description 1
- 239000002269 analeptic agent Substances 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 230000000844 anti-bacterial effect Effects 0.000 description 1
- 239000002518 antifoaming agent Substances 0.000 description 1
- 239000003429 antifungal agent Substances 0.000 description 1
- 229940121375 antifungal agent Drugs 0.000 description 1
- 210000000612 antigen-presenting cell Anatomy 0.000 description 1
- 239000004599 antimicrobial Substances 0.000 description 1
- 230000003078 antioxidant effect Effects 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 210000004507 artificial chromosome Anatomy 0.000 description 1
- 235000009582 asparagine Nutrition 0.000 description 1
- 229960001230 asparagine Drugs 0.000 description 1
- 235000003704 aspartic acid Nutrition 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 230000004888 barrier function Effects 0.000 description 1
- 239000011324 bead Substances 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 229960000686 benzalkonium chloride Drugs 0.000 description 1
- CADWTSSKOVRVJC-UHFFFAOYSA-N benzyl(dimethyl)azanium;chloride Chemical compound [Cl-].C[NH+](C)CC1=CC=CC=C1 CADWTSSKOVRVJC-UHFFFAOYSA-N 0.000 description 1
- MSWZFWKMSRAUBD-UHFFFAOYSA-N beta-D-galactosamine Natural products NC1C(O)OC(CO)C(O)C1O MSWZFWKMSRAUBD-UHFFFAOYSA-N 0.000 description 1
- MSWZFWKMSRAUBD-QZABAPFNSA-N beta-D-glucosamine Chemical group N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O MSWZFWKMSRAUBD-QZABAPFNSA-N 0.000 description 1
- OQFSQFPPLPISGP-UHFFFAOYSA-N beta-carboxyaspartic acid Natural products OC(=O)C(N)C(C(O)=O)C(O)=O OQFSQFPPLPISGP-UHFFFAOYSA-N 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 239000003139 biocide Substances 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 239000004067 bulking agent Substances 0.000 description 1
- 239000011575 calcium Substances 0.000 description 1
- 229910052791 calcium Inorganic materials 0.000 description 1
- XAAHAAMILDNBPS-UHFFFAOYSA-L calcium hydrogenphosphate dihydrate Chemical compound O.O.[Ca+2].OP([O-])([O-])=O XAAHAAMILDNBPS-UHFFFAOYSA-L 0.000 description 1
- 235000013539 calcium stearate Nutrition 0.000 description 1
- 239000008116 calcium stearate Substances 0.000 description 1
- 235000010948 carboxy methyl cellulose Nutrition 0.000 description 1
- 239000008112 carboxymethyl-cellulose Substances 0.000 description 1
- 229940105329 carboxymethylcellulose Drugs 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 125000002091 cationic group Chemical group 0.000 description 1
- 230000022131 cell cycle Effects 0.000 description 1
- 230000032823 cell division Effects 0.000 description 1
- 230000003915 cell function Effects 0.000 description 1
- 239000013592 cell lysate Substances 0.000 description 1
- 230000004663 cell proliferation Effects 0.000 description 1
- 239000002738 chelating agent Substances 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 230000035605 chemotaxis Effects 0.000 description 1
- 235000015111 chews Nutrition 0.000 description 1
- 229960003260 chlorhexidine Drugs 0.000 description 1
- 229960004926 chlorobutanol Drugs 0.000 description 1
- 230000002759 chromosomal effect Effects 0.000 description 1
- 210000000349 chromosome Anatomy 0.000 description 1
- 239000013599 cloning vector Substances 0.000 description 1
- 229940110456 cocoa butter Drugs 0.000 description 1
- 235000019868 cocoa butter Nutrition 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 238000013270 controlled release Methods 0.000 description 1
- 239000002826 coolant Substances 0.000 description 1
- 239000008120 corn starch Substances 0.000 description 1
- 230000000139 costimulatory effect Effects 0.000 description 1
- 229960001681 croscarmellose sodium Drugs 0.000 description 1
- 229960000913 crospovidone Drugs 0.000 description 1
- 235000010947 crosslinked sodium carboxy methyl cellulose Nutrition 0.000 description 1
- 239000002577 cryoprotective agent Substances 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 235000018417 cysteine Nutrition 0.000 description 1
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 1
- 230000009089 cytolysis Effects 0.000 description 1
- 230000002950 deficient Effects 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 229960002086 dextran Drugs 0.000 description 1
- 230000005750 disease progression Effects 0.000 description 1
- 239000006185 dispersion Substances 0.000 description 1
- 239000002612 dispersion medium Substances 0.000 description 1
- 238000010494 dissociation reaction Methods 0.000 description 1
- 230000005593 dissociations Effects 0.000 description 1
- 230000003828 downregulation Effects 0.000 description 1
- 230000035622 drinking Effects 0.000 description 1
- 239000003651 drinking water Substances 0.000 description 1
- 235000020188 drinking water Nutrition 0.000 description 1
- 238000012377 drug delivery Methods 0.000 description 1
- 239000012636 effector Substances 0.000 description 1
- 210000003162 effector t lymphocyte Anatomy 0.000 description 1
- 239000003792 electrolyte Substances 0.000 description 1
- 239000003974 emollient agent Substances 0.000 description 1
- 239000003995 emulsifying agent Substances 0.000 description 1
- 201000002491 encephalomyelitis Diseases 0.000 description 1
- 239000002702 enteric coating Substances 0.000 description 1
- 238000009505 enteric coating Methods 0.000 description 1
- 210000000981 epithelium Anatomy 0.000 description 1
- 210000003743 erythrocyte Anatomy 0.000 description 1
- BEFDCLMNVWHSGT-UHFFFAOYSA-N ethenylcyclopentane Chemical compound C=CC1CCCC1 BEFDCLMNVWHSGT-UHFFFAOYSA-N 0.000 description 1
- MVPICKVDHDWCJQ-UHFFFAOYSA-N ethyl 3-pyrrolidin-1-ylpropanoate Chemical compound CCOC(=O)CCN1CCCC1 MVPICKVDHDWCJQ-UHFFFAOYSA-N 0.000 description 1
- 208000012997 experimental autoimmune encephalomyelitis Diseases 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 239000013020 final formulation Substances 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 235000019264 food flavour enhancer Nutrition 0.000 description 1
- 235000003599 food sweetener Nutrition 0.000 description 1
- 238000004108 freeze drying Methods 0.000 description 1
- 239000007903 gelatin capsule Substances 0.000 description 1
- 238000001415 gene therapy Methods 0.000 description 1
- 239000000174 gluconic acid Substances 0.000 description 1
- 235000012208 gluconic acid Nutrition 0.000 description 1
- 229960002442 glucosamine Drugs 0.000 description 1
- 125000002791 glucosyl group Chemical group C1([C@H](O)[C@@H](O)[C@H](O)[C@H](O1)CO)* 0.000 description 1
- 235000013922 glutamic acid Nutrition 0.000 description 1
- 239000004220 glutamic acid Substances 0.000 description 1
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 description 1
- 229940075507 glyceryl monostearate Drugs 0.000 description 1
- 229940075529 glyceryl stearate Drugs 0.000 description 1
- 239000008187 granular material Substances 0.000 description 1
- 238000000227 grinding Methods 0.000 description 1
- 244000005709 gut microbiome Species 0.000 description 1
- 231100000869 headache Toxicity 0.000 description 1
- 229910001385 heavy metal Inorganic materials 0.000 description 1
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 1
- 235000001050 hortel pimenta Nutrition 0.000 description 1
- 239000003906 humectant Substances 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- WGCNASOHLSPBMP-UHFFFAOYSA-N hydroxyacetaldehyde Natural products OCC=O WGCNASOHLSPBMP-UHFFFAOYSA-N 0.000 description 1
- 235000010979 hydroxypropyl methyl cellulose Nutrition 0.000 description 1
- 239000001866 hydroxypropyl methyl cellulose Substances 0.000 description 1
- 229920003088 hydroxypropyl methyl cellulose Polymers 0.000 description 1
- UFVKGYZPFZQRLF-UHFFFAOYSA-N hydroxypropyl methyl cellulose Chemical compound OC1C(O)C(OC)OC(CO)C1OC1C(O)C(O)C(OC2C(C(O)C(OC3C(C(O)C(O)C(CO)O3)O)C(CO)O2)O)C(CO)O1 UFVKGYZPFZQRLF-UHFFFAOYSA-N 0.000 description 1
- 230000005934 immune activation Effects 0.000 description 1
- 230000003832 immune regulation Effects 0.000 description 1
- 230000002998 immunogenetic effect Effects 0.000 description 1
- 239000007943 implant Substances 0.000 description 1
- 238000010348 incorporation Methods 0.000 description 1
- 239000011261 inert gas Substances 0.000 description 1
- 230000004968 inflammatory condition Effects 0.000 description 1
- 230000028709 inflammatory response Effects 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 238000003780 insertion Methods 0.000 description 1
- 230000037431 insertion Effects 0.000 description 1
- 230000007358 intestinal barrier function Effects 0.000 description 1
- 210000000936 intestine Anatomy 0.000 description 1
- 230000004068 intracellular signaling Effects 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- AGPKZVBTJJNPAG-UHFFFAOYSA-N isoleucine Natural products CCC(C)C(N)C(O)=O AGPKZVBTJJNPAG-UHFFFAOYSA-N 0.000 description 1
- 229960000310 isoleucine Drugs 0.000 description 1
- FZWBNHMXJMCXLU-BLAUPYHCSA-N isomaltotriose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1OC[C@@H]1[C@@H](O)[C@H](O)[C@@H](O)[C@@H](OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C=O)O1 FZWBNHMXJMCXLU-BLAUPYHCSA-N 0.000 description 1
- 229960001375 lactose Drugs 0.000 description 1
- 239000003446 ligand Substances 0.000 description 1
- 230000000670 limiting effect Effects 0.000 description 1
- 239000007937 lozenge Substances 0.000 description 1
- 239000006166 lysate Substances 0.000 description 1
- 230000002101 lytic effect Effects 0.000 description 1
- 210000004962 mammalian cell Anatomy 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 230000001404 mediated effect Effects 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
- 108020004999 messenger RNA Proteins 0.000 description 1
- 229930182817 methionine Natural products 0.000 description 1
- 229920000609 methyl cellulose Polymers 0.000 description 1
- 229960001047 methyl salicylate Drugs 0.000 description 1
- 230000011987 methylation Effects 0.000 description 1
- 238000007069 methylation reaction Methods 0.000 description 1
- 235000010981 methylcellulose Nutrition 0.000 description 1
- 239000001923 methylcellulose Substances 0.000 description 1
- 239000011325 microbead Substances 0.000 description 1
- 238000009126 molecular therapy Methods 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 239000001788 mono and diglycerides of fatty acids Substances 0.000 description 1
- 238000010172 mouse model Methods 0.000 description 1
- 230000003232 mucoadhesive effect Effects 0.000 description 1
- 201000006417 multiple sclerosis Diseases 0.000 description 1
- 230000008693 nausea Effects 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 239000012457 nonaqueous media Substances 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 239000003921 oil Substances 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 125000005430 oxychloro group Chemical group 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 239000000137 peptide hydrolase inhibitor Substances 0.000 description 1
- WVDDGKGOMKODPV-ZQBYOMGUSA-N phenyl(114C)methanol Chemical compound O[14CH2]C1=CC=CC=C1 WVDDGKGOMKODPV-ZQBYOMGUSA-N 0.000 description 1
- COLNVLDHVKWLRT-UHFFFAOYSA-N phenylalanine Natural products OC(=O)C(N)CC1=CC=CC=C1 COLNVLDHVKWLRT-UHFFFAOYSA-N 0.000 description 1
- 229940096826 phenylmercuric acetate Drugs 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 230000001766 physiological effect Effects 0.000 description 1
- 239000006187 pill Substances 0.000 description 1
- 239000004014 plasticizer Substances 0.000 description 1
- 235000010482 polyoxyethylene sorbitan monooleate Nutrition 0.000 description 1
- 239000000244 polyoxyethylene sorbitan monooleate Substances 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 229920000136 polysorbate Polymers 0.000 description 1
- 229940068968 polysorbate 80 Drugs 0.000 description 1
- 229920000053 polysorbate 80 Polymers 0.000 description 1
- 235000013809 polyvinylpolypyrrolidone Nutrition 0.000 description 1
- 229920000523 polyvinylpolypyrrolidone Polymers 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 238000000159 protein binding assay Methods 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 238000003259 recombinant expression Methods 0.000 description 1
- 230000002829 reductive effect Effects 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 230000008844 regulatory mechanism Effects 0.000 description 1
- 230000003362 replicative effect Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 108091008146 restriction endonucleases Proteins 0.000 description 1
- 238000012552 review Methods 0.000 description 1
- CVHZOJJKTDOEJC-UHFFFAOYSA-N saccharin Chemical compound C1=CC=C2C(=O)NS(=O)(=O)C2=C1 CVHZOJJKTDOEJC-UHFFFAOYSA-N 0.000 description 1
- 229940081974 saccharin Drugs 0.000 description 1
- 235000019204 saccharin Nutrition 0.000 description 1
- 239000000901 saccharin and its Na,K and Ca salt Substances 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 239000000377 silicon dioxide Substances 0.000 description 1
- 235000012239 silicon dioxide Nutrition 0.000 description 1
- 235000010413 sodium alginate Nutrition 0.000 description 1
- 239000000661 sodium alginate Substances 0.000 description 1
- 229940005550 sodium alginate Drugs 0.000 description 1
- 235000019812 sodium carboxymethyl cellulose Nutrition 0.000 description 1
- 229920001027 sodium carboxymethylcellulose Polymers 0.000 description 1
- 229960002668 sodium chloride Drugs 0.000 description 1
- 239000008109 sodium starch glycolate Substances 0.000 description 1
- 229920003109 sodium starch glycolate Polymers 0.000 description 1
- 229940079832 sodium starch glycolate Drugs 0.000 description 1
- 229940045902 sodium stearyl fumarate Drugs 0.000 description 1
- 239000007909 solid dosage form Substances 0.000 description 1
- 229940075582 sorbic acid Drugs 0.000 description 1
- 235000010199 sorbic acid Nutrition 0.000 description 1
- 239000004334 sorbic acid Substances 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 239000007921 spray Substances 0.000 description 1
- 230000007480 spreading Effects 0.000 description 1
- 238000003892 spreading Methods 0.000 description 1
- 239000003381 stabilizer Substances 0.000 description 1
- 239000008223 sterile water Substances 0.000 description 1
- 150000008163 sugars Chemical class 0.000 description 1
- 239000002511 suppository base Substances 0.000 description 1
- 230000001629 suppression Effects 0.000 description 1
- 230000009747 swallowing Effects 0.000 description 1
- 239000003765 sweetening agent Substances 0.000 description 1
- 239000007916 tablet composition Substances 0.000 description 1
- 239000000454 talc Substances 0.000 description 1
- 229910052623 talc Inorganic materials 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 239000002562 thickening agent Substances 0.000 description 1
- RTKIYNMVFMVABJ-UHFFFAOYSA-L thimerosal Chemical compound [Na+].CC[Hg]SC1=CC=CC=C1C([O-])=O RTKIYNMVFMVABJ-UHFFFAOYSA-L 0.000 description 1
- 229940033663 thimerosal Drugs 0.000 description 1
- 239000010409 thin film Substances 0.000 description 1
- 238000002627 tracheal intubation Methods 0.000 description 1
- 238000013518 transcription Methods 0.000 description 1
- 230000035897 transcription Effects 0.000 description 1
- 230000002103 transcriptional effect Effects 0.000 description 1
- 229940074410 trehalose Drugs 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 1
- 241001515965 unidentified phage Species 0.000 description 1
- 230000003827 upregulation Effects 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- 239000002023 wood Substances 0.000 description 1
- 239000000811 xylitol Substances 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
- 235000010447 xylitol Nutrition 0.000 description 1
- XOOUIPVCVHRTMJ-UHFFFAOYSA-L zinc stearate Chemical compound [Zn+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O XOOUIPVCVHRTMJ-UHFFFAOYSA-L 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
- C07K14/70503—Immunoglobulin superfamily
- C07K14/70532—B7 molecules, e.g. CD80, CD86
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7088—Compounds having three or more nucleosides or nucleotides
- A61K31/711—Natural deoxyribonucleic acids, i.e. containing only 2'-deoxyriboses attached to adenine, guanine, cytosine or thymine and having 3'-5' phosphodiester links
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
- A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
- A61K9/51—Nanocapsules; Nanoparticles
- A61K9/5107—Excipients; Inactive ingredients
- A61K9/513—Organic macromolecular compounds; Dendrimers
- A61K9/5161—Polysaccharides, e.g. alginate, chitosan, cellulose derivatives; Cyclodextrin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/06—Anti-spasmodics, e.g. drugs for colics, esophagic dyskinesia
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
- A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
-
- 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
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
Definitions
- the present disclosure relates to methods and compositions for the amelioration of inflammatory disorders employing gene expression of PD-L1.
- Immune activation in general, and the T cell activation state in particular, are subject to complex overlapping regulatory mechanisms mediated by a host of costimulatory (e.g. CD28, ICOS, OX40, etc.) and co-inhibitory (e.g., CTLA-4, programmed death-1 (“PD-1”), etc.) molecules on T cells.
- PD-1 programmed death ligand-1
- PD-L1 also known as B7H1
- the present invention resolves the foregoing scientific uncertainties in the prior art by successfully employing localized intestinal expression of PD-L1 polypeptides, including soluble PD-L1 polypeptides, in an immune protective setting for the treatment of a variety of inflammatory disorders including, e.g., inflammatory bowel disease (e.g. ulcerative colitis, Crohn's disease, and the like) as well as graft-versus-host disease (GvHD) induced by organ or bone marrow transplant.
- inflammatory bowel disease e.g. ulcerative colitis, Crohn's disease, and the like
- GvHD graft-versus-host disease
- the invention provides a method of treating an inflammatory disorder in a patient in need thereof, comprising administering to the intestinal tract of said patient an expression vector comprising a PD-L1 nucleic acid.
- the PD-L1 polypeptide is a membrane-bound PD-L1 polypeptide.
- the PD-L1 polypeptide is human PD-L1 or splice variants thereof.
- the PD-L1 nucleic acid encodes for a PD-L1 polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 1.
- the PD-L1 polypeptide is a soluble PD-L1 polypeptide, e.g. comprises the signal sequence, IgV domain and IgC domain of PD-L1, and preferably human PD-L1 (i.e. amino acids 1-239 of SEQ ID NO: 1).
- the soluble PD-L1 polypeptide may lack all or part of the signal sequence (e.g. it may comprise amino acids 19-238 of SEQ ID NO: 1).
- the PD-L1 nucleic acid is delivered by an expression vector encapsulated in a nucleic acid delivery vehicle.
- Preferred nucleic acid delivery vehicles include chitosan or chitosan derivative nanoparticles.
- the chitosan derivative nanoparticles comprise chitosan coupled with arginine and/or gluconic acid.
- the chitosan derivative nanoparticles comprise chitosan coupled with arginine and a hydrophilic polyol.
- the hydrophilic polyol is glucose.
- the invention provides expression vectors comprising a PD-L1 nucleic acid to treat an inflammatory disorder in a patient in need thereof, wherein said expression vector is administered to the intestine of said subject.
- the PD-L1 nucleic acid comprises SEQ ID NO: 2.
- the PD-L1 nucleic acid comprises at least one synonymous mutation in SEQ ID NO: 2.
- the PD-L1 nucleic acid comprises a plurality of such mutations to assist in differentiation and, optionally, improved expression.
- the PD-L1 nucleic acid comprises SEQ ID NO: 3.
- the PD-L1 nucleic acid further comprises a heterologous sequence.
- the heterologous sequence may comprise an Fc domain, a protein tag, a conjugated therapeutic, or a combination thereof.
- the N-terminal region of the PD-L1 polypeptide is fused to a human IgG1 Fc region or portion thereof.
- the PD-L1 polypeptide may be fused to a human IgG1 Fc region or portion thereof by way of an amino acid sequence of (GGGGS)n (SEQ ID NO: 5).
- the IgG1 Fc is mutated to reduce antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cellular cytotoxicity (CDCC) by altering one or more of the following amino acids in the Fc domain: E233P, L234V, L235A, deletion of G236, A327G, A330S and P331S.
- the PD-L1 nucleic acid comprises SEQ ID NO: 4.
- Inflammatory disorders that can be advantageously treated by way of the subject invention include both chronic and acute conditions, including inflammation associated with infection (e.g. septic shock, sepsis, or systemic inflammatory response syndrome (SIRS)), ischemia-reperfusion injury, endotoxin lethality, inflammatory bowel disease, Crohn's disease, colitis, or resulting from over production of cytokines (e.g. TNF or IL-1) as well as GvHD.
