US20220048948A1 - Cd40 targeted peptides and uses thereof - Google Patents
Cd40 targeted peptides and uses thereof Download PDFInfo
- Publication number
- US20220048948A1 US20220048948A1 US17/380,303 US202117380303A US2022048948A1 US 20220048948 A1 US20220048948 A1 US 20220048948A1 US 202117380303 A US202117380303 A US 202117380303A US 2022048948 A1 US2022048948 A1 US 2022048948A1
- Authority
- US
- United States
- Prior art keywords
- peptide
- cell
- amino acid
- seq
- protein
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 108090000765 processed proteins & peptides Proteins 0.000 title claims abstract description 607
- 102000004196 processed proteins & peptides Human genes 0.000 title claims abstract description 232
- 238000000034 method Methods 0.000 claims abstract description 222
- 102100032937 CD40 ligand Human genes 0.000 claims abstract description 71
- 102100040245 Tumor necrosis factor receptor superfamily member 5 Human genes 0.000 claims abstract description 12
- 208000023275 Autoimmune disease Diseases 0.000 claims abstract description 8
- 101000868215 Homo sapiens CD40 ligand Proteins 0.000 claims abstract description 8
- 101150013553 CD40 gene Proteins 0.000 claims abstract 8
- 125000003275 alpha amino acid group Chemical group 0.000 claims description 122
- 239000003795 chemical substances by application Substances 0.000 claims description 99
- 229920001184 polypeptide Polymers 0.000 claims description 64
- 230000027455 binding Effects 0.000 claims description 62
- 239000008194 pharmaceutical composition Substances 0.000 claims description 50
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims description 47
- 230000004048 modification Effects 0.000 claims description 44
- 238000012986 modification Methods 0.000 claims description 44
- 210000003719 b-lymphocyte Anatomy 0.000 claims description 39
- 239000003814 drug Substances 0.000 claims description 29
- 201000010099 disease Diseases 0.000 claims description 28
- 210000001744 T-lymphocyte Anatomy 0.000 claims description 26
- 238000012384 transportation and delivery Methods 0.000 claims description 24
- 239000002502 liposome Substances 0.000 claims description 18
- 230000035755 proliferation Effects 0.000 claims description 14
- 239000003937 drug carrier Substances 0.000 claims description 12
- 239000000546 pharmaceutical excipient Substances 0.000 claims description 12
- 229940124597 therapeutic agent Drugs 0.000 claims description 12
- 230000003844 B-cell-activation Effects 0.000 claims description 11
- 239000003085 diluting agent Substances 0.000 claims description 10
- 230000005764 inhibitory process Effects 0.000 claims description 10
- 206010061218 Inflammation Diseases 0.000 claims description 9
- 238000007385 chemical modification Methods 0.000 claims description 9
- 230000013595 glycosylation Effects 0.000 claims description 9
- 238000006206 glycosylation reaction Methods 0.000 claims description 9
- 230000004054 inflammatory process Effects 0.000 claims description 9
- 238000012545 processing Methods 0.000 claims description 9
- 230000009467 reduction Effects 0.000 claims description 9
- 230000021736 acetylation Effects 0.000 claims description 7
- 238000006640 acetylation reaction Methods 0.000 claims description 7
- 230000004481 post-translational protein modification Effects 0.000 claims description 7
- 239000000032 diagnostic agent Substances 0.000 claims description 6
- 229940039227 diagnostic agent Drugs 0.000 claims description 6
- 230000001613 neoplastic effect Effects 0.000 claims description 6
- 230000006320 pegylation Effects 0.000 claims description 6
- 206010025323 Lymphomas Diseases 0.000 claims description 5
- 201000009030 Carcinoma Diseases 0.000 claims description 4
- 230000010933 acylation Effects 0.000 claims description 4
- 238000005917 acylation reaction Methods 0.000 claims description 4
- 230000009435 amidation Effects 0.000 claims description 4
- 238000007112 amidation reaction Methods 0.000 claims description 4
- 238000006664 bond formation reaction Methods 0.000 claims description 4
- 230000029226 lipidation Effects 0.000 claims description 4
- 239000002105 nanoparticle Substances 0.000 claims description 4
- 230000026731 phosphorylation Effects 0.000 claims description 4
- 238000006366 phosphorylation reaction Methods 0.000 claims description 4
- 239000006249 magnetic particle Substances 0.000 claims description 3
- 230000011987 methylation Effects 0.000 claims description 3
- 238000007069 methylation reaction Methods 0.000 claims description 3
- 230000002797 proteolythic effect Effects 0.000 claims description 3
- 238000010798 ubiquitination Methods 0.000 claims description 3
- 230000034512 ubiquitination Effects 0.000 claims description 3
- 230000005730 ADP ribosylation Effects 0.000 claims description 2
- 206010035226 Plasma cell myeloma Diseases 0.000 claims description 2
- 125000002252 acyl group Chemical group 0.000 claims description 2
- 230000021523 carboxylation Effects 0.000 claims description 2
- 238000006473 carboxylation reaction Methods 0.000 claims description 2
- 238000005805 hydroxylation reaction Methods 0.000 claims description 2
- 201000000050 myeloid neoplasm Diseases 0.000 claims description 2
- 230000007498 myristoylation Effects 0.000 claims description 2
- 230000011664 signaling Effects 0.000 claims description 2
- 230000019635 sulfation Effects 0.000 claims description 2
- 238000005670 sulfation reaction Methods 0.000 claims description 2
- 238000006177 thiolation reaction Methods 0.000 claims description 2
- 230000029936 alkylation Effects 0.000 claims 1
- 238000005804 alkylation reaction Methods 0.000 claims 1
- 230000033444 hydroxylation Effects 0.000 claims 1
- 230000013823 prenylation Effects 0.000 claims 1
- 239000000203 mixture Substances 0.000 abstract description 172
- 230000003993 interaction Effects 0.000 abstract description 67
- 238000002054 transplantation Methods 0.000 abstract description 35
- 230000028993 immune response Effects 0.000 abstract description 24
- 230000002401 inhibitory effect Effects 0.000 abstract description 21
- 238000011282 treatment Methods 0.000 abstract description 17
- 230000001506 immunosuppresive effect Effects 0.000 abstract description 14
- 206010062016 Immunosuppression Diseases 0.000 abstract description 13
- 230000001939 inductive effect Effects 0.000 abstract description 13
- 206010028980 Neoplasm Diseases 0.000 abstract description 5
- 201000011510 cancer Diseases 0.000 abstract description 5
- 208000037979 autoimmune inflammatory disease Diseases 0.000 abstract 1
- 210000004027 cell Anatomy 0.000 description 215
- 108010063916 CD40 Antigens Proteins 0.000 description 198
- 108090000623 proteins and genes Proteins 0.000 description 133
- 235000001014 amino acid Nutrition 0.000 description 123
- 229940024606 amino acid Drugs 0.000 description 112
- 150000001413 amino acids Chemical class 0.000 description 112
- 150000007523 nucleic acids Chemical group 0.000 description 105
- 102000004169 proteins and genes Human genes 0.000 description 100
- 235000018102 proteins Nutrition 0.000 description 90
- 210000000265 leukocyte Anatomy 0.000 description 83
- 230000001640 apoptogenic effect Effects 0.000 description 76
- 102000039446 nucleic acids Human genes 0.000 description 68
- 108020004707 nucleic acids Proteins 0.000 description 68
- 108010029697 CD40 Ligand Proteins 0.000 description 63
- 239000012634 fragment Substances 0.000 description 62
- 210000001519 tissue Anatomy 0.000 description 61
- 239000013598 vector Substances 0.000 description 60
- 241000282414 Homo sapiens Species 0.000 description 50
- 230000000694 effects Effects 0.000 description 50
- -1 siCD40 Chemical compound 0.000 description 50
- 238000006467 substitution reaction Methods 0.000 description 47
- 230000004071 biological effect Effects 0.000 description 43
- 210000000056 organ Anatomy 0.000 description 43
- 238000003556 assay Methods 0.000 description 39
- 125000000539 amino acid group Chemical group 0.000 description 37
- 241001465754 Metazoa Species 0.000 description 34
- 210000002865 immune cell Anatomy 0.000 description 31
- 239000000243 solution Substances 0.000 description 28
- 150000001875 compounds Chemical class 0.000 description 27
- 239000000126 substance Substances 0.000 description 27
- 150000002632 lipids Chemical class 0.000 description 26
- 229920001223 polyethylene glycol Polymers 0.000 description 25
- 238000009472 formulation Methods 0.000 description 24
- 230000006870 function Effects 0.000 description 23
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 23
- 208000027866 inflammatory disease Diseases 0.000 description 22
- 210000002540 macrophage Anatomy 0.000 description 20
- 230000001225 therapeutic effect Effects 0.000 description 20
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 19
- 102000004127 Cytokines Human genes 0.000 description 18
- 108090000695 Cytokines Proteins 0.000 description 18
- 208000035475 disorder Diseases 0.000 description 18
- 239000007788 liquid Substances 0.000 description 18
- 108700028369 Alleles Proteins 0.000 description 17
- 108020004414 DNA Proteins 0.000 description 17
- 230000004913 activation Effects 0.000 description 17
- 230000015572 biosynthetic process Effects 0.000 description 17
- 239000003431 cross linking reagent Substances 0.000 description 17
- 230000007423 decrease Effects 0.000 description 17
- 238000001727 in vivo Methods 0.000 description 17
- 210000004153 islets of langerhan Anatomy 0.000 description 17
- 239000002904 solvent Substances 0.000 description 17
- 239000000725 suspension Substances 0.000 description 17
- 238000007792 addition Methods 0.000 description 16
- 102000040430 polynucleotide Human genes 0.000 description 16
- 108091033319 polynucleotide Proteins 0.000 description 16
- 239000002157 polynucleotide Substances 0.000 description 16
- 230000002519 immonomodulatory effect Effects 0.000 description 15
- 238000002360 preparation method Methods 0.000 description 15
- 238000003786 synthesis reaction Methods 0.000 description 15
- 108091028043 Nucleic acid sequence Proteins 0.000 description 14
- 238000012217 deletion Methods 0.000 description 14
- 230000037430 deletion Effects 0.000 description 14
- 229940079593 drug Drugs 0.000 description 14
- 239000000843 powder Substances 0.000 description 14
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 13
- 108091054438 MHC class II family Proteins 0.000 description 13
- 241000282898 Sus scrofa Species 0.000 description 13
- 238000005516 engineering process Methods 0.000 description 13
- 239000007924 injection Substances 0.000 description 13
- 238000002347 injection Methods 0.000 description 13
- 238000001990 intravenous administration Methods 0.000 description 13
- 238000010647 peptide synthesis reaction Methods 0.000 description 13
- 239000011347 resin Substances 0.000 description 13
- 229920005989 resin Polymers 0.000 description 13
- 210000000130 stem cell Anatomy 0.000 description 13
- 102000043131 MHC class II family Human genes 0.000 description 12
- 241000124008 Mammalia Species 0.000 description 12
- 239000000427 antigen Substances 0.000 description 12
- 108091007433 antigens Proteins 0.000 description 12
- 102000036639 antigens Human genes 0.000 description 12
- 238000000338 in vitro Methods 0.000 description 12
- 230000001965 increasing effect Effects 0.000 description 12
- 239000000463 material Substances 0.000 description 12
- 125000003729 nucleotide group Chemical group 0.000 description 12
- 229920000642 polymer Polymers 0.000 description 12
- 239000003755 preservative agent Substances 0.000 description 12
- 235000002639 sodium chloride Nutrition 0.000 description 12
- 150000008574 D-amino acids Chemical class 0.000 description 11
- 239000000872 buffer Substances 0.000 description 11
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 11
- 238000013270 controlled release Methods 0.000 description 11
- 239000002552 dosage form Substances 0.000 description 11
- 239000013604 expression vector Substances 0.000 description 11
- 230000002209 hydrophobic effect Effects 0.000 description 11
- 230000001404 mediated effect Effects 0.000 description 11
- 241000283690 Bos taurus Species 0.000 description 10
- 239000002202 Polyethylene glycol Substances 0.000 description 10
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 10
- 241000700605 Viruses Species 0.000 description 10
- 239000000443 aerosol Substances 0.000 description 10
- 230000004075 alteration Effects 0.000 description 10
- 210000000612 antigen-presenting cell Anatomy 0.000 description 10
- 230000021615 conjugation Effects 0.000 description 10
- 235000019441 ethanol Nutrition 0.000 description 10
- 239000003446 ligand Substances 0.000 description 10
- 230000008569 process Effects 0.000 description 10
- 239000000047 product Substances 0.000 description 10
- DNIAPMSPPWPWGF-UHFFFAOYSA-N propylene glycol Substances CC(O)CO DNIAPMSPPWPWGF-UHFFFAOYSA-N 0.000 description 10
- 125000006239 protecting group Chemical group 0.000 description 10
- 239000003981 vehicle Substances 0.000 description 10
- 241001272173 Laurasiatheria Species 0.000 description 9
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 9
- 108700019146 Transgenes Proteins 0.000 description 9
- 230000008901 benefit Effects 0.000 description 9
- 210000004899 c-terminal region Anatomy 0.000 description 9
- 239000000839 emulsion Substances 0.000 description 9
- 238000004519 manufacturing process Methods 0.000 description 9
- 230000002441 reversible effect Effects 0.000 description 9
- 150000003839 salts Chemical class 0.000 description 9
- 239000007790 solid phase Substances 0.000 description 9
- 238000007920 subcutaneous administration Methods 0.000 description 9
- 230000014616 translation Effects 0.000 description 9
- 239000013603 viral vector Substances 0.000 description 9
- 241000282994 Cervidae Species 0.000 description 8
- 241000282412 Homo Species 0.000 description 8
- 108700018351 Major Histocompatibility Complex Proteins 0.000 description 8
- 108700008625 Reporter Genes Proteins 0.000 description 8
- IQFYYKKMVGJFEH-XLPZGREQSA-N Thymidine Chemical compound O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](CO)[C@@H](O)C1 IQFYYKKMVGJFEH-XLPZGREQSA-N 0.000 description 8
- 238000000684 flow cytometry Methods 0.000 description 8
- 108020001507 fusion proteins Proteins 0.000 description 8
- 102000037865 fusion proteins Human genes 0.000 description 8
- 210000004263 induced pluripotent stem cell Anatomy 0.000 description 8
- 238000001802 infusion Methods 0.000 description 8
- 230000000670 limiting effect Effects 0.000 description 8
- 241000894007 species Species 0.000 description 8
- 230000020382 suppression by virus of host antigen processing and presentation of peptide antigen via MHC class I Effects 0.000 description 8
- 238000002965 ELISA Methods 0.000 description 7
- 102000018713 Histocompatibility Antigens Class II Human genes 0.000 description 7
- AYFVYJQAPQTCCC-GBXIJSLDSA-N L-threonine Chemical compound C[C@@H](O)[C@H](N)C(O)=O AYFVYJQAPQTCCC-GBXIJSLDSA-N 0.000 description 7
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 7
- 108020004511 Recombinant DNA Proteins 0.000 description 7
- 241000282887 Suidae Species 0.000 description 7
- 108060008682 Tumor Necrosis Factor Proteins 0.000 description 7
- 102000000852 Tumor Necrosis Factor-alpha Human genes 0.000 description 7
- 239000000654 additive Substances 0.000 description 7
- 239000002775 capsule Substances 0.000 description 7
- 238000006243 chemical reaction Methods 0.000 description 7
- 239000003153 chemical reaction reagent Substances 0.000 description 7
- 210000004443 dendritic cell Anatomy 0.000 description 7
- 206010012601 diabetes mellitus Diseases 0.000 description 7
- 230000002255 enzymatic effect Effects 0.000 description 7
- 239000001257 hydrogen Substances 0.000 description 7
- 229910052739 hydrogen Inorganic materials 0.000 description 7
- 229940100601 interleukin-6 Drugs 0.000 description 7
- 238000007918 intramuscular administration Methods 0.000 description 7
- 239000002674 ointment Substances 0.000 description 7
- 239000007787 solid Substances 0.000 description 7
- 230000008685 targeting Effects 0.000 description 7
- 238000011191 terminal modification Methods 0.000 description 7
- 238000013518 transcription Methods 0.000 description 7
- 230000035897 transcription Effects 0.000 description 7
- 230000003612 virological effect Effects 0.000 description 7
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 6
- 102000040650 (ribonucleotides)n+m Human genes 0.000 description 6
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 6
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 6
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 6
- 102100028976 HLA class I histocompatibility antigen, B alpha chain Human genes 0.000 description 6
- 108010027412 Histocompatibility Antigens Class II Proteins 0.000 description 6
- HNDVDQJCIGZPNO-YFKPBYRVSA-N L-histidine Chemical compound OC(=O)[C@@H](N)CC1=CN=CN1 HNDVDQJCIGZPNO-YFKPBYRVSA-N 0.000 description 6
- AGPKZVBTJJNPAG-WHFBIAKZSA-N L-isoleucine Chemical compound CC[C@H](C)[C@H](N)C(O)=O AGPKZVBTJJNPAG-WHFBIAKZSA-N 0.000 description 6
- KZSNJWFQEVHDMF-BYPYZUCNSA-N L-valine Chemical compound CC(C)[C@H](N)C(O)=O KZSNJWFQEVHDMF-BYPYZUCNSA-N 0.000 description 6
- 102000043129 MHC class I family Human genes 0.000 description 6
- 108091054437 MHC class I family Proteins 0.000 description 6
- 101710163270 Nuclease Proteins 0.000 description 6
- AYFVYJQAPQTCCC-UHFFFAOYSA-N Threonine Natural products CC(O)C(N)C(O)=O AYFVYJQAPQTCCC-UHFFFAOYSA-N 0.000 description 6
- 239000004473 Threonine Substances 0.000 description 6
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 description 6
- DRTQHJPVMGBUCF-XVFCMESISA-N Uridine Chemical compound O[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1N1C(=O)NC(=O)C=C1 DRTQHJPVMGBUCF-XVFCMESISA-N 0.000 description 6
- KZSNJWFQEVHDMF-UHFFFAOYSA-N Valine Natural products CC(C)C(N)C(O)=O KZSNJWFQEVHDMF-UHFFFAOYSA-N 0.000 description 6
- 239000002253 acid Substances 0.000 description 6
- 239000004480 active ingredient Substances 0.000 description 6
- 239000013543 active substance Substances 0.000 description 6
- OIRDTQYFTABQOQ-KQYNXXCUSA-N adenosine Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O OIRDTQYFTABQOQ-KQYNXXCUSA-N 0.000 description 6
- 239000007864 aqueous solution Substances 0.000 description 6
- 210000000227 basophil cell of anterior lobe of hypophysis Anatomy 0.000 description 6
- 230000000903 blocking effect Effects 0.000 description 6
- 210000004369 blood Anatomy 0.000 description 6
- 239000008280 blood Substances 0.000 description 6
- 239000006071 cream Substances 0.000 description 6
- 235000018417 cysteine Nutrition 0.000 description 6
- 230000016396 cytokine production Effects 0.000 description 6
- 238000011161 development Methods 0.000 description 6
- 238000010494 dissociation reaction Methods 0.000 description 6
- 230000005593 dissociations Effects 0.000 description 6
- 230000005714 functional activity Effects 0.000 description 6
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 6
- 239000004615 ingredient Substances 0.000 description 6
- NOESYZHRGYRDHS-UHFFFAOYSA-N insulin Chemical compound N1C(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(NC(=O)CN)C(C)CC)CSSCC(C(NC(CO)C(=O)NC(CC(C)C)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CCC(N)=O)C(=O)NC(CC(C)C)C(=O)NC(CCC(O)=O)C(=O)NC(CC(N)=O)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CSSCC(NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2C=CC(O)=CC=2)NC(=O)C(CC(C)C)NC(=O)C(C)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2NC=NC=2)NC(=O)C(CO)NC(=O)CNC2=O)C(=O)NCC(=O)NC(CCC(O)=O)C(=O)NC(CCCNC(N)=N)C(=O)NCC(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC(O)=CC=3)C(=O)NC(C(C)O)C(=O)N3C(CCC3)C(=O)NC(CCCCN)C(=O)NC(C)C(O)=O)C(=O)NC(CC(N)=O)C(O)=O)=O)NC(=O)C(C(C)CC)NC(=O)C(CO)NC(=O)C(C(C)O)NC(=O)C1CSSCC2NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(N)CC=1C=CC=CC=1)C(C)C)CC1=CN=CN1 NOESYZHRGYRDHS-UHFFFAOYSA-N 0.000 description 6
- 229960000310 isoleucine Drugs 0.000 description 6
- AGPKZVBTJJNPAG-UHFFFAOYSA-N isoleucine Natural products CCC(C)C(N)C(O)=O AGPKZVBTJJNPAG-UHFFFAOYSA-N 0.000 description 6
- 238000005259 measurement Methods 0.000 description 6
- 239000002773 nucleotide Substances 0.000 description 6
- 210000000496 pancreas Anatomy 0.000 description 6
- 239000006201 parenteral dosage form Substances 0.000 description 6
- 230000002829 reductive effect Effects 0.000 description 6
- 230000001105 regulatory effect Effects 0.000 description 6
- 125000002652 ribonucleotide group Chemical group 0.000 description 6
- 239000011780 sodium chloride Substances 0.000 description 6
- 239000007921 spray Substances 0.000 description 6
- 238000013519 translation Methods 0.000 description 6
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 6
- 229960005486 vaccine Drugs 0.000 description 6
- 239000004474 valine Substances 0.000 description 6
- MTCFGRXMJLQNBG-REOHCLBHSA-N (2S)-2-Amino-3-hydroxypropansäure Chemical compound OC[C@H](N)C(O)=O MTCFGRXMJLQNBG-REOHCLBHSA-N 0.000 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 description 5
- 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 5
- 241000306001 Cetartiodactyla Species 0.000 description 5
- 108010069514 Cyclic Peptides Proteins 0.000 description 5