- infection e.g. septic shock, sepsis, or systemic inflammatory response syndrome (SIRS)
- ischemia-reperfusion injury e.g. ischemia-reperfusion injury, endotoxin lethality, inflammatory bowel disease, Crohn's disease, colitis, or resulting from over production of cytokines (e.g. TNF or IL-1) as well as GvHD.
- cytokines e.g. TNF or IL-1
- FIG. 1 The pVax-hPD-L1 vector (SEQ ID NO: 6). Plasmid sequence of the vector encoding a membrane-bound human PD-L1 polypeptide, the optimized human PD-L1 cDNA is highlighted.
- FIG. 2 The pVax-hPD-L1 Fc vector (SEQ ID NO: 7). Plasmid sequence of the vector encoding a soluble human PD-L1 Fc polypeptide, the optimized human PD-L1 cDNA is highlighted.
- FIGS. 3 A-B Human PD-L1 plasmid and in vitro expression.
- A Plasmid map of the optimized hPD-L1 DNA sequences cloned in a pVax backbone containing a plasmid replication origin (pUC ori;) under the control of a human cytomegalovirus immediate-early gene promoter (CMV;) with a kanamycin resistance gene.
- pUC ori a plasmid replication origin
- CMV human cytomegalovirus immediate-early gene promoter
- HEK293T cells were transfected with 1 ⁇ g or 2.5 ⁇ g of Fc control (White), PD-L1-Fc (Black), or PD-L1 (Grey) plasmid DNA using Lipofectamine 2000 (Invitrogen). PD-L1 protein content in cell culture supernatant or cell lysate was quantified 48 h post-transfection.
- FIGS. 4 A-B In vitro expression of PD-L1-Fc from polyplex transfection.
- HEK-293T were transfected with PD-L1-Fc polyplexes containing increasing concentrations of pVAX-opt-hPD-L1-Fc DNA, as indicated.
- A Supernatants were collected at 48 hours post-transfection and assayed for hPD-L1-Fc protein by ELISA. Data were normalized to total cellular protein.
- B Table of EC50 and maximum protein expression.
- FIGS. 5 A-D Purification of in vitro-generated Fc-fused constructs and expression.
- A Protein expression confirmed by coomassie gel staining: HEK293T cells were transfected with Fc control or PD-L1-Fc plasmid DNA using Lipofectamine 2000 (Invitrogen). Cell culture medium was exchanged for serum-free medium 24 h post-transfection. Cell culture supernatant was collected 72 h post-transfection and Fc-fused proteins were purified using Protein G HiTrap columns (GE). Buffer was exchanged for PBS using PD-10 desalting columns (GE), and protein was eluted in four equal fractions (eluate 1-4).
- HEK293T cells were transfected with Fc control or PD-L1-Fc plasmid DNA using Lipofectamine 2000 (Invitrogen).
- Cell culture medium was exchanged for serum-free medium 24 h post-transfection.
- Cell culture supernatant was collected 72 h post-transfection and Fc-fused proteins were purified using Protein G HiTrap columns (GE). Buffer was exchanged for PBS using PD-10 desalting columns (GE), and protein was eluted in four equal fractions (F1-4).
- PD-L1 protein concentration was determined by ELISA.
- NIH/3T3 cells were transfected with wild-type PD-L1 plasmid DNA or control (pVax) using Lipofectamine 2000 (Invitrogen) in a 6-well plate. Cells were washed and re-seeded in a 96-well flat-bottomed plate 24 h post-transfection. The following day (Day 2), and on the 3 subsequent days, PD-L1 expression at the cell surface was analyzed by flow cytometry.
- D NIH/3T3 cells were transfected with wild-type PD-L1 plasmid DNA or control (pVax) using Lipofectamine 2000 (Invitrogen) in a 6-well plate.
- FIGS. 6 A-B Expressed PD-L1-Fc signals through the human PD-1 receptor in a dose-dependent manner.
- Cells were transfected with PD-L1-Fc and purified from the cell culture supernatant.
- PathHunter PD-1 Signaling Assay cells were stimulated with increasing concentrations of PD-L1-Fc (40 min) and signal activation was measured by luminescence (RLU; Relative Luminescence Units).
- PathHunter PD-1 Signaling Assay cells were incubated with recombinant IgG1-Fc or PD-L1-Fc (50 ⁇ g/mL, 40 min) with and without anti-PD-1 (lo: 0.1 ⁇ g/mL, hi: 1.0 ⁇ g/mL) and signal activation was measured by luminescence (RLU; Relative Luminescence Units). Cell activation was assayed in triplicate and data is presented as RLU relative to unstimulated control. Data is presented as mean ⁇ standard deviation and is representative of 4 independent experiments. Data was analyzed using a student's t test and asterisks represent statistically significant differences (*, p ⁇ 0.05; **, p ⁇ 0.01; ***, p 0.001).
- FIGS. 7 A-B Plasmid DNA-generated PD-L1-Fc suppresses T cell activation in vitro.
- A Purified CD4+ T cells were isolated from three C57BL/6 mice and pooled. Cells were seeded into a 96-well flat-bottomed plate that was pre-coated with anti-CD3 (0.2 ⁇ g/ml) alone or with 5 ⁇ g/ml serum free media produced PD-L1-Fc. Recombinant IgG1-Fc and recombinant PD-L1-Fc were used as controls. Cells were stimulated for 3 days, and cell activation was measured by detecting cell size in the FSC-SSC gate by flow cytometry.
- NIH/3T3 cells were transfected with wild-type PD-L1 plasmid DNA or control (pVax;) in a 6-well plate. Forty-eight hours post-transfection, cells were re-seeded in a 96-well v-bottom plate, and incubated with recombinant human PD-1 at various concentrations. Cells were washed and stained with APC-conjugated anti-human PD-1 or isotype control, and PD-1 binding was analyzed by flow cytometry by assessing APC detection on transfected cells. The binding of PE-conjugated anti-human PD-L1 or isotype control was also assessed.
- FIG. 7 A discloses “GGGGS” as SEQ ID NO: 5 and “(GGGGS)3” as SEQ ID NO: 8.
- FIGS. 8 A-B Disease model optimization.
- A Male Rag1 ⁇ / ⁇ mice were injected intraperitoneally with na ⁇ ve T cells, defined as CD4+CD25 ⁇ CD45RBhi or left untreated. Transfer of na ⁇ ve T cells induced disease as observed by the bodyweight loss.
- B To induce acute graft versus host disease (GvHD), female BALB/c mice were irradiated with 700 cGY and transferred with allogeneic C57Bl6/J bone marrow (10 M cells) combined with 2.5M splenocytes. GvHD was successfully induced using these conditions as observed by the initial bodyweight loss observed within the first 10 days. Furthermore, a secondary bodyweight drop was observed starting approximately on day 20. The no transplant groups confirmed the successful irradiation and the BMT control confirms successful transfer of the allogeneic bone marrow.
- FIGS. 9 A-C Therapeutic efficacy of PD-L1 and PD-L1 Fc polyplexes in GvHD.
- GvHD was induced as previously demonstrated in FIG. 8 B .
- Mice were either left untreated or injected by enema weekly for 7 weeks with pVAX, PD-L1 or PD-L1 Fc polyplexes at a concentration of c1000. Animals reaching 75% of their initial bodyweight were euthanized. The bodyweight of the animals was rescued with the administration of PD-L1 and PD-L1 Fc polyplexes.
- FIGS. 10 A-C Therapeutic efficacy of PD-L1 Fc and PD-L1 polyplexes in GvHD.
- A GvHD was induced as previously demonstrated in FIG. 8 B . Mice were either left untreated or injected by enema weekly for 7 weeks with pVAX, or PD-L1 Fc polyplexes at a concentration of c1000. Animals reaching 75% of their initial bodyweight were euthanized. The bodyweight of the animals was rescued with the administration of PD-L1 Fc polyplexes.
- GvHD was induced as previously demonstrated in FIG. 8 B .
- mice were either left untreated or injected by enema weekly for 7 weeks with pVAX, or PD-L1 polyplexes at a concentration of c1000. Animals reaching 75% of their initial bodyweight were euthanized. The bodyweight of the animals was rescued with the administration of PD-L1 polyplexes.
- C Survival curve of mice following the induction of GvHD, all pVAX treated animals succumb to disease, approximately 40% of the PD-L1 Fc and 30% of the PD-L1, treated animals survived until day 75.
- FIGS. 11 A-B Therapeutic effects of PD-L1 and PD-L1 Fc in GvHD.
- GvHD was induced as previously demonstrated in FIG. 8 B . Mice were either left untreated or injected by enema weekly for 7 weeks with pVAX, PD-L1 or PD-L1 Fc polyplexes at a concentration of c1000. The bodyweight of the PD-L1 and PD-L1 Fc treated animals was greatly improved compared with the no treatment and pVAX treated animals starting on day 18.
- FIGS. 12 A-B Administration of recombinant PD-L1 Fc in a DSS-model of colitis.
- A Figure taken from Song et al, Gut 2015.
- Rag1 ⁇ / ⁇ mice were administered 2% DSS in their drinking water and injected with recombinant PD-L1 Fc protein or PBS intraperitoneally on day 2. Injection of recombinant PD-L1 Fc lead to a less severe bodyweight loss compared with PBS injected animals.
- FIGS. 13 A-E Therapeutic efficacy of PD-L1 and PD-L1 Fc in a model of T cell colitis.
- A-B T cell colitis was induced as per FIG. 8 A . Starting on day 15 post-transfer, mice were injected with pVAX or PD-L1 c1000 polyplexes weekly for the following 6 weeks via enema or left untreated. Animals that succumbed to disease were given a fixed score of 75% of their initial bodyweight. Injection of PD-L1 polyplexes dampened disease progression as observed by the steady bodyweight throughout the study.
- B Survival curve of mice following the induction of T cell colitis demonstrates that none of the PD-L1 treated animals succumbed to disease.
- C-E T cell colitis was induced as per FIG. 8 A .
- the bodyweight (C) and clinical signs (D) of the animals were monitored two to three times per week and the bodyweight at day 0 was used as the baseline bodyweight (100%; day 0 is the first day of intracolonic instillation corresponding to day 19 post-transfer). Animals received weekly intracolonic instillations for 7 weeks starting on day 19 post-transfer.
- E Survival of the mice following the induction of T cell colitis. Animals were euthanized at 75% of their initial bodyweight or if the activity was severely decreased.
- the bodyweight of the euthanized mice was represented as 75% of their initial bodyweight for the remainder of the experiment.
- the clinical score of the euthanized mice was represented as the maximum score of 8 until the termination of the study.
- FIGS. 14 A-B Administration of PD-L1 polyplex induces the expression of FoxP3 in regulatory T cells.
- the mean fluorescence intensity (MFI) of FoxP3 was examined in regulatory T cells, defined as CD4+CD25+ FoxP3+ cells, at endpoint in mesenteric lymph nodes and spleen.
- the expression of FoxP3 was higher in PD-L1 treated animals in both the (A) mesenteric lymph node (MLN) and (B) spleen compared with the pVax group.
- the present invention contemplates the intestinal expression of PD-L1 nucleic acids for the treatment of inflammatory disorders.
- the PD-L1 polypeptides contemplated herein include the full-length sequence, soluble fragments, and variants thereof.
- Full-length PD-L1 proteins generally comprise a signal sequence, an IgV domain, and an IgC domain, a transmembrane domain, and a cytoplasmic domain.
- the human sequences are available to the public at the GenBank database under NM_014143.3 and NP_054862.1.
- the sequence of human PD-L1 transcript variant 1 is the canonical sequence, and all positional information described with respect to known isoforms are determined from this sequence.
- the signal sequence is shown from about amino acid 1 to about amino acid 18, the IgV domain is shown from about amino acid 19 to about amino acid 134, the IgC domain is shown from about amino acid 135 to about amino acid 227, the transmembrane domain is shown from about amino acid 239 to about amino acid 259, and the cytoplasmic domain is shown from about amino acid 260 to about amino acid 290.
- At least five transcript (i.e., splice) variants encoding different human PD-L1 isoforms are known and are described, e.g. in U.S. Patent Publication No. 2016/0122829, the disclosure of which is expressly incorporated by reference herein.
- Nucleic acid and amino acid sequence information for PD-L1 in multiple species is well known in the art and readily available in public databases, including, for example, monkey PD-L1 (NM 001083889.1 and NP_001077358.1), chimpanzee PD-L1 (XM_0011401705.2 and XP_001140705.1), mouse PD-L1 (NM 021893.3 and NP_068693.1), rat PD-L1 (NM_001191954.1 and NP_001178883.1), chicken PD-L1 (XM_424811.3 and XP_424811.3), cow PD-L1 (NM_001163412.1 and NP_001156884.1), and dog PD-L1 (XM_541302.3 and XP_541302.3).
- the PD-L1 nucleic acids of the subject invention will generally comprise a nucleic acid sequence encoding a human PD-L1 polypeptide, wherein the PD-L1 polypeptide preferably comprises an amino acid sequence of SEQ ID NO: 1 or an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 1, or fragments thereof.
- the PD-L1 polypeptide is membrane-bound, i.e. full-length PD-L1 including the transmembrane and cytoplasmic domains.
- the PD-L1 polypeptide is a soluble PD-L1 polypeptide comprising at least the IgV domain and IgC domains of human PD-L1, and optionally further comprising the signal sequence.
- the PD-L1 polypeptide comprises amino acids 1-239 of SEQ ID NO: 1, which corresponds to nucleotides 1-717 of the full-length cDNA sequence.
- the soluble PD-L1 polypeptide may lack all or part of the signal sequence (e.g. it may comprise amino acids 19-239 of SEQ ID NO: 1). See, e.g., U.S. Patent Publication No. 2017/0189476, the disclosure of which is expressly incorporated by reference herein.
- PD-L1 nucleic acid is identical to the native PD-L1 nucleic acid sequence (SEQ ID NO: 2) or a fragment thereof.
- at least one synonymous nucleic acid substitution is made to allow differentiation and detection of the resulting transcript from endogenous human PD-L1 nucleotide following administration of the subject nucleic acids, and still more preferably a plurality of such synonymous mutations are made.
- the PD-L1 nucleic acid sequence is codon-optimized to improve expression.
- the PD-L1 nucleic acid preferably comprises the nucleic acid sequence of SEQ ID NO: 3 or a sequence that is at least about 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 3.
- the PD-L1 polypeptides of the subject invention may be fused to an Fc region or portion thereof.
- the PD-L1 nucleic acid comprises the nucleic sequence of SEQ ID NO: 4 or a nucleic acid sequence that is at least about 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 4.
- the PD-L1 nucleic acids of the subject invention may include exons 1-4 in their entirety, as well as the first 35 nucleotides of exon 5. It is noted that the ATG site (start codon) is found at position 13 in exon 2. Therefore, the PD-L1 nucleic acids of the subject invention may include the last 52 nucleotides of exon 2, all of exon 3 and 4, as well as the first 35 nucleotides of exon 5.
- nucleotide sequences may code for a given amino acid sequence. Such nucleotide sequences are considered functionally equivalent since they result in the production of the same amino acid sequence in all organisms (although certain organisms may translate some sequences more efficiently than they do others). Moreover, occasionally, a methylated variant of a purine or pyrimidine may be found in a given nucleotide sequence. Such methylations do not affect the coding relationship between the trinucleotide codon and the corresponding amino acid.
- nucleotide sequence of a DNA or RNA coding for a fusion protein or polypeptide of the invention can be used to derive the fusion protein or polypeptide amino acid sequence, using the genetic code to translate the DNA or RNA into an amino acid sequence.
- corresponding nucleotide sequences that can encode the fusion protein or polypeptide can be deduced from the genetic code (which, because of its redundancy, will produce multiple nucleic acid sequences for any given amino acid sequence).
- description and/or disclosure herein of a polynucleotide sequence which encodes a fusion protein or polypeptide should be considered to also include description and/or disclosure of the amino acid sequence encoded by the nucleotide sequence.
- description and/or disclosure of a fusion protein or polypeptide amino acid sequence herein should be considered to also include description and/or disclosure of all possible nucleotide sequences that can encode the amino acid sequence.
- the PD-L1 proteins of the present invention do not contain the signal sequence as such a sequence is usually cleaved prior to secretion of the polypeptide from the cell.
- the PD-L1 proteins are soluble, i.e. consist of the IgV domain and the IgC domain (i.e., the extracellular portion of the full-length, membrane-bound PD-L1) and can further comprise heterologous sequences, such as Fc domains, protein tags, conjugated therapeutics, and the like.
- heterologous sequences such as Fc domains, protein tags, conjugated therapeutics, and the like.
- Such soluble PD-L1 isoforms can be generated by alternative splicing in a number of ways well known to the skilled artisan.
- the soluble PD-L1 comprises the elimination of part of exon 5 and the entirety of exons 6 and 7 of full-length, membrane-bound PD-L1 cDNA, as illustrated in FIG. 2 herein.
- the soluble PD-L1 isoforms can be generated by fusing the N terminal region of PD-L1 to human IgG1 Fc and, in some embodiments, can be linked by amino acid sequences (GGGGS)n (SEQ ID NO: 5).
- the IgG1 Fc can be mutated to reduce antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cellular cytotoxicity (CDCC) by altering the following amino acids in the Fc domain: E233P, L234V, L235A, deletion of G236, A327G, A330S and P331S.
- the Fc domain may be of human origin and may also be derived from the Fc of human IgG1.
- an expression vector comprising a PD-L1 nucleic acid so as to achieve effective intestinal expression can be accomplished using any method known in the art.
- administration of the subject nucleic acid delivery vehicles is localized to the gut (i.e. not systemically administered).
- the nucleic acid delivery vehicle comprises a viral vector.
- the nucleic acid delivery vehicle comprises a cationic liposome.
- the nucleic acid delivery vehicle comprises chitosan.
- biological gene delivery vehicles such as, e.g., gut bacteria, bacteriophage, virus-like particles, biological liposomes, or the like, to deliver a PD-L1 polynucleotide to gut epithelium.
- BGV biological gene delivery vehicles
- the nucleic acid delivery vehicle comprises a chitosan derivative, e.g., a chitosan that incorporates an additional functionalization, e.g., with an attached ligand.
- a chitosan derivative e.g., a chitosan that incorporates an additional functionalization, e.g., with an attached ligand.
- Chitosan as used herein will be understood to include the broad category of chitosan-based polymers comprising covalently modified N-acetyl-D-glucosamine and/or D-glucosamine units, as well as chitosan-based polymers incorporating other units, or attached to other moieties. Derivatives are frequently based on a modification of the hydroxyl group or the amine group of glucosamine, such as done with arginine-functionalized chitosan.
- chitosan derivatives include, but are not limited to, trimethylated chitosan, PEGylated chitosan, thiolated chitosan, galactosylated chitosan, alkylated chitosan, PEI-incorporated chitosan, uronic acid modified chitosan, glycol chitosan, and the like.
- chitosan derivatives see, for example, pp. 63-74 of “Non-viral Gene Therapy”, K. Taira, K. Kataoka, T.
- Chitosans with any degree of deacetylation (DDA) greater than 50% are used in the present invention, with functionalization between 1% and 50%. (Percent functionalization is determined relative to the number of free amino moieties on the chitosan polymer.)
- the degrees of deacetylation and functionalization impart a specific charge density to the functionalized chitosan derivative.
- the resulting charge density affects solubility, nucleic acid binding and subsequent release, and interaction with mammalian cell membranes. Thus, in accordance with the present invention, these properties must be optimized for optimal efficacy.
- Exemplary chitosan derivatives are described in Baker et al; Ser. No. 11/657,382 filed on Jan. 24, 2007, which is incorporated by reference herein by reference.
- the dually derivatized chitosan described herein comprises chitosan having a degree of deacetylation of at least 50%. In one embodiment, the degree of deacetylation is at least 60%, more preferably at least 70%, more preferably at least 80%, more preferably at least 90%, and most preferably at least 95%. In a preferred embodiment, the dually derivatized chitosan described herein comprises chitosan having a degree of deacetylation of at least 98%.
- the chitosan derivatives described herein have a range of average molecular weights that are soluble at neutral and physiological pH, and include for the purposes of this invention molecular weights ranging from 3-110 kDa.
- Embodiments described herein feature lower average molecular weight of derivatized chitosans ( ⁇ 25 kDa, e.g., from about 5 kDa to about 25 kDa), which can have desirable delivery and transfection properties, and are small in size and have favorable solubility.
- a lower average molecular weight derivatized chitosan is generally more soluble than one with a higher molecular weight, the former thus producing a nucleic acid/chitosan complex that will release more easily the nucleic acid and provide increased transfection of cells.
- administering is accomplished using chitosan or a chitosan derivative as the nucleic acid delivery vehicle.
- the chitosan derivatives described herein are generated by functionalizing the resulting free amino groups with positively charged and/or hydrophilic moieties.
- PCT/CA2013/050218 and PCT/CA2014/050921 have a number of properties which are advantageous for a nucleic acid delivery vehicle including: they effectively bind and complex the negatively charged nucleic acids, they can be formed into nanoparticles of a controllable size, they can be taken up by the cells and they can release the nucleic acids at the appropriate time within the cells.
- dually derivatized-chitosan or “DD-chitosan” is used, which refers to chitosan that has been dually functionalized (“dually functionalized-chitosan” or “DF-chitosan), e.g., coupled with both a Arg and a hydrophilic polyol, both of which are covalently attached to chitosan.
- the Arg may be covalently attached to chitosan either as single amino acid or as a polypeptide.
- the hydrophilic polyol may be a sugar such as glucose.
- DD-chitosan nucleic acid polyplex or its grammatical equivalents is meant a complex comprising a plurality of DD-chitosan molecules and a plurality of nucleic acid molecules coding for PD-L1 or fragments thereof.
- the dually derivatized-chitosan is complexed with said nucleic acid coding for a PD-L1 polypeptide.
- the DD-chitosan PD-L1 nucleic acid polyplexes comprise a PD-L1 nucleic acid component and a DD-chitosan component.
- Chitosan, and DD-chitosan nucleic acid polyplexes may be prepared by any method known in the art. For example, functionalized chitosan and nucleotide feedstock concentrations may be adjusted to accommodate various amine-to-phosphate ratios (N/P), mixing ratios and target nucleotide concentrations.
- N/P amine-to-phosphate ratios
- a preferred method for polyplex formation is disclosed in WO 2009/039657, which is expressly incorporated herein in its entirety by reference.
- the present invention relates to expression vectors comprising a PD-L1 nucleic acid, a promoter, and transcriptional and translational stop signals.
- the various nucleotide and control sequences may be joined together to produce an expression vector that may include one or more convenient restriction sites to allow for insertion or substitution of the nucleic acid encoding for PD-L1 at such sites.
- the PD-L1 nucleic acid may be expressed by inserting the polynucleotide or a nucleic acid construct comprising the PD-L1 nucleic acid into an appropriate vector for expression.
- the coding sequence for PD-L1 is located in the vector such that it is operably linked with the appropriate control sequences for expression.
- the expression vector may be any vector (e.g., a plasmid or virus) that can be conveniently subjected to recombinant DNA procedures and can bring about expression of the nucleic acid.
- the choice of the vector will typically depend on the compatibility of the vector with the host cell into which the vector is to be introduced.
- the vector may be a linear or closed circular plasmid.
- the vector may be an autonomously replicating vector, i.e., a vector that exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g., a plasmid, an extrachromosomal element, a minichromosome, or an artificial chromosome.
- the vector may contain any means for assuring self-replication.
- the vector may be one that, when introduced into the host cell, is integrated into the genome and replicated together with the chromosome(s) into which it has been integrated.
- a single vector or plasmid or two or more vectors or plasmids that together contain the total nucleci acid to be introduced into the genome of the host cell, or a transposon may be used.
- the vector may optionally include one or more selectable markers that permit easy selection of transformed, transfected, transduced, or the like cells.
- a selectable marker is a gene the product of which provides for biocide or viral resistance, resistance to heavy metals, prototrophy to auxotrophs, and the like.
- the vector may further comprise an origin of replication enabling the vector to replicate autonomously in the host cell in question.
- the origin of replication may be any plasmid replicator mediating autonomous replication that functions in a cell.
- the term “origin of replication” or “plasmid replicator” means a polynucleotide that enables a plasmid or vector to replicate in vivo.
- the expression vectors of the present invention comprise a nucleic acid molecule encoding for PD-L1 or fragments thereof, comprising an expression control region operably linked to a coding region for a PD-L1 polypeptide as described herein.
- the nucleic acid is DNA or RNA, e.g., mRNA.
- the PD-L1 nucleic acid is an artificial nucleic acid.
- Preferred artificial nucleic acids include, but are not limited to, peptide nucleic acid (PNA), phosphorodiamidate morpholino oligo (PMO), locked nucleic acid (LNA), glycol nucleic acid (GNA) and threose nucleic acid (TNA).
- PNA peptide nucleic acid
- PMO phosphorodiamidate morpholino oligo
- LNA locked nucleic acid
- GNA glycol nucleic acid
- TAA threose nucleic acid
- the expression vector is pVAX_hPD-L1 as exemplified herein ( FIG. 1 ). In another specific embodiment, the expression vector is pVax_hPD-L1 Fc ( FIG. 2 ).
- the expression control region possesses constitutive activity. In a number of preferred embodiments, the expression control region does not have constitutive activity. This provides for the dynamic expression of the PD-L1 nucleic acid.
- dynamic expression is meant expression that changes over time. Dynamic expression may include several such periods of low or absent expression separated by periods of detectable expression.
- the PD-L1 nucleic acid is operably linked to a regulatable promoter. This provides for the regulatable expression of the nucleic acid molecule.
- Expression control regions comprise regulatory polynucleotides (sometimes referred to herein as elements), such as promoters and enhancers, which influence expression of an operably linked PD-L1 nucleic acid.
- Expression control elements included herein can be from bacteria, yeast, plant, or animal (mammalian or non-mammalian). Expression control regions include full-length promoter sequences, such as native promoter and enhancer elements, as well as subsequences or polynucleotide variants that retain all or part of full-length or non-variant function (e.g., retain some amount of nutrient regulation or cell/tissue-specific expression).
- the term “functional” and grammatical variants thereof, when used in reference to a nucleic acid sequence, subsequence or fragment means that the sequence has one or more functions of native nucleic acid sequence (e.g., non-variant or unmodified sequence).
- variant means a sequence substitution, deletion, or addition, or other modification (e.g., chemical derivatives such as modified forms resistant to nucleases).
- operable linkage refers to a physical juxtaposition of the components so described as to permit them to function in their intended manner.
- the relationship is such that the control element modulates expression of the nucleic acid.
- an expression control region that modulates transcription is juxtaposed near the 5′ end of the transcribed nucleic acid (i.e., “upstream”).
- Expression control regions can also be located at the 3′ end of the transcribed sequence (i.e., “downstream”) or within the transcript (e.g., in an intron).
- Expression control elements can be located at a distance away from the transcribed sequence (e.g., 100 to 500, 500 to 1000, 2000 to 5000, or more nucleotides from the nucleic acid).
- a specific example of an expression control element is a promoter, which is usually located 5′ of the transcribed sequence.
- Another example of an expression control element is an enhancer, which can be located 5′ or 3′ of the transcribed sequence, or within the transcribed sequence.
- Some expression control regions confer regulatable expression to an operably linked PD-L1 nucleic acid.
- a signal (sometimes referred to as a stimulus) can increase or decrease expression of a PD-L1 nucleic acid operably linked to such an expression control region.
- Such expression control regions that increase expression in response to a signal are often referred to as inducible.
- Such expression control regions that decrease expression in response to a signal are often referred to as repressible.
- the amount of increase or decrease conferred by such elements is proportional to the amount of signal present; the greater the amount of signal, the greater the increase or decrease in expression.
- Preferred inducible expression control regions include those comprising an inducible promoter that is stimulated with a small molecule chemical compound.
- an expression control region is responsive to a chemical that is orally deliverable but not normally found in food. Particular examples can be found, for example, in U.S. Pat. Nos. 5,989,910; 5,935,934; 6,015,709; and 6,004,941, all of which are incorporated herein by reference in their entirety.
- the expression vector further comprises an integration sequence.
- the expression vector comprises a single integration sequence.
- the expression vector comprises a first and a second integration sequence for integrating the PD-L1 nucleic acid or a portion thereof into the genome of a target cell.
- the integration sequence(s) is functional in combination with a means for integration that is selected from the group consisting of mariner, sleeping beauty, FLP, Cre, ⁇ C31, R, lambda, and means for integration from integrating viruses such as AAV, retroviruses, and lentiviruses.
- the subject composition further comprises a non-therapeutic construct in addition to the PD-L1 construct, wherein the non-therapeutic construct comprises a nucleic acid sequence encoding a means for integration operably linked to a second expression control region.
- This second expression control region and the expression control region operably linked to the PD-L1 nucleic acid may be the same or different.
- the encoded means for integration is preferably selected from the group consisting of mariner, sleeping beauty, FLP, Cre, ⁇ C31, R, lambda, and means for integration from integrating viruses such as AAV, retroviruses, and lentiviruses.
- the present invention also provides “pharmaceutically acceptable” or “physiologically acceptable” formulations comprising DD-chitosan nucleic acid polyplex compositions of the invention. Such formulations can be administered in vivo to a subject in order to practice treatment methods.
- the terms “pharmaceutically acceptable” and “physiologically acceptable” refer to carriers, diluents, excipients and the like that can be administered to a subject, preferably without producing excessive adverse side-effects (e.g., nausea, abdominal pain, headaches, etc.).
- Such preparations for administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions.
- compositions can be made from carriers, diluents, excipients, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with administration to a subject.
- Such formulations can be contained in a tablet (coated or uncoated), capsule (hard or soft), microbead, emulsion, powder, granule, crystal, suspension, syrup or elixir.
- Supplementary active compounds and preservatives, among other additives may also be present, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like.
- Excipients can include a salt, an isotonic agent, a serum protein, a buffer or other pH-controlling agent, an anti-oxidant, a thickener, an uncharged polymer, a preservative or a cryoprotectant.
- xcipients used in compositions of the invention may further include an isotonic agent and a buffer or other pH-controlling agent. These excipients may be added for the attainment of preferred ranges of pH (about 6.0-8.0) and osmolarity (about 50-400 mmol/L).
- suitable buffers are acetate, borate, carbonate, citrate, phosphate and sulfonated organic molecule buffer.
- Such buffers may be present in a composition in concentrations from 0.01 to 1.0% (w/v).
- An isotonic agent may be selected from any of those known in the art, e.g. mannitol, dextrose, glucose and sodium chloride, or other electrolytes.
- the isotonic agent is glucose or sodium chloride.
- the isotonic agents may be used in amounts that impart to the composition the same or a similar osmotic pressure as that of the biological environment into which it is introduced.
- the concentration of isotonic agent in the composition will depend upon the nature of the particular isotonic agent used and may range from about 0.1 to 10%.
- compositions of the invention may further contain a preservative.
- preservatives are polyhexamethylene-biguanidine, benzalkonium chloride, stabilized oxychloro complexes (such as those known as Purite®), phenylmercuric acetate, chlorobutanol, sorbic acid, chlorhexidine, benzyl alcohol, parabens, and thimerosal.
- compositions of the invention may also contain a cryopreservative agent.
- cryopreservatives are glucose, sucrose, mannitol, lactose, trehalose, sorbitol, colloidal silicon dioxide, dextran of molecular weight preferable below 100,000 g/mol, glycerol, and polyethylene glycols of molecular weights below 100,000 g/mol or mixtures thereof. Most preferred are glucose, trehalose and polyethylene glycol.
- cryopreservatives are present at concentrations from about 0.01 to 10%.
- a pharmaceutical formulation can be formulated to be compatible with its intended route of administration.
- a composition can be incorporated with excipients and used in the form of tablets, troches, capsules, e.g., gelatin capsules, or coatings, e.g., enteric coatings (Eudragit® or Sureteric®).
- Pharmaceutically compatible binding agents, and/or adjuvant materials can be included in oral formulations.
- the tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or other stearates; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or flavoring.
- a binder such as microcrystalline cellulose, gum tragacanth or gelatin
- an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch
- a lubricant such as magnesium stearate or other stearates
- a glidant such as colloidal silicon dioxide
- Formulations can also include carriers to protect the composition against rapid degradation or elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems.
- a controlled release formulation including implants and microencapsulated delivery systems.
- a time delay material such as glyceryl monostearate or glyceryl stearate alone, or in combination with a wax, may be employed.
- Suppositories and other rectally administrable formulations are also contemplated.
- rectal delivery see, for example, Song et al., Mucosal drug delivery: membranes, methodologies, and applications, Crit. Rev. Ther. Drug. Carrier Syst., 21:195-256, 2004; Wearley, Recent progress in protein and peptide delivery by noninvasive routes, Crit. Rev. Ther. Drug. Carrier Syst., 8:331-394, 1991.
- compositions may be administered orally.
- Oral administration may involve swallowing, so that the compound enters the gastrointestinal tract.
- Compositions of the invention may also be administered directly to the gastrointestinal tract.
- Syringes, endoscopes, cannulas, intubation tubes, catheters and other articles may be used for such administration.
- Formulations suitable for oral administration include solid formulations such as tablets, capsules, coated capsules containing particulates or coated particulates, liquids, or powders, lozenges (including liquid-filled), chews, multi- and nano-particulates, gels, films, ovules, and sprays.
- Liquid formulations include suspensions, solutions, syrups and elixirs. Liquid formulations may be prepared by the reconstitution of a solid.
- Tablet dosage forms generally contain a disintegrant.
- disintegrants include sodium starch glycolate, sodium carboxymethyl cellulose, calcium carboxymethyl cellulose, croscarmellose sodium, crospovidone, polyvinylpyrrolidone, methyl cellulose, microcrystalline cellulose, lower alkyl-substituted hydroxypropyl cellulose, starch, pregelatinised starch and sodium alginate.
- the disintegrant will comprise from 1 weight % to 25 weight %, preferably from 5 weight % to 20 weight % of the dosage form.
- Binders are generally used to impart cohesive qualities to a tablet formulation. Suitable binders include microcrystalline cellulose, gelatin, sugars, polyethylene glycol, natural and synthetic gums, polyvinylpyrrolidone, pregelatinised starch, hydroxypropyl cellulose and hydroxypropyl methylcellulose. Tablets may also contain diluents, such as lactose (monohydrate, spray-dried monohydrate, anhydrous and the like), mannitol, xylitol, dextrose, sucrose, sorbitol, microcrystalline cellulose, starch and dibasic calcium phosphate dihydrate.
- lactose monohydrate, spray-dried monohydrate, anhydrous and the like
- mannitol xylitol
- dextrose sucrose
- sorbitol microcrystalline cellulose
- starch dibasic calcium phosphate dihydrate
- Tablets may also optionally comprise surface active agents, such as sodium lauryl sulfate and polysorbate 80, and glidants such as silicon dioxide and talc.
- surface active agents such as sodium lauryl sulfate and polysorbate 80
- glidants such as silicon dioxide and talc.
- surface active agents may comprise from 0.2 weight % to 5 weight % of the tablet, and glidants may comprise from 0.2 weight % to 1 weight % of the tablet.
- Tablets also generally contain lubricants such as magnesium stearate, calcium stearate, zinc stearate, sodium stearyl fumarate, and mixtures of magnesium stearate with sodium lauryl sulphate.
- Lubricants generally comprise from 0.25 weight % to 10 weight %, preferably from 0.5 weight % to 3 weight % of the tablet.
- ingredients include anti-oxidants, colorants, flavoring agents, preservatives and taste-masking agents.
- Tablet blends may be compressed directly or by roller to form tablets. Tablet blends or portions of blends may alternatively be wet-, dry-, or melt-granulated, melt congealed, or extruded before tabletting.
- the final formulation may comprise one or more layers and may be coated or uncoated; it may even be encapsulated.
- Consumable oral films for human or veterinary use are typically pliable water-soluble or water-swellable thin film dosage forms which may be rapidly dissolving or mucoadhesive and typically comprise a film-forming polymer, a binder, a solvent, a humectant, a plasticiser, a stabiliser or emulsifier, a viscosity-modifying agent and a solvent. Some components of the formulation may perform more than one function.
- multiparticulate beads comprising a composition of the invention.
- ingredients include anti-oxidants, colorants, flavourings and flavour enhancers, preservatives, salivary stimulating agents, cooling agents, co-solvents (including oils), emollients, bulking agents, anti-foaming agents, surfactants and taste-masking agents.
- Films in accordance with the invention are typically prepared by evaporative drying of thin aqueous films coated onto a peelable backing support or paper. This may be done in a drying oven or tunnel, typically a combined coater dryer, or by freeze-drying or vacuuming.
- Solid formulations for oral administration may be formulated to be immediate and/or modified release.
- Modified release formulations include delayed-, sustained-, pulsed-, controlled-, targeted and programmed release.
- the compounds of the invention may be administered rectally or vaginally, for example, in the form of a suppository, pessary, or enema.
- Cocoa butter is a traditional suppository base, but various alternatives may be used as appropriate.
- Formulations for rectal/vaginal administration may be formulated to be immediate and/or modified release.
- Modified release formulations include delayed-, sustained-, pulsed-, controlled-, targeted and programmed release.
- the doses or “effective amount” for treating a subject are preferably sufficient to ameliorate one, several or all of the symptoms of the condition, to a measurable or detectable extent, although preventing or inhibiting a progression or worsening of the inflammatory disorder or condition, or a symptom, is a satisfactory outcome.
- the amount of PD-L1 protein produced to ameliorate a condition treatable by a method of the invention will depend on the condition and the desired outcome and can be readily ascertained by the skilled artisan. Appropriate amounts will depend upon the condition treated, the therapeutic effect desired, as well as the individual subject (e.g., the bioavailability within the subject, gender, age, etc.). The effective amount can be ascertained by measuring relevant physiological effects.
- the invention provides methods of treating non-human mammals, which involve administering a chitosan-based nanoparticle of the invention to a non-human mammal in need of treatment.
- the therapeutic polypeptide may inhibit the proliferation and differentiation of cells involved in an inflammatory response.
- These molecules can be used to treat inflammatory conditions, both chronic and acute conditions, including inflammation associated with infection (e.g. septic shock, sepsis, or systemic inflammatory response syndrome (SIRS)), ischemia-reperfusion injury, endotoxin lethality, inflammatory bowel disease (IBD), Crohn's disease, colitis, or resulting from over production of cytokines (e.g. TNF or IL-1.)
- Inflammatory disorders of particular interest for treatment in the present invention include, but are not limited to, Crohn's disease, and inflammatory bowel disease.
- a therapeutic composition of the present invention may also be used to treat and/or prevent organ rejection or graft-versus-host disease (GvHD).
- Organ rejection occurs by host immune cell destruction of the transplanted tissue through an immune response.
- an immune response is also involved in GvHD, but, in this case, the foreign transplanted immune cells destroy the host tissues.
- the administration of a therapeutic composition of the present invention that inhibits an immune response, particularly the proliferation, differentiation, or chemotaxis of T-cells may be an effective therapy in preventing organ rejection or GvHD.
- Plasmid contain a plasmid replication origin (pUC ori) under the control of a human cytomegalovirus immediate-early gene promoter (CMV) and with a kanamycin resistance gene (KAN). Designed plasmids were custom synthesized by DNA 2.0 (Newark, CA). Once received, DNA was reconstituted according to the manufacturer's recommendation. Briefly absorbent paper containing the DNA was put into a small 0.2 mL tube with perforated bottom (performed with 23G needle). The small tube was then put into a larger 1.5 mL tube.
- CMV human cytomegalovirus immediate-early gene promoter
- KAN kanamycin resistance gene
- E. coli DH5 ⁇ chemically competent bacteria. Briefly, to a 100 ⁇ l thawed E. coli DH5 ⁇ cells 10 ⁇ L of the eluted DNA was added in a sterile condition. After 30 mins incubation on ice, cells were heat shocked at 42 C for 30 secs and incubated on ice for 2 min.
- LB medium Luria-Bertani (LB) medium was added to the DH5 ⁇ transformed cells and incubated for 1 hour at 37 C under 180 rpm shaking. The culture is then plated on a LB-agar plate containing kanamycin (50 ⁇ g/ml) and incubated for 16 h at 37 C. The next day, a single isolated colony was used to inoculate 6 mL of LB Broth containing kanamycin (50 ⁇ g/ml). The culture was incubated on a shaker (180 rpm) at 37 C for 5-6 hours. The culture was subsequently used to inoculate 2.5 L of LB medium which was incubated at 37 C for 16 hours on shaker.
- LB Luria-Bertani
- Plasmid DNA was then isolated from this large bacterial culture using EndoFree Plasmid Giga Kit (QIAGEN) according to the manufacturer's instruction. Upon isolation PD-L1 and PD-L1-Fc DNA insert within the plasmid was verified using restriction enzymes and fragment size confirmed by agarose gel
- HEK-293T cells were seeded in 6-well tissue culture plates at a density of 800,000 cells per well in high glucose DMEM complete media (10% Fetal Bovine Serum, 50 Units/ml penicillin, 50 ⁇ g/ml streptomycin) and incubated overnight at 37° C. and 5% CO 2 .
- DMEM complete media 10% Fetal Bovine Serum, 50 Units/ml penicillin, 50 ⁇ g/ml streptomycin
- PD-L1-Fc polyplex was thawed briefly in a 37° C. water bath.
- Each polyplex was diluted in sterile water to a concentration of 12.5-200 ⁇ g/ml of DNA, corresponding to 0.5-8 ⁇ g of DNA per well, and kept on ice until transfection.
- HEK-293T supernatant was removed from each well and cells were gently washed with 2 ml of pre-warmed OptiMEM. 1 ml of pre-warmed OptiMEM was gently added to each well. 40 ⁇ l of diluted polyplex was added to the well and the plate was gently swirled to ensure proper mixing. The cells were incubated for 3 hours at 37° C. and 5% CO 2 . Following incubation, the cell culture medium was removed by pipetting and replaced with 2 ml of pre-warmed DMEM complete medium and the cells were incubated at 37° C. and 5% C02 for 48 hours.