- 102000001189 Cyclic Peptides Human genes 0.000 description 5
- 241000702421 Dependoparvovirus Species 0.000 description 5
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 5
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 5
- 108010088652 Histocompatibility Antigens Class I Proteins 0.000 description 5
- 108090000174 Interleukin-10 Proteins 0.000 description 5
- 102000003814 Interleukin-10 Human genes 0.000 description 5
- 108090001005 Interleukin-6 Proteins 0.000 description 5
- 102000004889 Interleukin-6 Human genes 0.000 description 5
- 150000008575 L-amino acids Chemical class 0.000 description 5
- KDXKERNSBIXSRK-YFKPBYRVSA-N L-lysine Chemical compound NCCCC[C@H](N)C(O)=O KDXKERNSBIXSRK-YFKPBYRVSA-N 0.000 description 5
- COLNVLDHVKWLRT-QMMMGPOBSA-N L-phenylalanine Chemical compound OC(=O)[C@@H](N)CC1=CC=CC=C1 COLNVLDHVKWLRT-QMMMGPOBSA-N 0.000 description 5
- 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 5
- 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 5
- 241000713666 Lentivirus Species 0.000 description 5
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 5
- 239000004472 Lysine Substances 0.000 description 5
- 230000006044 T cell activation Effects 0.000 description 5
- 230000006052 T cell proliferation Effects 0.000 description 5
- QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Natural products C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 description 5
- 238000004458 analytical method Methods 0.000 description 5
- 230000001580 bacterial effect Effects 0.000 description 5
- 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 5
- 150000001718 carbodiimides Chemical class 0.000 description 5
- 230000004663 cell proliferation Effects 0.000 description 5
- 230000008859 change Effects 0.000 description 5
- 238000003776 cleavage reaction Methods 0.000 description 5
- 239000002299 complementary DNA Substances 0.000 description 5
- 238000010168 coupling process Methods 0.000 description 5
- 238000001514 detection method Methods 0.000 description 5
- 235000014113 dietary fatty acids Nutrition 0.000 description 5
- 210000001671 embryonic stem cell Anatomy 0.000 description 5
- 239000003623 enhancer Substances 0.000 description 5
- 150000002148 esters Chemical group 0.000 description 5
- 229930195729 fatty acid Natural products 0.000 description 5
- 239000000194 fatty acid Substances 0.000 description 5
- 150000004665 fatty acids Chemical class 0.000 description 5
- 230000004927 fusion Effects 0.000 description 5
- 239000000499 gel Substances 0.000 description 5
- 238000012239 gene modification Methods 0.000 description 5
- 230000005017 genetic modification Effects 0.000 description 5
- 235000013617 genetically modified food Nutrition 0.000 description 5
- 229960001031 glucose Drugs 0.000 description 5
- 238000010348 incorporation Methods 0.000 description 5
- 239000003112 inhibitor Substances 0.000 description 5
- 238000003780 insertion Methods 0.000 description 5
- 230000037431 insertion Effects 0.000 description 5
- 229940076144 interleukin-10 Drugs 0.000 description 5
- 238000007912 intraperitoneal administration Methods 0.000 description 5
- 150000002500 ions Chemical class 0.000 description 5
- 239000008101 lactose Substances 0.000 description 5
- 108091005601 modified peptides Proteins 0.000 description 5
- 238000010369 molecular cloning Methods 0.000 description 5
- 210000001616 monocyte Anatomy 0.000 description 5
- 210000000440 neutrophil Anatomy 0.000 description 5
- 210000003819 peripheral blood mononuclear cell Anatomy 0.000 description 5
- COLNVLDHVKWLRT-UHFFFAOYSA-N phenylalanine Natural products OC(=O)C(N)CC1=CC=CC=C1 COLNVLDHVKWLRT-UHFFFAOYSA-N 0.000 description 5
- 239000013612 plasmid Substances 0.000 description 5
- 229910052700 potassium Inorganic materials 0.000 description 5
- 239000003380 propellant Substances 0.000 description 5
- 238000000746 purification Methods 0.000 description 5
- 102000005962 receptors Human genes 0.000 description 5
- 108020003175 receptors Proteins 0.000 description 5
- 230000007017 scission Effects 0.000 description 5
- 230000000638 stimulation Effects 0.000 description 5
- 235000000346 sugar Nutrition 0.000 description 5
- 229910052717 sulfur Inorganic materials 0.000 description 5
- 238000013268 sustained release Methods 0.000 description 5
- 239000012730 sustained-release form Substances 0.000 description 5
- 239000003826 tablet Substances 0.000 description 5
- 238000011200 topical administration Methods 0.000 description 5
- 241000701161 unidentified adenovirus Species 0.000 description 5
- MZOFCQQQCNRIBI-VMXHOPILSA-N (3s)-4-[[(2s)-1-[[(2s)-1-[[(1s)-1-carboxy-2-hydroxyethyl]amino]-4-methyl-1-oxopentan-2-yl]amino]-5-(diaminomethylideneamino)-1-oxopentan-2-yl]amino]-3-[[2-[[(2s)-2,6-diaminohexanoyl]amino]acetyl]amino]-4-oxobutanoic acid Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CC(O)=O)NC(=O)CNC(=O)[C@@H](N)CCCCN MZOFCQQQCNRIBI-VMXHOPILSA-N 0.000 description 4
- FUOOLUPWFVMBKG-UHFFFAOYSA-N 2-Aminoisobutyric acid Chemical compound CC(C)(N)C(O)=O FUOOLUPWFVMBKG-UHFFFAOYSA-N 0.000 description 4
- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 description 4
- 241000894006 Bacteria Species 0.000 description 4
- FERIUCNNQQJTOY-UHFFFAOYSA-N Butyric acid Chemical group CCCC(O)=O FERIUCNNQQJTOY-UHFFFAOYSA-N 0.000 description 4
- 102000014914 Carrier Proteins Human genes 0.000 description 4
- 108010078791 Carrier Proteins Proteins 0.000 description 4
- 241000282693 Cercopithecidae Species 0.000 description 4
- QOSSAOTZNIDXMA-UHFFFAOYSA-N Dicylcohexylcarbodiimide Chemical compound C1CCCCC1N=C=NC1CCCCC1 QOSSAOTZNIDXMA-UHFFFAOYSA-N 0.000 description 4
- 102000004190 Enzymes Human genes 0.000 description 4
- 108090000790 Enzymes Proteins 0.000 description 4
- 102000005720 Glutathione transferase Human genes 0.000 description 4
- 108010070675 Glutathione transferase Proteins 0.000 description 4
- 108010043121 Green Fluorescent Proteins Proteins 0.000 description 4
- NYHBQMYGNKIUIF-UUOKFMHZSA-N Guanosine Chemical compound C1=NC=2C(=O)NC(N)=NC=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O NYHBQMYGNKIUIF-UUOKFMHZSA-N 0.000 description 4
- 241000238631 Hexapoda Species 0.000 description 4
- 108010065805 Interleukin-12 Proteins 0.000 description 4
- 102000013462 Interleukin-12 Human genes 0.000 description 4
- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 description 4
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 description 4
- ROHFNLRQFUQHCH-YFKPBYRVSA-N L-leucine Chemical compound CC(C)C[C@H](N)C(O)=O ROHFNLRQFUQHCH-YFKPBYRVSA-N 0.000 description 4
- ROHFNLRQFUQHCH-UHFFFAOYSA-N Leucine Natural products CC(C)CC(N)C(O)=O ROHFNLRQFUQHCH-UHFFFAOYSA-N 0.000 description 4
- 108010015302 Matrix metalloproteinase-9 Proteins 0.000 description 4
- 102000005741 Metalloproteases Human genes 0.000 description 4
- 108010006035 Metalloproteases Proteins 0.000 description 4
- 241000699666 Mus <mouse, genus> Species 0.000 description 4
- 101001024425 Mus musculus Ig gamma-2A chain C region secreted form Proteins 0.000 description 4
- GXCLVBGFBYZDAG-UHFFFAOYSA-N N-[2-(1H-indol-3-yl)ethyl]-N-methylprop-2-en-1-amine Chemical compound CN(CCC1=CNC2=C1C=CC=C2)CC=C GXCLVBGFBYZDAG-UHFFFAOYSA-N 0.000 description 4
- 102000035195 Peptidases Human genes 0.000 description 4
- 108091005804 Peptidases Proteins 0.000 description 4
- 241000283089 Perissodactyla Species 0.000 description 4
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Natural products OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 4
- 108010076504 Protein Sorting Signals Proteins 0.000 description 4
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 description 4
- 108010071390 Serum Albumin Proteins 0.000 description 4
- 102000007562 Serum Albumin Human genes 0.000 description 4
- 230000002378 acidificating effect Effects 0.000 description 4
- 235000004279 alanine Nutrition 0.000 description 4
- 125000003277 amino group Chemical group 0.000 description 4
- 239000011324 bead Substances 0.000 description 4
- IQFYYKKMVGJFEH-UHFFFAOYSA-N beta-L-thymidine Natural products O=C1NC(=O)C(C)=CN1C1OC(CO)C(O)C1 IQFYYKKMVGJFEH-UHFFFAOYSA-N 0.000 description 4
- 229960002685 biotin Drugs 0.000 description 4
- 239000011616 biotin Substances 0.000 description 4
- 238000004113 cell culture Methods 0.000 description 4
- 230000008878 coupling Effects 0.000 description 4
- 238000005859 coupling reaction Methods 0.000 description 4
- 125000000151 cysteine group Chemical class N[C@@H](CS)C(=O)* 0.000 description 4
- 230000003247 decreasing effect Effects 0.000 description 4
- 230000004069 differentiation Effects 0.000 description 4
- 229940088598 enzyme Drugs 0.000 description 4
- 238000001476 gene delivery Methods 0.000 description 4
- 238000010353 genetic engineering Methods 0.000 description 4
- 230000005847 immunogenicity Effects 0.000 description 4
- 230000016784 immunoglobulin production Effects 0.000 description 4
- 238000002513 implantation Methods 0.000 description 4
- 230000003834 intracellular effect Effects 0.000 description 4
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 4
- 125000005647 linker group Chemical group 0.000 description 4
- 230000007774 longterm Effects 0.000 description 4
- 210000004072 lung Anatomy 0.000 description 4
- 239000003550 marker Substances 0.000 description 4
- 210000004379 membrane Anatomy 0.000 description 4
- 239000012528 membrane Substances 0.000 description 4
- 108020004999 messenger RNA Proteins 0.000 description 4
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 4
- 239000000693 micelle Substances 0.000 description 4
- 239000006199 nebulizer Substances 0.000 description 4
- 238000007911 parenteral administration Methods 0.000 description 4
- 239000000816 peptidomimetic Substances 0.000 description 4
- 150000003904 phospholipids Chemical class 0.000 description 4
- 230000002335 preservative effect Effects 0.000 description 4
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 4
- 230000000069 prophylactic effect Effects 0.000 description 4
- 229960004063 propylene glycol Drugs 0.000 description 4
- 230000017854 proteolysis Effects 0.000 description 4
- 230000002685 pulmonary effect Effects 0.000 description 4
- 230000004044 response Effects 0.000 description 4
- 230000001177 retroviral effect Effects 0.000 description 4
- 238000007363 ring formation reaction Methods 0.000 description 4
- 239000000523 sample Substances 0.000 description 4
- 230000028327 secretion Effects 0.000 description 4
- 239000000758 substrate Substances 0.000 description 4
- 239000000375 suspending agent Substances 0.000 description 4
- 208000024891 symptom Diseases 0.000 description 4
- 230000009885 systemic effect Effects 0.000 description 4
- 238000002560 therapeutic procedure Methods 0.000 description 4
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 3
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 3
- 239000004475 Arginine Substances 0.000 description 3
- DCXYFEDJOCDNAF-UHFFFAOYSA-N Asparagine Natural products OC(=O)C(N)CC(N)=O DCXYFEDJOCDNAF-UHFFFAOYSA-N 0.000 description 3
- WVDDGKGOMKODPV-UHFFFAOYSA-N Benzyl alcohol Chemical compound OCC1=CC=CC=C1 WVDDGKGOMKODPV-UHFFFAOYSA-N 0.000 description 3
- 239000002126 C01EB10 - Adenosine Substances 0.000 description 3
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 3
- 241000282836 Camelus dromedarius Species 0.000 description 3
- 241000282472 Canis lupus familiaris Species 0.000 description 3
- 241001466804 Carnivora Species 0.000 description 3
- 108010051109 Cell-Penetrating Peptides Proteins 0.000 description 3
- 102000020313 Cell-Penetrating Peptides Human genes 0.000 description 3
- 102000019034 Chemokines Human genes 0.000 description 3
- 108010012236 Chemokines Proteins 0.000 description 3
- KRKNYBCHXYNGOX-UHFFFAOYSA-K Citrate Chemical compound [O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O KRKNYBCHXYNGOX-UHFFFAOYSA-K 0.000 description 3
- 241000283073 Equus caballus Species 0.000 description 3
- 239000005977 Ethylene Substances 0.000 description 3
- 241000282326 Felis catus Species 0.000 description 3
- 241000282819 Giraffa Species 0.000 description 3
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 description 3
- 239000004471 Glycine Substances 0.000 description 3
- 102000004144 Green Fluorescent Proteins Human genes 0.000 description 3
- 206010020751 Hypersensitivity Diseases 0.000 description 3
- 102000004877 Insulin Human genes 0.000 description 3
- 108090001061 Insulin Proteins 0.000 description 3
- 108010002350 Interleukin-2 Proteins 0.000 description 3
- 102000000588 Interleukin-2 Human genes 0.000 description 3
- 102000004388 Interleukin-4 Human genes 0.000 description 3
- 108090000978 Interleukin-4 Proteins 0.000 description 3
- 108090001007 Interleukin-8 Proteins 0.000 description 3
- 102000004890 Interleukin-8 Human genes 0.000 description 3
- XUJNEKJLAYXESH-REOHCLBHSA-N L-Cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 description 3
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 description 3
- ODKSFYDXXFIFQN-BYPYZUCNSA-P L-argininium(2+) Chemical compound NC(=[NH2+])NCCC[C@H]([NH3+])C(O)=O ODKSFYDXXFIFQN-BYPYZUCNSA-P 0.000 description 3
- DCXYFEDJOCDNAF-REOHCLBHSA-N L-asparagine Chemical compound OC(=O)[C@@H](N)CC(N)=O DCXYFEDJOCDNAF-REOHCLBHSA-N 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
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 description 3
- 239000000232 Lipid Bilayer Substances 0.000 description 3
- 108060001084 Luciferase Proteins 0.000 description 3
- 239000005089 Luciferase Substances 0.000 description 3
- 108091060545 Nonsense suppressor Proteins 0.000 description 3
- MUBZPKHOEPUJKR-UHFFFAOYSA-N Oxalic acid Chemical compound OC(=O)C(O)=O MUBZPKHOEPUJKR-UHFFFAOYSA-N 0.000 description 3
- 241000288906 Primates Species 0.000 description 3
- 241000700159 Rattus Species 0.000 description 3
- 241000282798 Rhinocerotidae Species 0.000 description 3
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 3
- 230000005867 T cell response Effects 0.000 description 3
- 241000283068 Tapiridae Species 0.000 description 3
- FEWJPZIEWOKRBE-UHFFFAOYSA-N Tartaric acid Natural products [H+].[H+].[O-]C(=O)C(O)C(O)C([O-])=O FEWJPZIEWOKRBE-UHFFFAOYSA-N 0.000 description 3
- 206010052779 Transplant rejections Diseases 0.000 description 3
- 150000007513 acids Chemical class 0.000 description 3
- 230000009471 action Effects 0.000 description 3
- 229960005305 adenosine Drugs 0.000 description 3
- 239000000556 agonist Substances 0.000 description 3
- QWCKQJZIFLGMSD-UHFFFAOYSA-N alpha-aminobutyric acid Chemical compound CCC(N)C(O)=O QWCKQJZIFLGMSD-UHFFFAOYSA-N 0.000 description 3
- 239000003708 ampul Substances 0.000 description 3
- 239000003963 antioxidant agent Substances 0.000 description 3
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 3
- 235000009582 asparagine Nutrition 0.000 description 3
- 229960001230 asparagine Drugs 0.000 description 3
- 235000003704 aspartic acid Nutrition 0.000 description 3
- DRTQHJPVMGBUCF-PSQAKQOGSA-N beta-L-uridine Natural products O[C@H]1[C@@H](O)[C@H](CO)O[C@@H]1N1C(=O)NC(=O)C=C1 DRTQHJPVMGBUCF-PSQAKQOGSA-N 0.000 description 3
- 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 3
- 235000020958 biotin Nutrition 0.000 description 3
- 210000001772 blood platelet Anatomy 0.000 description 3
- 239000011575 calcium Substances 0.000 description 3
- 229910052791 calcium Inorganic materials 0.000 description 3
- 239000000969 carrier Substances 0.000 description 3
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 3
- 230000009918 complex formation Effects 0.000 description 3
- 125000004122 cyclic group Chemical group 0.000 description 3
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 3
- 238000001212 derivatisation Methods 0.000 description 3
- 239000003995 emulsifying agent Substances 0.000 description 3
- 210000002889 endothelial cell Anatomy 0.000 description 3
- 230000002708 enhancing effect Effects 0.000 description 3
- 230000032050 esterification Effects 0.000 description 3
- 238000005886 esterification reaction Methods 0.000 description 3
- 210000003527 eukaryotic cell Anatomy 0.000 description 3
- 229910052731 fluorine Inorganic materials 0.000 description 3
- 238000001415 gene therapy Methods 0.000 description 3
- 239000008103 glucose Substances 0.000 description 3
- 235000013922 glutamic acid Nutrition 0.000 description 3
- 239000004220 glutamic acid Substances 0.000 description 3
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 description 3
- 239000005090 green fluorescent protein Substances 0.000 description 3
- 230000010247 heart contraction Effects 0.000 description 3
- 210000002443 helper t lymphocyte Anatomy 0.000 description 3
- 238000004128 high performance liquid chromatography Methods 0.000 description 3
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 3
- 230000001900 immune effect Effects 0.000 description 3
- 230000001976 improved effect Effects 0.000 description 3
- 230000006872 improvement Effects 0.000 description 3
- 208000015181 infectious disease Diseases 0.000 description 3
- 230000002757 inflammatory effect Effects 0.000 description 3
- 229940125396 insulin Drugs 0.000 description 3
- 238000000185 intracerebroventricular administration Methods 0.000 description 3
- 238000007917 intracranial administration Methods 0.000 description 3
- 238000007913 intrathecal administration Methods 0.000 description 3
- 230000000366 juvenile effect Effects 0.000 description 3
- 239000008176 lyophilized powder Substances 0.000 description 3
- 201000001441 melanoma Diseases 0.000 description 3
- 229930182817 methionine Natural products 0.000 description 3
- 239000004005 microsphere Substances 0.000 description 3
- 230000035772 mutation Effects 0.000 description 3
- 210000000822 natural killer cell Anatomy 0.000 description 3
- 230000007935 neutral effect Effects 0.000 description 3
- 229910052757 nitrogen Inorganic materials 0.000 description 3
- 230000003204 osmotic effect Effects 0.000 description 3
- 230000036961 partial effect Effects 0.000 description 3
- 239000002245 particle Substances 0.000 description 3
- 230000007030 peptide scission Effects 0.000 description 3
- 230000009984 peri-natal effect Effects 0.000 description 3
- 210000005259 peripheral blood Anatomy 0.000 description 3
- 239000011886 peripheral blood Substances 0.000 description 3
- 125000002467 phosphate group Chemical group [H]OP(=O)(O[H])O[*] 0.000 description 3
- 235000013772 propylene glycol Nutrition 0.000 description 3
- QELSKZZBTMNZEB-UHFFFAOYSA-N propylparaben Chemical compound CCCOC(=O)C1=CC=C(O)C=C1 QELSKZZBTMNZEB-UHFFFAOYSA-N 0.000 description 3
- 230000010076 replication Effects 0.000 description 3
- 210000002966 serum Anatomy 0.000 description 3
- 210000003491 skin Anatomy 0.000 description 3
- 239000003381 stabilizer Substances 0.000 description 3
- 239000008174 sterile solution Substances 0.000 description 3
- 150000008163 sugars Chemical class 0.000 description 3
- 239000004094 surface-active agent Substances 0.000 description 3
- 230000004083 survival effect Effects 0.000 description 3
- 230000001839 systemic circulation Effects 0.000 description 3
- 235000002906 tartaric acid Nutrition 0.000 description 3
- 239000002562 thickening agent Substances 0.000 description 3
- 230000000699 topical effect Effects 0.000 description 3
- 230000001988 toxicity Effects 0.000 description 3
- 231100000419 toxicity Toxicity 0.000 description 3
- 238000012546 transfer Methods 0.000 description 3
- 230000009261 transgenic effect Effects 0.000 description 3
- 241001430294 unidentified retrovirus Species 0.000 description 3
- DRTQHJPVMGBUCF-UHFFFAOYSA-N uracil arabinoside Natural products OC1C(O)C(CO)OC1N1C(=O)NC(=O)C=C1 DRTQHJPVMGBUCF-UHFFFAOYSA-N 0.000 description 3
- 229940045145 uridine Drugs 0.000 description 3
- 229920003169 water-soluble polymer Polymers 0.000 description 3
- 239000000080 wetting agent Substances 0.000 description 3
- 210000005253 yeast cell Anatomy 0.000 description 3
- UHPQFNXOFFPHJW-UHFFFAOYSA-N (4-methylphenyl)-phenylmethanamine Chemical compound C1=CC(C)=CC=C1C(N)C1=CC=CC=C1 UHPQFNXOFFPHJW-UHFFFAOYSA-N 0.000 description 2
- 125000003088 (fluoren-9-ylmethoxy)carbonyl group Chemical group 0.000 description 2
- LMDZBCPBFSXMTL-UHFFFAOYSA-N 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide Chemical compound CCN=C=NCCCN(C)C LMDZBCPBFSXMTL-UHFFFAOYSA-N 0.000 description 2
- MIJDSYMOBYNHOT-UHFFFAOYSA-N 2-(ethylamino)ethanol Chemical compound CCNCCO MIJDSYMOBYNHOT-UHFFFAOYSA-N 0.000 description 2
- AQTFKGDWFRRIHR-UHFFFAOYSA-L 3-[18-(2-carboxylatoethyl)-8,13-bis(ethenyl)-3,7,12,17-tetramethylporphyrin-21,24-diid-2-yl]propanoate;cobalt(2+);hydron Chemical compound [Co+2].[N-]1C(C=C2C(=C(C)C(C=C3C(=C(C)C(=C4)[N-]3)C=C)=N2)C=C)=C(C)C(CCC(O)=O)=C1C=C1C(CCC(O)=O)=C(C)C4=N1 AQTFKGDWFRRIHR-UHFFFAOYSA-L 0.000 description 2
- 101710169336 5'-deoxyadenosine deaminase Proteins 0.000 description 2
- WOVKYSAHUYNSMH-RRKCRQDMSA-N 5-bromodeoxyuridine Chemical compound C1[C@H](O)[C@@H](CO)O[C@H]1N1C(=O)NC(=O)C(Br)=C1 WOVKYSAHUYNSMH-RRKCRQDMSA-N 0.000 description 2
- 239000013607 AAV vector Substances 0.000 description 2
- 206010001052 Acute respiratory distress syndrome Diseases 0.000 description 2
- 102000055025 Adenosine deaminases Human genes 0.000 description 2
- 102000009027 Albumins Human genes 0.000 description 2