- the cell culture medium was removed from wells transfected with PD-L1-Fc polyplex or untransfected cells as a control.
- Cell debris was removed from the supernatant by centrifugation (1500 rpm, 5 min, 4° C.) and the supernatant was stored at ⁇ 80° C. until analysis.
- cell culture supernatant was removed from the wells, the transfected cells were washed with cold PBS, and 500 l of lysis buffer (50 mM Tris pH 8.0, 1% TritonX-100, 100 mM NaCl, 1 mM EDTA, 10% glycerol plus cOmplete Protease Inhibitor Cocktail) was added to the wells.
- lysis buffer 50 mM Tris pH 8.0, 1% TritonX-100, 100 mM NaCl, 1 mM EDTA, 10% glycerol plus cOmplete Protease Inhibitor Cocktail
- the plate was left on ice for 2-3 minutes and cells were collected using a cell scraper, transferred to a 1.5 ml microcentrifuge tube and left on ice for 30 min. Cell debris was removed from the lysate by centrifugation (13000 rpm, 10 min, 4° C.) and total protein was measured by Lowry assay.
- the PD-L1 protein concentration was measured by ELISA, as per the manufacturer's protocol. Briefly, 96 well plates were coated with anti-human PD-L1 capture antibody and incubated overnight at 25° C. The following day, plates were washed with 0.05% Tween®20 in PBS, blocked with 1% bovine serum albumin (BSA) in PBS for 1 hour and followed by an additional wash step. Samples or standards were added to the plate and incubated at 25° C. for 2 hours. A biotinylated goat anti-human PD-L1 detection antibody was added to the wells for 2 hours, followed by washes and the addition of streptavidin-HRP for 20 min.
- BSA bovine serum albumin
- PD-L1 protein levels were calculated using lyophilized standard protein provided by the manufacturer. Protein samples were fitted to a standard 4 parameter logistic curve. In some instances, the PD-L1 protein levels were normalized to total protein in the transfected cells and represented as ng of PD-L1-Fc/mg of total cellular protein.
- HEK-293T were transfected with increasing amounts of DNA contained within the DD-X polymer.
- the quantification of PD-L1 protein showed a dose-dependent increase in hPD-L1-Fc expression with increasing amounts of pVAX-opt-hPD-L1-Fc plasmid DNA ( FIG. 4 A ).
- the EC50 was similar between independent transfections, although the maximum expression of PD-L1-Fc (ng/mg) showed some variability between transfections ( FIG. 4 B ).
- HEK293T cells were plated in T75 flasks at 5 ⁇ 10 6 cells/flask in DMEM (10% FBS, Multicell) and incubated at 37 C. The following day, cells were transfected with PD-L1-Fc plasmid DNA (9 ug/flask; 3 ml per flask) using Lipofectamine 2000 (Invitrogen), as recommended by the manufacturer, and incubated at 37° C. After 24 h, DMEM medium was exchanged with 12 ml EXCELL serum-free medium (Sigma Aldrich). After 48 h (72 h post transfection), supernatant was collected and stored at ⁇ 20° C. until purification.
- Fc-fused proteins were purified using Protein G HiTrap columns (GE), as recommended by the manufacturer. Buffer was then exchanged using PD-10 desalting columns, and protein was eluted in 4 fractions of PBS (Gibco). Presence of protein was detected by Coomassie stain. PDL1 concentration in each of the fractions was measured by ELISA (Duoset, R&D), as recommended by the manufacturer.
- anti-mouse CD3 0.2 ⁇ g/ml, eBioscience
- anti-mouse CD28 1 ⁇ g/ml, eBioscience
- PDL1-Fc or commercial rhPDL1-Fc were added to wells of a 96-well flat-bottom plate in PBS (50 ⁇ l/well).
- rhIgG1-Fc (Adipogen) was used as a negative control.
- mice Jackson Laboratories
- Splenocytes were isolated by grinding the spleens on the frosted ends of microscope slides.
- Red blood cells were lysed for 1 min in 5 ml of in house made ACK lysis buffer, and the lysis was stopped by adding 45 ml PBS (10% FBS).
- PBS 50% FBS
- cells from the 3 mice were resuspended in supplemented RPMI medium (10% FBS, NEAA, L-glutamine, 2-ME; Multicell), pooled, counted, and adjusted to an appropriate concentration.
- a portion of cells were stained with CellTrace Violet (Life Technologies), as recommended by the manufacturer.
- the PBS was removed from the aCD3/aCD28 96-well plate carefully by pipetting. Cells were then seeded into the wells in the RPMI medium (200,000 cells/well), and incubated at 37 C for 3 or 4 days. At either time point, the cells were transferred to a new 96-well v-bottom plate and stained with a fixable viability dye (eBioscience) for 30 mins in the dark and on ice. Cells were washed twice with cold FACS buffer (PBS 2% FBS) and acquired on the flow cytometer (BD LSR II). Cell proliferation was measured by monitoring cell division peaks in the CellTrace Violet channel. Cell activation was measured by detecting cell size in the FSC-SSC gate.
- HEK-293T cells were plated in 30 ml DMEM (Multicell) complete (10% FBS, 100 U/ml Penicillin, 100 ⁇ g/ml Streptomycin) in a T175 flask and incubated overnight at 37° C. and 5% CO 2 . The following day, medium was aspirated from the flask and cells were washed once with 10 ml PBS (Multicell). Cells were transfected with 49 ⁇ g of PD-L1-Fc plasmid using Lipofectamine 2000 (Life Technologies; 100 ⁇ l per flask) in 5.5 ml of OptiMEM (Gibco). Cells were incubated (37° C.
- Buffer was exchanged for PBS using PD-10 desalting columns (GE) as recommended by the manufacturer.
- PD-L1-Fc was stored at ⁇ 80° C. until protein was concentrated and quantified.
- PD-L1-Fc protein was concentrated using Amicon Ultra-4 centrifugal filters (Millipore Sigma) as recommended by the manufacturer. Protein was immediately quantified by PD-L1 ELISA (R&D). In some cases, PD-L1-Fc concentration was diluted to 1 mg/mL in PBS and stored at ⁇ 80° C.
- the PathHunter cells were cultured as recommended by the manufacturer. Cells were centrifuged and re-suspended in pre-warmed AssayComplete Cell Plating Reagent. Cells were seeded into the wells of a white 96-well flat-bottomed tissue culture plate (50 ⁇ l/well; 20,000 cells/well) and incubated at room temperature for 15 min. AssayComplete Cell Plating Reagent (10 ⁇ l/well) was added to wells with or without anti-PD-1 (clone NAT105, Abcam).
- Purified PD-L1-Fc protein or recombinant non-lytic human IgG1-Fc (Adipogen) were prepared in AssayComplete Plating Reagent and added to the cells (50 ⁇ l/well). Cells were incubated for 40 min at 37° C. and 5% C02. PathHunter Bioassay Reagent 1 was added to the cells (10 ⁇ l/well) and the plate was placed on a plate shaker for 1 min at 350 rpm. Cells were incubated for 15 min at room temperature and in the dark. PathHunter Bioassay Reagent 2 (40 ⁇ l) was added to each well and cells were incubated for 1 hour at room temperature and in the dark.
- Luminescence was read on a luminometer (enSpire—Perkin Elmer). Data was analyzed as luminescence (RLU; Relative Fluorescence Units) relative to unstimulated controls. Data was presented as mean ⁇ standard deviation. Data was analyzed using a student's t test and asterisks represent statistically significant differences (*, p ⁇ 0.05; **, p ⁇ 0.01; ***, p ⁇ 0.001).
- NIH/3T3 cells were seeded (400,000 cells/well; 2 ml; DMEM 10% FBS) into a 6-well plate and incubated overnight at 37 C. The following day, cells were transfected with wild-type PD-L1 construct plasmid or pVax control with Lipofectamine 2000, as recommended by the manufacturer. Twenty-four hours after transfection, cells were washed with PBS, and resuspended by adding enzyme-free dissociation buffer (EFDB, Gibco; 2 ml/well), incubating at 37 C for 5 mins, and gently pipetting the cells off of the plate.
- EFDB enzyme-free dissociation buffer
- Cells were centrifuged at 1500 rpm for 5 mins and resuspended in DMEM (10% FBS). Cells were re-seeded (10,000 cells/well) into a 96-well flat-bottomed plate, and cultured for 2-5 days. At each time point, supernatant was removed, cells were resuspended in EFDB as described above, and cells were distributed into a 96-well v-bottomed plate for FACS staining. Cells were stained with anti-human PD-L1-PE (BioLegend) at 0.2 ng/well and a fixable viability dye eFluor 780 (eBioscience) as recommended by supplier for 30 mins on ice and in the dark. Cells were washed twice, resuspended in FACS buffer, and acquired on the flow cytometer.
- DMEM 50% FBS
- a PD-1 binding assay was employed.
- NIH/3T3 cells were transfected in 6-well plates as described above. Forty-eight hours after transfection, cells were resuspended in EFDB as described above, centrifuged, resuspended in FACS Buffer, and seeded into a 96-well v-bottomed plate. Cells were centrifuged at 1500 rpm for 5 mins, decanted, resuspended (50 ul) with or without rhPD-1 (R&D) at various concentrations, and incubated at 37 C for 30 mins.
- R&D rhPD-1
- mice 10 weeks old female BALB/c SPF mice (Charles River) received a total body irradiation of 700 cGy in split dose 2 ⁇ 350 cGy (RS2000 Biological Research X-ray source) first irradiation was given in the afternoon of day ⁇ 1 and the second in the morning of day 0. 8-10 weeks old male C57Bl6/j mice (Charles River) were used for donor cells.
- mice were treated once a week by enema for the following 6 weeks (for a total of 7 dosing) or left untreated. Animals were monitored daily for body weight and clinical signs. An animal with more than 25% body weight loss and or clinical scoring of 8 or more was terminated.
- mice that received PD-L1 or PD-L1-Fc also showed a reduction in the clinical signs associated with GvHD ( FIG. 9 B ) and had improved survival relative to untreated and pVAX control treated animals ( FIGS. 9 C, 10 C and 11 B ).
- T cells traffic through the gut, they are exposed to the PD-L1 polypeptide and become “tolerized.” Upon leaving the gut, the T cells go into circulation and can suppress effector T cell function.
- the tolerized T cells may result in upregulation of a regulatory T cell subset and/or a decrease or suppression of pathogenic effector cells.
- PD-L1 has been shown to upregulate molecules that are important for epithelial repair. Without being bound by theory, it is also contemplated herein that given that there is damage to the barrier function of the gastrointestinal tract following irradiation and disease, it follows that increased PD-L1 leading to increased epithelial repair may also help restore intestinal barrier function.
- CD4+ T cells were isolated from the spleens of 6-8-week-old female C57BL/6 mice via negative selection using the magnetic activation cell sorter (MACS) CD4 T-cell isolation kit (Miltenyi Biotec, Auburn, CA). Enriched cells were subsequently sorted for CD4+CD25-CD45RBhigh naive T cells using FACS (FACSAriaTM, BD Biosciences, San Jose, CA). 5 ⁇ 105 CD4+CD25-CD45RBhigh cells were transferred intraperitoneally (IP) to recipient 6-8-week-old female B10-RAG2-deficient mice (Jackson).
- IP intraperitoneally
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Health & Medical Sciences (AREA)
- Immunology (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Public Health (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- Veterinary Medicine (AREA)
- Zoology (AREA)
- Molecular Biology (AREA)
- Biochemistry (AREA)
- Wood Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Biotechnology (AREA)
- Biophysics (AREA)
- General Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Physics & Mathematics (AREA)
- Microbiology (AREA)
- Analytical Chemistry (AREA)
- Epidemiology (AREA)
- Transplantation (AREA)
- Cell Biology (AREA)
- Toxicology (AREA)
- Gastroenterology & Hepatology (AREA)
- Plant Pathology (AREA)
- Pathology (AREA)
- Nanotechnology (AREA)
- Optics & Photonics (AREA)
- Peptides Or Proteins (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
Provided herein are methods and compositions for the amelioration of inflammatory disorders comprising the intestinal expression of programmed death ligand 1.
Description
- This application is a continuation of U.S. patent application Ser. No. 16/348,834, filed May 9, 2019, which is a National Stage application of International Application No. PCT/CA2017/051340, filed Nov. 9, 2017, which claims the benefit of U.S. Provisional Application No. 62/419,899, filed Nov. 9, 2016. The entire contents of the above-identified applications are hereby fully incorporated herein by reference.
- The contents of the electronic sequence listing (189650.xml; Size: 22,074 bytes; and Date of Creation: Sep. 18, 2023) are herein incorporated by reference in their entireties.
- The present disclosure relates to methods and compositions for the amelioration of inflammatory disorders employing gene expression of PD-L1.
- Immune activation in general, and the T cell activation state in particular, are subject to complex overlapping regulatory mechanisms mediated by a host of costimulatory (e.g. CD28, ICOS, OX40, etc.) and co-inhibitory (e.g., CTLA-4, programmed death-1 (“PD-1”), etc.) molecules on T cells. Programmed death ligand-1 (“PD-L1”, also known as B7H1) is constitutively expressed by dendritic cells and other antigen presenting cells and via its interaction with PD-1 appears to mediate T cell down-regulation. Patsoukis et al., Selective effects of PD-1 on Akt and Ras pathways regulate molecular components of the cell cycle and inhibit T cell proliferation, Sci. Signal 5: ra46 (2012).
- Unfortunately, however, the effect of PD-L1 interaction with CD80 is less clear, as is the interaction between these two pathways. In some settings, blockade of PD-1 by way of soluble PD-L1 can potently enhance the T cell response, and enhance graft-versus-host disease (“GvHD”). Blazar et al., Blockade of Programmed Death-1 Engagement Accelerates Graft-Versus-Host Lethality by an IFN-γ-Dependent Mechanism J. Immunol. 171:1272-77 (2003). Conversely, other researchers were able to ameliorate GvHD with a soluble form of PD-L1 but only in a PD-L1 knock-out background. Deng et al. B7H1/CD80 Interaction Augments PD-1 Dependent T Cell Apoptosis and Ameliorates Graft-versus-Host Disease, J. Immunol. 194:560-74 (2105). Moreover, it appears that role of peripheral dendritic cells on the T cell response is more complicated than once thought. For example, while immature peripheral dendritic cells normally do not initiate T cell differentiation, populations of dendritic cells have been observed infiltrating the central nervous system (CNS) and facilitating epitope spreading in experimental autoimmune encephalomyelitis (EAE) mouse models. Spagnuolo et al., Involvement of Immune Regulation in Multiple Sclerosis, Immunology and Immunogenetics Insights 9:1-10.
- Accordingly, there is a clear need in the art to better understand the role of PD-L1 in T cell activation in immune protective settings, to better exploit the therapeutic potential of PD-L1 in inflammatory disorders.
- The present invention resolves the foregoing scientific uncertainties in the prior art by successfully employing localized intestinal expression of PD-L1 polypeptides, including soluble PD-L1 polypeptides, in an immune protective setting for the treatment of a variety of inflammatory disorders including, e.g., inflammatory bowel disease (e.g. ulcerative colitis, Crohn's disease, and the like) as well as graft-versus-host disease (GvHD) induced by organ or bone marrow transplant. In one aspect, therefore, the invention provides a method of treating an inflammatory disorder in a patient in need thereof, comprising administering to the intestinal tract of said patient an expression vector comprising a PD-L1 nucleic acid.
- In one embodiment, the PD-L1 polypeptide is a membrane-bound PD-L1 polypeptide. In preferred embodiments, the PD-L1 polypeptide is human PD-L1 or splice variants thereof. In one such embodiment, the PD-L1 nucleic acid encodes for a PD-L1 polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 1.
- In alternative embodiments, the PD-L1 polypeptide is a soluble PD-L1 polypeptide, e.g. comprises the signal sequence, IgV domain and IgC domain of PD-L1, and preferably human PD-L1 (i.e. amino acids 1-239 of SEQ ID NO: 1). In some embodiments, the soluble PD-L1 polypeptide may lack all or part of the signal sequence (e.g. it may comprise amino acids 19-238 of SEQ ID NO: 1).
- In some embodiments, the PD-L1 nucleic acid is delivered by an expression vector encapsulated in a nucleic acid delivery vehicle. Preferred nucleic acid delivery vehicles include chitosan or chitosan derivative nanoparticles. In one such embodiment, the chitosan derivative nanoparticles comprise chitosan coupled with arginine and/or gluconic acid. In another such embodiment, the chitosan derivative nanoparticles comprise chitosan coupled with arginine and a hydrophilic polyol. In one embodiment, the hydrophilic polyol is glucose.
- In another aspect, the invention provides expression vectors comprising a PD-L1 nucleic acid to treat an inflammatory disorder in a patient in need thereof, wherein said expression vector is administered to the intestine of said subject. In some embodiments, the PD-L1 nucleic acid comprises SEQ ID NO: 2. Preferably, the PD-L1 nucleic acid comprises at least one synonymous mutation in SEQ ID NO: 2. Still more preferably, the PD-L1 nucleic acid comprises a plurality of such mutations to assist in differentiation and, optionally, improved expression. In an exemplary embodiment, the PD-L1 nucleic acid comprises SEQ ID NO: 3.
- In some embodiments, the PD-L1 nucleic acid further comprises a heterologous sequence. The heterologous sequence may comprise an Fc domain, a protein tag, a conjugated therapeutic, or a combination thereof. In one embodiment, the N-terminal region of the PD-L1 polypeptide is fused to a human IgG1 Fc region or portion thereof. The PD-L1 polypeptide may be fused to a human IgG1 Fc region or portion thereof by way of an amino acid sequence of (GGGGS)n (SEQ ID NO: 5). In some embodiments, the IgG1 Fc is mutated to reduce antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cellular cytotoxicity (CDCC) by altering one or more of the following amino acids in the Fc domain: E233P, L234V, L235A, deletion of G236, A327G, A330S and P331S. In an exemplary embodiment, the PD-L1 nucleic acid comprises SEQ ID NO: 4.
- Inflammatory disorders that can be advantageously treated by way of the subject invention include both chronic and acute conditions, including inflammation associated with infection (e.g. septic shock, sepsis, or systemic inflammatory response syndrome (SIRS)), ischemia-reperfusion injury, endotoxin lethality, inflammatory bowel disease, Crohn's disease, colitis, or resulting from over production of cytokines (e.g. TNF or IL-1) as well as GvHD.
- All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.