- 108010088751 Albumins Proteins 0.000 description 2
- QGZKDVFQNNGYKY-UHFFFAOYSA-O Ammonium Chemical compound [NH4+] QGZKDVFQNNGYKY-UHFFFAOYSA-O 0.000 description 2
- 235000002198 Annona diversifolia Nutrition 0.000 description 2
- 241000282815 Antilocapra americana Species 0.000 description 2
- 201000001320 Atherosclerosis Diseases 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 108090001008 Avidin Proteins 0.000 description 2
- 244000063299 Bacillus subtilis Species 0.000 description 2
- 235000014469 Bacillus subtilis Nutrition 0.000 description 2
- DWRXFEITVBNRMK-UHFFFAOYSA-N Beta-D-1-Arabinofuranosylthymine Natural products O=C1NC(=O)C(C)=CN1C1C(O)C(O)C(CO)O1 DWRXFEITVBNRMK-UHFFFAOYSA-N 0.000 description 2
- 102100026189 Beta-galactosidase Human genes 0.000 description 2
- 102100023995 Beta-nerve growth factor Human genes 0.000 description 2
- 241000282817 Bovidae Species 0.000 description 2
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 2
- 208000007204 Brain death Diseases 0.000 description 2
- 102100023701 C-C motif chemokine 18 Human genes 0.000 description 2
- 102100032367 C-C motif chemokine 5 Human genes 0.000 description 2
- 241000282832 Camelidae Species 0.000 description 2
- 241000282421 Canidae Species 0.000 description 2
- 241000282465 Canis Species 0.000 description 2
- 241000283707 Capra Species 0.000 description 2
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 2
- 241000288673 Chiroptera Species 0.000 description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-M Chloride anion Chemical compound [Cl-] VEXZGXHMUGYJMC-UHFFFAOYSA-M 0.000 description 2
- 208000006545 Chronic Obstructive Pulmonary Disease Diseases 0.000 description 2
- 108020004705 Codon Proteins 0.000 description 2
- 108700010070 Codon Usage Proteins 0.000 description 2
- 102000029816 Collagenase Human genes 0.000 description 2
- 108060005980 Collagenase Proteins 0.000 description 2
- 229920002261 Corn starch Polymers 0.000 description 2
- 201000003883 Cystic fibrosis Diseases 0.000 description 2
- 241000701022 Cytomegalovirus Species 0.000 description 2
- BWGNESOTFCXPMA-UHFFFAOYSA-N Dihydrogen disulfide Chemical compound SS BWGNESOTFCXPMA-UHFFFAOYSA-N 0.000 description 2
- 108010024212 E-Selectin Proteins 0.000 description 2
- 102100023471 E-selectin Human genes 0.000 description 2
- 238000012286 ELISA Assay Methods 0.000 description 2
- 102100025137 Early activation antigen CD69 Human genes 0.000 description 2
- LVGKNOAMLMIIKO-UHFFFAOYSA-N Elaidinsaeure-aethylester Natural products CCCCCCCCC=CCCCCCCCC(=O)OCC LVGKNOAMLMIIKO-UHFFFAOYSA-N 0.000 description 2
- 238000011510 Elispot assay Methods 0.000 description 2
- 241000283086 Equidae Species 0.000 description 2
- 241000588724 Escherichia coli Species 0.000 description 2
- VGGSQFUCUMXWEO-UHFFFAOYSA-N Ethene Chemical compound C=C VGGSQFUCUMXWEO-UHFFFAOYSA-N 0.000 description 2
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 description 2
- 239000004606 Fillers/Extenders Substances 0.000 description 2
- KRHYYFGTRYWZRS-UHFFFAOYSA-N Fluorane Chemical compound F KRHYYFGTRYWZRS-UHFFFAOYSA-N 0.000 description 2
- 238000012413 Fluorescence activated cell sorting analysis Methods 0.000 description 2
- 108010010803 Gelatin Proteins 0.000 description 2
- 108700028146 Genetic Enhancer Elements Proteins 0.000 description 2
- 241000282818 Giraffidae Species 0.000 description 2
- 102000006395 Globulins Human genes 0.000 description 2
- 108010044091 Globulins Proteins 0.000 description 2
- AEMRFAOFKBGASW-UHFFFAOYSA-N Glycolic acid Chemical compound OCC(O)=O AEMRFAOFKBGASW-UHFFFAOYSA-N 0.000 description 2
- 208000015023 Graves' disease Diseases 0.000 description 2
- 102000003745 Hepatocyte Growth Factor Human genes 0.000 description 2
- 108090000100 Hepatocyte Growth Factor Proteins 0.000 description 2
- 241000282824 Hippopotamidae Species 0.000 description 2
- 101000978371 Homo sapiens C-C motif chemokine 18 Proteins 0.000 description 2
- 101100099884 Homo sapiens CD40 gene Proteins 0.000 description 2
- 101000934374 Homo sapiens Early activation antigen CD69 Proteins 0.000 description 2
- 101001057504 Homo sapiens Interferon-stimulated gene 20 kDa protein Proteins 0.000 description 2
- 101001055144 Homo sapiens Interleukin-2 receptor subunit alpha Proteins 0.000 description 2
- 108091006905 Human Serum Albumin Proteins 0.000 description 2
- 102000008100 Human Serum Albumin Human genes 0.000 description 2
- 241000725303 Human immunodeficiency virus Species 0.000 description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 2
- PMMYEEVYMWASQN-DMTCNVIQSA-N Hydroxyproline Chemical compound O[C@H]1CN[C@H](C(O)=O)C1 PMMYEEVYMWASQN-DMTCNVIQSA-N 0.000 description 2
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 2
- 108010021625 Immunoglobulin Fragments Proteins 0.000 description 2
- 102000008394 Immunoglobulin Fragments Human genes 0.000 description 2
- 108010064593 Intercellular Adhesion Molecule-1 Proteins 0.000 description 2
- 102100037877 Intercellular adhesion molecule 1 Human genes 0.000 description 2
- 102100037850 Interferon gamma Human genes 0.000 description 2
- 108010074328 Interferon-gamma Proteins 0.000 description 2
- 102100027268 Interferon-stimulated gene 20 kDa protein Human genes 0.000 description 2
- ONIBWKKTOPOVIA-BYPYZUCNSA-N L-Proline Chemical compound OC(=O)[C@@H]1CCCN1 ONIBWKKTOPOVIA-BYPYZUCNSA-N 0.000 description 2
- 241000282838 Lama Species 0.000 description 2
- 241000282560 Macaca mulatta Species 0.000 description 2
- 241000829100 Macaca mulatta polyomavirus 1 Species 0.000 description 2
- 101710175625 Maltose/maltodextrin-binding periplasmic protein Proteins 0.000 description 2
- 102000001776 Matrix metalloproteinase-9 Human genes 0.000 description 2
- 102100030412 Matrix metalloproteinase-9 Human genes 0.000 description 2
- 102000012750 Membrane Glycoproteins Human genes 0.000 description 2
- 108010090054 Membrane Glycoproteins Proteins 0.000 description 2
- BAVYZALUXZFZLV-UHFFFAOYSA-N Methylamine Chemical compound NC BAVYZALUXZFZLV-UHFFFAOYSA-N 0.000 description 2
- 229920000168 Microcrystalline cellulose Polymers 0.000 description 2
- 101710151805 Mitochondrial intermediate peptidase 1 Proteins 0.000 description 2
- 101001013152 Mycobacterium avium Major membrane protein 1 Proteins 0.000 description 2
- 101001013151 Mycobacterium leprae (strain TN) Major membrane protein I Proteins 0.000 description 2
- 108010057466 NF-kappa B Proteins 0.000 description 2
- 102000003945 NF-kappa B Human genes 0.000 description 2
- UFWIBTONFRDIAS-UHFFFAOYSA-N Naphthalene Chemical compound C1=CC=CC2=CC=CC=C21 UFWIBTONFRDIAS-UHFFFAOYSA-N 0.000 description 2
- 108010025020 Nerve Growth Factor Proteins 0.000 description 2
- MWUXSHHQAYIFBG-UHFFFAOYSA-N Nitric oxide Chemical compound O=[N] MWUXSHHQAYIFBG-UHFFFAOYSA-N 0.000 description 2
- 108091034117 Oligonucleotide Proteins 0.000 description 2
- 241000283973 Oryctolagus cuniculus Species 0.000 description 2
- 241000282577 Pan troglodytes Species 0.000 description 2
- 235000019483 Peanut oil Nutrition 0.000 description 2
- 241001494479 Pecora Species 0.000 description 2
- 241000009328 Perro Species 0.000 description 2
- 241000283966 Pholidota <mammal> Species 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 2
- 108010039918 Polylysine Proteins 0.000 description 2
- 239000004793 Polystyrene Substances 0.000 description 2
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 2
- RJKFOVLPORLFTN-LEKSSAKUSA-N Progesterone Chemical compound C1CC2=CC(=O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H](C(=O)C)[C@@]1(C)CC2 RJKFOVLPORLFTN-LEKSSAKUSA-N 0.000 description 2
- ONIBWKKTOPOVIA-UHFFFAOYSA-N Proline Natural products OC(=O)C1CCCN1 ONIBWKKTOPOVIA-UHFFFAOYSA-N 0.000 description 2
- ATUOYWHBWRKTHZ-UHFFFAOYSA-N Propane Chemical compound CCC ATUOYWHBWRKTHZ-UHFFFAOYSA-N 0.000 description 2
- 239000004365 Protease Substances 0.000 description 2
- 201000004681 Psoriasis Diseases 0.000 description 2
- LCTONWCANYUPML-UHFFFAOYSA-N Pyruvic acid Chemical compound CC(=O)C(O)=O LCTONWCANYUPML-UHFFFAOYSA-N 0.000 description 2
- 208000013616 Respiratory Distress Syndrome Diseases 0.000 description 2
- 108091028664 Ribonucleotide Proteins 0.000 description 2
- 239000008156 Ringer's lactate solution Substances 0.000 description 2
- 241000283984 Rodentia Species 0.000 description 2
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 2
- 102000002669 Small Ubiquitin-Related Modifier Proteins Human genes 0.000 description 2
- 108010043401 Small Ubiquitin-Related Modifier Proteins Proteins 0.000 description 2
- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical compound [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 description 2
- 239000004141 Sodium laurylsulphate Substances 0.000 description 2
- 229920002125 Sokalan® Polymers 0.000 description 2
- 229920002472 Starch Polymers 0.000 description 2
- PPBRXRYQALVLMV-UHFFFAOYSA-N Styrene Chemical compound C=CC1=CC=CC=C1 PPBRXRYQALVLMV-UHFFFAOYSA-N 0.000 description 2
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 2
- 230000024932 T cell mediated immunity Effects 0.000 description 2
- 241000282910 Tayassuidae Species 0.000 description 2
- 241000282851 Tragulidae Species 0.000 description 2
- 108020004566 Transfer RNA Proteins 0.000 description 2
- 102000004887 Transforming Growth Factor beta Human genes 0.000 description 2
- 108090001012 Transforming Growth Factor beta Proteins 0.000 description 2
- 229940122618 Trypsin inhibitor Drugs 0.000 description 2
- 101710162629 Trypsin inhibitor Proteins 0.000 description 2
- 206010067584 Type 1 diabetes mellitus Diseases 0.000 description 2
- 108010000134 Vascular Cell Adhesion Molecule-1 Proteins 0.000 description 2
- 102100023543 Vascular cell adhesion protein 1 Human genes 0.000 description 2
- QYSXJUFSXHHAJI-XFEUOLMDSA-N Vitamin D3 Natural products C1(/[C@@H]2CC[C@@H]([C@]2(CCC1)C)[C@H](C)CCCC(C)C)=C/C=C1\C[C@@H](O)CCC1=C QYSXJUFSXHHAJI-XFEUOLMDSA-N 0.000 description 2
- 235000011054 acetic acid Nutrition 0.000 description 2
- 230000013564 activation of immune response Effects 0.000 description 2
- 230000033289 adaptive immune response Effects 0.000 description 2
- 230000000996 additive effect Effects 0.000 description 2
- 201000000028 adult respiratory distress syndrome Diseases 0.000 description 2
- 230000002411 adverse Effects 0.000 description 2
- 150000001299 aldehydes Chemical class 0.000 description 2
- 125000000217 alkyl group Chemical group 0.000 description 2
- 208000026935 allergic disease Diseases 0.000 description 2
- 230000000735 allogeneic effect Effects 0.000 description 2
- 238000011316 allogeneic transplantation Methods 0.000 description 2
- 150000001412 amines Chemical class 0.000 description 2
- 210000004102 animal cell Anatomy 0.000 description 2
- 150000001450 anions Chemical class 0.000 description 2
- 238000013459 approach Methods 0.000 description 2
- 239000012736 aqueous medium Substances 0.000 description 2
- 239000007900 aqueous suspension Substances 0.000 description 2
- 239000008135 aqueous vehicle Substances 0.000 description 2
- 125000003118 aryl group Chemical group 0.000 description 2
- 208000006673 asthma Diseases 0.000 description 2
- 230000001363 autoimmune Effects 0.000 description 2
- 230000006399 behavior Effects 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- WPYMKLBDIGXBTP-UHFFFAOYSA-N benzoic acid Chemical compound OC(=O)C1=CC=CC=C1 WPYMKLBDIGXBTP-UHFFFAOYSA-N 0.000 description 2
- SESFRYSPDFLNCH-UHFFFAOYSA-N benzyl benzoate Chemical compound C=1C=CC=CC=1C(=O)OCC1=CC=CC=C1 SESFRYSPDFLNCH-UHFFFAOYSA-N 0.000 description 2
- 108010005774 beta-Galactosidase Proteins 0.000 description 2
- SQVRNKJHWKZAKO-UHFFFAOYSA-N beta-N-Acetyl-D-neuraminic acid Natural products CC(=O)NC1C(O)CC(O)(C(O)=O)OC1C(O)C(O)CO SQVRNKJHWKZAKO-UHFFFAOYSA-N 0.000 description 2
- UCMIRNVEIXFBKS-UHFFFAOYSA-N beta-alanine Chemical compound NCCC(O)=O UCMIRNVEIXFBKS-UHFFFAOYSA-N 0.000 description 2
- 239000011230 binding agent Substances 0.000 description 2
- 238000010170 biological method Methods 0.000 description 2
- 230000033228 biological regulation Effects 0.000 description 2
- 230000008499 blood brain barrier function Effects 0.000 description 2
- 230000036765 blood level Effects 0.000 description 2
- 210000001218 blood-brain barrier Anatomy 0.000 description 2
- 210000001185 bone marrow Anatomy 0.000 description 2
- 229940098773 bovine serum albumin Drugs 0.000 description 2
- 239000004067 bulking agent Substances 0.000 description 2
- 125000000484 butyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 2
- 239000001506 calcium phosphate Substances 0.000 description 2
- 229910000389 calcium phosphate Inorganic materials 0.000 description 2
- 235000011010 calcium phosphates Nutrition 0.000 description 2
- 125000002843 carboxylic acid group Chemical group 0.000 description 2
- 210000000748 cardiovascular system Anatomy 0.000 description 2
- 230000015556 catabolic process Effects 0.000 description 2
- YCIMNLLNPGFGHC-UHFFFAOYSA-N catechol Chemical compound OC1=CC=CC=C1O YCIMNLLNPGFGHC-UHFFFAOYSA-N 0.000 description 2
- 210000000170 cell membrane Anatomy 0.000 description 2
- 235000010980 cellulose Nutrition 0.000 description 2
- 229920002678 cellulose Polymers 0.000 description 2
- 239000001913 cellulose Substances 0.000 description 2
- 239000005081 chemiluminescent agent Substances 0.000 description 2
- OSASVXMJTNOKOY-UHFFFAOYSA-N chlorobutanol Chemical compound CC(C)(O)C(Cl)(Cl)Cl OSASVXMJTNOKOY-UHFFFAOYSA-N 0.000 description 2
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 description 2
- 229960002424 collagenase Drugs 0.000 description 2
- 239000003086 colorant Substances 0.000 description 2
- 239000002131 composite material Substances 0.000 description 2
- 235000008504 concentrate Nutrition 0.000 description 2
- 239000012141 concentrate Substances 0.000 description 2
- 238000009833 condensation Methods 0.000 description 2
- 230000005494 condensation Effects 0.000 description 2
- 238000007796 conventional method Methods 0.000 description 2
- 235000005687 corn oil Nutrition 0.000 description 2
- 239000002285 corn oil Substances 0.000 description 2
- 235000012343 cottonseed oil Nutrition 0.000 description 2
- 239000002385 cottonseed oil Substances 0.000 description 2
- VFLDPWHFBUODDF-FCXRPNKRSA-N curcumin Chemical group C1=C(O)C(OC)=CC(\C=C\C(=O)CC(=O)\C=C\C=2C=C(OC)C(O)=CC=2)=C1 VFLDPWHFBUODDF-FCXRPNKRSA-N 0.000 description 2
- XVOYSCVBGLVSOL-UHFFFAOYSA-N cysteic acid Chemical compound OC(=O)C(N)CS(O)(=O)=O XVOYSCVBGLVSOL-UHFFFAOYSA-N 0.000 description 2
- OPTASPLRGRRNAP-UHFFFAOYSA-N cytosine Chemical compound NC=1C=CNC(=O)N=1 OPTASPLRGRRNAP-UHFFFAOYSA-N 0.000 description 2
- 230000001472 cytotoxic effect Effects 0.000 description 2
- 230000003013 cytotoxicity Effects 0.000 description 2
- 231100000135 cytotoxicity Toxicity 0.000 description 2
- 238000006731 degradation reaction Methods 0.000 description 2
- 239000003405 delayed action preparation Substances 0.000 description 2
- 239000003599 detergent Substances 0.000 description 2
- 239000008121 dextrose Substances 0.000 description 2
- RNPXCFINMKSQPQ-UHFFFAOYSA-N dicetyl hydrogen phosphate Chemical compound CCCCCCCCCCCCCCCCOP(O)(=O)OCCCCCCCCCCCCCCCC RNPXCFINMKSQPQ-UHFFFAOYSA-N 0.000 description 2
- FFYPMLJYZAEMQB-UHFFFAOYSA-N diethyl pyrocarbonate Chemical compound CCOC(=O)OC(=O)OCC FFYPMLJYZAEMQB-UHFFFAOYSA-N 0.000 description 2
- XBDQKXXYIPTUBI-UHFFFAOYSA-N dimethylselenoniopropionate Natural products CCC(O)=O XBDQKXXYIPTUBI-UHFFFAOYSA-N 0.000 description 2
- 239000002270 dispersing agent Substances 0.000 description 2
- PMMYEEVYMWASQN-UHFFFAOYSA-N dl-hydroxyproline Natural products OC1C[NH2+]C(C([O-])=O)C1 PMMYEEVYMWASQN-UHFFFAOYSA-N 0.000 description 2
- 239000006196 drop Substances 0.000 description 2
- 239000000975 dye Substances 0.000 description 2
- 230000002124 endocrine Effects 0.000 description 2
- 238000003114 enzyme-linked immunosorbent spot assay Methods 0.000 description 2
- 210000002919 epithelial cell Anatomy 0.000 description 2
- 239000003797 essential amino acid Substances 0.000 description 2
- 235000020776 essential amino acid Nutrition 0.000 description 2
- BEFDCLMNVWHSGT-UHFFFAOYSA-N ethenylcyclopentane Chemical compound C=CC1CCCC1 BEFDCLMNVWHSGT-UHFFFAOYSA-N 0.000 description 2
- MMXKVMNBHPAILY-UHFFFAOYSA-N ethyl laurate Chemical compound CCCCCCCCCCCC(=O)OCC MMXKVMNBHPAILY-UHFFFAOYSA-N 0.000 description 2
- LVGKNOAMLMIIKO-QXMHVHEDSA-N ethyl oleate Chemical compound CCCCCCCC\C=C/CCCCCCCC(=O)OCC LVGKNOAMLMIIKO-QXMHVHEDSA-N 0.000 description 2
- 229940093471 ethyl oleate Drugs 0.000 description 2
- 238000013265 extended release Methods 0.000 description 2
- 230000001605 fetal effect Effects 0.000 description 2
- 210000002950 fibroblast Anatomy 0.000 description 2
- 239000000945 filler Substances 0.000 description 2
- 239000000796 flavoring agent Substances 0.000 description 2
- 239000007850 fluorescent dye Substances 0.000 description 2
- 239000006260 foam Substances 0.000 description 2
- 125000002485 formyl group Chemical group [H]C(*)=O 0.000 description 2
- BTCSSZJGUNDROE-UHFFFAOYSA-N gamma-aminobutyric acid Chemical compound NCCCC(O)=O BTCSSZJGUNDROE-UHFFFAOYSA-N 0.000 description 2
- 229920000159 gelatin Polymers 0.000 description 2
- 239000008273 gelatin Substances 0.000 description 2
- 235000019322 gelatine Nutrition 0.000 description 2
- 235000011852 gelatine desserts Nutrition 0.000 description 2
- 238000010362 genome editing Methods 0.000 description 2
- 125000005456 glyceride group Chemical group 0.000 description 2
- 235000011187 glycerol Nutrition 0.000 description 2
- 230000012010 growth Effects 0.000 description 2
- 125000005843 halogen group Chemical group 0.000 description 2
- 210000002216 heart Anatomy 0.000 description 2
- 210000003958 hematopoietic stem cell Anatomy 0.000 description 2
- 210000003494 hepatocyte Anatomy 0.000 description 2
- 229940088597 hormone Drugs 0.000 description 2
- 239000005556 hormone Substances 0.000 description 2
- 230000028996 humoral immune response Effects 0.000 description 2
- 150000004679 hydroxides Chemical class 0.000 description 2
- NPZTUJOABDZTLV-UHFFFAOYSA-N hydroxybenzotriazole Substances O=C1C=CC=C2NNN=C12 NPZTUJOABDZTLV-UHFFFAOYSA-N 0.000 description 2
- 229960002591 hydroxyproline Drugs 0.000 description 2
- 235000010979 hydroxypropyl methyl cellulose Nutrition 0.000 description 2
- 229920003088 hydroxypropyl methyl cellulose Polymers 0.000 description 2
- 239000001866 hydroxypropyl methyl cellulose Substances 0.000 description 2
- 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 2
- 230000009610 hypersensitivity Effects 0.000 description 2
- 210000000987 immune system Anatomy 0.000 description 2
- 230000002163 immunogen Effects 0.000 description 2
- 108020004201 indoleamine 2,3-dioxygenase Proteins 0.000 description 2
- 102000006639 indoleamine 2,3-dioxygenase Human genes 0.000 description 2
- 230000006698 induction Effects 0.000 description 2
- 230000000977 initiatory effect Effects 0.000 description 2
- 230000010354 integration Effects 0.000 description 2
- 230000000968 intestinal effect Effects 0.000 description 2
- 238000010255 intramuscular injection Methods 0.000 description 2
- 238000010253 intravenous injection Methods 0.000 description 2
- NNPPMTNAJDCUHE-UHFFFAOYSA-N isobutane Chemical compound CC(C)C NNPPMTNAJDCUHE-UHFFFAOYSA-N 0.000 description 2
- JJWLVOIRVHMVIS-UHFFFAOYSA-N isopropylamine Chemical compound CC(C)N JJWLVOIRVHMVIS-UHFFFAOYSA-N 0.000 description 2
- 210000003734 kidney Anatomy 0.000 description 2
- 238000002372 labelling Methods 0.000 description 2
- 239000004310 lactic acid Substances 0.000 description 2
- 235000014655 lactic acid Nutrition 0.000 description 2
- 210000004185 liver Anatomy 0.000 description 2
- 239000006210 lotion Substances 0.000 description 2
- 239000000314 lubricant Substances 0.000 description 2
- 206010025135 lupus erythematosus Diseases 0.000 description 2
- RLSSMJSEOOYNOY-UHFFFAOYSA-N m-cresol Chemical compound CC1=CC=CC(O)=C1 RLSSMJSEOOYNOY-UHFFFAOYSA-N 0.000 description 2
- 229940124302 mTOR inhibitor Drugs 0.000 description 2
- 229920002521 macromolecule Polymers 0.000 description 2
- HQKMJHAJHXVSDF-UHFFFAOYSA-L magnesium stearate Chemical compound [Mg+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O HQKMJHAJHXVSDF-UHFFFAOYSA-L 0.000 description 2
- 210000004962 mammalian cell Anatomy 0.000 description 2
- 239000003628 mammalian target of rapamycin inhibitor Substances 0.000 description 2
- 239000002609 medium Substances 0.000 description 2
- 230000015654 memory Effects 0.000 description 2
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 description 2
- 229920000609 methyl cellulose Polymers 0.000 description 2
- 235000010270 methyl p-hydroxybenzoate Nutrition 0.000 description 2
- 239000001923 methylcellulose Substances 0.000 description 2
- 235000010981 methylcellulose Nutrition 0.000 description 2
- 235000019813 microcrystalline cellulose Nutrition 0.000 description 2
- 239000008108 microcrystalline cellulose Substances 0.000 description 2
- 229940016286 microcrystalline cellulose Drugs 0.000 description 2
- 230000003232 mucoadhesive effect Effects 0.000 description 2
- 210000005170 neoplastic cell Anatomy 0.000 description 2
- 229940053128 nerve growth factor Drugs 0.000 description 2
- 239000002687 nonaqueous vehicle Substances 0.000 description 2
- 239000002777 nucleoside Substances 0.000 description 2
- 150000003833 nucleoside derivatives Chemical class 0.000 description 2
- 239000003921 oil Substances 0.000 description 2
- 235000019198 oils Nutrition 0.000 description 2
- 238000004806 packaging method and process Methods 0.000 description 2
- 239000000312 peanut oil Substances 0.000 description 2
- AQIXEPGDORPWBJ-UHFFFAOYSA-N pentan-3-ol Chemical compound CCC(O)CC AQIXEPGDORPWBJ-UHFFFAOYSA-N 0.000 description 2
- 239000000863 peptide conjugate Substances 0.000 description 2
- 239000012071 phase Substances 0.000 description 2
- 229910052698 phosphorus Inorganic materials 0.000 description 2
- BZQFBWGGLXLEPQ-REOHCLBHSA-N phosphoserine Chemical compound OC(=O)[C@@H](N)COP(O)(O)=O BZQFBWGGLXLEPQ-REOHCLBHSA-N 0.000 description 2
- OXNIZHLAWKMVMX-UHFFFAOYSA-N picric acid Chemical compound OC1=C([N+]([O-])=O)C=C([N+]([O-])=O)C=C1[N+]([O-])=O OXNIZHLAWKMVMX-UHFFFAOYSA-N 0.000 description 2
- 229920003023 plastic Polymers 0.000 description 2
- 239000004033 plastic Substances 0.000 description 2
- 229920000191 poly(N-vinyl pyrrolidone) Polymers 0.000 description 2
- 229920002187 poly[N-2-(hydroxypropyl) methacrylamide] polymer Polymers 0.000 description 2
- 229920000656 polylysine Polymers 0.000 description 2
- 229920005862 polyol Polymers 0.000 description 2
- 150000003077 polyols Chemical class 0.000 description 2
- 229920002451 polyvinyl alcohol Polymers 0.000 description 2
- 239000011591 potassium Substances 0.000 description 2
- KMUONIBRACKNSN-UHFFFAOYSA-N potassium dichromate Chemical compound [K+].[K+].[O-][Cr](=O)(=O)O[Cr]([O-])(=O)=O KMUONIBRACKNSN-UHFFFAOYSA-N 0.000 description 2
- 229920001592 potato starch Polymers 0.000 description 2
- MFDFERRIHVXMIY-UHFFFAOYSA-N procaine Chemical compound CCN(CC)CCOC(=O)C1=CC=C(N)C=C1 MFDFERRIHVXMIY-UHFFFAOYSA-N 0.000 description 2
- 229960004919 procaine Drugs 0.000 description 2
- 230000002062 proliferating effect Effects 0.000 description 2
- 230000002035 prolonged effect Effects 0.000 description 2
- 235000010232 propyl p-hydroxybenzoate Nutrition 0.000 description 2
- 238000001742 protein purification Methods 0.000 description 2
- 230000006337 proteolytic cleavage Effects 0.000 description 2
- 230000003362 replicative effect Effects 0.000 description 2
- GHMLBKRAJCXXBS-UHFFFAOYSA-N resorcinol Chemical compound OC1=CC=CC(O)=C1 GHMLBKRAJCXXBS-UHFFFAOYSA-N 0.000 description 2
- 210000002345 respiratory system Anatomy 0.000 description 2
- 108091008146 restriction endonucleases Proteins 0.000 description 2
- 238000003757 reverse transcription PCR Methods 0.000 description 2
- 238000012552 review Methods 0.000 description 2
- 206010039073 rheumatoid arthritis Diseases 0.000 description 2
- 239000002336 ribonucleotide Substances 0.000 description 2
- YGSDEFSMJLZEOE-UHFFFAOYSA-N salicylic acid Chemical compound OC(=O)C1=CC=CC=C1O YGSDEFSMJLZEOE-UHFFFAOYSA-N 0.000 description 2
- FSYKKLYZXJSNPZ-UHFFFAOYSA-N sarcosine Chemical compound C[NH2+]CC([O-])=O FSYKKLYZXJSNPZ-UHFFFAOYSA-N 0.000 description 2
- 230000003248 secreting effect Effects 0.000 description 2
- 239000008159 sesame oil Substances 0.000 description 2
- 235000011803 sesame oil Nutrition 0.000 description 2
- 210000000329 smooth muscle myocyte Anatomy 0.000 description 2
- 239000011734 sodium Substances 0.000 description 2
- 229910052708 sodium Inorganic materials 0.000 description 2
- 235000019333 sodium laurylsulphate Nutrition 0.000 description 2
- 238000010532 solid phase synthesis reaction Methods 0.000 description 2
- 239000004334 sorbic acid Substances 0.000 description 2
- 235000010199 sorbic acid Nutrition 0.000 description 2
- 229940075582 sorbic acid Drugs 0.000 description 2
- 235000010356 sorbitol Nutrition 0.000 description 2
- 239000000600 sorbitol Substances 0.000 description 2
- 230000006641 stabilisation Effects 0.000 description 2
- 238000011105 stabilization Methods 0.000 description 2
- 235000019698 starch Nutrition 0.000 description 2
- 239000008223 sterile water Substances 0.000 description 2
- 238000003860 storage Methods 0.000 description 2
- 125000001424 substituent group Chemical group 0.000 description 2
- 239000000829 suppository Substances 0.000 description 2
- 230000001629 suppression Effects 0.000 description 2
- 230000009897 systematic effect Effects 0.000 description 2
- 239000000454 talc Substances 0.000 description 2
- 235000012222 talc Nutrition 0.000 description 2
- 229910052623 talc Inorganic materials 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- ZRKFYGHZFMAOKI-QMGMOQQFSA-N tgfbeta Chemical compound C([C@H](NC(=O)[C@H](C(C)C)NC(=O)CNC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CC(C)C)NC(=O)CNC(=O)[C@H](C)NC(=O)[C@H](CO)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@@H](NC(=O)[C@H](C)NC(=O)[C@H](C)NC(=O)[C@@H](NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](N)CCSC)C(C)C)[C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](C)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C)C(=O)N[C@@H](CC(C)C)C(=O)N1[C@@H](CCC1)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(O)=O)C1=CC=C(O)C=C1 ZRKFYGHZFMAOKI-QMGMOQQFSA-N 0.000 description 2
- 238000011287 therapeutic dose Methods 0.000 description 2
- 238000004809 thin layer chromatography Methods 0.000 description 2
- RYYWUUFWQRZTIU-UHFFFAOYSA-K thiophosphate Chemical compound [O-]P([O-])([O-])=S RYYWUUFWQRZTIU-UHFFFAOYSA-K 0.000 description 2
- 229940104230 thymidine Drugs 0.000 description 2
- 239000006208 topical dosage form Substances 0.000 description 2
- 231100000331 toxic Toxicity 0.000 description 2
- 230000002588 toxic effect Effects 0.000 description 2
- QORWJWZARLRLPR-UHFFFAOYSA-H tricalcium bis(phosphate) Chemical compound [Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O QORWJWZARLRLPR-UHFFFAOYSA-H 0.000 description 2
- 229950002929 trinitrophenol Drugs 0.000 description 2
- 239000002753 trypsin inhibitor Substances 0.000 description 2
- 241001529453 unidentified herpesvirus Species 0.000 description 2
- 238000011144 upstream manufacturing Methods 0.000 description 2
- 210000002845 virion Anatomy 0.000 description 2
- QYSXJUFSXHHAJI-YRZJJWOYSA-N vitamin D3 Chemical compound C1(/[C@@H]2CC[C@@H]([C@]2(CCC1)C)[C@H](C)CCCC(C)C)=C\C=C1\C[C@@H](O)CCC1=C QYSXJUFSXHHAJI-YRZJJWOYSA-N 0.000 description 2
- 239000011647 vitamin D3 Substances 0.000 description 2
- 235000005282 vitamin D3 Nutrition 0.000 description 2
- 229940021056 vitamin d3 Drugs 0.000 description 2
- 238000001262 western blot Methods 0.000 description 2
- 238000002689 xenotransplantation Methods 0.000 description 2
- JIAARYAFYJHUJI-UHFFFAOYSA-L zinc dichloride Chemical compound [Cl-].[Cl-].[Zn+2] JIAARYAFYJHUJI-UHFFFAOYSA-L 0.000 description 2
- JWDFQMWEFLOOED-UHFFFAOYSA-N (2,5-dioxopyrrolidin-1-yl) 3-(pyridin-2-yldisulfanyl)propanoate Chemical compound O=C1CCC(=O)N1OC(=O)CCSSC1=CC=CC=N1 JWDFQMWEFLOOED-UHFFFAOYSA-N 0.000 description 1
- PZFZLRNAOHUQPH-DJBVYZKNSA-N (2r)-3-[(2r)-2,3-di(hexadecanoyloxy)propyl]sulfanyl-2-(hexadecanoylamino)propanoic acid Chemical compound CCCCCCCCCCCCCCCC(=O)N[C@H](C(O)=O)CSC[C@H](OC(=O)CCCCCCCCCCCCCCC)COC(=O)CCCCCCCCCCCCCCC PZFZLRNAOHUQPH-DJBVYZKNSA-N 0.000 description 1
- LNAZSHAWQACDHT-XIYTZBAFSA-N (2r,3r,4s,5r,6s)-4,5-dimethoxy-2-(methoxymethyl)-3-[(2s,3r,4s,5r,6r)-3,4,5-trimethoxy-6-(methoxymethyl)oxan-2-yl]oxy-6-[(2r,3r,4s,5r,6r)-4,5,6-trimethoxy-2-(methoxymethyl)oxan-3-yl]oxyoxane Chemical compound CO[C@@H]1[C@@H](OC)[C@H](OC)[C@@H](COC)O[C@H]1O[C@H]1[C@H](OC)[C@@H](OC)[C@H](O[C@H]2[C@@H]([C@@H](OC)[C@H](OC)O[C@@H]2COC)OC)O[C@@H]1COC LNAZSHAWQACDHT-XIYTZBAFSA-N 0.000 description 1
- BVAUMRCGVHUWOZ-ZETCQYMHSA-N (2s)-2-(cyclohexylazaniumyl)propanoate Chemical compound OC(=O)[C@H](C)NC1CCCCC1 BVAUMRCGVHUWOZ-ZETCQYMHSA-N 0.000 description 1
- MRTPISKDZDHEQI-YFKPBYRVSA-N (2s)-2-(tert-butylamino)propanoic acid Chemical compound OC(=O)[C@H](C)NC(C)(C)C MRTPISKDZDHEQI-YFKPBYRVSA-N 0.000 description 1
- NPDBDJFLKKQMCM-SCSAIBSYSA-N (2s)-2-amino-3,3-dimethylbutanoic acid Chemical compound CC(C)(C)[C@H](N)C(O)=O NPDBDJFLKKQMCM-SCSAIBSYSA-N 0.000 description 1
- BJEPYKJPYRNKOW-REOHCLBHSA-N (S)-malic acid Chemical compound OC(=O)[C@@H](O)CC(O)=O BJEPYKJPYRNKOW-REOHCLBHSA-N 0.000 description 1
- DDMOUSALMHHKOS-UHFFFAOYSA-N 1,2-dichloro-1,1,2,2-tetrafluoroethane Chemical compound FC(F)(Cl)C(F)(F)Cl DDMOUSALMHHKOS-UHFFFAOYSA-N 0.000 description 1
- TZCPCKNHXULUIY-RGULYWFUSA-N 1,2-distearoyl-sn-glycero-3-phosphoserine Chemical compound CCCCCCCCCCCCCCCCCC(=O)OC[C@H](COP(O)(=O)OC[C@H](N)C(O)=O)OC(=O)CCCCCCCCCCCCCCCCC TZCPCKNHXULUIY-RGULYWFUSA-N 0.000 description 1
- BDNKZNFMNDZQMI-UHFFFAOYSA-N 1,3-diisopropylcarbodiimide Chemical compound CC(C)N=C=NC(C)C BDNKZNFMNDZQMI-UHFFFAOYSA-N 0.000 description 1
- AZQWKYJCGOJGHM-UHFFFAOYSA-N 1,4-benzoquinone Chemical compound O=C1C=CC(=O)C=C1 AZQWKYJCGOJGHM-UHFFFAOYSA-N 0.000 description 1
- GZEFTKHSACGIBG-UGKPPGOTSA-N 1-[(2r,3r,4s,5r)-3,4-dihydroxy-5-(hydroxymethyl)-2-propyloxolan-2-yl]pyrimidine-2,4-dione Chemical compound C1=CC(=O)NC(=O)N1[C@]1(CCC)O[C@H](CO)[C@@H](O)[C@H]1O GZEFTKHSACGIBG-UGKPPGOTSA-N 0.000 description 1
- UHDGCWIWMRVCDJ-UHFFFAOYSA-N 1-beta-D-Xylofuranosyl-NH-Cytosine Natural products O=C1N=C(N)C=CN1C1C(O)C(O)C(CO)O1 UHDGCWIWMRVCDJ-UHFFFAOYSA-N 0.000 description 1
- XLPJNCYCZORXHG-UHFFFAOYSA-N 1-morpholin-4-ylprop-2-en-1-one Chemical compound C=CC(=O)N1CCOCC1 XLPJNCYCZORXHG-UHFFFAOYSA-N 0.000 description 1
- IIZPXYDJLKNOIY-JXPKJXOSSA-N 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCC\C=C/C\C=C/C\C=C/C\C=C/CCCCC IIZPXYDJLKNOIY-JXPKJXOSSA-N 0.000 description 1
- IQFYYKKMVGJFEH-BIIVOSGPSA-N 2'-deoxythymidine Natural products O=C1NC(=O)C(C)=CN1[C@@H]1O[C@@H](CO)[C@@H](O)C1 IQFYYKKMVGJFEH-BIIVOSGPSA-N 0.000 description 1
- OGNSCSPNOLGXSM-UHFFFAOYSA-N 2,4-diaminobutyric acid Chemical compound NCCC(N)C(O)=O OGNSCSPNOLGXSM-UHFFFAOYSA-N 0.000 description 1
- SXGZJKUKBWWHRA-UHFFFAOYSA-N 2-(N-morpholiniumyl)ethanesulfonate Chemical compound [O-]S(=O)(=O)CC[NH+]1CCOCC1 SXGZJKUKBWWHRA-UHFFFAOYSA-N 0.000 description 1
- FADQCEBBTITJBI-UHFFFAOYSA-N 2-[(2-methoxyphenyl)methoxymethyl]oxirane Chemical compound COC1=CC=CC=C1COCC1OC1 FADQCEBBTITJBI-UHFFFAOYSA-N 0.000 description 1
- YEDUAINPPJYDJZ-UHFFFAOYSA-N 2-hydroxybenzothiazole Chemical compound C1=CC=C2SC(O)=NC2=C1 YEDUAINPPJYDJZ-UHFFFAOYSA-N 0.000 description 1
- FAWLNURBQMTKEB-URDPEVQOSA-N 213546-53-3 Chemical compound N([C@@H](C)C(=O)N[C@H](C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C)C(=O)N1CCC[C@H]1C(=O)N[C@H](C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCSC)C(=O)N1[C@@H](CCC1)C(O)=O)C(C)C)C(C)C)C(=O)[C@@H]1CCCN1C(=O)[C@H](CC(C)C)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](C)NC(=O)[C@@H](NC(=O)[C@H](C)NC(=O)[C@H](C)N)C(C)C FAWLNURBQMTKEB-URDPEVQOSA-N 0.000 description 1
- JMTMSDXUXJISAY-UHFFFAOYSA-N 2H-benzotriazol-4-ol Chemical compound OC1=CC=CC2=C1N=NN2 JMTMSDXUXJISAY-UHFFFAOYSA-N 0.000 description 1
- BRMWTNUJHUMWMS-UHFFFAOYSA-N 3-Methylhistidine Natural products CN1C=NC(CC(N)C(O)=O)=C1 BRMWTNUJHUMWMS-UHFFFAOYSA-N 0.000 description 1
- BMYNFMYTOJXKLE-UHFFFAOYSA-N 3-azaniumyl-2-hydroxypropanoate Chemical compound NCC(O)C(O)=O BMYNFMYTOJXKLE-UHFFFAOYSA-N 0.000 description 1
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 description 1
- 229960000549 4-dimethylaminophenol Drugs 0.000 description 1
- VHYFNPMBLIVWCW-UHFFFAOYSA-N 4-dimethylaminopyridine Substances CN(C)C1=CC=NC=C1 VHYFNPMBLIVWCW-UHFFFAOYSA-N 0.000 description 1
- HIQIXEFWDLTDED-UHFFFAOYSA-N 4-hydroxy-1-piperidin-4-ylpyrrolidin-2-one Chemical compound O=C1CC(O)CN1C1CCNCC1 HIQIXEFWDLTDED-UHFFFAOYSA-N 0.000 description 1
- 108020005029 5' Flanking Region Proteins 0.000 description 1
- AGFIRQJZCNVMCW-UAKXSSHOSA-N 5-bromouridine Chemical compound O[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1N1C(=O)NC(=O)C(Br)=C1 AGFIRQJZCNVMCW-UAKXSSHOSA-N 0.000 description 1
- 229940117976 5-hydroxylysine Drugs 0.000 description 1
- SLXKOJJOQWFEFD-UHFFFAOYSA-N 6-aminohexanoic acid Chemical compound NCCCCCC(O)=O SLXKOJJOQWFEFD-UHFFFAOYSA-N 0.000 description 1
- VDABVNMGKGUPEY-UHFFFAOYSA-N 6-carboxyfluorescein succinimidyl ester Chemical compound C=1C(O)=CC=C2C=1OC1=CC(O)=CC=C1C2(C1=C2)OC(=O)C1=CC=C2C(=O)ON1C(=O)CCC1=O VDABVNMGKGUPEY-UHFFFAOYSA-N 0.000 description 1
- ASUCSHXLTWZYBA-UMMCILCDSA-N 8-Bromoguanosine Chemical compound C1=2NC(N)=NC(=O)C=2N=C(Br)N1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O ASUCSHXLTWZYBA-UMMCILCDSA-N 0.000 description 1
- HDZZVAMISRMYHH-UHFFFAOYSA-N 9beta-Ribofuranosyl-7-deazaadenin Natural products C1=CC=2C(N)=NC=NC=2N1C1OC(CO)C(O)C1O HDZZVAMISRMYHH-UHFFFAOYSA-N 0.000 description 1
- 208000002874 Acne Vulgaris Diseases 0.000 description 1
- HGINCPLSRVDWNT-UHFFFAOYSA-N Acrolein Chemical compound C=CC=O HGINCPLSRVDWNT-UHFFFAOYSA-N 0.000 description 1
- 241000251468 Actinopterygii Species 0.000 description 1
- 102000007469 Actins Human genes 0.000 description 1
- 108010085238 Actins Proteins 0.000 description 1
- 229920001817 Agar Polymers 0.000 description 1
- 102000002260 Alkaline Phosphatase Human genes 0.000 description 1
- 108020004774 Alkaline Phosphatase Proteins 0.000 description 1
- 208000035285 Allergic Seasonal Rhinitis Diseases 0.000 description 1
- 235000019489 Almond oil Nutrition 0.000 description 1
- 241000710929 Alphavirus Species 0.000 description 1
- 102000052866 Amino Acyl-tRNA Synthetases Human genes 0.000 description 1
- 108700028939 Amino Acyl-tRNA Synthetases Proteins 0.000 description 1
- 108090000672 Annexin A5 Proteins 0.000 description 1
- 102000004121 Annexin A5 Human genes 0.000 description 1
- 108091023037 Aptamer Proteins 0.000 description 1
- 241000228212 Aspergillus Species 0.000 description 1
- 241000416162 Astragalus gummifer Species 0.000 description 1
- 241000282672 Ateles sp. Species 0.000 description 1
- 241000972773 Aulopiformes Species 0.000 description 1
- 241000271566 Aves Species 0.000 description 1
- 241000714230 Avian leukemia virus Species 0.000 description 1
- 208000003950 B-cell lymphoma Diseases 0.000 description 1
- 210000002237 B-cell of pancreatic islet Anatomy 0.000 description 1
- 102100024222 B-lymphocyte antigen CD19 Human genes 0.000 description 1
- 102100022005 B-lymphocyte antigen CD20 Human genes 0.000 description 1
- WOVKYSAHUYNSMH-UHFFFAOYSA-N BROMODEOXYURIDINE Natural products C1C(O)C(CO)OC1N1C(=O)NC(=O)C(Br)=C1 WOVKYSAHUYNSMH-UHFFFAOYSA-N 0.000 description 1
- 208000023328 Basedow disease Diseases 0.000 description 1
- 239000005711 Benzoic acid Substances 0.000 description 1
- 208000008439 Biliary Liver Cirrhosis Diseases 0.000 description 1
- 208000033222 Biliary cirrhosis primary Diseases 0.000 description 1
- 241000283726 Bison Species 0.000 description 1
- 101000800130 Bos taurus Thyroglobulin Proteins 0.000 description 1
- 101800001415 Bri23 peptide Proteins 0.000 description 1
- 102100023698 C-C motif chemokine 17 Human genes 0.000 description 1
- 125000001433 C-terminal amino-acid group Chemical group 0.000 description 1
- 101800000655 C-terminal peptide Proteins 0.000 description 1
- 102400000107 C-terminal peptide Human genes 0.000 description 1
- 102100027207 CD27 antigen Human genes 0.000 description 1
- 102000017420 CD3 protein, epsilon/gamma/delta subunit Human genes 0.000 description 1
- 108050005493 CD3 protein, epsilon/gamma/delta subunit Proteins 0.000 description 1
- 210000004366 CD4-positive T-lymphocyte Anatomy 0.000 description 1
- 101100289995 Caenorhabditis elegans mac-1 gene Proteins 0.000 description 1
- 241000282461 Canis lupus Species 0.000 description 1
- 239000004215 Carbon black (E152) Substances 0.000 description 1
- BVKZGUZCCUSVTD-UHFFFAOYSA-L Carbonate Chemical compound [O-]C([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-L 0.000 description 1
- 208000024172 Cardiovascular disease Diseases 0.000 description 1
- 241000283153 Cetacea Species 0.000 description 1
- 108010055166 Chemokine CCL5 Proteins 0.000 description 1
- 108010035563 Chloramphenicol O-acetyltransferase Proteins 0.000 description 1
- 108010049048 Cholera Toxin Proteins 0.000 description 1
- 102000009016 Cholera Toxin Human genes 0.000 description 1
- 108091026890 Coding region Proteins 0.000 description 1
- 206010009900 Colitis ulcerative Diseases 0.000 description 1
- 108010035532 Collagen Proteins 0.000 description 1
- 102000008186 Collagen Human genes 0.000 description 1
- 208000035473 Communicable disease Diseases 0.000 description 1
- 108010028780 Complement C3 Proteins 0.000 description 1
- 102000016918 Complement C3 Human genes 0.000 description 1
- 108010028773 Complement C5 Proteins 0.000 description 1
- 102100031506 Complement C5 Human genes 0.000 description 1
- 108020004635 Complementary DNA Proteins 0.000 description 1
- 108010026206 Conalbumin Proteins 0.000 description 1
- 108091035707 Consensus sequence Proteins 0.000 description 1
- 102000004420 Creatine Kinase Human genes 0.000 description 1
- 108010042126 Creatine kinase Proteins 0.000 description 1
- 241000699800 Cricetinae Species 0.000 description 1
- 208000014997 Crohn colitis Diseases 0.000 description 1
- 208000011231 Crohn disease Diseases 0.000 description 1
- MIKUYHXYGGJMLM-GIMIYPNGSA-N Crotonoside Natural products C1=NC2=C(N)NC(=O)N=C2N1[C@H]1O[C@@H](CO)[C@H](O)[C@@H]1O MIKUYHXYGGJMLM-GIMIYPNGSA-N 0.000 description 1
- 229920000858 Cyclodextrin Polymers 0.000 description 1
- UHDGCWIWMRVCDJ-PSQAKQOGSA-N Cytidine Natural products O=C1N=C(N)C=CN1[C@@H]1[C@@H](O)[C@@H](O)[C@H](CO)O1 UHDGCWIWMRVCDJ-PSQAKQOGSA-N 0.000 description 1
- FBPFZTCFMRRESA-FSIIMWSLSA-N D-Glucitol Natural products OC[C@H](O)[C@H](O)[C@@H](O)[C@H](O)CO FBPFZTCFMRRESA-FSIIMWSLSA-N 0.000 description 1
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 description 1
- FBPFZTCFMRRESA-JGWLITMVSA-N D-glucitol Chemical compound OC[C@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-JGWLITMVSA-N 0.000 description 1
- NYHBQMYGNKIUIF-UHFFFAOYSA-N D-guanosine Natural products C1=2NC(N)=NC(=O)C=2N=CN1C1OC(CO)C(O)C1O NYHBQMYGNKIUIF-UHFFFAOYSA-N 0.000 description 1
- 102000053602 DNA Human genes 0.000 description 1
- 230000006820 DNA synthesis Effects 0.000 description 1
- 201000004624 Dermatitis Diseases 0.000 description 1
- 206010012438 Dermatitis atopic Diseases 0.000 description 1
- 206010012442 Dermatitis contact Diseases 0.000 description 1
- 229920002307 Dextran Polymers 0.000 description 1
- FEWJPZIEWOKRBE-JCYAYHJZSA-N Dextrotartaric acid Chemical compound OC(=O)[C@H](O)[C@@H](O)C(O)=O FEWJPZIEWOKRBE-JCYAYHJZSA-N 0.000 description 1
- 239000004338 Dichlorodifluoromethane Substances 0.000 description 1
- 101100058679 Dictyostelium discoideum btg gene Proteins 0.000 description 1
- SHIBSTMRCDJXLN-UHFFFAOYSA-N Digoxigenin Natural products C1CC(C2C(C3(C)CCC(O)CC3CC2)CC2O)(O)C2(C)C1C1=CC(=O)OC1 SHIBSTMRCDJXLN-UHFFFAOYSA-N 0.000 description 1
- GZDFHIJNHHMENY-UHFFFAOYSA-N Dimethyl dicarbonate Chemical compound COC(=O)OC(=O)OC GZDFHIJNHHMENY-UHFFFAOYSA-N 0.000 description 1
- 241000271571 Dromaius novaehollandiae Species 0.000 description 1
- 241000255581 Drosophila <fruit fly, genus> Species 0.000 description 1
- 102000016942 Elastin Human genes 0.000 description 1
- 108010014258 Elastin Proteins 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 206010014561 Emphysema Diseases 0.000 description 1
- 241000289659 Erinaceidae Species 0.000 description 1
- 101000759376 Escherichia phage Mu Tail sheath protein Proteins 0.000 description 1
- 239000001856 Ethyl cellulose Substances 0.000 description 1
- ZZSNKZQZMQGXPY-UHFFFAOYSA-N Ethyl cellulose Chemical compound CCOCC1OC(OC)C(OCC)C(OCC)C1OC1C(O)C(O)C(OC)C(CO)O1 ZZSNKZQZMQGXPY-UHFFFAOYSA-N 0.000 description 1
- IAYPIBMASNFSPL-UHFFFAOYSA-N Ethylene oxide Chemical compound C1CO1 IAYPIBMASNFSPL-UHFFFAOYSA-N 0.000 description 1
- 241000206602 Eukaryota Species 0.000 description 1
- 241000289695 Eutheria Species 0.000 description 1
- 241000282324 Felis Species 0.000 description 1
- 108010067306 Fibronectins Proteins 0.000 description 1
- 102000016359 Fibronectins Human genes 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 241000287828 Gallus gallus Species 0.000 description 1
- 208000007882 Gastritis Diseases 0.000 description 1
- 108700039691 Genetic Promoter Regions Proteins 0.000 description 1
- 206010071602 Genetic polymorphism Diseases 0.000 description 1
- AZKVWQKMDGGDSV-BCMRRPTOSA-N Genipin Chemical compound COC(=O)C1=CO[C@@H](O)[C@@H]2C(CO)=CC[C@H]12 AZKVWQKMDGGDSV-BCMRRPTOSA-N 0.000 description 1
- 244000068988 Glycine max Species 0.000 description 1
- 235000010469 Glycine max Nutrition 0.000 description 1
- 208000003807 Graves Disease Diseases 0.000 description 1
- 229940122853 Growth hormone antagonist Drugs 0.000 description 1
- 208000035895 Guillain-Barré syndrome Diseases 0.000 description 1
- 102100028972 HLA class I histocompatibility antigen, A alpha chain Human genes 0.000 description 1
- 108010075704 HLA-A Antigens Proteins 0.000 description 1
- 108010035452 HLA-A1 Antigen Proteins 0.000 description 1
- 108010086377 HLA-A3 Antigen Proteins 0.000 description 1
- 108010058607 HLA-B Antigens Proteins 0.000 description 1
- 108010091938 HLA-B7 Antigen Proteins 0.000 description 1
- 208000030836 Hashimoto thyroiditis Diseases 0.000 description 1
- 244000020551 Helianthus annuus Species 0.000 description 1
- 235000003222 Helianthus annuus Nutrition 0.000 description 1
- 208000018565 Hemochromatosis Diseases 0.000 description 1