- The present disclosure is disclosed with reference to the accompanying drawings, wherein:
-
FIG. 1 : The pVax-hPD-L1 vector (SEQ ID NO: 6). Plasmid sequence of the vector encoding a membrane-bound human PD-L1 polypeptide, the optimized human PD-L1 cDNA is highlighted. -
FIG. 2 : The pVax-hPD-L1 Fc vector (SEQ ID NO: 7). Plasmid sequence of the vector encoding a soluble human PD-L1 Fc polypeptide, the optimized human PD-L1 cDNA is highlighted. -
FIGS. 3A-B : Human PD-L1 plasmid and in vitro expression. (A) Plasmid map of the optimized hPD-L1 DNA sequences cloned in a pVax backbone containing a plasmid replication origin (pUC ori;) under the control of a human cytomegalovirus immediate-early gene promoter (CMV;) with a kanamycin resistance gene. (B) In vitro PD-L1 and PD-L1-Fc expression: HEK293T cells were transfected with 1 μg or 2.5 μg of Fc control (White), PD-L1-Fc (Black), or PD-L1 (Grey) plasmid DNA using Lipofectamine 2000 (Invitrogen). PD-L1 protein content in cell culture supernatant or cell lysate was quantified 48 h post-transfection. -
FIGS. 4A-B : In vitro expression of PD-L1-Fc from polyplex transfection. HEK-293T were transfected with PD-L1-Fc polyplexes containing increasing concentrations of pVAX-opt-hPD-L1-Fc DNA, as indicated. (A) Supernatants were collected at 48 hours post-transfection and assayed for hPD-L1-Fc protein by ELISA. Data were normalized to total cellular protein. (B) Table of EC50 and maximum protein expression. -
FIGS. 5A-D : Purification of in vitro-generated Fc-fused constructs and expression. (A) Protein expression confirmed by coomassie gel staining: HEK293T cells were transfected with Fc control or PD-L1-Fc plasmid DNA using Lipofectamine 2000 (Invitrogen). Cell culture medium was exchanged for serum-free medium 24 h post-transfection. Cell culture supernatant was collected 72 h post-transfection and Fc-fused proteins were purified using Protein G HiTrap columns (GE). Buffer was exchanged for PBS using PD-10 desalting columns (GE), and protein was eluted in four equal fractions (eluate 1-4). (B) HEK293T cells were transfected with Fc control or PD-L1-Fc plasmid DNA using Lipofectamine 2000 (Invitrogen). Cell culture medium was exchanged for serum-free medium 24 h post-transfection. Cell culture supernatant was collected 72 h post-transfection and Fc-fused proteins were purified using Protein G HiTrap columns (GE). Buffer was exchanged for PBS using PD-10 desalting columns (GE), and protein was eluted in four equal fractions (F1-4). PD-L1 protein concentration was determined by ELISA. (C) NIH/3T3 cells were transfected with wild-type PD-L1 plasmid DNA or control (pVax) using Lipofectamine 2000 (Invitrogen) in a 6-well plate. Cells were washed and re-seeded in a 96-well flat-bottomed plate 24 h post-transfection. The following day (Day 2), and on the 3 subsequent days, PD-L1 expression at the cell surface was analyzed by flow cytometry. (D) NIH/3T3 cells were transfected with wild-type PD-L1 plasmid DNA or control (pVax) using Lipofectamine 2000 (Invitrogen) in a 6-well plate. Cells were washed and re-seeded in a 96-well flat-bottomed plate 24 h post-transfection. The following day (Day 2), and on the 3 subsequent days, PD-L1 expression levels at the cell surface was analyzed by flow cytometry. -
FIGS. 6A-B : Expressed PD-L1-Fc signals through the human PD-1 receptor in a dose-dependent manner. Cells were transfected with PD-L1-Fc and purified from the cell culture supernatant. (A) PathHunter PD-1 Signaling Assay cells were stimulated with increasing concentrations of PD-L1-Fc (40 min) and signal activation was measured by luminescence (RLU; Relative Luminescence Units). (B) PathHunter PD-1 Signaling Assay cells were incubated with recombinant IgG1-Fc or PD-L1-Fc (50 μg/mL, 40 min) with and without anti-PD-1 (lo: 0.1 μg/mL, hi: 1.0 μg/mL) and signal activation was measured by luminescence (RLU; Relative Luminescence Units). Cell activation was assayed in triplicate and data is presented as RLU relative to unstimulated control. Data is presented as mean±standard deviation and is representative of 4 independent experiments. Data was analyzed using a student's t test and asterisks represent statistically significant differences (*, p<0.05; **, p<0.01; ***, p 0.001). -
FIGS. 7A-B : Plasmid DNA-generated PD-L1-Fc suppresses T cell activation in vitro. (A) Purified CD4+ T cells were isolated from three C57BL/6 mice and pooled. Cells were seeded into a 96-well flat-bottomed plate that was pre-coated with anti-CD3 (0.2 μg/ml) alone or with 5 μg/ml serum free media produced PD-L1-Fc. Recombinant IgG1-Fc and recombinant PD-L1-Fc were used as controls. Cells were stimulated for 3 days, and cell activation was measured by detecting cell size in the FSC-SSC gate by flow cytometry. (B) NIH/3T3 cells were transfected with wild-type PD-L1 plasmid DNA or control (pVax;) in a 6-well plate. Forty-eight hours post-transfection, cells were re-seeded in a 96-well v-bottom plate, and incubated with recombinant human PD-1 at various concentrations. Cells were washed and stained with APC-conjugated anti-human PD-1 or isotype control, and PD-1 binding was analyzed by flow cytometry by assessing APC detection on transfected cells. The binding of PE-conjugated anti-human PD-L1 or isotype control was also assessed. A reduction in the binding of anti-human PD-L1-PE is predicted to be the result of steric hindrance of antibody binding given that the recombinant PD-1 has bound to the PD-L1 expressed on the NIH/3T3 cells.FIG. 7A discloses “GGGGS” as SEQ ID NO: 5 and “(GGGGS)3” as SEQ ID NO: 8. -
FIGS. 8A-B : Disease model optimization. (A) Male Rag1−/− mice were injected intraperitoneally with naïve T cells, defined as CD4+CD25−CD45RBhi or left untreated. Transfer of naïve T cells induced disease as observed by the bodyweight loss. (B) To induce acute graft versus host disease (GvHD), female BALB/c mice were irradiated with 700 cGY and transferred with allogeneic C57Bl6/J bone marrow (10 M cells) combined with 2.5M splenocytes. GvHD was successfully induced using these conditions as observed by the initial bodyweight loss observed within the first 10 days. Furthermore, a secondary bodyweight drop was observed starting approximately onday 20. The no transplant groups confirmed the successful irradiation and the BMT control confirms successful transfer of the allogeneic bone marrow. -
FIGS. 9A-C : Therapeutic efficacy of PD-L1 and PD-L1 Fc polyplexes in GvHD. (A) GvHD was induced as previously demonstrated inFIG. 8B . Mice were either left untreated or injected by enema weekly for 7 weeks with pVAX, PD-L1 or PD-L1 Fc polyplexes at a concentration of c1000. Animals reaching 75% of their initial bodyweight were euthanized. The bodyweight of the animals was rescued with the administration of PD-L1 and PD-L1 Fc polyplexes. (B) The clinical signs of disease were reduced in PD-L1 or PD-L1-Fc treated mice and the average clinical signs observed onday 21 were also decreased in PD-L1 and PD-L1-Fc treated mice. (C) Survival curve of mice following the induction of GvHD, all untreated and pVAX treated animals succumb to disease, approximately 40% of the PD-L1 Fc and 30% of the PD-L1, treated animals survived untilday 75. -
FIGS. 10A-C : Therapeutic efficacy of PD-L1 Fc and PD-L1 polyplexes in GvHD. (A) GvHD was induced as previously demonstrated inFIG. 8B . Mice were either left untreated or injected by enema weekly for 7 weeks with pVAX, or PD-L1 Fc polyplexes at a concentration of c1000. Animals reaching 75% of their initial bodyweight were euthanized. The bodyweight of the animals was rescued with the administration of PD-L1 Fc polyplexes. (B) GvHD was induced as previously demonstrated inFIG. 8B . Mice were either left untreated or injected by enema weekly for 7 weeks with pVAX, or PD-L1 polyplexes at a concentration of c1000. Animals reaching 75% of their initial bodyweight were euthanized. The bodyweight of the animals was rescued with the administration of PD-L1 polyplexes. (C) Survival curve of mice following the induction of GvHD, all pVAX treated animals succumb to disease, approximately 40% of the PD-L1 Fc and 30% of the PD-L1, treated animals survived untilday 75. -
FIGS. 11A-B : Therapeutic effects of PD-L1 and PD-L1 Fc in GvHD. (A) GvHD was induced as previously demonstrated inFIG. 8B . Mice were either left untreated or injected by enema weekly for 7 weeks with pVAX, PD-L1 or PD-L1 Fc polyplexes at a concentration of c1000. The bodyweight of the PD-L1 and PD-L1 Fc treated animals was greatly improved compared with the no treatment and pVAX treated animals starting on day 18. (B) Survival curve of mice following the induction of GvHD, all pVAX treated as well as untreated animals succumb to disease. On the other hand, 50% of the animals treated with PD-L1 Fe and 25% of the animals administered with PD-L1 survive untilday 45. -
FIGS. 12A-B : Administration of recombinant PD-L1 Fc in a DSS-model of colitis. (A) Figure taken from Song et al, Gut 2015. Rag1−/− mice were administered 2% DSS in their drinking water and injected with recombinant PD-L1 Fc protein or PBS intraperitoneally onday 2. Injection of recombinant PD-L1 Fc lead to a less severe bodyweight loss compared with PBS injected animals. (B) Rag1−/− mice were administered 4% DSS in their drinking and injected intraperitoneally with recombinant PD-L1 Fc or PBS onday 3 andday 6. The bodyweight of the animals was equivalent throughout the study. However, no therapeutic effect was observed with PD-L1-Fc soluble protein and accordingly the Song et al. results were not reproduceable. -
FIGS. 13A-E : Therapeutic efficacy of PD-L1 and PD-L1 Fc in a model of T cell colitis. (A-B) T cell colitis was induced as perFIG. 8A . Starting onday 15 post-transfer, mice were injected with pVAX or PD-L1 c1000 polyplexes weekly for the following 6 weeks via enema or left untreated. Animals that succumbed to disease were given a fixed score of 75% of their initial bodyweight. Injection of PD-L1 polyplexes dampened disease progression as observed by the steady bodyweight throughout the study. (B) Survival curve of mice following the induction of T cell colitis demonstrates that none of the PD-L1 treated animals succumbed to disease. On the other hand, 1 pVAX treated animal and 2 untreated animals had to be euthanized. (C-E) T cell colitis was induced as perFIG. 8A . The bodyweight (C) and clinical signs (D) of the animals were monitored two to three times per week and the bodyweight atday 0 was used as the baseline bodyweight (100%;day 0 is the first day of intracolonic instillation corresponding to day 19 post-transfer). Animals received weekly intracolonic instillations for 7 weeks starting on day 19 post-transfer. (E) Survival of the mice following the induction of T cell colitis. Animals were euthanized at 75% of their initial bodyweight or if the activity was severely decreased. For graphing purposes, the bodyweight of the euthanized mice was represented as 75% of their initial bodyweight for the remainder of the experiment. Similarly, the clinical score of the euthanized mice was represented as the maximum score of 8 until the termination of the study. For (C-E), Sucrose n=8, pVAX n=9, PD-L1-Fc n=9. -
FIGS. 14A-B : Administration of PD-L1 polyplex induces the expression of FoxP3 in regulatory T cells. The mean fluorescence intensity (MFI) of FoxP3 was examined in regulatory T cells, defined as CD4+CD25+ FoxP3+ cells, at endpoint in mesenteric lymph nodes and spleen. The expression of FoxP3 was higher in PD-L1 treated animals in both the (A) mesenteric lymph node (MLN) and (B) spleen compared with the pVax group. - The present invention contemplates the intestinal expression of PD-L1 nucleic acids for the treatment of inflammatory disorders.
- The PD-L1 polypeptides contemplated herein include the full-length sequence, soluble fragments, and variants thereof.
- Full-length PD-L1 proteins generally comprise a signal sequence, an IgV domain, and an IgC domain, a transmembrane domain, and a cytoplasmic domain. The human sequences are available to the public at the GenBank database under NM_014143.3 and NP_054862.1. The sequence of human PD-
L1 transcript variant 1 is the canonical sequence, and all positional information described with respect to known isoforms are determined from this sequence. In this isoform, the signal sequence is shown from aboutamino acid 1 to about amino acid 18, the IgV domain is shown from about amino acid 19 to about amino acid 134, the IgC domain is shown from about amino acid 135 to about amino acid 227, the transmembrane domain is shown from about amino acid 239 to about amino acid 259, and the cytoplasmic domain is shown from about amino acid 260 to about amino acid 290. At least five transcript (i.e., splice) variants encoding different human PD-L1 isoforms are known and are described, e.g. in U.S. Patent Publication No. 2016/0122829, the disclosure of which is expressly incorporated by reference herein. - Nucleic acid and amino acid sequence information for PD-L1 in multiple species is well known in the art and readily available in public databases, including, for example, monkey PD-L1 (NM 001083889.1 and NP_001077358.1), chimpanzee PD-L1 (XM_0011401705.2 and XP_001140705.1), mouse PD-L1 (NM 021893.3 and NP_068693.1), rat PD-L1 (NM_001191954.1 and NP_001178883.1), chicken PD-L1 (XM_424811.3 and XP_424811.3), cow PD-L1 (NM_001163412.1 and NP_001156884.1), and dog PD-L1 (XM_541302.3 and XP_541302.3).
- The PD-L1 nucleic acids of the subject invention will generally comprise a nucleic acid sequence encoding a human PD-L1 polypeptide, wherein the PD-L1 polypeptide preferably comprises an amino acid sequence of SEQ ID NO: 1 or an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 1, or fragments thereof. In some embodiments, the PD-L1 polypeptide is membrane-bound, i.e. full-length PD-L1 including the transmembrane and cytoplasmic domains. In alternative embodiments, the PD-L1 polypeptide is a soluble PD-L1 polypeptide comprising at least the IgV domain and IgC domains of human PD-L1, and optionally further comprising the signal sequence. In one embodiment, the PD-L1 polypeptide comprises amino acids 1-239 of SEQ ID NO: 1, which corresponds to nucleotides 1-717 of the full-length cDNA sequence. In other embodiments, the soluble PD-L1 polypeptide may lack all or part of the signal sequence (e.g. it may comprise amino acids 19-239 of SEQ ID NO: 1). See, e.g., U.S. Patent Publication No. 2017/0189476, the disclosure of which is expressly incorporated by reference herein.
-
TABLE 1 Amino Acid sequence for human PD-L1 MRIFAVFIFMTYWHLLNAFTVTVPKDLYVVEYGSNMTIECKFPVEKQLD LAALIVYWEMEDKNIIQFVHGEEDLKVQHSSYRQRARLLKDQLSLGNAA LQITDVKLQDAGVYRCMISYGGADYKRITVKVNAPYNKINQRILVVDPV TSEHELTCQAEGYPKAEVIWTSSDHQVLSGKTTTTNSKREEKLFNVTST LRINTTTNEIFYCTFRRLDPEENHTAELVIPELPLAHPPNERTHLVILG AILLCLGVALTFIFRLRKGRMMDVKKCGIQDTNSKKQSDTHLEET - In some embodiments, PD-L1 nucleic acid is identical to the native PD-L1 nucleic acid sequence (SEQ ID NO: 2) or a fragment thereof. Preferably, at least one synonymous nucleic acid substitution is made to allow differentiation and detection of the resulting transcript from endogenous human PD-L1 nucleotide following administration of the subject nucleic acids, and still more preferably a plurality of such synonymous mutations are made. In particularly preferred embodiments, the PD-L1 nucleic acid sequence is codon-optimized to improve expression. In an exemplary embodiment, the PD-L1 nucleic acid preferably comprises the nucleic acid sequence of SEQ ID NO: 3 or a sequence that is at least about 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 3.
- In some embodiments, the PD-L1 polypeptides of the subject invention may be fused to an Fc region or portion thereof. In an exemplary embodiment, the PD-L1 nucleic acid comprises the nucleic sequence of SEQ ID NO: 4 or a nucleic acid sequence that is at least about 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 4.
- The PD-L1 nucleic acids of the subject invention may include exons 1-4 in their entirety, as well as the first 35 nucleotides of
exon 5. It is noted that the ATG site (start codon) is found at position 13 inexon 2. Therefore, the PD-L1 nucleic acids of the subject invention may include the last 52 nucleotides ofexon 2, all ofexon exon 5. -
TABLE 2 Nucleic Acid Sequence for human PD-L1 ATGAGGATATTTGCTGTCTTTATATTCATGACCTACTGGCATTTGCTGA ACGCATTTACTGTCACGGTTCCCAAGGACCTATATGTGGTAGAGTATGG TAGCAATATGACAATTGAATGCAAATTCCCAGTAGAAAAACAATTAGAC CTGGCTGCACTAATTGTCTATTGGGAAATGGAGGATAAGAACATTATTC AATTTGTGCATGGAGAGGAAGACCTGAAGGTTCAGCATAGTAGCTACAG ACAGAGGGCCCGGCTGTTGAAGGACCAGCTCTCCCTGGGAAATGCTGCA CTTCAGATCACAGATGTGAAATTGCAGGATGCAGGGGTGTACCGCTGCA TGATCAGCTATGGTGGTGCCGACTACAAGCGAATTACTGTGAAAGTCAA TGCCCCATACAACAAAATCAACCAAAGAATTTTGGTTGTGGATCCAGTC ACCTCTGAACATGAACTGACATGTCAGGCTGAGGGCTACCCCAAGGCCG AAGTCATCTGGACAAGCAGTGACCATCAAGTCCTGAGTGGTAAGACCAC CACCACCAATTCCAAGAGAGAGGAGAAGCTTTTCAATGTGACCAGCACA CTGAGAATCAACACAACAACTAATGAGATTTTCTACTGCACTTTTAGGA GATTAGATCCTGAGGAAAACCATACAGCTGAATTGGTCATCCCAGAACT ACCTCTGGCACATCCTCCAAATGAAAGGACTCACTTGGTAATTCTGGGA GCCATCTTATTATGCCTTGGTGTAGCACTGACATTCATCTTCCGTTTAA GAAAAGGGAGAATGATGGATGTGAAAAAATGTGGCATCCAAGATACAAA CTCAAAGAAGCAAAGTGATACACATTTGGAGGAGACGTAA (SEQ ID NO. 2) -
TABLE 3 Codon Optimized Nucleic Acid Sequence of Membrane-Bound PD-L1 ATGAGAATCTTCGCGGTGTTCATCTTCATGACCTACTGGCACCTCCTGA ACGCTTTCACTGTGACCGTGCCTAAGGACCTCTACGTCGTGGAATACGG CTCCAACATGACCATCGAGTGCAAATTCCCAGTGGAGAAGCAGCTGGAC CTGGCTGCCCTGATCGTGTACTGGGAAATGGAGGACAAGAACATCATCC AATTCGTGCATGGGGAGGAGGACCTGAAGGTCCAGCATTCGTCATATCG GCAAAGAGCCAGGCTGCTGAAGGATCAGCTGTCCCTCGGCAATGCGGCA CTGCAGATTACCGATGTGAAGCTGCAGGACGCCGGAGTCTACCGGTGCA TGATTTCCTACGGCGGAGCAGACTACAAGCGCATTACCGTGAAGGTCAA CGCTCCCTACAACAAGATCAACCAGCGGATTCTGGTGGTCGACCCTGTG ACCTCCGAGCATGAGCTGACCTGTCAAGCCGAAGGTTACCCGAAAGCGG AAGTGATCTGGACGTCGAGCGACCACCAGGTCTTGAGCGGAAAGACGAC CACTACTAACAGCAAGCGGGAAGAGAAACTGTTTAACGTGACCAGCACT CTTCGGATCAACACCACCACTAACGAGATTTTCTACTGTACCTTTCGCC GGCTTGACCCGGAAGAAAATCACACCGCCGAGCTCGTGATCCCCGAGCT GCCCCTCGCCCACCCTCCTAACGAAAGAACCCACCTGGTCATCTTGGGG GCCATCCTGCTGTGCCTGGGAGTGGCCCTGACCTTCATTTTTAGGCTCC GAAAGGGCCGCATGATGGACGTGAAGAAATGCGGAATCCAGGACACTAA CTCCAAGAAGCAGTCCGATACTCACCTGGAAGAAACCTAG (SEQ ID NO. 3) -
TABLE 4 Codon Optimized Nucleic Acid Sequence of Soluble PD-L1 Fused With an Fc Fragment ATGAGAATCTTCGCGGTGTTCATCTTCATGACCTACTGGCACCTCCTGA ACGCTTTCACTGTGACCGTGCCTAAGGACCTCTACGTCGTGGAATACGG CTCCAACATGACCATCGAGTGCAAATTCCCAGTGGAGAAGCAGCTGGAC CTGGCTGCCCTGATCGTGTACTGGGAAATGGAGGACAAGAACATCATCC AATTCGTGCATGGGGAGGAGGACCTGAAGGTCCAGCATTCGTCATATCG GCAAAGAGCCAGGCTGCTGAAGGATCAGCTGTCCCTCGGCAATGCGGCA CTGCAGATTACCGATGTGAAGCTGCAGGACGCCGGAGTCTACCGGTGCA TGATTTCCTACGGCGGAGCAGACTACAAGCGCATTACCGTGAAGGTCAA CGCTCCCTACAACAAGATCAACCAGCGGATTCTGGTGGTCGACCCTGTG ACCTCCGAGCATGAGCTGACCTGTCAAGCCGAAGGTTACCCGAAAGCGG AAGTGATCTGGACGTCGAGCGACCACCAGGTCTTGAGCGGAAAGACGAC CACTACTAACAGCAAGCGGGAAGAGAAACTGTTTAACGTGACCAGCACT CTTCGGATCAACACCACCACTAACGAGATTTTCTACTGTACCTTTCGCC GGCTTGACCCGGAAGAAAATCACACCGCCGAGCTCGTGATCCCCGAGCT GCCCCTCGCCCACCCTCCTAACGAAAGAACTCCCAAGTCTTGCGATAAG ACCCACACATGCCCGCCATGCCCAGCCCCGCCCGTGGCGGGCCCCTCCG TGTTTCTTTTCCCGCCGAAGCCTAAGGATACCCTGATGATCTCCCGCAC CCCCGAAGTCACTTGTGTGGTGGTGGACGTCAGCCACGAAGATCCGGAA GTCAAGTTCAATTGGTACGTGGACGGGGTCGAAGTGCACAACGCCAAGA CCAAGCCCCGCGAGGAACAGTACAACTCAACGTACCGGGTGGTGTCCGT GCTGACCGTGCTGCATCAGGACTGGCTGAACGGAAAGGAGTACAAGTGC AAAGTGTCCAACAAGGGACTGCCGAGCTCGATCGAAAAGACCATTTCGA AGGCCAAGGGGCAGCCTAGGGAGCCACAGGTCTATACCCTCCCGCCCTC ACGAGATGAACTGACCAAGAACCAAGTGTCATTGACTTGCCTCGTGAAG GGCTTCTACCCTTCCGACATCGCCGTGGAATGGGAATCCAACGGACAGC CGGAGAACAACTACAAGACTACTCCGCCCGTGCTTGACTCCGACGGTTC GTTCTTCCTGTACTCCAAGCTGACCGTGGATAAGTCCCGCTGGCAACAG GGCAACGTGTTCTCCTGCTCCGTGATGCACGAAGCCCTGCACAACCACT ACACCCAGAAGTCCCTCTCGTTGAGCCCTGGAAAATAG (SEQ ID NO. 4) - There is a known and definite correspondence between the amino acid sequence of a particular protein and the nucleotide sequences that can code for the protein, as defined by the genetic code. Likewise, there is a known and definite correspondence between the nucleotide sequence of a particular nucleic acid and the amino acid sequence encoded by that nucleic acid, as defined by the genetic code.