- 102000001554 Hemoglobins Human genes 0.000 description 1
- 108010054147 Hemoglobins Proteins 0.000 description 1
- 101000980825 Homo sapiens B-lymphocyte antigen CD19 Proteins 0.000 description 1
- 101000897405 Homo sapiens B-lymphocyte antigen CD20 Proteins 0.000 description 1
- 101000978362 Homo sapiens C-C motif chemokine 17 Proteins 0.000 description 1
- 101000797762 Homo sapiens C-C motif chemokine 5 Proteins 0.000 description 1
- 101000914511 Homo sapiens CD27 antigen Proteins 0.000 description 1
- 101100059511 Homo sapiens CD40LG gene Proteins 0.000 description 1
- 101001055222 Homo sapiens Interleukin-8 Proteins 0.000 description 1
- 101000914484 Homo sapiens T-lymphocyte activation antigen CD80 Proteins 0.000 description 1
- 101000611185 Homo sapiens Tumor necrosis factor receptor superfamily member 5 Proteins 0.000 description 1
- 101000851376 Homo sapiens Tumor necrosis factor receptor superfamily member 8 Proteins 0.000 description 1
- 102000002265 Human Growth Hormone Human genes 0.000 description 1
- 108010000521 Human Growth Hormone Proteins 0.000 description 1
- 239000000854 Human Growth Hormone Substances 0.000 description 1
- 241000701044 Human gammaherpesvirus 4 Species 0.000 description 1
- 241000713772 Human immunodeficiency virus 1 Species 0.000 description 1
- 102000004157 Hydrolases Human genes 0.000 description 1
- 108090000604 Hydrolases Proteins 0.000 description 1
- 229940124790 IL-6 inhibitor Drugs 0.000 description 1
- 108060003951 Immunoglobulin Proteins 0.000 description 1
- 208000022559 Inflammatory bowel disease Diseases 0.000 description 1
- 108090000723 Insulin-Like Growth Factor I Proteins 0.000 description 1
- 102100026720 Interferon beta Human genes 0.000 description 1
- 108090000467 Interferon-beta Proteins 0.000 description 1
- 102000000589 Interleukin-1 Human genes 0.000 description 1
- 108010002352 Interleukin-1 Proteins 0.000 description 1
- 102000003777 Interleukin-1 beta Human genes 0.000 description 1
- 108090000193 Interleukin-1 beta Proteins 0.000 description 1
- 108010002616 Interleukin-5 Proteins 0.000 description 1
- 102000000743 Interleukin-5 Human genes 0.000 description 1
- 102100026236 Interleukin-8 Human genes 0.000 description 1
- SNDPXSYFESPGGJ-BYPYZUCNSA-N L-2-aminopentanoic acid Chemical compound CCC[C@H](N)C(O)=O SNDPXSYFESPGGJ-BYPYZUCNSA-N 0.000 description 1
- AHLPHDHHMVZTML-BYPYZUCNSA-N L-Ornithine Chemical compound NCCC[C@H](N)C(O)=O AHLPHDHHMVZTML-BYPYZUCNSA-N 0.000 description 1
- ZGUNAGUHMKGQNY-ZETCQYMHSA-N L-alpha-phenylglycine zwitterion Chemical compound OC(=O)[C@@H](N)C1=CC=CC=C1 ZGUNAGUHMKGQNY-ZETCQYMHSA-N 0.000 description 1
- RHGKLRLOHDJJDR-BYPYZUCNSA-N L-citrulline Chemical compound NC(=O)NCCC[C@H]([NH3+])C([O-])=O RHGKLRLOHDJJDR-BYPYZUCNSA-N 0.000 description 1
- XIGSAGMEBXLVJJ-YFKPBYRVSA-N L-homocitrulline Chemical compound NC(=O)NCCCC[C@H]([NH3+])C([O-])=O XIGSAGMEBXLVJJ-YFKPBYRVSA-N 0.000 description 1
- SNDPXSYFESPGGJ-UHFFFAOYSA-N L-norVal-OH Natural products CCCC(N)C(O)=O SNDPXSYFESPGGJ-UHFFFAOYSA-N 0.000 description 1
- LRQKBLKVPFOOQJ-YFKPBYRVSA-N L-norleucine Chemical compound CCCC[C@H]([NH3+])C([O-])=O LRQKBLKVPFOOQJ-YFKPBYRVSA-N 0.000 description 1
- RNKSNIBMTUYWSH-YFKPBYRVSA-N L-prolylglycine Chemical compound [O-]C(=O)CNC(=O)[C@@H]1CCC[NH2+]1 RNKSNIBMTUYWSH-YFKPBYRVSA-N 0.000 description 1
- 240000007472 Leucaena leucocephala Species 0.000 description 1
- 235000010643 Leucaena leucocephala Nutrition 0.000 description 1
- NNJVILVZKWQKPM-UHFFFAOYSA-N Lidocaine Chemical compound CCN(CC)CC(=O)NC1=C(C)C=CC=C1C NNJVILVZKWQKPM-UHFFFAOYSA-N 0.000 description 1
- 241000239220 Limulus polyphemus Species 0.000 description 1
- 108090001030 Lipoproteins Proteins 0.000 description 1
- 102000004895 Lipoproteins Human genes 0.000 description 1
- 241000282553 Macaca Species 0.000 description 1
- FYYHWMGAXLPEAU-UHFFFAOYSA-N Magnesium Chemical compound [Mg] FYYHWMGAXLPEAU-UHFFFAOYSA-N 0.000 description 1
- 235000019759 Maize starch Nutrition 0.000 description 1
- PEEHTFAAVSWFBL-UHFFFAOYSA-N Maleimide Chemical compound O=C1NC(=O)C=C1 PEEHTFAAVSWFBL-UHFFFAOYSA-N 0.000 description 1
- 229930195725 Mannitol Natural products 0.000 description 1
- 241000283923 Marmota monax Species 0.000 description 1
- 208000014413 Maternally-inherited diabetes and deafness Diseases 0.000 description 1
- 102000000380 Matrix Metalloproteinase 1 Human genes 0.000 description 1
- 108010016113 Matrix Metalloproteinase 1 Proteins 0.000 description 1
- 108700011259 MicroRNAs Proteins 0.000 description 1
- 206010049567 Miller Fisher syndrome Diseases 0.000 description 1
- 239000004909 Moisturizer Substances 0.000 description 1
- 241000713333 Mouse mammary tumor virus Species 0.000 description 1
- 241000714177 Murine leukemia virus Species 0.000 description 1
- 241000699670 Mus sp. Species 0.000 description 1
- 241000282339 Mustela Species 0.000 description 1
- 101710135898 Myc proto-oncogene protein Proteins 0.000 description 1
- 102100038895 Myc proto-oncogene protein Human genes 0.000 description 1
- 102000003505 Myosin Human genes 0.000 description 1
- 108060008487 Myosin Proteins 0.000 description 1
- DTERQYGMUDWYAZ-ZETCQYMHSA-N N(6)-acetyl-L-lysine Chemical compound CC(=O)NCCCC[C@H]([NH3+])C([O-])=O DTERQYGMUDWYAZ-ZETCQYMHSA-N 0.000 description 1
- VQAYFKKCNSOZKM-IOSLPCCCSA-N N(6)-methyladenosine Chemical compound C1=NC=2C(NC)=NC=NC=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O VQAYFKKCNSOZKM-IOSLPCCCSA-N 0.000 description 1
- JDHILDINMRGULE-LURJTMIESA-N N(pros)-methyl-L-histidine Chemical compound CN1C=NC=C1C[C@H](N)C(O)=O JDHILDINMRGULE-LURJTMIESA-N 0.000 description 1
- JGFZNNIVVJXRND-UHFFFAOYSA-N N,N-Diisopropylethylamine (DIPEA) Chemical compound CCN(C(C)C)C(C)C JGFZNNIVVJXRND-UHFFFAOYSA-N 0.000 description 1
- WHNWPMSKXPGLAX-UHFFFAOYSA-N N-Vinyl-2-pyrrolidone Chemical compound C=CN1CCCC1=O WHNWPMSKXPGLAX-UHFFFAOYSA-N 0.000 description 1
- JJIHLJJYMXLCOY-BYPYZUCNSA-N N-acetyl-L-serine Chemical compound CC(=O)N[C@@H](CO)C(O)=O JJIHLJJYMXLCOY-BYPYZUCNSA-N 0.000 description 1
- SQVRNKJHWKZAKO-PFQGKNLYSA-N N-acetyl-beta-neuraminic acid Chemical compound CC(=O)N[C@@H]1[C@@H](O)C[C@@](O)(C(O)=O)O[C@H]1[C@H](O)[C@H](O)CO SQVRNKJHWKZAKO-PFQGKNLYSA-N 0.000 description 1
- PYUSHNKNPOHWEZ-YFKPBYRVSA-N N-formyl-L-methionine Chemical compound CSCC[C@@H](C(O)=O)NC=O PYUSHNKNPOHWEZ-YFKPBYRVSA-N 0.000 description 1
- 125000001429 N-terminal alpha-amino-acid group Chemical group 0.000 description 1
- 229910002651 NO3 Inorganic materials 0.000 description 1
- VQAYFKKCNSOZKM-UHFFFAOYSA-N NSC 29409 Natural products C1=NC=2C(NC)=NC=NC=2N1C1OC(CO)C(O)C1O VQAYFKKCNSOZKM-UHFFFAOYSA-N 0.000 description 1
- 108091007491 NSP3 Papain-like protease domains Proteins 0.000 description 1
- RHGKLRLOHDJJDR-UHFFFAOYSA-N Ndelta-carbamoyl-DL-ornithine Natural products OC(=O)C(N)CCCNC(N)=O RHGKLRLOHDJJDR-UHFFFAOYSA-N 0.000 description 1
- NHNBFGGVMKEFGY-UHFFFAOYSA-N Nitrate Chemical compound [O-][N+]([O-])=O NHNBFGGVMKEFGY-UHFFFAOYSA-N 0.000 description 1
- 101710089543 Nitric oxide synthase, inducible Proteins 0.000 description 1
- 102100029438 Nitric oxide synthase, inducible Human genes 0.000 description 1
- 238000000636 Northern blotting Methods 0.000 description 1
- 108091005461 Nucleic proteins Proteins 0.000 description 1
- BZQFBWGGLXLEPQ-UHFFFAOYSA-N O-phosphoryl-L-serine Natural products OC(=O)C(N)COP(O)(O)=O BZQFBWGGLXLEPQ-UHFFFAOYSA-N 0.000 description 1
- 108010038807 Oligopeptides Proteins 0.000 description 1
- 102000015636 Oligopeptides Human genes 0.000 description 1
- 108700026244 Open Reading Frames Proteins 0.000 description 1
- AHLPHDHHMVZTML-UHFFFAOYSA-N Orn-delta-NH2 Natural products NCCCC(N)C(O)=O AHLPHDHHMVZTML-UHFFFAOYSA-N 0.000 description 1
- UTJLXEIPEHZYQJ-UHFFFAOYSA-N Ornithine Natural products OC(=O)C(C)CCCN UTJLXEIPEHZYQJ-UHFFFAOYSA-N 0.000 description 1
- 108010058846 Ovalbumin Proteins 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 241000282579 Pan Species 0.000 description 1
- 241000238124 Paralithodes camtschaticus Species 0.000 description 1
- 241000237988 Patellidae Species 0.000 description 1
- 229920002732 Polyanhydride Polymers 0.000 description 1
- 239000004721 Polyphenylene oxide Substances 0.000 description 1
- 208000012654 Primary biliary cholangitis Diseases 0.000 description 1
- 108010057464 Prolactin Proteins 0.000 description 1
- 102100024819 Prolactin Human genes 0.000 description 1
- 108010029485 Protein Isoforms Proteins 0.000 description 1
- 102000001708 Protein Isoforms Human genes 0.000 description 1
- 241000589517 Pseudomonas aeruginosa Species 0.000 description 1
- 201000001263 Psoriatic Arthritis Diseases 0.000 description 1
- 208000036824 Psoriatic arthropathy Diseases 0.000 description 1
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 1
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 1
- 241000714474 Rous sarcoma virus Species 0.000 description 1
- 235000019485 Safflower oil Nutrition 0.000 description 1
- 241000277331 Salmonidae Species 0.000 description 1
- 108010077895 Sarcosine Proteins 0.000 description 1
- 241000700584 Simplexvirus Species 0.000 description 1
- 108091027967 Small hairpin RNA Proteins 0.000 description 1
- 108020004459 Small interfering RNA Proteins 0.000 description 1
- BCKXLBQYZLBQEK-KVVVOXFISA-M Sodium oleate Chemical compound [Na+].CCCCCCCC\C=C/CCCCCCCC([O-])=O BCKXLBQYZLBQEK-KVVVOXFISA-M 0.000 description 1
- 102000013275 Somatomedins Human genes 0.000 description 1
- 241000288726 Soricidae Species 0.000 description 1
- 238000002105 Southern blotting Methods 0.000 description 1
- 241000256248 Spodoptera Species 0.000 description 1
- 241000295644 Staphylococcaceae Species 0.000 description 1
- 241000187747 Streptomyces Species 0.000 description 1
- 241000272534 Struthio camelus Species 0.000 description 1
- KDYFGRWQOYBRFD-UHFFFAOYSA-N Succinic acid Natural products OC(=O)CCC(O)=O KDYFGRWQOYBRFD-UHFFFAOYSA-N 0.000 description 1
- 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 1
- 229930006000 Sucrose Natural products 0.000 description 1
- 241001493546 Suina Species 0.000 description 1
- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical group [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 description 1
- 241000282890 Sus Species 0.000 description 1
- 241000282894 Sus scrofa domesticus Species 0.000 description 1
- 230000037453 T cell priming Effects 0.000 description 1
- 102100027222 T-lymphocyte activation antigen CD80 Human genes 0.000 description 1
- 239000004098 Tetracycline Substances 0.000 description 1
- 102100036407 Thioredoxin Human genes 0.000 description 1
- 102000006601 Thymidine Kinase Human genes 0.000 description 1
- 108020004440 Thymidine kinase Proteins 0.000 description 1
- 108010034949 Thyroglobulin Proteins 0.000 description 1
- 102000009843 Thyroglobulin Human genes 0.000 description 1
- 229920001615 Tragacanth Polymers 0.000 description 1
- 101710150448 Transcriptional regulator Myc Proteins 0.000 description 1
- 102000004338 Transferrin Human genes 0.000 description 1
- 108090000901 Transferrin Proteins 0.000 description 1
- DFBIRQPKNDILPW-CIVMWXNOSA-N Triptolide Chemical compound O=C1OCC([C@@H]2C3)=C1CC[C@]2(C)[C@]12O[C@H]1[C@@H]1O[C@]1(C(C)C)[C@@H](O)[C@]21[C@H]3O1 DFBIRQPKNDILPW-CIVMWXNOSA-N 0.000 description 1
- 239000007983 Tris buffer Substances 0.000 description 1
- 108060008683 Tumor Necrosis Factor Receptor Proteins 0.000 description 1
- 101710165474 Tumor necrosis factor receptor superfamily member 5 Proteins 0.000 description 1
- 102100036857 Tumor necrosis factor receptor superfamily member 8 Human genes 0.000 description 1
- 206010054094 Tumour necrosis Diseases 0.000 description 1
- 208000035896 Twin-reversed arterial perfusion sequence Diseases 0.000 description 1
- 208000006391 Type 1 Hyper-IgM Immunodeficiency Syndrome Diseases 0.000 description 1
- 208000025865 Ulcer Diseases 0.000 description 1
- 201000006704 Ulcerative Colitis Diseases 0.000 description 1
- 206010047115 Vasculitis Diseases 0.000 description 1
- 241000251539 Vertebrata <Metazoa> Species 0.000 description 1
- 241000282485 Vulpes vulpes Species 0.000 description 1
- 201000001696 X-linked hyper IgM syndrome Diseases 0.000 description 1
- 241000269370 Xenopus <genus> Species 0.000 description 1
- ATBOMIWRCZXYSZ-XZBBILGWSA-N [1-[2,3-dihydroxypropoxy(hydroxy)phosphoryl]oxy-3-hexadecanoyloxypropan-2-yl] (9e,12e)-octadeca-9,12-dienoate Chemical compound CCCCCCCCCCCCCCCC(=O)OCC(COP(O)(=O)OCC(O)CO)OC(=O)CCCCCCC\C=C\C\C=C\CCCCC ATBOMIWRCZXYSZ-XZBBILGWSA-N 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 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 1
- 150000008065 acid anhydrides Chemical class 0.000 description 1
- 206010000496 acne Diseases 0.000 description 1
- 239000008186 active pharmaceutical agent Substances 0.000 description 1
- 230000001154 acute effect Effects 0.000 description 1
- 206010069351 acute lung injury Diseases 0.000 description 1
- 150000003838 adenosines Chemical class 0.000 description 1
- 239000002671 adjuvant Substances 0.000 description 1
- 238000012382 advanced drug delivery Methods 0.000 description 1
- 239000004479 aerosol dispenser Substances 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 238000007818 agglutination assay Methods 0.000 description 1
- 125000003295 alanine group Chemical group N[C@@H](C)C(=O)* 0.000 description 1
- 150000001295 alanines Chemical class 0.000 description 1
- 150000001298 alcohols Chemical class 0.000 description 1
- 235000010443 alginic acid Nutrition 0.000 description 1
- 239000000783 alginic acid Substances 0.000 description 1
- 229920000615 alginic acid Polymers 0.000 description 1
- 229960001126 alginic acid Drugs 0.000 description 1
- 150000004781 alginic acids Chemical class 0.000 description 1
- 150000001338 aliphatic hydrocarbons Chemical class 0.000 description 1
- 125000003342 alkenyl group Chemical group 0.000 description 1
- 229940100198 alkylating agent Drugs 0.000 description 1
- 239000002168 alkylating agent Substances 0.000 description 1
- 125000000304 alkynyl group Chemical group 0.000 description 1
- 230000002009 allergenic effect Effects 0.000 description 1
- 230000007815 allergy Effects 0.000 description 1
- 239000008168 almond oil Substances 0.000 description 1
- 108010050122 alpha 1-Antitrypsin Proteins 0.000 description 1
- 102000015395 alpha 1-Antitrypsin Human genes 0.000 description 1
- 229940024142 alpha 1-antitrypsin Drugs 0.000 description 1
- AWUCVROLDVIAJX-UHFFFAOYSA-N alpha-glycerophosphate Natural products OCC(O)COP(O)(O)=O AWUCVROLDVIAJX-UHFFFAOYSA-N 0.000 description 1
- BJEPYKJPYRNKOW-UHFFFAOYSA-N alpha-hydroxysuccinic acid Natural products OC(=O)C(O)CC(O)=O BJEPYKJPYRNKOW-UHFFFAOYSA-N 0.000 description 1
- WNROFYMDJYEPJX-UHFFFAOYSA-K aluminium hydroxide Chemical compound [OH-].[OH-].[OH-].[Al+3] WNROFYMDJYEPJX-UHFFFAOYSA-K 0.000 description 1
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 1
- 150000001414 amino alcohols Chemical class 0.000 description 1
- 229960002684 aminocaproic acid Drugs 0.000 description 1
- 239000012491 analyte Substances 0.000 description 1
- 150000008064 anhydrides Chemical class 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 239000003945 anionic surfactant Substances 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 230000000844 anti-bacterial effect Effects 0.000 description 1
- 230000003110 anti-inflammatory effect Effects 0.000 description 1
- 230000000845 anti-microbial effect Effects 0.000 description 1
- 230000000259 anti-tumor effect Effects 0.000 description 1
- 229940121375 antifungal agent Drugs 0.000 description 1
- 239000003429 antifungal agent Substances 0.000 description 1
- 230000030741 antigen processing and presentation Effects 0.000 description 1
- 239000002246 antineoplastic agent Substances 0.000 description 1
- 239000000074 antisense oligonucleotide Substances 0.000 description 1
- 238000012230 antisense oligonucleotides Methods 0.000 description 1
- 239000011260 aqueous acid Substances 0.000 description 1
- 239000012062 aqueous buffer Substances 0.000 description 1
- 239000000823 artificial membrane Substances 0.000 description 1
- 229940009098 aspartate Drugs 0.000 description 1
- 201000008937 atopic dermatitis Diseases 0.000 description 1
- 239000012752 auxiliary agent Substances 0.000 description 1
- DMLAVOWQYNRWNQ-UHFFFAOYSA-N azobenzene Chemical compound C1=CC=CC=C1N=NC1=CC=CC=C1 DMLAVOWQYNRWNQ-UHFFFAOYSA-N 0.000 description 1
- UREZNYTWGJKWBI-UHFFFAOYSA-M benzethonium chloride Chemical compound [Cl-].C1=CC(C(C)(C)CC(C)(C)C)=CC=C1OCCOCC[N+](C)(C)CC1=CC=CC=C1 UREZNYTWGJKWBI-UHFFFAOYSA-M 0.000 description 1
- 229960001950 benzethonium chloride Drugs 0.000 description 1
- 235000010233 benzoic acid Nutrition 0.000 description 1
- 235000019445 benzyl alcohol Nutrition 0.000 description 1
- 229960002903 benzyl benzoate Drugs 0.000 description 1
- 125000001797 benzyl group Chemical group [H]C1=C([H])C([H])=C(C([H])=C1[H])C([H])([H])* 0.000 description 1
- 239000003012 bilayer membrane Substances 0.000 description 1
- 229920000249 biocompatible polymer Polymers 0.000 description 1
- 230000031018 biological processes and functions Effects 0.000 description 1
- HUTDDBSSHVOYJR-UHFFFAOYSA-H bis[(2-oxo-1,3,2$l^{5},4$l^{2}-dioxaphosphaplumbetan-2-yl)oxy]lead Chemical compound [Pb+2].[Pb+2].[Pb+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O HUTDDBSSHVOYJR-UHFFFAOYSA-H 0.000 description 1
- 210000003995 blood forming stem cell Anatomy 0.000 description 1
- 210000001124 body fluid Anatomy 0.000 description 1
- 239000010839 body fluid Substances 0.000 description 1
- KGBXLFKZBHKPEV-UHFFFAOYSA-N boric acid Chemical compound OB(O)O KGBXLFKZBHKPEV-UHFFFAOYSA-N 0.000 description 1
- 239000004327 boric acid Substances 0.000 description 1
- 229960002645 boric acid Drugs 0.000 description 1
- 235000010338 boric acid Nutrition 0.000 description 1
- 206010006451 bronchitis Diseases 0.000 description 1
- 229950004398 broxuridine Drugs 0.000 description 1
- 239000000337 buffer salt Substances 0.000 description 1
- 239000008366 buffered solution Substances 0.000 description 1
- 239000006172 buffering agent Substances 0.000 description 1
- 239000001273 butane Substances 0.000 description 1
- KDYFGRWQOYBRFD-NUQCWPJISA-N butanedioic acid Chemical compound O[14C](=O)CC[14C](O)=O KDYFGRWQOYBRFD-NUQCWPJISA-N 0.000 description 1
- LRHPLDYGYMQRHN-UHFFFAOYSA-N butyl alcohol Substances CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 description 1
- 238000010804 cDNA synthesis Methods 0.000 description 1
- BQRGNLJZBFXNCZ-UHFFFAOYSA-N calcein am Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC(CN(CC(=O)OCOC(C)=O)CC(=O)OCOC(C)=O)=C(OC(C)=O)C=C1OC1=C2C=C(CN(CC(=O)OCOC(C)=O)CC(=O)OCOC(=O)C)C(OC(C)=O)=C1 BQRGNLJZBFXNCZ-UHFFFAOYSA-N 0.000 description 1
- FUFJGUQYACFECW-UHFFFAOYSA-L calcium hydrogenphosphate Chemical compound [Ca+2].OP([O-])([O-])=O FUFJGUQYACFECW-UHFFFAOYSA-L 0.000 description 1
- BPKIGYQJPYCAOW-FFJTTWKXSA-I calcium;potassium;disodium;(2s)-2-hydroxypropanoate;dichloride;dihydroxide;hydrate Chemical compound O.[OH-].[OH-].[Na+].[Na+].[Cl-].[Cl-].[K+].[Ca+2].C[C@H](O)C([O-])=O BPKIGYQJPYCAOW-FFJTTWKXSA-I 0.000 description 1
- BMLSTPRTEKLIPM-UHFFFAOYSA-I calcium;potassium;disodium;hydrogen carbonate;dichloride;dihydroxide;hydrate Chemical compound O.[OH-].[OH-].[Na+].[Na+].[Cl-].[Cl-].[K+].[Ca+2].OC([O-])=O BMLSTPRTEKLIPM-UHFFFAOYSA-I 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 239000001569 carbon dioxide Substances 0.000 description 1
- 229910002092 carbon dioxide Inorganic materials 0.000 description 1
- 229960004424 carbon dioxide Drugs 0.000 description 1
- 239000001768 carboxy methyl cellulose Substances 0.000 description 1
- UHBYWPGGCSDKFX-UHFFFAOYSA-N carboxyglutamic acid Chemical compound OC(=O)C(N)CC(C(O)=O)C(O)=O UHBYWPGGCSDKFX-UHFFFAOYSA-N 0.000 description 1
- 230000001925 catabolic effect Effects 0.000 description 1
- 241001233037 catfish Species 0.000 description 1
- 239000003093 cationic surfactant Substances 0.000 description 1
- 150000001768 cations Chemical class 0.000 description 1
- 230000020411 cell activation Effects 0.000 description 1
- 230000003915 cell function Effects 0.000 description 1
- 238000001516 cell proliferation assay Methods 0.000 description 1
- 230000007969 cellular immunity Effects 0.000 description 1
- 230000005754 cellular signaling Effects 0.000 description 1
- 229920002301 cellulose acetate Polymers 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 239000002738 chelating agent Substances 0.000 description 1
- 239000003638 chemical reducing agent Substances 0.000 description 1
- 239000002975 chemoattractant Substances 0.000 description 1
- 150000001805 chlorine compounds Chemical class 0.000 description 1
- 229960004926 chlorobutanol Drugs 0.000 description 1
- 235000012000 cholesterol Nutrition 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 230000001684 chronic effect Effects 0.000 description 1
- 235000015165 citric acid Nutrition 0.000 description 1
- 229960002173 citrulline Drugs 0.000 description 1
- 235000013477 citrulline Nutrition 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 238000012761 co-transfection Methods 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 229940110456 cocoa butter Drugs 0.000 description 1
- 235000019868 cocoa butter Nutrition 0.000 description 1
- 206010009887 colitis Diseases 0.000 description 1
- 229920001436 collagen Polymers 0.000 description 1
- 238000001246 colloidal dispersion Methods 0.000 description 1
- 239000000084 colloidal system Substances 0.000 description 1
- 238000004040 coloring Methods 0.000 description 1
- 238000002648 combination therapy Methods 0.000 description 1
- 230000002860 competitive effect Effects 0.000 description 1
- 238000006482 condensation reaction Methods 0.000 description 1
- 239000000562 conjugate Substances 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 208000010247 contact dermatitis Diseases 0.000 description 1
- 239000000356 contaminant Substances 0.000 description 1
- 239000012059 conventional drug carrier Substances 0.000 description 1
- 239000008120 corn starch Substances 0.000 description 1
- 230000000139 costimulatory effect Effects 0.000 description 1
- 229940109262 curcumin Drugs 0.000 description 1
- 235000012754 curcumin Nutrition 0.000 description 1
- 239000004148 curcumin Substances 0.000 description 1
- MGNCLNQXLYJVJD-UHFFFAOYSA-N cyanuric chloride Chemical group ClC1=NC(Cl)=NC(Cl)=N1 MGNCLNQXLYJVJD-UHFFFAOYSA-N 0.000 description 1
- 229940097362 cyclodextrins Drugs 0.000 description 1
- HPXRVTGHNJAIIH-UHFFFAOYSA-N cyclohexanol Chemical compound OC1CCCCC1 HPXRVTGHNJAIIH-UHFFFAOYSA-N 0.000 description 1
- UHDGCWIWMRVCDJ-ZAKLUEHWSA-N cytidine Chemical compound O=C1N=C(N)C=CN1[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O1 UHDGCWIWMRVCDJ-ZAKLUEHWSA-N 0.000 description 1
- 238000007822 cytometric assay Methods 0.000 description 1
- 210000000805 cytoplasm Anatomy 0.000 description 1
- 229940104302 cytosine Drugs 0.000 description 1
- 230000001086 cytosolic effect Effects 0.000 description 1
- 210000001151 cytotoxic T lymphocyte Anatomy 0.000 description 1
- 231100000599 cytotoxic agent Toxicity 0.000 description 1
- 238000002784 cytotoxicity assay Methods 0.000 description 1
- 231100000263 cytotoxicity test Toxicity 0.000 description 1
- 239000002619 cytotoxin Substances 0.000 description 1
- 230000003111 delayed effect Effects 0.000 description 1
- 238000002716 delivery method Methods 0.000 description 1
- YSMODUONRAFBET-UHFFFAOYSA-N delta-DL-hydroxylysine Natural products NCC(O)CCC(N)C(O)=O YSMODUONRAFBET-UHFFFAOYSA-N 0.000 description 1
- KXGVEGMKQFWNSR-LLQZFEROSA-N deoxycholic acid Chemical compound C([C@H]1CC2)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC(O)=O)C)[C@@]2(C)[C@@H](O)C1 KXGVEGMKQFWNSR-LLQZFEROSA-N 0.000 description 1
- 229960003964 deoxycholic acid Drugs 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 230000000779 depleting effect Effects 0.000 description 1
- 238000010511 deprotection reaction Methods 0.000 description 1
- 239000002274 desiccant Substances 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 229950006137 dexfosfoserine Drugs 0.000 description 1
- 239000008356 dextrose and sodium chloride injection Substances 0.000 description 1
- 239000008355 dextrose injection Substances 0.000 description 1
- 235000019700 dicalcium phosphate Nutrition 0.000 description 1
- 229940093541 dicetylphosphate Drugs 0.000 description 1
- PXBRQCKWGAHEHS-UHFFFAOYSA-N dichlorodifluoromethane Chemical compound FC(F)(Cl)Cl PXBRQCKWGAHEHS-UHFFFAOYSA-N 0.000 description 1
- 235000019404 dichlorodifluoromethane Nutrition 0.000 description 1
- 229940042935 dichlorodifluoromethane Drugs 0.000 description 1
- 229940087091 dichlorotetrafluoroethane Drugs 0.000 description 1
- VFLDPWHFBUODDF-UHFFFAOYSA-N diferuloylmethane Natural products C1=C(O)C(OC)=CC(C=CC(=O)CC(=O)C=CC=2C=C(OC)C(O)=CC=2)=C1 VFLDPWHFBUODDF-UHFFFAOYSA-N 0.000 description 1
- QONQRTHLHBTMGP-UHFFFAOYSA-N digitoxigenin Natural products CC12CCC(C3(CCC(O)CC3CC3)C)C3C11OC1CC2C1=CC(=O)OC1 QONQRTHLHBTMGP-UHFFFAOYSA-N 0.000 description 1
- SHIBSTMRCDJXLN-KCZCNTNESA-N digoxigenin Chemical compound C1([C@@H]2[C@@]3([C@@](CC2)(O)[C@H]2[C@@H]([C@@]4(C)CC[C@H](O)C[C@H]4CC2)C[C@H]3O)C)=CC(=O)OC1 SHIBSTMRCDJXLN-KCZCNTNESA-N 0.000 description 1
- 125000005442 diisocyanate group Chemical group 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 238000006471 dimerization reaction Methods 0.000 description 1
- 230000003467 diminishing effect Effects 0.000 description 1
- BPHQZTVXXXJVHI-UHFFFAOYSA-N dimyristoyl phosphatidylglycerol Chemical compound CCCCCCCCCCCCCC(=O)OCC(COP(O)(=O)OCC(O)CO)OC(=O)CCCCCCCCCCCCC BPHQZTVXXXJVHI-UHFFFAOYSA-N 0.000 description 1
- XEYBRNLFEZDVAW-ARSRFYASSA-N dinoprostone Chemical compound CCCCC[C@H](O)\C=C\[C@H]1[C@H](O)CC(=O)[C@@H]1C\C=C/CCCC(O)=O XEYBRNLFEZDVAW-ARSRFYASSA-N 0.000 description 1
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 1
- 150000002016 disaccharides Chemical class 0.000 description 1
- 208000016097 disease of metabolism Diseases 0.000 description 1
- 239000007884 disintegrant Substances 0.000 description 1
- BFMYDTVEBKDAKJ-UHFFFAOYSA-L disodium;(2',7'-dibromo-3',6'-dioxido-3-oxospiro[2-benzofuran-1,9'-xanthene]-4'-yl)mercury;hydrate Chemical compound O.[Na+].[Na+].O1C(=O)C2=CC=CC=C2C21C1=CC(Br)=C([O-])C([Hg])=C1OC1=C2C=C(Br)C([O-])=C1 BFMYDTVEBKDAKJ-UHFFFAOYSA-L 0.000 description 1
- 239000006185 dispersion Substances 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 230000006334 disulfide bridging Effects 0.000 description 1
- 238000012377 drug delivery Methods 0.000 description 1
- 229940088679 drug related substance Drugs 0.000 description 1
- 239000000428 dust Substances 0.000 description 1
- 239000012636 effector Substances 0.000 description 1
- 229920002549 elastin Polymers 0.000 description 1
- 238000004520 electroporation Methods 0.000 description 1
- 230000008030 elimination Effects 0.000 description 1
- 238000003379 elimination reaction Methods 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 238000001976 enzyme digestion Methods 0.000 description 1
- YSMODUONRAFBET-UHNVWZDZSA-N erythro-5-hydroxy-L-lysine Chemical compound NC[C@H](O)CC[C@H](N)C(O)=O YSMODUONRAFBET-UHNVWZDZSA-N 0.000 description 1
- 235000019325 ethyl cellulose Nutrition 0.000 description 1
- 229920001249 ethyl cellulose Polymers 0.000 description 1
- 210000003722 extracellular fluid Anatomy 0.000 description 1
- 210000001723 extracellular space Anatomy 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 239000003925 fat Substances 0.000 description 1
- 235000019197 fats Nutrition 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 235000019688 fish Nutrition 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 description 1
- 238000001943 fluorescence-activated cell sorting Methods 0.000 description 1
- 108091006047 fluorescent proteins Proteins 0.000 description 1
- 102000034287 fluorescent proteins Human genes 0.000 description 1
- 239000011737 fluorine Substances 0.000 description 1
- 239000011888 foil Substances 0.000 description 1
- 235000013355 food flavoring agent Nutrition 0.000 description 1
- 235000003599 food sweetener Nutrition 0.000 description 1
- 235000019253 formic acid Nutrition 0.000 description 1
- 125000000524 functional group Chemical group 0.000 description 1
- 230000002538 fungal effect Effects 0.000 description 1
- 229960003692 gamma aminobutyric acid Drugs 0.000 description 1
- 230000006251 gamma-carboxylation Effects 0.000 description 1
- 150000002270 gangliosides Chemical class 0.000 description 1
- 239000007789 gas Substances 0.000 description 1
- 238000001502 gel electrophoresis Methods 0.000 description 1
- 239000007897 gelcap Substances 0.000 description 1
- 238000003209 gene knockout Methods 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- AZKVWQKMDGGDSV-UHFFFAOYSA-N genipin Natural products COC(=O)C1=COC(O)C2C(CO)=CCC12 AZKVWQKMDGGDSV-UHFFFAOYSA-N 0.000 description 1
- 210000004602 germ cell Anatomy 0.000 description 1
- 210000002980 germ line cell Anatomy 0.000 description 1
- 208000004104 gestational diabetes Diseases 0.000 description 1
- 239000003862 glucocorticoid Substances 0.000 description 1
- 229930195712 glutamate Natural products 0.000 description 1
- KWIUHFFTVRNATP-UHFFFAOYSA-N glycine betaine Chemical group C[N+](C)(C)CC([O-])=O KWIUHFFTVRNATP-UHFFFAOYSA-N 0.000 description 1
- 150000002334 glycols Chemical class 0.000 description 1
- 230000001279 glycosylating effect Effects 0.000 description 1
- LEQAOMBKQFMDFZ-UHFFFAOYSA-N glyoxal Chemical group O=CC=O LEQAOMBKQFMDFZ-UHFFFAOYSA-N 0.000 description 1
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
- 239000008187 granular material Substances 0.000 description 1
- 239000003966 growth inhibitor Substances 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 229940029575 guanosine Drugs 0.000 description 1
- 230000003394 haemopoietic effect Effects 0.000 description 1
- 230000003862 health status Effects 0.000 description 1
- 108060003552 hemocyanin Proteins 0.000 description 1
- 238000004896 high resolution mass spectrometry Methods 0.000 description 1
- 210000005260 human cell Anatomy 0.000 description 1
- 235000020256 human milk Nutrition 0.000 description 1
- 210000004251 human milk Anatomy 0.000 description 1
- 239000003906 humectant Substances 0.000 description 1
- 230000004727 humoral immunity Effects 0.000 description 1
- 238000009396 hybridization Methods 0.000 description 1
- 229930195733 hydrocarbon Natural products 0.000 description 1
- 150000002430 hydrocarbons Chemical class 0.000 description 1
- 235000011167 hydrochloric acid Nutrition 0.000 description 1
- 239000000017 hydrogel Substances 0.000 description 1
- 230000003301 hydrolyzing effect Effects 0.000 description 1
- WGCNASOHLSPBMP-UHFFFAOYSA-N hydroxyacetaldehyde Natural products OCC=O WGCNASOHLSPBMP-UHFFFAOYSA-N 0.000 description 1
- 208000026095 hyper-IgM syndrome type 1 Diseases 0.000 description 1
- 230000006058 immune tolerance Effects 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 230000003053 immunization Effects 0.000 description 1
- 238000002649 immunization Methods 0.000 description 1
- 238000003018 immunoassay Methods 0.000 description 1
- 238000003119 immunoblot Methods 0.000 description 1
- 102000018358 immunoglobulin Human genes 0.000 description 1
- 229940027941 immunoglobulin g Drugs 0.000 description 1
- 230000006054 immunological memory Effects 0.000 description 1
- 238000001114 immunoprecipitation Methods 0.000 description 1
- 230000004957 immunoregulator effect Effects 0.000 description 1
- 239000003547 immunosorbent Substances 0.000 description 1
- 230000003308 immunostimulating effect Effects 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 238000000099 in vitro assay Methods 0.000 description 1
- 238000012606 in vitro cell culture Methods 0.000 description 1
- 238000005462 in vivo assay Methods 0.000 description 1
- 230000002458 infectious effect Effects 0.000 description 1
- 230000036512 infertility Effects 0.000 description 1
- 230000004968 inflammatory condition Effects 0.000 description 1
- 208000030603 inherited susceptibility to asthma Diseases 0.000 description 1
- 230000000266 injurious effect Effects 0.000 description 1
- 230000015788 innate immune response Effects 0.000 description 1
- 238000001361 intraarterial administration Methods 0.000 description 1
- 239000007927 intramuscular injection Substances 0.000 description 1
- 238000005342 ion exchange Methods 0.000 description 1
- 239000003456 ion exchange resin Substances 0.000 description 1
- 229920003303 ion-exchange polymer Polymers 0.000 description 1
- 150000008040 ionic compounds Chemical class 0.000 description 1
- 230000002262 irrigation Effects 0.000 description 1
- 238000003973 irrigation Methods 0.000 description 1
- 208000028867 ischemia Diseases 0.000 description 1
- 239000001282 iso-butane Substances 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 230000006122 isoprenylation Effects 0.000 description 1
- 229940074928 isopropyl myristate Drugs 0.000 description 1
- 229960000448 lactic acid Drugs 0.000 description 1
- 239000004816 latex Substances 0.000 description 1
- 229920000126 latex Polymers 0.000 description 1
- 239000010410 layer Substances 0.000 description 1
- 229940067606 lecithin Drugs 0.000 description 1
- 235000010445 lecithin Nutrition 0.000 description 1
- 239000000787 lecithin Substances 0.000 description 1
- 229960004194 lidocaine Drugs 0.000 description 1
- 239000000865 liniment Substances 0.000 description 1
- 125000003473 lipid group Chemical group 0.000 description 1
- 238000001638 lipofection Methods 0.000 description 1
- 239000003589 local anesthetic agent Substances 0.000 description 1
- 231100000053 low toxicity Toxicity 0.000 description 1
- 238000004020 luminiscence type Methods 0.000 description 1
- 210000004698 lymphocyte Anatomy 0.000 description 1
- 125000003588 lysine group Chemical group [H]N([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])(N([H])[H])C(*)=O 0.000 description 1
- 229920001427 mPEG Polymers 0.000 description 1
- 239000011777 magnesium Substances 0.000 description 1
- 229910052749 magnesium Inorganic materials 0.000 description 1
- VTHJTEIRLNZDEV-UHFFFAOYSA-L magnesium dihydroxide Chemical compound [OH-].[OH-].[Mg+2] VTHJTEIRLNZDEV-UHFFFAOYSA-L 0.000 description 1
- 239000000347 magnesium hydroxide Substances 0.000 description 1
- 229910001862 magnesium hydroxide Inorganic materials 0.000 description 1
- 235000019359 magnesium stearate Nutrition 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- FPYJFEHAWHCUMM-UHFFFAOYSA-N maleic anhydride Chemical compound O=C1OC(=O)C=C1 FPYJFEHAWHCUMM-UHFFFAOYSA-N 0.000 description 1
- 239000001630 malic acid Substances 0.000 description 1
- 235000011090 malic acid Nutrition 0.000 description 1
- 150000002690 malonic acid derivatives Chemical class 0.000 description 1
- 239000000594 mannitol Substances 0.000 description 1
- 235000010355 mannitol Nutrition 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 229960004961 mechlorethamine Drugs 0.000 description 1
- HAWPXGHAZFHHAD-UHFFFAOYSA-N mechlorethamine Chemical class ClCCN(C)CCCl HAWPXGHAZFHHAD-UHFFFAOYSA-N 0.000 description 1
- 229960002523 mercuric chloride Drugs 0.000 description 1
- LWJROJCJINYWOX-UHFFFAOYSA-L mercury dichloride Chemical compound Cl[Hg]Cl LWJROJCJINYWOX-UHFFFAOYSA-L 0.000 description 1
- 208000030159 metabolic disease Diseases 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 150000004702 methyl esters Chemical group 0.000 description 1
- 239000004292 methyl p-hydroxybenzoate Substances 0.000 description 1
- 229960002216 methylparaben Drugs 0.000 description 1
- 239000002679 microRNA Substances 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 238000000520 microinjection Methods 0.000 description 1
- 239000011859 microparticle Substances 0.000 description 1
- 230000003228 microsomal effect Effects 0.000 description 1
- 230000005012 migration Effects 0.000 description 1
- 238000013508 migration Methods 0.000 description 1
- 230000003278 mimic effect Effects 0.000 description 1
- 150000007522 mineralic acids Chemical class 0.000 description 1
- 244000309715 mini pig Species 0.000 description 1
- 239000003595 mist Substances 0.000 description 1
- 238000007799 mixed lymphocyte reaction assay Methods 0.000 description 1
- 230000001333 moisturizer Effects 0.000 description 1
- 238000000329 molecular dynamics simulation Methods 0.000 description 1
- 230000004001 molecular interaction Effects 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 208000029077 monogenic diabetes Diseases 0.000 description 1
- 201000006417 multiple sclerosis Diseases 0.000 description 1
- 238000007837 multiplex assay Methods 0.000 description 1
- 210000003205 muscle Anatomy 0.000 description 1
- 201000006938 muscular dystrophy Diseases 0.000 description 1
- 206010028417 myasthenia gravis Diseases 0.000 description 1
- IJDNQMDRQITEOD-UHFFFAOYSA-N n-butane Chemical compound CCCC IJDNQMDRQITEOD-UHFFFAOYSA-N 0.000 description 1
- OFBQJSOFQDEBGM-UHFFFAOYSA-N n-pentane Natural products CCCCC OFBQJSOFQDEBGM-UHFFFAOYSA-N 0.000 description 1
- 239000002088 nanocapsule Substances 0.000 description 1
- 239000002086 nanomaterial Substances 0.000 description 1
- 229940075566 naphthalene Drugs 0.000 description 1
- 239000007922 nasal spray Substances 0.000 description 1
- 229940097496 nasal spray Drugs 0.000 description 1
- 230000037125 natural defense Effects 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 230000017128 negative regulation of NF-kappaB transcription factor activity Effects 0.000 description 1
- 230000010807 negative regulation of binding Effects 0.000 description 1
- 239000012457 nonaqueous media Substances 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
- QIQXTHQIDYTFRH-UHFFFAOYSA-N octadecanoic acid Chemical compound CCCCCCCCCCCCCCCCCC(O)=O QIQXTHQIDYTFRH-UHFFFAOYSA-N 0.000 description 1
- 239000004006 olive oil Substances 0.000 description 1
- 235000008390 olive oil Nutrition 0.000 description 1
- 150000007524 organic acids Chemical class 0.000 description 1
- 235000005985 organic acids Nutrition 0.000 description 1
- 150000002894 organic compounds Chemical class 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- 229960003104 ornithine Drugs 0.000 description 1
- 239000012285 osmium tetroxide Substances 0.000 description 1
- 229910000489 osmium tetroxide Inorganic materials 0.000 description 1
- 229940092253 ovalbumin Drugs 0.000 description 1
- 235000006408 oxalic acid Nutrition 0.000 description 1
- LXCFILQKKLGQFO-UHFFFAOYSA-N p-hydroxybenzoic acid methyl ester Natural products COC(=O)C1=CC=C(O)C=C1 LXCFILQKKLGQFO-UHFFFAOYSA-N 0.000 description 1
- 239000006179 pH buffering agent Substances 0.000 description 1
- 210000002571 pancreatic alpha cell Anatomy 0.000 description 1
- 210000001285 pancreatic epsilon cell Anatomy 0.000 description 1
- FJKROLUGYXJWQN-UHFFFAOYSA-N papa-hydroxy-benzoic acid Natural products OC(=O)C1=CC=C(O)C=C1 FJKROLUGYXJWQN-UHFFFAOYSA-N 0.000 description 1
- 230000005298 paramagnetic effect Effects 0.000 description 1
- 244000045947 parasite Species 0.000 description 1
- 238000005192 partition Methods 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 230000007918 pathogenicity Effects 0.000 description 1
- 230000001575 pathological effect Effects 0.000 description 1
- 230000037361 pathway Effects 0.000 description 1
- 125000000538 pentafluorophenyl group Chemical group FC1=C(F)C(F)=C(*)C(F)=C1F 0.000 description 1
- 201000001245 periodontitis Diseases 0.000 description 1
- 239000008177 pharmaceutical agent Substances 0.000 description 1
- 230000003285 pharmacodynamic effect Effects 0.000 description 1
- 229960003742 phenol Drugs 0.000 description 1
- 239000002953 phosphate buffered saline Substances 0.000 description 1
- WTJKGGKOPKCXLL-RRHRGVEJSA-N phosphatidylcholine Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCCCCCCC=CCCCCCCCC WTJKGGKOPKCXLL-RRHRGVEJSA-N 0.000 description 1
- 150000004713 phosphodiesters Chemical class 0.000 description 1
- 235000011007 phosphoric acid Nutrition 0.000 description 1
- 125000005642 phosphothioate group Chemical group 0.000 description 1
- USRGIUJOYOXOQJ-GBXIJSLDSA-N phosphothreonine Chemical compound OP(=O)(O)O[C@H](C)[C@H](N)C(O)=O USRGIUJOYOXOQJ-GBXIJSLDSA-N 0.000 description 1
- DCWXELXMIBXGTH-UHFFFAOYSA-N phosphotyrosine Chemical group OC(=O)C(N)CC1=CC=C(OP(O)(O)=O)C=C1 DCWXELXMIBXGTH-UHFFFAOYSA-N 0.000 description 1
- 239000007856 photoaffinity label Substances 0.000 description 1
- 238000000053 physical method Methods 0.000 description 1
- 230000001766 physiological effect Effects 0.000 description 1
- 230000035790 physiological processes and functions Effects 0.000 description 1
- 230000006461 physiological response Effects 0.000 description 1
- 239000002504 physiological saline solution Substances 0.000 description 1
- 239000006187 pill Substances 0.000 description 1
- 210000002381 plasma Anatomy 0.000 description 1
- 230000036470 plasma concentration Effects 0.000 description 1
- 239000013600 plasmid vector Substances 0.000 description 1
- 210000001778 pluripotent stem cell Anatomy 0.000 description 1
- 239000013573 pollen allergen Substances 0.000 description 1
- 238000002264 polyacrylamide gel electrophoresis Methods 0.000 description 1
- 230000008488 polyadenylation Effects 0.000 description 1
- 229920006122 polyamide resin Polymers 0.000 description 1
- 239000004417 polycarbonate Substances 0.000 description 1
- 229920000515 polycarbonate Polymers 0.000 description 1
- 229920000728 polyester Polymers 0.000 description 1
- 229920000570 polyether Polymers 0.000 description 1
- 238000003752 polymerase chain reaction Methods 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 150000004804 polysaccharides Chemical class 0.000 description 1
- 230000006267 polysialylation Effects 0.000 description 1
- 229920002223 polystyrene Polymers 0.000 description 1
- 229920005990 polystyrene resin Polymers 0.000 description 1
- 235000013855 polyvinylpyrrolidone Nutrition 0.000 description 1
- 229920000036 polyvinylpyrrolidone Polymers 0.000 description 1
- 239000001267 polyvinylpyrrolidone Substances 0.000 description 1
- 230000001124 posttranscriptional effect Effects 0.000 description 1
- 230000001323 posttranslational effect Effects 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 230000035935 pregnancy Effects 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 239000000186 progesterone Substances 0.000 description 1
- 229960003387 progesterone Drugs 0.000 description 1
- 230000000770 proinflammatory effect Effects 0.000 description 1