-
TABLE 3 GENETIC CODE Alanine (Ala, A) GCA, GCC, GCG, GCT Arginine (Arg, R) AGA, ACG, CGA, CGC, CGG, CGT Asparagine (Asn, N) AAC, AAT Aspartic acid (Asp, D) GAC, GAT Cysteine (Cys, C) TGC, TGT Glutamic acid (Glu, E) GAA, GAG Glutamine (Gln, Q) CAA, CAG Glycine (Gly, G) GGA, GGC, GGG, GGT Histidine (His, H) CAC, CAT Isoleucine (Ile, I) ATA, ATC, ATT Leucine (Leu, L) CTA, CTC, CTG, CTT, TTA, TTG Lysine (Lys, K) AAA, AAG Methionine (Met, M) ATG Phenylalanine (Phe, F) TTC, TTT Proline (Pro, P) CCA, CCC, CCG, CCT Serine (Ser, S) AGC, AGT, TCA, TCC, TCG, TCT Threonine (Thr, T) ACA, ACC, ACG, ACT Tryptophan (Trp, W) TGG Tyrosine (Tyr, Y) TAC, TAT Valine (Val, V) GTA, GTC, GTG, GTT - An important and well known feature of the genetic code is its redundancy, whereby, for most of the amino acids used to make proteins, more than one coding nucleotide triplet may be employed. Therefore, a number of different nucleotide sequences may code for a given amino acid sequence. Such nucleotide sequences are considered functionally equivalent since they result in the production of the same amino acid sequence in all organisms (although certain organisms may translate some sequences more efficiently than they do others). Moreover, occasionally, a methylated variant of a purine or pyrimidine may be found in a given nucleotide sequence. Such methylations do not affect the coding relationship between the trinucleotide codon and the corresponding amino acid.
- In view of the foregoing, the nucleotide sequence of a DNA or RNA coding for a fusion protein or polypeptide of the invention (or any portion thereof) can be used to derive the fusion protein or polypeptide amino acid sequence, using the genetic code to translate the DNA or RNA into an amino acid sequence. Likewise, for a fusion protein or polypeptide amino acid sequence, corresponding nucleotide sequences that can encode the fusion protein or polypeptide can be deduced from the genetic code (which, because of its redundancy, will produce multiple nucleic acid sequences for any given amino acid sequence). Thus, description and/or disclosure herein of a polynucleotide sequence which encodes a fusion protein or polypeptide should be considered to also include description and/or disclosure of the amino acid sequence encoded by the nucleotide sequence. Similarly, description and/or disclosure of a fusion protein or polypeptide amino acid sequence herein should be considered to also include description and/or disclosure of all possible nucleotide sequences that can encode the amino acid sequence.
- In some embodiments, the PD-L1 proteins of the present invention do not contain the signal sequence as such a sequence is usually cleaved prior to secretion of the polypeptide from the cell. In other embodiments, the PD-L1 proteins are soluble, i.e. consist of the IgV domain and the IgC domain (i.e., the extracellular portion of the full-length, membrane-bound PD-L1) and can further comprise heterologous sequences, such as Fc domains, protein tags, conjugated therapeutics, and the like. Such soluble PD-L1 isoforms can be generated by alternative splicing in a number of ways well known to the skilled artisan.
- In preferred embodiments, the soluble PD-L1 comprises the elimination of part of
exon 5 and the entirety ofexons FIG. 2 herein. The soluble PD-L1 isoforms can be generated by fusing the N terminal region of PD-L1 to human IgG1 Fc and, in some embodiments, can be linked by amino acid sequences (GGGGS)n (SEQ ID NO: 5). In preferred embodiments, the IgG1 Fc can be mutated to reduce antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cellular cytotoxicity (CDCC) by altering the following amino acids in the Fc domain: E233P, L234V, L235A, deletion of G236, A327G, A330S and P331S. The Fc domain may be of human origin and may also be derived from the Fc of human IgG1. - Administration of an expression vector comprising a PD-L1 nucleic acid so as to achieve effective intestinal expression can be accomplished using any method known in the art. In preferred embodiments, administration of the subject nucleic acid delivery vehicles is localized to the gut (i.e. not systemically administered). In one embodiment, the nucleic acid delivery vehicle comprises a viral vector. In another embodiment, the nucleic acid delivery vehicle comprises a cationic liposome. In preferred embodiments, the nucleic acid delivery vehicle comprises chitosan. In alternative embodiments, biological gene delivery vehicles (BGV) are used such as, e.g., gut bacteria, bacteriophage, virus-like particles, biological liposomes, or the like, to deliver a PD-L1 polynucleotide to gut epithelium. For a review of BGVs currently under investigation, see Seow and Wood, Biological Gene Delivery Vehicles Beyond Viral Vectors, Molecular Therapy 17:767-777 (2009), the contents of which are herein incorporated in their entirety.
- In particular embodiments, the nucleic acid delivery vehicle comprises a chitosan derivative, e.g., a chitosan that incorporates an additional functionalization, e.g., with an attached ligand. “Chitosan” as used herein will be understood to include the broad category of chitosan-based polymers comprising covalently modified N-acetyl-D-glucosamine and/or D-glucosamine units, as well as chitosan-based polymers incorporating other units, or attached to other moieties. Derivatives are frequently based on a modification of the hydroxyl group or the amine group of glucosamine, such as done with arginine-functionalized chitosan. Examples of chitosan derivatives include, but are not limited to, trimethylated chitosan, PEGylated chitosan, thiolated chitosan, galactosylated chitosan, alkylated chitosan, PEI-incorporated chitosan, uronic acid modified chitosan, glycol chitosan, and the like. For further teaching on chitosan derivatives, see, for example, pp. 63-74 of “Non-viral Gene Therapy”, K. Taira, K. Kataoka, T. Niidome (editors), Springer-Verlag Tokyo, 2005, ISBN 4-431-25122-7; Zhu et al., Chinese Science Bulletin, December 2007, vol. 52 (23), pp. 3207-3215; and Varma et al., Carbohydrate Polymers 55 (2004) 77-93
- Chitosans with any degree of deacetylation (DDA) greater than 50% are used in the present invention, with functionalization between 1% and 50%. (Percent functionalization is determined relative to the number of free amino moieties on the chitosan polymer.) The degrees of deacetylation and functionalization impart a specific charge density to the functionalized chitosan derivative. The resulting charge density affects solubility, nucleic acid binding and subsequent release, and interaction with mammalian cell membranes. Thus, in accordance with the present invention, these properties must be optimized for optimal efficacy. Exemplary chitosan derivatives are described in Baker et al; Ser. No. 11/657,382 filed on Jan. 24, 2007, which is incorporated by reference herein by reference. In one embodiment, the dually derivatized chitosan described herein comprises chitosan having a degree of deacetylation of at least 50%. In one embodiment, the degree of deacetylation is at least 60%, more preferably at least 70%, more preferably at least 80%, more preferably at least 90%, and most preferably at least 95%. In a preferred embodiment, the dually derivatized chitosan described herein comprises chitosan having a degree of deacetylation of at least 98%.
- The chitosan derivatives described herein have a range of average molecular weights that are soluble at neutral and physiological pH, and include for the purposes of this invention molecular weights ranging from 3-110 kDa. Embodiments described herein feature lower average molecular weight of derivatized chitosans (<25 kDa, e.g., from about 5 kDa to about 25 kDa), which can have desirable delivery and transfection properties, and are small in size and have favorable solubility. A lower average molecular weight derivatized chitosan is generally more soluble than one with a higher molecular weight, the former thus producing a nucleic acid/chitosan complex that will release more easily the nucleic acid and provide increased transfection of cells.
- In a preferred embodiment, administration of an expression vector comprising a PD-L1 nucleic acid is accomplished using chitosan or a chitosan derivative as the nucleic acid delivery vehicle. The chitosan derivatives described herein are generated by functionalizing the resulting free amino groups with positively charged and/or hydrophilic moieties. The derivatized chitosans described in International Patent Application Nos. PCT/CA2013/050218 and PCT/CA2014/050921, the contents of which are herein incorporated by reference in their entirety, have a number of properties which are advantageous for a nucleic acid delivery vehicle including: they effectively bind and complex the negatively charged nucleic acids, they can be formed into nanoparticles of a controllable size, they can be taken up by the cells and they can release the nucleic acids at the appropriate time within the cells.
- In preferred embodiments, “dually derivatized-chitosan” or “DD-chitosan” is used, which refers to chitosan that has been dually functionalized (“dually functionalized-chitosan” or “DF-chitosan), e.g., coupled with both a Arg and a hydrophilic polyol, both of which are covalently attached to chitosan. The Arg may be covalently attached to chitosan either as single amino acid or as a polypeptide. The hydrophilic polyol may be a sugar such as glucose. By “DD-chitosan nucleic acid polyplex” or its grammatical equivalents is meant a complex comprising a plurality of DD-chitosan molecules and a plurality of nucleic acid molecules coding for PD-L1 or fragments thereof. In a preferred embodiment, the dually derivatized-chitosan is complexed with said nucleic acid coding for a PD-L1 polypeptide.
- The DD-chitosan PD-L1 nucleic acid polyplexes comprise a PD-L1 nucleic acid component and a DD-chitosan component. Chitosan, and DD-chitosan nucleic acid polyplexes may be prepared by any method known in the art. For example, functionalized chitosan and nucleotide feedstock concentrations may be adjusted to accommodate various amine-to-phosphate ratios (N/P), mixing ratios and target nucleotide concentrations. A preferred method for polyplex formation is disclosed in WO 2009/039657, which is expressly incorporated herein in its entirety by reference.
- The present invention relates to expression vectors comprising a PD-L1 nucleic acid, a promoter, and transcriptional and translational stop signals. The various nucleotide and control sequences may be joined together to produce an expression vector that may include one or more convenient restriction sites to allow for insertion or substitution of the nucleic acid encoding for PD-L1 at such sites. Alternatively, the PD-L1 nucleic acid may be expressed by inserting the polynucleotide or a nucleic acid construct comprising the PD-L1 nucleic acid into an appropriate vector for expression. In creating the expression vector, the coding sequence for PD-L1 is located in the vector such that it is operably linked with the appropriate control sequences for expression.
- The expression vector may be any vector (e.g., a plasmid or virus) that can be conveniently subjected to recombinant DNA procedures and can bring about expression of the nucleic acid. The choice of the vector will typically depend on the compatibility of the vector with the host cell into which the vector is to be introduced. The vector may be a linear or closed circular plasmid. The vector may be an autonomously replicating vector, i.e., a vector that exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g., a plasmid, an extrachromosomal element, a minichromosome, or an artificial chromosome. The vector may contain any means for assuring self-replication. Alternatively, the vector may be one that, when introduced into the host cell, is integrated into the genome and replicated together with the chromosome(s) into which it has been integrated. Furthermore, a single vector or plasmid or two or more vectors or plasmids that together contain the total nucleci acid to be introduced into the genome of the host cell, or a transposon, may be used.
- The vector may optionally include one or more selectable markers that permit easy selection of transformed, transfected, transduced, or the like cells. A selectable marker is a gene the product of which provides for biocide or viral resistance, resistance to heavy metals, prototrophy to auxotrophs, and the like. For autonomous replication, the vector may further comprise an origin of replication enabling the vector to replicate autonomously in the host cell in question. The origin of replication may be any plasmid replicator mediating autonomous replication that functions in a cell. The term “origin of replication” or “plasmid replicator” means a polynucleotide that enables a plasmid or vector to replicate in vivo.
- In preferred embodiments, the expression vectors of the present invention comprise a nucleic acid molecule encoding for PD-L1 or fragments thereof, comprising an expression control region operably linked to a coding region for a PD-L1 polypeptide as described herein. In some embodiments, the nucleic acid is DNA or RNA, e.g., mRNA. In some embodiments, the PD-L1 nucleic acid is an artificial nucleic acid. Preferred artificial nucleic acids include, but are not limited to, peptide nucleic acid (PNA), phosphorodiamidate morpholino oligo (PMO), locked nucleic acid (LNA), glycol nucleic acid (GNA) and threose nucleic acid (TNA).
- In one specific embodiment, the expression vector is pVAX_hPD-L1 as exemplified herein (
FIG. 1 ). In another specific embodiment, the expression vector is pVax_hPD-L1 Fc (FIG. 2 ). - In some embodiments, the expression control region possesses constitutive activity. In a number of preferred embodiments, the expression control region does not have constitutive activity. This provides for the dynamic expression of the PD-L1 nucleic acid. By “dynamic” expression is meant expression that changes over time. Dynamic expression may include several such periods of low or absent expression separated by periods of detectable expression. In a number of preferred embodiments, the PD-L1 nucleic acid is operably linked to a regulatable promoter. This provides for the regulatable expression of the nucleic acid molecule. Expression control regions comprise regulatory polynucleotides (sometimes referred to herein as elements), such as promoters and enhancers, which influence expression of an operably linked PD-L1 nucleic acid.
- Expression control elements included herein can be from bacteria, yeast, plant, or animal (mammalian or non-mammalian). Expression control regions include full-length promoter sequences, such as native promoter and enhancer elements, as well as subsequences or polynucleotide variants that retain all or part of full-length or non-variant function (e.g., retain some amount of nutrient regulation or cell/tissue-specific expression). As used herein, the term “functional” and grammatical variants thereof, when used in reference to a nucleic acid sequence, subsequence or fragment, means that the sequence has one or more functions of native nucleic acid sequence (e.g., non-variant or unmodified sequence). As used herein, the term “variant” means a sequence substitution, deletion, or addition, or other modification (e.g., chemical derivatives such as modified forms resistant to nucleases).
- As used herein, the term “operable linkage” refers to a physical juxtaposition of the components so described as to permit them to function in their intended manner. In the example of an expression control element in operable linkage with a nucleic acid, the relationship is such that the control element modulates expression of the nucleic acid. Typically, an expression control region that modulates transcription is juxtaposed near the 5′ end of the transcribed nucleic acid (i.e., “upstream”). Expression control regions can also be located at the 3′ end of the transcribed sequence (i.e., “downstream”) or within the transcript (e.g., in an intron). Expression control elements can be located at a distance away from the transcribed sequence (e.g., 100 to 500, 500 to 1000, 2000 to 5000, or more nucleotides from the nucleic acid). A specific example of an expression control element is a promoter, which is usually located 5′ of the transcribed sequence. Another example of an expression control element is an enhancer, which can be located 5′ or 3′ of the transcribed sequence, or within the transcribed sequence.
- Some expression control regions confer regulatable expression to an operably linked PD-L1 nucleic acid. A signal (sometimes referred to as a stimulus) can increase or decrease expression of a PD-L1 nucleic acid operably linked to such an expression control region. Such expression control regions that increase expression in response to a signal are often referred to as inducible. Such expression control regions that decrease expression in response to a signal are often referred to as repressible. Typically, the amount of increase or decrease conferred by such elements is proportional to the amount of signal present; the greater the amount of signal, the greater the increase or decrease in expression.
- Numerous regulatable promoters are known in the art. Preferred inducible expression control regions include those comprising an inducible promoter that is stimulated with a small molecule chemical compound. In one embodiment, an expression control region is responsive to a chemical that is orally deliverable but not normally found in food. Particular examples can be found, for example, in U.S. Pat. Nos. 5,989,910; 5,935,934; 6,015,709; and 6,004,941, all of which are incorporated herein by reference in their entirety.
- In one embodiment, the expression vector further comprises an integration sequence. In one embodiment, the expression vector comprises a single integration sequence. In another embodiment, the expression vector comprises a first and a second integration sequence for integrating the PD-L1 nucleic acid or a portion thereof into the genome of a target cell. In a preferred embodiment, the integration sequence(s) is functional in combination with a means for integration that is selected from the group consisting of mariner, sleeping beauty, FLP, Cre, ΦC31, R, lambda, and means for integration from integrating viruses such as AAV, retroviruses, and lentiviruses.
- In one embodiment, the subject composition further comprises a non-therapeutic construct in addition to the PD-L1 construct, wherein the non-therapeutic construct comprises a nucleic acid sequence encoding a means for integration operably linked to a second expression control region. This second expression control region and the expression control region operably linked to the PD-L1 nucleic acid may be the same or different. The encoded means for integration is preferably selected from the group consisting of mariner, sleeping beauty, FLP, Cre, ΦC31, R, lambda, and means for integration from integrating viruses such as AAV, retroviruses, and lentiviruses.
- The procedures used to ligate the elements described above to construct the recombinant expression vectors of the present invention are well known to one skilled in the art see, e.g., Sambrook et al., 1989, supra). For further teaching, see WO2008020318, which is expressly incorporated herein in its entirety by reference.
- The present invention also provides “pharmaceutically acceptable” or “physiologically acceptable” formulations comprising DD-chitosan nucleic acid polyplex compositions of the invention. Such formulations can be administered in vivo to a subject in order to practice treatment methods.
- As used herein, the terms “pharmaceutically acceptable” and “physiologically acceptable” refer to carriers, diluents, excipients and the like that can be administered to a subject, preferably without producing excessive adverse side-effects (e.g., nausea, abdominal pain, headaches, etc.). Such preparations for administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions.
- Pharmaceutical formulations can be made from carriers, diluents, excipients, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with administration to a subject. Such formulations can be contained in a tablet (coated or uncoated), capsule (hard or soft), microbead, emulsion, powder, granule, crystal, suspension, syrup or elixir. Supplementary active compounds and preservatives, among other additives, may also be present, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like.
- Excipients can include a salt, an isotonic agent, a serum protein, a buffer or other pH-controlling agent, an anti-oxidant, a thickener, an uncharged polymer, a preservative or a cryoprotectant. xcipients used in compositions of the invention may further include an isotonic agent and a buffer or other pH-controlling agent. These excipients may be added for the attainment of preferred ranges of pH (about 6.0-8.0) and osmolarity (about 50-400 mmol/L). Examples of suitable buffers are acetate, borate, carbonate, citrate, phosphate and sulfonated organic molecule buffer. Such buffers may be present in a composition in concentrations from 0.01 to 1.0% (w/v). An isotonic agent may be selected from any of those known in the art, e.g. mannitol, dextrose, glucose and sodium chloride, or other electrolytes. Preferably, the isotonic agent is glucose or sodium chloride. The isotonic agents may be used in amounts that impart to the composition the same or a similar osmotic pressure as that of the biological environment into which it is introduced. The concentration of isotonic agent in the composition will depend upon the nature of the particular isotonic agent used and may range from about 0.1 to 10%. When glucose is used, it is preferably used in a concentration of from 1 to 5% w/v, more particularly 5% w/v. When the isotonic agent is sodium chloride, it is preferably employed in amounts of up to T % w/v, in particular 0.9% w/v. The compositions of the invention may further contain a preservative. Examples preservatives are polyhexamethylene-biguanidine, benzalkonium chloride, stabilized oxychloro complexes (such as those known as Purite®), phenylmercuric acetate, chlorobutanol, sorbic acid, chlorhexidine, benzyl alcohol, parabens, and thimerosal. Typically, such preservatives are present at concentrations from about 0.001 to 1.0%. Furthermore, the compositions of the invention may also contain a cryopreservative agent. Preferred cryopreservatives are glucose, sucrose, mannitol, lactose, trehalose, sorbitol, colloidal silicon dioxide, dextran of molecular weight preferable below 100,000 g/mol, glycerol, and polyethylene glycols of molecular weights below 100,000 g/mol or mixtures thereof. Most preferred are glucose, trehalose and polyethylene glycol. Typically, such cryopreservatives are present at concentrations from about 0.01 to 10%.
- A pharmaceutical formulation can be formulated to be compatible with its intended route of administration. For example, for oral administration, a composition can be incorporated with excipients and used in the form of tablets, troches, capsules, e.g., gelatin capsules, or coatings, e.g., enteric coatings (Eudragit® or Sureteric®). Pharmaceutically compatible binding agents, and/or adjuvant materials can be included in oral formulations. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or other stearates; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or flavoring.
- Formulations can also include carriers to protect the composition against rapid degradation or elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. For example, a time delay material such as glyceryl monostearate or glyceryl stearate alone, or in combination with a wax, may be employed.
- Suppositories and other rectally administrable formulations (e.g., those administrable by enema) are also contemplated. Further regarding rectal delivery, see, for example, Song et al., Mucosal drug delivery: membranes, methodologies, and applications, Crit. Rev. Ther. Drug. Carrier Syst., 21:195-256, 2004; Wearley, Recent progress in protein and peptide delivery by noninvasive routes, Crit. Rev. Ther. Drug. Carrier Syst., 8:331-394, 1991.
- Additional pharmaceutical formulations appropriate for administration are known in the art and are applicable in the methods and compositions of the invention (see, e.g., Remington's Pharmaceutical Sciences (1990) 18th ed., Mack Publishing Co., Easton, Pa.; The Merck Index (1996) 12th ed., Merck Publishing Group, Whitehouse, N.J.; and Pharmaceutical Principles of Solid Dosage Forms, Technonic Publishing Co., Inc., Lancaster, Pa., (1993)).
- The subject compositions may be administered orally. Oral administration may involve swallowing, so that the compound enters the gastrointestinal tract. Compositions of the invention may also be administered directly to the gastrointestinal tract. Syringes, endoscopes, cannulas, intubation tubes, catheters and other articles may be used for such administration.
- Formulations suitable for oral administration include solid formulations such as tablets, capsules, coated capsules containing particulates or coated particulates, liquids, or powders, lozenges (including liquid-filled), chews, multi- and nano-particulates, gels, films, ovules, and sprays.
Liquid formulations include suspensions, solutions, syrups and elixirs. Liquid formulations may be prepared by the reconstitution of a solid. - Tablet dosage forms generally contain a disintegrant. Examples of disintegrants include sodium starch glycolate, sodium carboxymethyl cellulose, calcium carboxymethyl cellulose, croscarmellose sodium, crospovidone, polyvinylpyrrolidone, methyl cellulose, microcrystalline cellulose, lower alkyl-substituted hydroxypropyl cellulose, starch, pregelatinised starch and sodium alginate. Generally, the disintegrant will comprise from 1 weight % to 25 weight %, preferably from 5 weight % to 20 weight % of the dosage form.
- Binders are generally used to impart cohesive qualities to a tablet formulation. Suitable binders include microcrystalline cellulose, gelatin, sugars, polyethylene glycol, natural and synthetic gums, polyvinylpyrrolidone, pregelatinised starch, hydroxypropyl cellulose and hydroxypropyl methylcellulose. Tablets may also contain diluents, such as lactose (monohydrate, spray-dried monohydrate, anhydrous and the like), mannitol, xylitol, dextrose, sucrose, sorbitol, microcrystalline cellulose, starch and dibasic calcium phosphate dihydrate.
- Tablets may also optionally comprise surface active agents, such as sodium lauryl sulfate and
polysorbate 80, and glidants such as silicon dioxide and talc. When present, surface active agents may comprise from 0.2 weight % to 5 weight % of the tablet, and glidants may comprise from 0.2 weight % to 1 weight % of the tablet. - Tablets also generally contain lubricants such as magnesium stearate, calcium stearate, zinc stearate, sodium stearyl fumarate, and mixtures of magnesium stearate with sodium lauryl sulphate. Lubricants generally comprise from 0.25 weight % to 10 weight %, preferably from 0.5 weight % to 3 weight % of the tablet.
- Other possible ingredients include anti-oxidants, colorants, flavoring agents, preservatives and taste-masking agents.
- Tablet blends may be compressed directly or by roller to form tablets. Tablet blends or portions of blends may alternatively be wet-, dry-, or melt-granulated, melt congealed, or extruded before tabletting. The final formulation may comprise one or more layers and may be coated or uncoated; it may even be encapsulated.
- The formulation of tablets is discussed in Pharmaceutical Dosage Forms: Tablets, Vol. 1, by H. Lieberman and L. Lachman (Marcel Dekker, New York, 1980).
- Consumable oral films for human or veterinary use are typically pliable water-soluble or water-swellable thin film dosage forms which may be rapidly dissolving or mucoadhesive and typically comprise a film-forming polymer, a binder, a solvent, a humectant, a plasticiser, a stabiliser or emulsifier, a viscosity-modifying agent and a solvent. Some components of the formulation may perform more than one function.
- Also included in the invention are multiparticulate beads comprising a composition of the invention.
- Other possible ingredients include anti-oxidants, colorants, flavourings and flavour enhancers, preservatives, salivary stimulating agents, cooling agents, co-solvents (including oils), emollients, bulking agents, anti-foaming agents, surfactants and taste-masking agents.
- Films in accordance with the invention are typically prepared by evaporative drying of thin aqueous films coated onto a peelable backing support or paper. This may be done in a drying oven or tunnel, typically a combined coater dryer, or by freeze-drying or vacuuming.
- Solid formulations for oral administration may be formulated to be immediate and/or modified release. Modified release formulations include delayed-, sustained-, pulsed-, controlled-, targeted and programmed release.
- Other suitable release technologies such as high energy dispersions and osmotic and coated particles are known.
- The compounds of the invention may be administered rectally or vaginally, for example, in the form of a suppository, pessary, or enema. Cocoa butter is a traditional suppository base, but various alternatives may be used as appropriate.
- Formulations for rectal/vaginal administration may be formulated to be immediate and/or modified release. Modified release formulations include delayed-, sustained-, pulsed-, controlled-, targeted and programmed release.
- The doses or “effective amount” for treating a subject are preferably sufficient to ameliorate one, several or all of the symptoms of the condition, to a measurable or detectable extent, although preventing or inhibiting a progression or worsening of the inflammatory disorder or condition, or a symptom, is a satisfactory outcome. Thus, the amount of PD-L1 protein produced to ameliorate a condition treatable by a method of the invention will depend on the condition and the desired outcome and can be readily ascertained by the skilled artisan. Appropriate amounts will depend upon the condition treated, the therapeutic effect desired, as well as the individual subject (e.g., the bioavailability within the subject, gender, age, etc.). The effective amount can be ascertained by measuring relevant physiological effects.
- Veterinary applications are also contemplated by the present invention. Accordingly, in one embodiment, the invention provides methods of treating non-human mammals, which involve administering a chitosan-based nanoparticle of the invention to a non-human mammal in need of treatment.
- The subject compositions and methods find advantageous use in modulating inflammation. For example, the therapeutic polypeptide may inhibit the proliferation and differentiation of cells involved in an inflammatory response. These molecules can be used to treat inflammatory conditions, both chronic and acute conditions, including inflammation associated with infection (e.g. septic shock, sepsis, or systemic inflammatory response syndrome (SIRS)), ischemia-reperfusion injury, endotoxin lethality, inflammatory bowel disease (IBD), Crohn's disease, colitis, or resulting from over production of cytokines (e.g. TNF or IL-1.) Inflammatory disorders of particular interest for treatment in the present invention include, but are not limited to, Crohn's disease, and inflammatory bowel disease.
- A therapeutic composition of the present invention may also be used to treat and/or prevent organ rejection or graft-versus-host disease (GvHD). Organ rejection occurs by host immune cell destruction of the transplanted tissue through an immune response. Similarly, an immune response is also involved in GvHD, but, in this case, the foreign transplanted immune cells destroy the host tissues. The administration of a therapeutic composition of the present invention that inhibits an immune response, particularly the proliferation, differentiation, or chemotaxis of T-cells, may be an effective therapy in preventing organ rejection or GvHD.
- The examples set out herein illustrate several embodiments of the present disclosure but should not be construed as limiting the scope of the present disclosure in any manner.
- Plasmid Construction and Production
- Both optimized PD-L1 and PD-L1-Fc DNA sequences were cloned in pVAX backbone cloning vector. Plasmid contain a plasmid replication origin (pUC ori) under the control of a human cytomegalovirus immediate-early gene promoter (CMV) and with a kanamycin resistance gene (KAN). Designed plasmids were custom synthesized by DNA 2.0 (Newark, CA). Once received, DNA was reconstituted according to the manufacturer's recommendation. Briefly absorbent paper containing the DNA was put into a small 0.2 mL tube with perforated bottom (performed with 23G needle). The small tube was then put into a larger 1.5 mL tube. 200 μL of water was added to the absorbent paper, after 1 min incubation @ RT, the tube was centrifuged at maximum speed for 1 min. Solubilized DNA was recovered in the 1.5 ml tube. The DNA was then used to transform E. coli DH5α, chemically competent bacteria. Briefly, to a 100 μl thawed E.
coli DH5α cells 10 μL of the eluted DNA was added in a sterile condition. After 30 mins incubation on ice, cells were heat shocked at 42 C for 30 secs and incubated on ice for 2 min. 30 μL of Luria-Bertani (LB) medium was added to the DH5α transformed cells and incubated for 1 hour at 37 C under 180 rpm shaking. The culture is then plated on a LB-agar plate containing kanamycin (50 μg/ml) and incubated for 16 h at 37 C. The next day, a single isolated colony was used to inoculate 6 mL of LB Broth containing kanamycin (50 μg/ml). The culture was incubated on a shaker (180 rpm) at 37 C for 5-6 hours. The culture was subsequently used to inoculate 2.5 L of LB medium which was incubated at 37 C for 16 hours on shaker. Plasmid DNA was then isolated from this large bacterial culture using EndoFree Plasmid Giga Kit (QIAGEN) according to the manufacturer's instruction. Upon isolation PD-L1 and PD-L1-Fc DNA insert within the plasmid was verified using restriction enzymes and fragment size confirmed by agarose gel - In Vitro PD-L1 and PD-L1-Fc Expression
- To measure in vitro expression from the polyplex, HEK-293T cells were seeded in 6-well tissue culture plates at a density of 800,000 cells per well in high glucose DMEM complete media (10% Fetal Bovine Serum, 50 Units/ml penicillin, 50 μg/ml streptomycin) and incubated overnight at 37° C. and 5% CO2. The following day, PD-L1-Fc polyplex was thawed briefly in a 37° C. water bath. Each polyplex was diluted in sterile water to a concentration of 12.5-200 μg/ml of DNA, corresponding to 0.5-8 μg of DNA per well, and kept on ice until transfection. HEK-293T supernatant was removed from each well and cells were gently washed with 2 ml of pre-warmed OptiMEM. 1 ml of pre-warmed OptiMEM was gently added to each well. 40 μl of diluted polyplex was added to the well and the plate was gently swirled to ensure proper mixing. The cells were incubated for 3 hours at 37° C. and 5% CO2. Following incubation, the cell culture medium was removed by pipetting and replaced with 2 ml of pre-warmed DMEM complete medium and the cells were incubated at 37° C. and 5% C02 for 48 hours. At 48 hours post-transfection, the cell culture medium was removed from wells transfected with PD-L1-Fc polyplex or untransfected cells as a control. Cell debris was removed from the supernatant by centrifugation (1500 rpm, 5 min, 4° C.) and the supernatant was stored at −80° C. until analysis.
- To quantify the total cellular protein, cell culture supernatant was removed from the wells, the transfected cells were washed with cold PBS, and 500 l of lysis buffer (50 mM Tris pH 8.0, 1% TritonX-100, 100 mM NaCl, 1 mM EDTA, 10% glycerol plus cOmplete Protease Inhibitor Cocktail) was added to the wells. The plate was left on ice for 2-3 minutes and cells were collected using a cell scraper, transferred to a 1.5 ml microcentrifuge tube and left on ice for 30 min. Cell debris was removed from the lysate by centrifugation (13000 rpm, 10 min, 4° C.) and total protein was measured by Lowry assay.
- The PD-L1 protein concentration was measured by ELISA, as per the manufacturer's protocol. Briefly, 96 well plates were coated with anti-human PD-L1 capture antibody and incubated overnight at 25° C. The following day, plates were washed with 0.05
% Tween® 20 in PBS, blocked with 1% bovine serum albumin (BSA) in PBS for 1 hour and followed by an additional wash step. Samples or standards were added to the plate and incubated at 25° C. for 2 hours. A biotinylated goat anti-human PD-L1 detection antibody was added to the wells for 2 hours, followed by washes and the addition of streptavidin-HRP for 20 min. The wells were washed and a tetramethylbenzidine solution was added for 20 min to detect HRP. The reaction was terminated by the addition of 2 N sulfuric acid and plates were read on a SpectraMAX Plus (ENG0214) using the SoftMax Pro software at 450 nm. PD-L1 protein levels were calculated using lyophilized standard protein provided by the manufacturer. Protein samples were fitted to a standard 4 parameter logistic curve. In some instances, the PD-L1 protein levels were normalized to total protein in the transfected cells and represented as ng of PD-L1-Fc/mg of total cellular protein. - To assess the transfection potency of PD-L1-Fc polyplexes, HEK-293T were transfected with increasing amounts of DNA contained within the DD-X polymer. The quantification of PD-L1 protein showed a dose-dependent increase in hPD-L1-Fc expression with increasing amounts of pVAX-opt-hPD-L1-Fc plasmid DNA (
FIG. 4A ). The EC50 was similar between independent transfections, although the maximum expression of PD-L1-Fc (ng/mg) showed some variability between transfections (FIG. 4B ). - HEK293T cells were plated in T75 flasks at 5×106 cells/flask in DMEM (10% FBS, Multicell) and incubated at 37 C. The following day, cells were transfected with PD-L1-Fc plasmid DNA (9 ug/flask; 3 ml per flask) using Lipofectamine 2000 (Invitrogen), as recommended by the manufacturer, and incubated at 37° C. After 24 h, DMEM medium was exchanged with 12 ml EXCELL serum-free medium (Sigma Aldrich). After 48 h (72 h post transfection), supernatant was collected and stored at −20° C. until purification. Fc-fused proteins were purified using Protein G HiTrap columns (GE), as recommended by the manufacturer. Buffer was then exchanged using PD-10 desalting columns, and protein was eluted in 4 fractions of PBS (Gibco). Presence of protein was detected by Coomassie stain. PDL1 concentration in each of the fractions was measured by ELISA (Duoset, R&D), as recommended by the manufacturer.
- In Vitro PD-L1-Fc Functionality Assay
- To assess functionality of Fc-fused PD-L1 construct, anti-mouse CD3 (0.2 μg/ml, eBioscience) and/or anti-mouse CD28 (1 μg/ml, eBioscience) with or without 5 μg/ml in vitro serum free media produced PDL1-Fc or commercial rhPDL1-Fc (Adipogen) were added to wells of a 96-well flat-bottom plate in PBS (50 μl/well). rhIgG1-Fc (Adipogen) was used as a negative control.
- The plate was put on plate-shaker on medium speed for 1 h and then incubated overnight at 4 C. The following day, spleens were removed from 3 mice (Jackson Laboratories). Splenocytes were isolated by grinding the spleens on the frosted ends of microscope slides. Red blood cells were lysed for 1 min in 5 ml of in house made ACK lysis buffer, and the lysis was stopped by adding 45 ml PBS (10% FBS). After centrifugation and decanting, cells from the 3 mice were resuspended in supplemented RPMI medium (10% FBS, NEAA, L-glutamine, 2-ME; Multicell), pooled, counted, and adjusted to an appropriate concentration. A portion of cells were stained with CellTrace Violet (Life Technologies), as recommended by the manufacturer. The PBS was removed from the aCD3/aCD28 96-well plate carefully by pipetting. Cells were then seeded into the wells in the RPMI medium (200,000 cells/well), and incubated at 37 C for 3 or 4 days. At either time point, the cells were transferred to a new 96-well v-bottom plate and stained with a fixable viability dye (eBioscience) for 30 mins in the dark and on ice. Cells were washed twice with cold FACS buffer (
PBS 2% FBS) and acquired on the flow cytometer (BD LSR II). Cell proliferation was measured by monitoring cell division peaks in the CellTrace Violet channel. Cell activation was measured by detecting cell size in the FSC-SSC gate. - To determine if opt-hPD-L1-Fc binds to the human PD-1 receptor and activates an intracellular signal, the PathHunter Jurkat PD-1 (SHP2) Signaling Assay (DiscoverX) was used. Prior to testing this assay, a large-scale batch of purified PD-L1-Fc protein was generated. Briefly, PD-L1-Fc was expressed from construct by transfecting HEK-293T cells with PD-L1-Fc plasmid DNA. Briefly, 3.9×105 HEK-293T cells were plated in 30 ml DMEM (Multicell) complete (10% FBS, 100 U/ml Penicillin, 100 μg/ml Streptomycin) in a T175 flask and incubated overnight at 37° C. and 5% CO2. The following day, medium was aspirated from the flask and cells were washed once with 10 ml PBS (Multicell). Cells were transfected with 49 μg of PD-L1-Fc plasmid using Lipofectamine 2000 (Life Technologies; 100 μl per flask) in 5.5 ml of OptiMEM (Gibco). Cells were incubated (37° C. and 5% CO2) for 2 h, and then 40 ml of DMEM complete was added to the flask. Cells were incubated for 24 h, medium was aspirated, and cells were washed with 10 ml PBS. EXCELL serum-free media (45 ml; Sigma) was added to the flask and cells were incubated at 37° C. and 5% CO2. After 24 h (48 h post-transfection), supernatant was removed and centrifuged for 5 min at 1500 rpm to remove cell debris. Supernatant was transferred to a new 50 ml tube and stored at −80° C. PD-L1-Fc was purified using Protein G HiTrap columns (GE) as recommended by the manufacturer. Buffer was exchanged for PBS using PD-10 desalting columns (GE) as recommended by the manufacturer. PD-L1-Fc was stored at −80° C. until protein was concentrated and quantified. PD-L1-Fc protein was concentrated using Amicon Ultra-4 centrifugal filters (Millipore Sigma) as recommended by the manufacturer. Protein was immediately quantified by PD-L1 ELISA (R&D). In some cases, PD-L1-Fc concentration was diluted to 1 mg/mL in PBS and stored at −80° C.
- To assess intracellular activation through the human PD-L1 receptor, the PathHunter cells were cultured as recommended by the manufacturer. Cells were centrifuged and re-suspended in pre-warmed AssayComplete Cell Plating Reagent. Cells were seeded into the wells of a white 96-well flat-bottomed tissue culture plate (50 μl/well; 20,000 cells/well) and incubated at room temperature for 15 min. AssayComplete Cell Plating Reagent (10 μl/well) was added to wells with or without anti-PD-1 (clone NAT105, Abcam). Purified PD-L1-Fc protein or recombinant non-lytic human IgG1-Fc (Adipogen) were prepared in AssayComplete Plating Reagent and added to the cells (50 μl/well). Cells were incubated for 40 min at 37° C. and 5% C02.