- 210000001236 prokaryotic cell Anatomy 0.000 description 1
- 229940097325 prolactin Drugs 0.000 description 1
- 108010029020 prolylglycine Proteins 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 239000001294 propane Substances 0.000 description 1
- 235000019260 propionic acid Nutrition 0.000 description 1
- 239000004405 propyl p-hydroxybenzoate Substances 0.000 description 1
- QQONPFPTGQHPMA-UHFFFAOYSA-N propylene Natural products CC=C QQONPFPTGQHPMA-UHFFFAOYSA-N 0.000 description 1
- 125000004805 propylene group Chemical group [H]C([H])([H])C([H])([*:1])C([H])([H])[*:2] 0.000 description 1
- 229960003415 propylparaben Drugs 0.000 description 1
- 235000019833 protease Nutrition 0.000 description 1
- 235000019419 proteases Nutrition 0.000 description 1
- 230000012846 protein folding Effects 0.000 description 1
- 230000005664 protein glycosylation in endoplasmic reticulum Effects 0.000 description 1
- 238000001243 protein synthesis Methods 0.000 description 1
- 230000012743 protein tagging Effects 0.000 description 1
- 229940024999 proteolytic enzymes for treatment of wounds and ulcers Drugs 0.000 description 1
- 238000012383 pulmonary drug delivery Methods 0.000 description 1
- 239000002213 purine nucleotide Substances 0.000 description 1
- 150000003212 purines Chemical class 0.000 description 1
- 239000002719 pyrimidine nucleotide Substances 0.000 description 1
- 150000003230 pyrimidines Chemical class 0.000 description 1
- 229940107700 pyruvic acid Drugs 0.000 description 1
- 125000001453 quaternary ammonium group Chemical group 0.000 description 1
- IUVKMZGDUIUOCP-BTNSXGMBSA-N quinbolone Chemical compound O([C@H]1CC[C@H]2[C@H]3[C@@H]([C@]4(C=CC(=O)C=C4CC3)C)CC[C@@]21C)C1=CCCC1 IUVKMZGDUIUOCP-BTNSXGMBSA-N 0.000 description 1
- 238000003127 radioimmunoassay Methods 0.000 description 1
- 230000008707 rearrangement Effects 0.000 description 1
- 238000003259 recombinant expression Methods 0.000 description 1
- 238000010188 recombinant method Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
- 238000004007 reversed phase HPLC Methods 0.000 description 1
- 125000000548 ribosyl group Chemical group C1([C@H](O)[C@H](O)[C@H](O1)CO)* 0.000 description 1
- 235000005713 safflower oil Nutrition 0.000 description 1
- 239000003813 safflower oil Substances 0.000 description 1
- 229960004889 salicylic acid Drugs 0.000 description 1
- 235000019515 salmon Nutrition 0.000 description 1
- 229940043230 sarcosine Drugs 0.000 description 1
- 150000003335 secondary amines Chemical class 0.000 description 1
- 230000001953 sensory effect Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 239000002453 shampoo Substances 0.000 description 1
- SQVRNKJHWKZAKO-OQPLDHBCSA-N sialic acid Chemical compound CC(=O)N[C@@H]1[C@@H](O)C[C@@](O)(C(O)=O)OC1[C@H](O)[C@H](O)CO SQVRNKJHWKZAKO-OQPLDHBCSA-N 0.000 description 1
- 239000002924 silencing RNA Substances 0.000 description 1
- 239000000377 silicon dioxide Substances 0.000 description 1
- 238000002741 site-directed mutagenesis Methods 0.000 description 1
- 239000004055 small Interfering RNA Substances 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- HELHAJAZNSDZJO-OLXYHTOASA-L sodium L-tartrate Chemical compound [Na+].[Na+].[O-]C(=O)[C@H](O)[C@@H](O)C([O-])=O HELHAJAZNSDZJO-OLXYHTOASA-L 0.000 description 1
- 235000019812 sodium carboxymethyl cellulose Nutrition 0.000 description 1
- 229920001027 sodium carboxymethylcellulose Polymers 0.000 description 1
- 239000008354 sodium chloride injection Substances 0.000 description 1
- 229910001415 sodium ion Inorganic materials 0.000 description 1
- 239000008109 sodium starch glycolate Substances 0.000 description 1
- 229940079832 sodium starch glycolate Drugs 0.000 description 1
- 229920003109 sodium starch glycolate Polymers 0.000 description 1
- 239000001433 sodium tartrate Substances 0.000 description 1
- 229960002167 sodium tartrate Drugs 0.000 description 1
- 235000011004 sodium tartrates Nutrition 0.000 description 1
- 239000008247 solid mixture Substances 0.000 description 1
- 238000007614 solvation Methods 0.000 description 1
- 210000002325 somatostatin-secreting cell Anatomy 0.000 description 1
- 239000003549 soybean oil Substances 0.000 description 1
- 235000012424 soybean oil Nutrition 0.000 description 1
- 210000000952 spleen Anatomy 0.000 description 1
- 230000002269 spontaneous effect Effects 0.000 description 1
- 230000000087 stabilizing effect Effects 0.000 description 1
- 238000012289 standard assay Methods 0.000 description 1
- 238000010561 standard procedure Methods 0.000 description 1
- 229940032147 starch Drugs 0.000 description 1
- 239000008107 starch Substances 0.000 description 1
- SFVFIFLLYFPGHH-UHFFFAOYSA-M stearalkonium chloride Chemical compound [Cl-].CCCCCCCCCCCCCCCCCC[N+](C)(C)CC1=CC=CC=C1 SFVFIFLLYFPGHH-UHFFFAOYSA-M 0.000 description 1
- 239000008227 sterile water for injection Substances 0.000 description 1
- 239000011550 stock solution Substances 0.000 description 1
- 230000035322 succinylation Effects 0.000 description 1
- 238000010613 succinylation reaction Methods 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 229960004793 sucrose Drugs 0.000 description 1
- 238000006277 sulfonation reaction Methods 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 239000013589 supplement Substances 0.000 description 1
- 230000008093 supporting effect Effects 0.000 description 1
- 238000002198 surface plasmon resonance spectroscopy Methods 0.000 description 1
- 239000003765 sweetening agent Substances 0.000 description 1
- 230000002195 synergetic effect Effects 0.000 description 1
- 238000001308 synthesis method Methods 0.000 description 1
- 229920001059 synthetic polymer Polymers 0.000 description 1
- 239000006188 syrup Substances 0.000 description 1
- 235000020357 syrup Nutrition 0.000 description 1
- 238000012385 systemic delivery Methods 0.000 description 1
- 239000011975 tartaric acid Substances 0.000 description 1
- 125000000999 tert-butyl group Chemical group [H]C([H])([H])C(*)(C([H])([H])[H])C([H])([H])[H] 0.000 description 1
- 229930101283 tetracycline Natural products 0.000 description 1
- 229960002180 tetracycline Drugs 0.000 description 1
- 235000019364 tetracycline Nutrition 0.000 description 1
- 150000003522 tetracyclines Chemical class 0.000 description 1
- 229940126585 therapeutic drug Drugs 0.000 description 1
- 125000003396 thiol group Chemical group [H]S* 0.000 description 1
- 108060008226 thioredoxin Proteins 0.000 description 1
- 229940094937 thioredoxin Drugs 0.000 description 1
- 229960002175 thyroglobulin Drugs 0.000 description 1
- 125000002088 tosyl group Chemical group [H]C1=C([H])C(=C([H])C([H])=C1C([H])([H])[H])S(*)(=O)=O 0.000 description 1
- 235000010487 tragacanth Nutrition 0.000 description 1
- 239000000196 tragacanth Substances 0.000 description 1
- 229940116362 tragacanth Drugs 0.000 description 1
- FGMPLJWBKKVCDB-UHFFFAOYSA-N trans-L-hydroxy-proline Natural products ON1CCCC1C(O)=O FGMPLJWBKKVCDB-UHFFFAOYSA-N 0.000 description 1
- 230000005026 transcription initiation Effects 0.000 description 1
- 230000002103 transcriptional effect Effects 0.000 description 1
- 230000037317 transdermal delivery Effects 0.000 description 1
- 238000001890 transfection Methods 0.000 description 1
- 239000012581 transferrin Substances 0.000 description 1
- 230000001131 transforming effect Effects 0.000 description 1
- 230000001052 transient effect Effects 0.000 description 1
- 230000032258 transport Effects 0.000 description 1
- PBKWZFANFUTEPS-CWUSWOHSSA-N transportan Chemical compound C([C@@H](C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(=O)NCC(=O)N[C@@H](CCCCN)C(=O)N[C@H](C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C)C(=O)N[C@@H](C)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC(C)C)C(N)=O)[C@@H](C)CC)NC(=O)CNC(=O)[C@H](C)NC(=O)[C@H](CO)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](NC(=O)[C@H](CC=1C2=CC=CC=C2NC=1)NC(=O)CN)[C@@H](C)O)C1=CC=C(O)C=C1 PBKWZFANFUTEPS-CWUSWOHSSA-N 0.000 description 1
- 108010062760 transportan Proteins 0.000 description 1
- CYRMSUTZVYGINF-UHFFFAOYSA-N trichlorofluoromethane Chemical compound FC(Cl)(Cl)Cl CYRMSUTZVYGINF-UHFFFAOYSA-N 0.000 description 1
- 229940029284 trichlorofluoromethane Drugs 0.000 description 1
- YKUJZZHGTWVWHA-UHFFFAOYSA-N triptolide Natural products COC12CC3OC3(C(C)C)C(O)C14OC4CC5C6=C(CCC25C)C(=O)OC6 YKUJZZHGTWVWHA-UHFFFAOYSA-N 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
- 125000002221 trityl group Chemical group [H]C1=C([H])C([H])=C([H])C([H])=C1C([*])(C1=C(C(=C(C(=C1[H])[H])[H])[H])[H])C1=C([H])C([H])=C([H])C([H])=C1[H] 0.000 description 1
- HDZZVAMISRMYHH-KCGFPETGSA-N tubercidin Chemical compound C1=CC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O HDZZVAMISRMYHH-KCGFPETGSA-N 0.000 description 1
- 229940046728 tumor necrosis factor alpha inhibitor Drugs 0.000 description 1
- 239000002452 tumor necrosis factor alpha inhibitor Substances 0.000 description 1
- 239000002451 tumor necrosis factor inhibitor Substances 0.000 description 1
- 102000003298 tumor necrosis factor receptor Human genes 0.000 description 1
- 235000015112 vegetable and seed oil Nutrition 0.000 description 1
- 239000008158 vegetable oil Substances 0.000 description 1
- 238000012800 visualization Methods 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- 239000008215 water for injection Substances 0.000 description 1
- 229920003176 water-insoluble polymer Polymers 0.000 description 1
- 239000008136 water-miscible vehicle Substances 0.000 description 1
- 239000001993 wax Substances 0.000 description 1
- 238000009736 wetting Methods 0.000 description 1
- 239000011592 zinc chloride Substances 0.000 description 1
- 235000005074 zinc chloride 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
- NWONKYPBYAMBJT-UHFFFAOYSA-L zinc sulfate Chemical compound [Zn+2].[O-]S([O-])(=O)=O NWONKYPBYAMBJT-UHFFFAOYSA-L 0.000 description 1
- 239000011686 zinc sulphate Substances 0.000 description 1
- 235000009529 zinc sulphate Nutrition 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
-
- 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
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
- A61K31/4353—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems
- A61K31/436—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems the heterocyclic ring system containing a six-membered ring having oxygen as a ring hetero atom, e.g. rapamycin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/14—Blood; Artificial blood
- A61K35/15—Cells of the myeloid line, e.g. granulocytes, basophils, eosinophils, neutrophils, leucocytes, monocytes, macrophages or mast cells; Myeloid precursor cells; Antigen-presenting cells, e.g. dendritic cells
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/37—Digestive system
- A61K35/39—Pancreas; Islets of Langerhans
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
- A61K38/10—Peptides having 12 to 20 amino acids
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/177—Receptors; Cell surface antigens; Cell surface determinants
- A61K38/1793—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/46—Cellular immunotherapy
- A61K39/461—Cellular immunotherapy characterised by the cell type used
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/46—Cellular immunotherapy
- A61K39/461—Cellular immunotherapy characterised by the cell type used
- A61K39/4612—B-cells
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/46—Cellular immunotherapy
- A61K39/462—Cellular immunotherapy characterized by the effect or the function of the cells
- A61K39/4621—Cellular immunotherapy characterized by the effect or the function of the cells immunosuppressive or immunotolerising
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/46—Cellular immunotherapy
- A61K39/462—Cellular immunotherapy characterized by the effect or the function of the cells
- A61K39/4622—Antigen presenting cells
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/46—Cellular immunotherapy
- A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
- A61K39/4643—Vertebrate antigens
- A61K39/46433—Antigens related to auto-immune diseases; Preparations to induce self-tolerance
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/46—Cellular immunotherapy
- A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
- A61K39/4643—Vertebrate antigens
- A61K39/46434—Antigens related to induction of tolerance to non-self
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/46—Cellular immunotherapy
- A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
- A61K39/4643—Vertebrate antigens
- A61K39/4644—Cancer antigens
- A61K39/464402—Receptors, cell surface antigens or cell surface determinants
- A61K39/464429—Molecules with a "CD" designation not provided for elsewhere
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
- A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
- A61K47/6901—Conjugates being cells, cell fragments, viruses, ghosts, red blood cells or viral vectors
-
- 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
- A61K9/0012—Galenical forms characterised by the site of application
- A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
-
- 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
- A61K9/10—Dispersions; Emulsions
- A61K9/127—Liposomes
-
- 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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
- A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
-
- 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
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
- A61K2239/26—Universal/off- the- shelf cellular immunotherapy; Allogenic cells or means to avoid rejection
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
- A61K2239/31—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterized by the route of administration
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Definitions
- CD40 ligand (CD40L), or CD154, binds to i) its classical receptor CD40 on B cells and other antigen presenting cells, ii) ⁇ M ⁇ 2, also known as Mac-1, on neutrophils, monocytes, and macrophages, iii) ⁇ 5 ⁇ 1 on monocytes, and ⁇ IIb ⁇ 3 on platelets.
- CD40L classical receptor CD40 on B cells and other antigen presenting cells
- ⁇ M ⁇ 2 also known as Mac-1
- neutrophils neutrophils
- monocytes monocytes
- macrophages iii) ⁇ 5 ⁇ 1 on monocytes
- ⁇ IIb ⁇ 3 on platelets.
- the interaction of CD154 with CD40 is implicated in the activation of CD40 expressing immune cells such as B-cell, T-cell, neutrophil, monocyte, macrophage, dendritic cell, and platelet.
- a pharmaceutical composition comprising a peptide or a portion thereof, wherein the peptide comprises an amino acid sequence with at least 85%, or at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-6 and 8-17, and a pharmaceutically acceptable carrier, wherein said peptide binds a CD40 protein or portion thereof.
- a pharmaceutical composition comprising a peptide, wherein the peptide comprises an amino acid sequence with at least 90% sequence identity to the sequences set forth in any one of SEQ ID NOs: 1-6 and 8-17, and a pharmaceutically acceptable carrier, wherein said peptide binds a CD40 protein or a portion thereof.
- the peptide comprises the amino acid sequence of SEQ ID NO: 1. In some embodiments, the peptide comprises the amino acid sequence of SEQ ID NO: 2. In some embodiments, the peptide comprises the amino acid sequence of SEQ ID NO: 3. In some embodiments, the peptide comprises the amino acid sequence of SEQ ID NO: 4. In some embodiments, the peptide comprises the amino acid sequence of SEQ ID NO: 5. In some embodiments, the peptide comprises the amino acid sequence of SEQ ID NO: 6. In some embodiments, the peptide comprises the amino acid sequence of SEQ ID NO: 8. In some embodiments, the peptide comprises the amino acid sequence of SEQ ID NO: 9. In some embodiments, the peptide comprises the amino acid sequence of SEQ ID NO: 10.
- the peptide comprises the amino acid sequence of SEQ ID NO: 11. In some embodiments, the peptide comprises the amino acid sequence of SEQ ID NO: 12. In some embodiments, the peptide comprises the amino acid sequence of SEQ ID NO: 13. In some embodiments, the peptide comprises the amino acid sequence of SEQ ID NO: 14. In some embodiments, the peptide comprises the amino acid sequence of SEQ ID NO: 15. In some embodiments, the peptide comprises the amino acid sequence of SEQ ID NO: 16. In some embodiments, the peptide comprises the amino acid sequence of SEQ ID NO: 17.
- the peptide is from 4 to 20 amino acids in length. In some embodiments, the peptide comprises at least one non-naturally occurring amino acid. In some embodiments, the peptide comprises a carboxy terminus that is amidated. In some embodiments, the peptide is in a cyclic form. In some embodiments, the peptide further comprises PEG. In some embodiments, the peptide is comprises a lipophilic molecular group. In some embodiments, the peptide is recombinant or synthesized. In some embodiments, the peptide is fused to a carrier polypeptide.
- the peptide is further conjugated to a detectable agent, a peptide tag, a magnetic particle, a diagnostic agent, a therapeutic agent, or a combination thereof.
- the pharmaceutical composition disclosed herein comprises a detectable agent that is an enzyme—substrate agent, a fluorescent agent, a chemiluminescent agent, or a radioisotope.
- the pharmaceutical composition disclosed herein comprises a therapeutic agent that is an anti-inflammatory, immunosuppressive, immunomodulatory, or an anti-tumor agent.
- the peptide is in a soluble form.
- the peptide is formulated in a liposome or a nanoparticle delivery system. In some embodiments, the peptide is formulated with a biocompatible polymer. In some embodiments, the pharmaceutical composition of the aspects above further comprises a pharmaceutically acceptable diluent, additive or excipient. In some embodiments, the pharmaceutical composition of the aspects disclosed above is formulated for administration via a subcutaneous, intravenous, intradermal, intraperitoneal, oral, intramuscular, intracerebroventricular, intranasal, intracranial, intracelial, intracerebellar, intrathecal, transdermal, pulmonary, or topical administration route. In some embodiments, the pharmaceutical composition of the aspects disclosed above is formulated for administration via intravenous administration route. In some embodiments, the pharmaceutical composition further comprises a ganglioside and/or a phosphotidylserine.
- the pharmaceutical composition further comprises saccharides selected from the group consisting of cyclodextrins, disaccharides, polysaccharides, and a combination thereof.
- the pharmaceutical composition of the aspects disclosed above is in a solution form or in a lyophilized form.
- the pharmaceutical composition is contained in a delivery device selected from the group consisting of a syringe, a blunt tip syringe, a catheter, an inhaler, a nebulizer, a nasal spray pump, a nasal irrigation pump or nasal lavage pump, and an implantable pump.
- the pharmaceutical composition has a shelf life of at least 2 days, 2 weeks, 1 month to 2 years at room temperature.
- the pharmaceutical composition has a shelf life of at least 2 days, 2 weeks, 1 month to 2 years at 4° C.
- the peptide or portion thereof binds a CD40 protein or a portion of the CD40 protein.
- the peptide or portion thereof, wherein the peptide or the functional thereof is in an amount sufficient to inhibit binding of the CD40 protein to a CD154.
- the technology herein relates to a complex comprising a peptide or a portion thereof non-covalently bound to a CD40 protein or a portion of the CD40 protein, wherein the peptide comprises an amino acid sequence with at least 85% or at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-6 and 8-17.
- the CD40 protein is mammalian. In some embodiments, the CD40 protein is human. In some embodiments, the CD40 protein is recombinant.
- an isolated nucleic acid fragment encoding a peptide or a portion thereof, wherein the peptide comprises an amino acid sequence with at least 85% or at least 90% sequence identity to an amino acid sequence set forth in any one of SEQ ID NOs: 1-6 and 8-17.
- the isolated nucleic acid fragment of the aspects disclosed above further comprising a nucleotide sequence encoding an agent, wherein the agent is a targeting agent, a detectable agent, a diagnostic agent, or a therapeutic agent.
- a vector comprising the isolated nucleic acid fragment of any one of aspects disclosed above.
- the isolated nucleic acid fragment is operably linked to a regulatory control sequence.
- a host cell comprising the isolated nucleic acid fragment of aspects above or the vector of aspects above.
- a preparatory regimen for transplantation of a cell, tissue or organ from a recipient to a donor comprising an anti-CD40 agent; wherein the anti-CD40 agent comprises a peptide containing an amino acid sequence with at least 85% or at least 90% sequence identity to the sequences set forth in any one of SEQ ID NOs. 1-6 and 8-17.
- a preparatory regimen for transplantation of a cell, tissue or organ from a recipient to a donor comprising an anti-CD40 agent; wherein the anti-CD40 agent comprises a peptide containing an amino acid sequence with at least 90% sequence identity to the sequences set forth in any one of SEQ ID NOs. 8-17.