PathHunter Bioassay Reagent 1 was added to the cells (10 μl/well) and the plate was placed on a plate shaker for 1 min at 350 rpm. Cells were incubated for 15 min at room temperature and in the dark. PathHunter Bioassay Reagent 2 (40 μl) was added to each well and cells were incubated for 1 hour at room temperature and in the dark. Luminescence was read on a luminometer (enSpire—Perkin Elmer). Data was analyzed as luminescence (RLU; Relative Fluorescence Units) relative to unstimulated controls. Data was presented as mean±standard deviation. Data was analyzed using a student's t test and asterisks represent statistically significant differences (*, p≤0.05; **, p<0.01; ***, p<0.001). - The results of the foregoing are summarized here. To determine whether protein expressed from the pVAX-opt-hPD-L1-Fc plasmid will bind to the human PD-1 receptor and initiate intracellular signaling, the PathHunter Jurkat PD-1 (SHP2) Signaling Assay (DiscoverX) was used. Signaling through the PD-1 receptor increased with increasing doses of purified PDL1-Fc and appeared to plateau at the maximum dose of 14.7 μM (783 μg/ml;
FIG. 6A ). To ensure that this dose-dependent induction of luminescence was due to a PDL1-PD1-specific interaction, a competition assay was utilized (FIG. 6B ). Stimulation of the PathHunter cells with purified PDL1-Fc, but not recombinant IgG1-Fc, resulted in an increase in luminescence, indicating that signal induction was not caused by binding of PD-1 to the Fc fragment fused to the PD-L1 protein. Furthermore, PDL1-Fc-induced luminescence was inhibited when cells were incubated with anti-human PD-1 prior to stimulation with PD-L1-Fc, indicating that the binding and signaling through the PD-1 receptor was due to a PDL1-PD1-specific interaction. - PD-L1 Wild-Type Membrane Bound In Vitro Expression and Functionality Assay
- To determine hPD-L1 expression kinetic at the cell surface, NIH/3T3 cells were seeded (400,000 cells/well; 2 ml; DMEM 10% FBS) into a 6-well plate and incubated overnight at 37 C. The following day, cells were transfected with wild-type PD-L1 construct plasmid or pVax control with
Lipofectamine 2000, as recommended by the manufacturer. Twenty-four hours after transfection, cells were washed with PBS, and resuspended by adding enzyme-free dissociation buffer (EFDB, Gibco; 2 ml/well), incubating at 37 C for 5 mins, and gently pipetting the cells off of the plate. Cells were centrifuged at 1500 rpm for 5 mins and resuspended in DMEM (10% FBS). Cells were re-seeded (10,000 cells/well) into a 96-well flat-bottomed plate, and cultured for 2-5 days. At each time point, supernatant was removed, cells were resuspended in EFDB as described above, and cells were distributed into a 96-well v-bottomed plate for FACS staining. Cells were stained with anti-human PD-L1-PE (BioLegend) at 0.2 ng/well and a fixable viability dye eFluor 780 (eBioscience) as recommended by supplier for 30 mins on ice and in the dark. Cells were washed twice, resuspended in FACS buffer, and acquired on the flow cytometer. - To test functionality of the hPD-L1 expressed at the NIH/3T3 cell surface, a PD-1 binding assay was employed. NIH/3T3 cells were transfected in 6-well plates as described above. Forty-eight hours after transfection, cells were resuspended in EFDB as described above, centrifuged, resuspended in FACS Buffer, and seeded into a 96-well v-bottomed plate. Cells were centrifuged at 1500 rpm for 5 mins, decanted, resuspended (50 ul) with or without rhPD-1 (R&D) at various concentrations, and incubated at 37 C for 30 mins. Cells were centrifuged, washed twice with FACS buffer, and stained with anti-human PDL1-PE (0.2 ng/well) or isotype control, anti-human PD-1-APC (eBioscience) at 0.8 ng/well or isotype control, and a viability dye for 30 mins on ice and in the dark. Cells were then washed twice, resuspended in FACS buffer, and acquired using BD LSR II Flow Cytometer. Data were analyzed using FlowJo 10.2 [software (Tree Star, Or. CA)
- In Vivo Efficacy Studies
- GvHD Model
- 10 weeks old female BALB/c SPF mice (Charles River) received a total body irradiation of 700 cGy in
split dose 2×350 cGy (RS2000 Biological Research X-ray source) first irradiation was given in the afternoon of day −1 and the second in the morning ofday 0. 8-10 weeks old male C57Bl6/j mice (Charles River) were used for donor cells. - Study includes 3 control groups (n=5): non-irradiation group, no transplant group, 10 million BMT only group; and 4 treatment groups (n=8) receiving 10 million BMT and 2.5 million splenocytes. Starting from
day 1, mice were treated once a week by enema for the following 6 weeks (for a total of 7 dosing) or left untreated. Animals were monitored daily for body weight and clinical signs. An animal with more than 25% body weight loss and or clinical scoring of 8 or more was terminated. - Weekly intracolonic administration of the PD-L1 or PD-L1-Fc polyplex decreased the bodyweight loss compared to pVAX control treated animals (
FIGS. 9A, 10A, 10B and 11A ). Mice that received PD-L1 or PD-L1-Fc also showed a reduction in the clinical signs associated with GvHD (FIG. 9B ) and had improved survival relative to untreated and pVAX control treated animals (FIGS. 9C, 10C and 11B ). Without being bound by theory, it is contemplated herein that as T cells traffic through the gut, they are exposed to the PD-L1 polypeptide and become “tolerized.” Upon leaving the gut, the T cells go into circulation and can suppress effector T cell function. The tolerized T cells may result in upregulation of a regulatory T cell subset and/or a decrease or suppression of pathogenic effector cells. Moreover, PD-L1 has been shown to upregulate molecules that are important for epithelial repair. Without being bound by theory, it is also contemplated herein that given that there is damage to the barrier function of the gastrointestinal tract following irradiation and disease, it follows that increased PD-L1 leading to increased epithelial repair may also help restore intestinal barrier function. - T Cell Colitis Model
- Total CD4+ T cells were isolated from the spleens of 6-8-week-old female C57BL/6 mice via negative selection using the magnetic activation cell sorter (MACS) CD4 T-cell isolation kit (Miltenyi Biotec, Auburn, CA). Enriched cells were subsequently sorted for CD4+CD25-CD45RBhigh naive T cells using FACS (FACSAria™, BD Biosciences, San Jose, CA). 5×105 CD4+CD25-CD45RBhigh cells were transferred intraperitoneally (IP) to recipient 6-8-week-old female B10-RAG2-deficient mice (Jackson).
- Animals bodyweight and clinical signs were monitored 2-3/week. On day 14, presence of CD4 T cell was confirmed by flow cytometry staining for CD4. Weekly enema treatment started on
day 15 or 19 post T cell transfer (total of 6 or 7 treatments). Animals were monitored twice per week for body weight and clinical signs. An animal with more than 25% body weight loss or severe clinical signs is terminated. - Weekly administration of the PD-L1-Fc polyplex by intracolonic instillation decreased the bodyweight loss and clinical signs of disease in PD-L1-Fc treated mice compared to pVAX and sucrose control mice (
FIG. 13C-D ). Furthermore, treatment with the PD-L1-Fc polyplex improved the survival of the mice relative to control groups (FIG. 13E ). - While the present disclosure has been described with reference to preferred embodiments, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted for elements thereof to adapt to particular situations without departing from the scope of the present disclosure. Therefore, it is intended that the present disclosure not be limited to the particular embodiments disclosed as the best mode contemplated for carrying out the present disclosure, but that the present disclosure will include all embodiments falling within the scope and spirit of the appended claims.
Claims (23)
1.-13. (canceled)
14. An expression vector comprising a PD-L1 nucleic acid, wherein the sequence of said PD-L1 nucleic acid comprises at least one synonymous substitution compared to SEQ ID NO: 2 for detection after administration, and preferably a plurality of synonymous substitutions.
15. The expression vector according to claim 14 , wherein said PD-L1 nucleic acid sequence is codon-optimized for expression.
16. The expression vector according to claim 14 , wherein said PD-L1 nucleic acid further comprises a nucleic acid sequence that encodes a heterologous sequence, preferably wherein said heterologous sequence comprises an Fc domain, a protein tag, a conjugated therapeutic, or a combination thereof.
17. (canceled)
18. The expression vector according to claim 16 , wherein said Fc domain comprises a human IgG1 Fc region or a portion thereof.
19. The expression vector according to claim 18 , wherein said PD-L1 nucleic acid comprises the sequence of SEQ ID NO: 4 or SEQ ID NO: 3.
20.-24. (canceled)
25. A chitosan derivative nanoparticle comprising an encapsulated expression vector, wherein the expression vector comprises a PD-L1 nucleic acid encoding a human PD-L1 polypeptide for localized intestinal expression of the human PD-L1 polypeptide, wherein the sequence of the PD-L1 nucleic acid comprises a plurality of synonymous substitutions compared to SEQ ID NO 2 for detection after administration.
26. The chitosan derivative nanoparticle of claim 25 , comprising chitosan coupled with arginine and a hydrophilic polyol.
27. The chitosan derivative nanoparticle of claim 26 , wherein the hydrophilic polyol is glucose.
28. The chitosan derivative nanoparticle of claim 25 , wherein the PD-L1 nucleic acid comprises a nucleic acid sequence that is at least about 85% or 95% identical to SEQ ID NO: 3 or about 85% or 95% identical to SEQ ID NO: 4.
29. The chitosan derivative nanoparticle of claim 25 , wherein the PD-L1 polypeptide is a soluble human PD-L1 polypeptide comprising or consisting of the IgV domain and the IgC domain of human PD-L1 (amino acids 19-239 of SEQ ID NO: 1).
30. The chitosan derivative nanoparticle of claim 25 , wherein the PD-L1 polypeptide is a soluble human PD-L1 polypeptide comprising or consisting of the signal sequence, the IgV domain and the IgC domain of human PD-L1 (amino acids 1-239 of SEQ ID NO: 1).
31. The chitosan derivative nanoparticle of claim 25 , wherein the PD-L1 polypeptide is N-terminally fused to a human IgG1 Fc region.
32. The chitosan derivative nanoparticle of claim 31 , wherein the PD-L1 polypeptide is fused to the human IgG1 Fc region via an amino acid sequence of (GGGGS)n (SEQ ID NO: 5).
33. The chitosan derivative nanoparticle of claim 31 , wherein the human IgG1 Fc is mutated to reduce antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cellular cytotoxicity (CDCC) by altering one or more of the following amino acids in the Fc domain: E233P, L234V, L235A, deletion of G236, A327G, A330S and P331S.
34. The chitosan derivative nanoparticle of claim 33 , wherein the PD-L1 nucleic acid comprises the sequence of SEQ ID NO: 4.
35. The chitosan derivative nanoparticle of claim 25 , wherein the PD-L1 polypeptide is a membrane-bound PD-L1 polypeptide comprising or consisting of the signal sequence, IgV domain, IgC domain and the transmembrane domain of human PD-L1 (amino acids 1-259 of SEQ ID NO: 1).
36. The chitosan derivative nanoparticle of claim 35 , wherein the PD-L1 polypeptide further comprises the cytoplasmic domain of human PD-L1.
37. The chitosan derivative nanoparticle of claim 35 , wherein the PD-L1 nucleic acid comprises the sequence of SEQ ID NO: 3.
38. A method for treating an inflammatory disorder in a patient in need thereof, comprising administering the chitosan derivative nanoparticle of claim 25 to the gastrointestinal tract of the patient.
39. The method of claim 38 , wherein the inflammatory disorder is selected from the group consisting of inflammatory bowel disease, ulcerative colitis, and Crohns disease.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US18/121,557 US20230416337A1 (en) | 2016-11-09 | 2023-03-14 | Intestinal expression of programmed death ligand 1 |
Applications Claiming Priority (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US201662419899P | 2016-11-09 | 2016-11-09 | |
PCT/CA2017/051340 WO2018085935A1 (en) | 2016-11-09 | 2017-11-09 | Intestinal expression of programmed death ligand 1 |
US201916348834A | 2019-05-09 | 2019-05-09 | |
US18/121,557 US20230416337A1 (en) | 2016-11-09 | 2023-03-14 | Intestinal expression of programmed death ligand 1 |
Related Parent Applications (2)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US16/348,834 Continuation US11603398B2 (en) | 2016-11-09 | 2017-11-09 | Intestinal expression of programmed death ligand 1 |
PCT/CA2017/051340 Continuation WO2018085935A1 (en) | 2016-11-09 | 2017-11-09 | Intestinal expression of programmed death ligand 1 |
Publications (1)
Publication Number | Publication Date |
---|---|
US20230416337A1 true US20230416337A1 (en) | 2023-12-28 |
Family
ID=62110197
Family Applications (2)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US16/348,834 Active 2038-04-12 US11603398B2 (en) | 2016-11-09 | 2017-11-09 | Intestinal expression of programmed death ligand 1 |
US18/121,557 Pending US20230416337A1 (en) | 2016-11-09 | 2023-03-14 | Intestinal expression of programmed death ligand 1 |
Family Applications Before (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US16/348,834 Active 2038-04-12 US11603398B2 (en) | 2016-11-09 | 2017-11-09 | Intestinal expression of programmed death ligand 1 |
Country Status (8)
Country | Link |
---|---|
US (2) | US11603398B2 (en) |
EP (1) | EP3538659A4 (en) |
JP (1) | JP2019534300A (en) |
CN (1) | CN110520534A (en) |
AU (1) | AU2017356350B2 (en) |
CA (1) | CA3043124A1 (en) |
IL (1) | IL266540A (en) |
WO (1) | WO2018085935A1 (en) |
Families Citing this family (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
AU2021205337A1 (en) * | 2020-01-10 | 2022-07-21 | Modernatx, Inc. | Methods of making tolerogenic dendritic cells |
WO2021150997A2 (en) * | 2020-01-22 | 2021-07-29 | Engene, Inc. | Localized expression of therapeutic nucleic acids in lung epithelial cells |
CN113061192B (en) * | 2021-04-12 | 2023-08-22 | 佰思巢(上海)生物科技有限公司 | PDL1 fusion protein with high affinity to PD-1 receptor and application thereof as T cell inhibitor |
Family Cites Families (26)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CA2462593A1 (en) * | 2001-10-03 | 2003-04-10 | Kam W. Leong | Compositions for oral gene therapy and methods of using same |
ATE482694T1 (en) * | 2002-07-05 | 2010-10-15 | Temrel Ltd | COMPOSITION FOR CONTROLLED DELIVERY OF ACTIVE INGREDIENTS |
EP1734933B1 (en) * | 2004-03-26 | 2010-05-26 | LEK Pharmaceuticals d.d. | Gastroresistant pharmaceutical dosage form comprising n-(2-(2-phthalimidoethoxy)-acetyl)-l-alanyl-d-glutamic acid (lk-423) |
CN101084438B (en) * | 2004-10-06 | 2013-12-25 | 梅约医学教育与研究基金会 | B7-H1 and methods of diagnosis, prognosis, and treatment of cancer |
WO2008020318A2 (en) * | 2006-03-30 | 2008-02-21 | Engene, Inc. | Non-viral compositions and methods for transfecting gut cells in vivo |
US20100035973A1 (en) * | 2006-07-17 | 2010-02-11 | Nationwide Children's Hospital, Inc. | Disruption of programmed death 1 (pd-1) ligand to adjuvant adeno-associated virus vector vaccines |
RS53072B (en) * | 2007-06-18 | 2014-04-30 | Merck Sharp & Dohme B.V. | Antibodies to human programmed death receptor pd-1 |
US8062852B2 (en) * | 2007-10-01 | 2011-11-22 | The Children's Hospital And Regional Medical Center | Detection and treatment of autoimmune disorders |
WO2009149191A2 (en) * | 2008-06-03 | 2009-12-10 | University Of Rochester | Methods of treating inflammatory intestinal disease and managing symptoms thereof |
EP2391387A1 (en) * | 2009-01-29 | 2011-12-07 | Synedgen, Inc. | Nucleic acid delivery using modified chitosans |
WO2010102278A1 (en) * | 2009-03-06 | 2010-09-10 | President And Fellows Of Harvard College | Methods and compositions for the generation and maintenance of regulatory t cells |
KR101342641B1 (en) * | 2009-06-25 | 2013-12-20 | 주식회사 바이오리더스 | Composition for Adjuvant Comprising Poly-Gamma-Glutamic Acid-Chitosan Nanoparticle |
WO2011060218A1 (en) | 2009-11-13 | 2011-05-19 | The United States Of America, As Represented By The Secretary, Department Of Health And Human Services | Modulated programmed death ligand-1 |
WO2011127344A2 (en) * | 2010-04-08 | 2011-10-13 | University Of Virginia Patent Foundation | Method to detect and treat infectious or inflammatory diarrhea |
US9644179B2 (en) * | 2011-11-23 | 2017-05-09 | The Trustees Of The University Of Pennsylvania | Use of PDL1 expressing cells to convert T cells into regulatory T cells |
JP6138813B2 (en) * | 2011-11-28 | 2017-05-31 | メルク パテント ゲゼルシャフト ミット ベシュレンクテル ハフツングMerck Patent Gesellschaft mit beschraenkter Haftung | Anti-PD-L1 antibody and use thereof |
KR102223224B1 (en) * | 2012-03-21 | 2021-03-08 | 엔진 인코포레이티드 | Dually derivatized chitosan nanoparticles and methods of making and using the same for gene transfer in vivo |
CA2925198C (en) * | 2013-09-25 | 2021-12-07 | Engene, Inc. | Dually derivatized chitosan nanoparticles and methods of making and using the same for gene transfer in vivo |
GB201319548D0 (en) * | 2013-11-05 | 2013-12-18 | Medical Res Council | Amorphous magnesium-substituted calcium phosphate compositions and their uses |
US20150139994A1 (en) * | 2013-11-12 | 2015-05-21 | The Regents Of The University Of California | Compositions and methods for preventing allogeneic immune rejection |
EP2881463A1 (en) * | 2013-12-09 | 2015-06-10 | DRK-Blutspendedienst Baden-Württemberg-Hessen gGmbH | Factor IX variants with clotting activity in absence of their cofactor and/or with increased F.IX clotting activity and their use for treating bleeding disorders |
AU2014363430A1 (en) * | 2013-12-13 | 2016-06-16 | Conaris Research Institute Ag | A pharmaceutical composition containing combinations of nicotinamide and 5-aminosalicylic acid for beneficially influencing the intestinal microbiota and/or treating gastrointestinal inflammation |
KR20150135148A (en) * | 2014-05-23 | 2015-12-02 | 주식회사 제넥신 | PD-L1 Fused protein and use thereof |
WO2015178746A1 (en) * | 2014-05-23 | 2015-11-26 | 주식회사 제넥신 | Pd-l1 fusion protein and use thereof |
US20160220636A1 (en) * | 2015-01-29 | 2016-08-04 | Sarah Bacus | Method of treating auto-immune or inflammatory diseases using b7 family proteins of derivatives thereof |
EP3324982A4 (en) * | 2015-07-21 | 2019-01-09 | The Children's Medical Center Corporation | Pd-l1 expressing hematopoietic stem cells and uses |
-
2017
- 2017-11-09 CA CA3043124A patent/CA3043124A1/en active Pending
- 2017-11-09 WO PCT/CA2017/051340 patent/WO2018085935A1/en unknown
- 2017-11-09 CN CN201780072387.3A patent/CN110520534A/en active Pending
- 2017-11-09 AU AU2017356350A patent/AU2017356350B2/en active Active
- 2017-11-09 EP EP17869214.1A patent/EP3538659A4/en active Pending
- 2017-11-09 US US16/348,834 patent/US11603398B2/en active Active
- 2017-11-09 JP JP2019524031A patent/JP2019534300A/en active Pending
-
2019
- 2019-05-08 IL IL266540A patent/IL266540A/en unknown
-
2023
- 2023-03-14 US US18/121,557 patent/US20230416337A1/en active Pending
Also Published As
Publication number | Publication date |
---|---|
EP3538659A1 (en) | 2019-09-18 |
WO2018085935A1 (en) | 2018-05-17 |
IL266540A (en) | 2019-07-31 |
CA3043124A1 (en) | 2018-05-17 |
AU2017356350B2 (en) | 2024-02-29 |
US20190263887A1 (en) | 2019-08-29 |
CN110520534A (en) | 2019-11-29 |
EP3538659A4 (en) | 2020-07-15 |
JP2019534300A (en) | 2019-11-28 |
AU2017356350A1 (en) | 2019-05-30 |
US11603398B2 (en) | 2023-03-14 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US20230416337A1 (en) | Intestinal expression of programmed death ligand 1 | |
JP2022177007A (en) | Mrna combination therapy for treatment of cancer | |
US20170002060A1 (en) | Polynucleotides for the in vivo production of antibodies | |
JP2021193112A (en) | Structure, manufacture and uses of human-derived cell-permeable peptides conjugated with specific biologically active cargo peptides | |
KR102271498B1 (en) | Administration of kynurenine depleting enzymes for tumor therapy | |
EP3092250A1 (en) | Polynucleotides for the in vivo production of antibodies | |
EP3353309A1 (en) | Compositions and methods for genome editing | |
KR20190071000A (en) | Cd20-binding immunotoxins for inducing cellular internalization and methods using same | |
TW202344273A (en) | Use of antibody drug conjugate | |
CN114072157A (en) | Engineered chimeric fusion protein compositions and methods of use thereof | |
JP2001514858A (en) | Conjugates containing the homeodomain of Antennapedia | |
HU228973B1 (en) | Polymer conjugates of neublastin and methods of using same | |
CN113453723A (en) | Joint | |
CN116583294A (en) | Anti-inflammatory cytokines and methods of use thereof | |
WO2015000181A1 (en) | Novel recombinant fusion protein, preparation method therefor and use thereof | |
US20170096670A1 (en) | Dna aptamers specific to cd200r1 and their therapeutic uses | |
WO2012122941A1 (en) | Polypeptide drug against hepatitis b virus x protein | |
WO2021228052A1 (en) | Biological macromolecular target-specific complement inhibitor, preparation method therefor, and application thereof | |
WO2015038891A2 (en) | Functional peptide analogs of pedf | |
WO2012094905A1 (en) | Dna vaccine based on b7-1-pe40kdel exotoxin fusion gene and use thereof | |
KR101466875B1 (en) | The therapy for autoimmune disease using minicircle vector designed to express TNFR2 | |
WO1998052972A1 (en) | Novel protein, its gene, reagents for inducing apoptosis, and anticancer agents | |
LIU et al. | Protective effect of human CD40-Ig fusion protein in a murine model of acute graft-versus-host disease | |
WO2012122942A1 (en) | Polypeptide drug against hepatitis b virus x protein | |
US12006511B2 (en) | Recombinant PD-L1 peptides and methods of use |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
AS | Assignment |
Owner name: ENGENE, INC., QUEBEC Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:DAUPHINEE, SHAUNA MARIE;MCCARTHY, CONNOR DANIEL ALEXANDER;DUPAUL-CHICOINE, JERMEY;AND OTHERS;SIGNING DATES FROM 20201105 TO 20201130;REEL/FRAME:063394/0231 |
|
STPP | Information on status: patent application and granting procedure in general |
Free format text: DOCKETED NEW CASE - READY FOR EXAMINATION |