- the preparatory regimen can further comprise instructions for administration of said anti-CD40 agent to said recipient.
- said anti-CD40 agent is administered to said recipient between about 10 days before and about 30 days after said transplantation.
- said anti-CD40 agent is administered to said recipient at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days before said transplantation.
- said anti-CD40 agent is administered to said recipient at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 15, 17, 18, 19, 20, 21, 22, 23, 24, or 25 days before said transplantation.
- said anti-CD40 agent is administered to said recipient subcutaneously, intravenously, intradermally, intraperitoneally, orally, intramuscularly, intracerebroventricularly, intranasally, intracranially, intracelially, intracerebellarly, intrathecally, or transdermally, or topically.
- said anti-CD40 agent is administered to said recipient at a dose of between about 1 mg/kg and 100 mg/kg. In one embodiment, the anti-CD40 agent is administered to said recipient at a dose of 50 mg/kg.
- the preparatory regimen provides long term tolerance to said transplant cell, tissue, or organ.
- the preparatory regimen further comprises administering an effective amount of apoptotic donor leukocytes.
- the donor leukocytes are fixed in a cross-linking agent.
- the apoptotic leukocytes are mammalian leukocytes.
- the apoptotic leukocytes are porcine leukocytes.
- the apoptotic leukocytes are human leukocytes.
- the apoptotic leukocytes are from a cadaveric donor, a brain dead donor, a non-heart beating donor, or a living donor.
- the apoptotic leukocytes are ex vivo expanded leukocytes. In one embodiment, the apoptotic leukocytes are isolated from a spleen, or peripheral blood. In one embodiment, the apoptotic leukocytes comprise B-lymphocytes. In one embodiment, the apoptotic leukocytes comprise cells that have been differentiated from stem cells or induced pluripotent stem cells ex vivo. In some embodiments, the stem cells are derived from the donor of said transplant cell, tissue, or organ.
- the apoptotic leukocytes and the recipient are matched for at least one of MHC class I A allele, MHC class I B allele, MHC class II DR allele, MHC class II DQ allele, or MHC class II DP allele.
- the apoptotic leukocytes and the transplant are matched for at least one of MHC class I A allele, MHC class I B allele, MHC class II DR allele, MHC class II DQ allele, or MHC class II DP allele.
- the apoptotic leucocytes and the transplant are haploidentical. the apoptotic leucocytes are from the donor of the transplant.
- transplant cell, tissue, or organ is a kidney, liver, heart, lung, pancreas, islet cell, small bowel, bone marrow, hematopoietic stem cell, embryonic stem cell-derived islet beta cell, induced pluripotent stem cell-derived islet beta cell, embryonic stem cell-derived islet, induced pluripotent stem cell-derived islet, a stem cell derived cell, tissue or organ, or a combination thereof.
- recipient and the donor of said transplant cell, tissue, or organ are matched for at least one MEW class I A allele, MEW class I B allele, MHC class II DR allele, MEW class II DQ allele, or MHC class II DP allele.
- a preparatory regimen for transplanting a cell, tissue or organ transplant to a recipient comprising: one or more anti-CD40 peptides conjugated to the surface of an apoptotic leukocyte, wherein the one or more anti-CD40 peptides, comprise an amino acid sequence with at least 85% or at least 90% sequence identity to a sequence selected from SEQ ID NOs: 1-6 and 8-17.
- a preparatory regimen for transplanting a cell, tissue or organ transplant to a recipient comprising: one or more anti-CD40 peptides conjugated to the surface of an apoptotic leukocyte, wherein the one or more anti-CD40 peptides, comprises an amino acid sequence with at least 90% sequence identity to a sequence selected from SEQ ID NOs: 8-17.
- the apoptotic leukocyte is further conjugated to one or more peptides derived from a MEW class II molecule of the recipient.
- the apoptotic leukocyte is further conjugated to one or more peptides derived from a MEW class II molecule of the recipient.
- the one or more peptides derived from the MHC class II molecule comprises peptides derived from a DR ⁇ -chain, a DQ ⁇ -chain, or a DP ⁇ -chain, or a combination thereof. In one embodiment, the one or more peptides derived from the MHC class II molecule comprise a sequence from a hypervariable region. In one embodiment, the one or more peptides conjugated to the surface of the leukocyte are synthesized in-vitro or recombinantly produced. In one embodiment, the apoptotic leukocyte is MHC class II matched to the donor and MEW class II mismatched to the recipient.
- the apoptotic leukocyte is conjugated to one or more peptides derived from a MEW class I molecule of the donor of the cell, tissue or organ transplant.
- the MEW class I molecule is expressed in the donor of the transplant.
- the MHC class I molecule is HLA-A1, HLA-A3, HLA-B7, or HLAB8.
- the MEW class I molecule is encoded by HLA-A*02, 24, 01 or HLA-B*35, 44, 51.
- the apoptotic leukocyte is MEW class I matched to the recipient, or MHC class II matched to the recipient, or both.
- the preparatory regimen further comprises administering one or more agents for short-term immunosuppression of said recipient.
- the one or more agents for short term immunosuppression is an anti-CD40 agent, wherein the anti-CD40 agent comprises a peptide containing an amino acid sequence with at least 85% or at least 90% sequence identity to an amino acid sequence set forth in any one of SEQ ID NOs: 1-6 and 8-17.
- the one or more agents for short term immunosuppression is an anti-CD40 agent, wherein the anti-CD40 agent comprises a peptide containing an amino acid sequence with at least 90% sequence identity to an amino acid sequence set forth in any one of SEQ ID NOs: 8-17.
- the one or more agents for short term immunosuppression comprise an mTOR inhibitor, an anti-tumor necrosis factor agent or an anti-tumor necrosis factor receptor agent, an anti-interleukin 6 agent or an anti-interleukin 6 receptor agent, an anti-CD40 agent, or a combination thereof.
- the preparatory regimen further comprises instructions for administration of said one or more agents for short term immunosuppression to said transplant recipient.
- the apoptotic leukocytes have further been contacted with an amount of one or more immunomodulatory molecules. In one embodiment, said amount of one or more immunomodulatory molecules is sufficient to modify a function of antigen-presenting cells in said recipient.
- said one or more immunomodulatory molecules are selected from IFN- ⁇ , an NF-kB inhibitor, vitamin D3, siCD40, cobalt protoporphyrin, insulin B9-23, all or a portion of a cluster of differentiation protein, or a combination thereof.
- said NF-kB inhibitor is curcumin, triptolide, Bay-117085, or a combination thereof.
- the one or more peptides are conjugated to the surface of the apoptotic leukocytes by treatment with the crosslinking agent.
- said crosslinking agent comprises a carbodiimide.
- said carbodiimide comprises 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (ECDI); N,N′-diisopropylcarbodiimide (DIC); N,N′-dicyclohexylcarbodiimide (DCC); or a combination thereof.
- said crosslinking agent does not comprise a carbodiimide.
- said crosslinking agent comprises genipin, acrylic aldehyde, diformyl, osmium tetroxide, a diimidoester, mercuric chloride, zinc sulphate, zinc chloride, trinitrophenol (picric acid), potassium dichromate, ethanol, methanol, acetone, acetic acid, or a combination thereof.
- said diimidoester is selected from cyanuric chloride, diisocyanate, diethylpyrocarbonate (DEPC), a maleimide, benzoquinone, or a combination thereof.
- said apoptotic leukocytes are fixed for a predetermined amount of time.
- said predetermined time is at least about 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 60 minutes, 75, minutes, 90 minutes, 120 minutes, 150 minutes, 180 minutes, 210 minutes, or 240 minutes.
- said apoptotic leukocytes are contacted with an amount of one or more immunomodulatory molecules, before fixation.
- a tolerizing regimen for post-transplant stabilization of a cell, tissue or organ transplant in a recipient comprising administering to the recipient an effective amount of an anti-CD40 peptide, wherein the anti-CD40 peptide comprises an amino acid sequence with at least 85%. or at least 90% sequence identity to a sequence selected from SEQ ID NOs. 1-6 and 8-17.
- a tolerizing regimen for post-transplant stabilization of cell, tissue or organ transplant in a recipient comprising administering to the recipient an effective amount of an anti-CD40 peptide, wherein the anti-CD40 peptide comprises an amino acid sequence with at least 90% sequence identity to a sequence selected from SEQ ID NOs. 8-17.
- the tolerizing regimen further comprises apoptotic leukocytes.
- the apoptotic leukocytes are expanded at least about 3 fold, 5 fold, 10 fold, 50 fold, 100 fold, 150 fold, 200 fold or 250 fold relative to the starting population prior to contacting with said crosslinking agent.
- the apoptotic leukocytes and the anti-CD40 peptide are administered separately.
- the apoptotic leukocytes and the anti-CD40 peptide are administered simultaneously.
- a method of inhibiting an interaction between a CD40 protein and a CD154 protein comprising contacting the CD40 protein with an anti-CD40 agent, wherein the anti-CD40 agent comprises a peptide containing an amino acid sequence with at least 85% or at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-6 and 8-17, thereby inhibiting the interaction between the CD40 protein and the CD154 protein.
- a method of inhibiting an interaction between a CD40 protein and a CD154 protein comprising contacting the CD40 protein with an anti-CD40 agent, wherein the anti-CD40 agent comprises a peptide containing an amino acid sequence with at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 8-17, thereby inhibiting the interaction between the CD40 protein and the CD154 protein.
- the peptide or the functional fragment thereof binds the CD40 protein.
- the CD40 protein is mammalian.
- the inhibition is greater than that exhibited by contacting a peptide or a functional fragment thereof comprising a sequence set forth in SEQ ID NO: 7.
- the CD40 protein is human. In one embodiment, the CD154 protein is mammalian. In one embodiment, the CD154 protein is human. In one embodiment, the CD40 protein is expressed by an immune cell. In one embodiment, the immune cell is selected from the group consisting of B-cell, T-cell, neutrophil, monocyte, macrophage, dendritic cell, and platelet. In one embodiment, the contacting inhibits B-cell activation, T-cell proliferation, T-cell activation, B-cell proliferation, macrophage activation, cytokine production, or a combination thereof.
- the CD40 protein is expressed by an endothelial cell, an epithelial cell, a fibroblast cell, a smooth muscle cell, a B lymphoma cell, a melanoma cell, or a carcinoma cell.
- the contacting comprises administering the peptide or a functional fragment thereof to a subject.
- the subject is a human.
- provided herein is a method of transplantation of a cell or tissue transplant from a mammalian donor in a recipient, the method comprising administering an effective amount of the pharmaceutical composition of any one the aspects described above.
- a method of transplantation of a cell, tissue or organ from a donor to a recipient the method comprising administering an effective amount of an anti-CD40 agent; wherein the anti-CD40 agent comprises a peptide containing an amino acid sequence with at least 85% or at least 90% sequence identity to the sequences set forth in any one of SEQ ID NOs. 1-6 and 8-17 to the recipient.
- a method of transplantation of a cell, tissue or organ from a donor to a recipient comprising administering an effective amount of an anti-CD40 agent; wherein the anti-CD40 agent comprises a peptide containing an amino acid sequence with at least 90% sequence identity to the sequences set forth in any one of SEQ ID NOs. 8-17 to the recipient.
- the peptide is conjugated to apoptotic leukocytes. In one embodiment, the method further comprises administering apoptotic leukocytes. In one embodiment, the method further comprises administering apoptotic leukocytes fixed with a cross-linking agent. In one embodiment, the method further comprises administering the apoptotic leukocytes and the peptide separately. In one embodiment, the method further comprises administering the apoptotic leukocytes and the peptide simultaneously. In one embodiment, the method further comprises the method further comprises transplanting the cell, tissue or organ in the recipient.
- the cell or tissue transplant is a kidney, liver, heart, lung, pancreas, islet cell, small bowel, bone marrow, hematopoietic stem cell, embryonic or induced pluripotent stem cell-derived islet beta cell, embryonic or induced pluripotent stem cell-derived islet, embryonic or pluripotent stem-cell derived hepatocyte or a combination thereof.
- the cell or tissue transplant is a xenotransplant. In one embodiment, the cell or tissue transplant is an allotransplant.
- the administering is subcutaneous, intravenous, intradermal, intraperitoneal, oral, intramuscular, intracerebroventricular, intranasal, intracranial, intracelial, intracerebellar, intrathecal, transdermal, pulmonary, or topical administration.
- the administering is intravenous administration.
- the administering is performed prior to, during, and after the transplantation.
- the administering is performed at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more days prior to the transplantation.
- the administering is performed during the transplantation.
- the administering is performed at least 1, 2, 3, 4, 5, 6, 7, 10, or more days after the transplantation.
- the tolerizing agent comprises mammalian leukocytes fixed with a crosslinking agent.
- the mammalian leukocytes are from the mammalian donor or from a donor that is MEW matched to the mammalian donor.
- the mammalian donor is genetically modified.
- administering an immunomodulatory agent to the recipient is provided.
- the immunomodulatory agent is an anti-CD40 agent, anti-CD40L agent, a B-cell depleting or modulating agent, an mTOR inhibitor, a TNF-alpha inhibitor, a IL-6 inhibitor, alpha 1antitrypsin inhibitor, dehydroxymethylepoxyquinomycin (DHMEQ), a nitrogen mustard alkylating agent, a complement C3 or C5 inhibitor, IFN ⁇ , an NF ⁇ B inhibitor, vitamin D3, siCD40, cobalt protoporphyrin, insulin B9-23, a cluster of differentiation protein, or any combination thereof.
- the cell or tissue transplant survives for at least 30 days, 50 days, 100 days, 1 year or more in the recipient.
- the recipient is a mammal. In one embodiment, the recipient is a human.
- provided herein is a method for inducing immunosuppression in a subject in need thereof, the method comprising administering to the subject an effective amount of the pharmaceutical composition in any one of the aspects described above.
- a method of inducing immunosuppression in a subject in need thereof the method comprising; administering to the recipient an effective amount of an anti-CD40 agent, wherein the agent is a peptide comprising an amino acid sequence with at least 85% or at least 90% sequence identity to the sequences set forth in any one of SEQ ID NOs. 1-6 and 8-17.
- a method of inducing immunosuppression in a subject in need thereof comprising; administering to the recipient an effective amount of an anti-CD40 agent, wherein the agent is a peptide comprising an amino acid sequence with at least 90% sequence identity to the sequences set forth in any one of SEQ ID NOs. 8-17.
- the method comprises further administering apoptotic leukocytes with the anti-CD40 agent.
- the peptide is conjugated to apoptotic leukocytes.
- the apoptotic leukocytes fixed with a cross-linking agent.
- the apoptotic leukocytes and the peptides are administered separately.
- the apoptotic leukocytes and the peptides are administered simultaneously.
- the method further comprises transplanting a cell, tissue or organ in the subject.
- the immune response comprises B-cell activation, T-cell proliferation, B-cell proliferation, macrophage activation, cytokine production, or a combination thereof.
- the subject has undergone, is undergoing or will be undergoing an allotransplant. In one embodiment, the subject has undergone, is undergoing or will be undergoing a xenotransplant. In one embodiment, the subject is a human.
- the subject is suffering from or is at a risk of developing an inflammatory disease.
- the inflammatory disease is an autoimmune disease.
- provided herein is a method of inducing immune tolerance in a subject in need thereof, the method comprising administering to the subject an effective amount of the pharmaceutical composition of any one of the aspects described above.
- a method of inducing tolerance to a cell, tissue or organ transplant in a recipient the method comprising, administering to the recipient an effective amount of a composition comprising an anti-CD40 agent, wherein the agent comprises a peptide containing an amino acid sequence with at least 85% or at least 90% sequence identity to any one of the sequences set forth in SEQ ID NOs. 1-6 and 8-17.
- a method of inducing tolerance to a cell, tissue or organ transplant in a recipient comprising, administering to the recipient an effective amount of a composition comprising an anti-CD40 agent, wherein the anti-CD40 agent comprises a peptide containing an amino acid sequence with at least 90% sequence identity to the sequences set forth any one of SEQ ID NOs. 8-17.
- the anti-CD40 agent is conjugated to apoptotic leukocytes. In one embodiment, the method further comprises administering apoptotic leukocytes. In one embodiment, the method further comprises administering apoptotic leukocytes fixed with a cross-linking agent. In one embodiment, the method further comprises administering the apoptotic leukocytes and the anti-CD40 agent separately. In one embodiment, the method further comprises administering the apoptotic leukocytes and the anti-CD40 agent simultaneously. In one embodiment, the method further comprises apoptotic leukocytes conjugated to one or more peptides derived from a MHC class II molecule of the recipient.
- the apoptotic leukocytes comprise cells that have been differentiated from stem cells ex vivo, wherein the stem cells are derived from a donor of the cell, tissue, or organ transplant.
- the apoptotic leukocytes are MHC matched to the recipient.
- the apoptotic leukocytes are conjugated to one or more peptides derived from a MHC class I molecule of a donor of the cell, tissue or organ transplant.
- the method further comprises transplanting the cell, tissue or organ transplant. In one embodiment, the transplanting is performed at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more days after administering the composition.
- the method further comprises administering at least one booster dose of the composition.
- the booster dose is administered at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 15, 17, 20, 21, or 24 days after the transplanting.
- said tolerance is for a period of at least one month. In one embodiment, said tolerance is for a period of at least 100 days. In one embodiment, said tolerance is for a period of at least one year.
- a method of post-transplant immune tolerizing a subject comprising administering to said subject: an anti-CD40 agent, wherein the agent comprises a peptide comprising an amino acid sequence with at least 85% or at least 90% sequence identity to the sequences set forth in any one of SEQ ID NOs: 1-6 and 8-17.
- a method of post-transplant immune tolerizing a subject comprising administering to said subject: an anti-CD40 agent, wherein the agent comprises a peptide comprising an amino acid sequence with at least 90% sequence identity to the sequences set forth in any one of SEQ ID NOs: 8-17.
- the anti-CD40 agent is conjugated to apoptotic leukocytes.
- the anti-CD40 agent is conjugated to apoptotic leukocytes. In one embodiment, the method further comprises administering apoptotic leukocytes. In one embodiment, the method further comprises administering apoptotic leukocytes fixed with a cross-linking agent. In one embodiment, the method further comprises administering the apoptotic leukocytes and the anti-CD40 agent separately. In one embodiment, the method further comprises administering the apoptotic leukocytes and the anti-CD40 agent simultaneously. In one embodiment, the apoptotic leukocytes are further conjugated to one or more peptides derived from a MHC class II molecule of the recipient.
- the apoptotic leukocytes comprise cells that have been differentiated from stem cells ex vivo, wherein the stem cells are derived from a donor of the cell, tissue, or organ transplant.
- the apoptotic leukocytes are MHC matched to the recipient.
- the apoptotic leukocytes are conjugated to one or more peptides derived from a MHC class I molecule of a donor of the cell, tissue or organ transplant.
- the method further comprises transplanting the cell, tissue or organ transplant.
- the method further comprises apoptotic leukocytes isolated from peripheral blood.
- the method further comprises apoptotic leukocytes enriched for B cells by positive or negative selection.
- said apoptotic leukocytes are expanded at least 200 fold relative to a starting population prior to said modulating with said carbodiimide crosslinking agent.
- provided herein is a method of treating a subject suffering from an inflammatory disease, the method comprising administering to the subject an effective amount of the pharmaceutical composition of any one of the aspects described above.
- a method of treating a subject suffering from or is at a risk of developing a neoplastic disease characterized by a neoplastic cell that expresses CD40 the method comprising administering to the subject an effective amount of the pharmaceutical composition of any one of the aspects described above.
- the inflammatory disease is an autoimmune disease.
- the inflammatory disease is selected from the group consisting of type I diabetes, multiple sclerosis, rheumatoid arthritis, systemic lupus, erythematosa, chronic obstructive pulmonary disease, atherosclerosis, Crohn's colitis, ulcerative gastritis, primary biliary cirrhosis, Guillain-Barre syndrome, Psoriasis, Graves' disease, Hashimoto's Thyroiditis, Myasthenia Gravis, Vasculitis, acute lung injury, bronchial asthma and acute respiratory distress syndrome.
- a method of treating a subject suffering from or is at a risk of developing a neoplastic disease characterized by a neoplastic cell that expresses CD40 comprising administering to the subject an effective amount of the pharmaceutical composition of any of the aspects described above.
- the neoplastic disease is a lymphoma, a myeloma or a carcinoma.
- the lymphoma is a B cell lymphoma.
- the subject is a mammal. In one embodiment, the subject is human.
- the donor cells are islet cells, stem cell, induced pluripotent stem cell-derived islet cells or a combination thereof.
- the donor cells are autologous, allogeneic, and xenogeneic.
- kits for transplantation of a cell, tissue or organ transplant in a recipient comprising; (a) a first container comprising a first composition comprising apoptotic leukocytes fixed in a crosslinking agent, an optionally a container comprising a composition comprising an anti-CD40 agent, wherein the agent is a peptide comprising an amino acid sequence with at least 85% or at least 90% sequence identity to the sequences set forth in any one of SEQ ID NOs:1-6 and 8-17.
- kits for transplantation of a cell, tissue or organ transplant in a recipient comprising; (a) a first container comprising a first composition comprising apoptotic leukocytes fixed in a crosslinking agent, an optionally a container comprising a composition comprising an anti-CD40 agent, wherein the agent is a peptide comprising an amino acid sequence with at least 90% sequence identity to the sequences set forth in any one of SEQ ID NOs:8-17.
- the kit of any one of claims Error! Reference source not found.- 227 wherein the anti-CD40 agent is conjugated to apoptotic leukocytes.
- the apoptotic leukocytes is further conjugated to one or more peptides derived from a MHC class II molecule of the recipient. In one embodiment, the apoptotic leukocytes are MHC matched to the recipient. In one embodiment, the apoptotic leukocytes are further conjugated to one or more peptides derived from a MHC class I molecule of a donor of the cell, tissue or organ transplant. In one embodiment, the kit further comprises the cell, tissue or organ transplant. In one embodiment, the first container and the optional second container is a bottle, a vial, a syringe, or a test tube. In one embodiment, the first container and the optional second container is a multi-use container
- a transplant kit comprising a preparatory regimen and a tolerizing regimen: said preparatory regimen comprising an anti-CD40 agent, wherein the agent comprises one or more peptides comprising an amino acid sequence with at least 85% or at least 90% sequence identity to the sequences set forth in any one of SEQ ID NOs: 8-17; said tolerizing regimen comprising: an anti-CD40 agent, wherein the agent comprises one or more peptides comprising an amino acid sequence with at least 85% or at least 90% sequence identity to the sequences set forth in any one of the sequences set forth in SEQ ID NOs: 8-17; wherein said preparatory regimen is administered to a subject prior to transplantation, and said tolerizing regimen is administered post-transplantation to said subject.
- a transplant kit comprising a preparatory regimen and a tolerizing regimen: said preparatory regimen comprising an anti-CD40 agent, wherein the agent comprises one or more peptides comprising an amino acid sequence with at least 85% or at least 90% sequence identity to the sequences set forth in any one of SEQ ID NOs: 1-6; said tolerizing regimen comprising: an anti-CD40 agent, wherein the agent comprises one or more peptides comprising an amino acid sequence with at least 85% or at least 90% sequence identity to the sequences set forth in any one of the sequences set forth in SEQ ID NOs: 1-6; wherein said preparatory regimen is administered to a subject prior to transplantation, and said tolerizing regimen is administered post-transplantation to said subject
- the transplant kit further comprises the transplant kit further comprises a cell, tissue or organ transplant.
- the anti-CD40 agent is conjugated to apoptotic leukocytes.
- the transplant kit further comprises apoptotic leukocytes.
- the transplant kit further comprises apoptotic leukocytes fixed with a cross-linking agent.
- the transplant kit further comprises apoptotic leukocytes conjugated with one or more peptides derived from a MHC class II molecule of the recipient.
- the transplant kit further comprises apoptotic leukocytes conjugated one or more peptides derived from a MHC class I molecule of a donor of the cell, tissue or organ transplant.
- the transplant kit further comprises apoptotic leukocytes that have been differentiated from stem cells ex vivo, wherein the stem cells are derived from a donor of the cell, tissue, or organ transplant.
- the transplant kit further comprises apoptotic leukocytes that are MHC matched to the recipient.
- the transplant kit further comprises instructions for transplanting the cell, tissue or organ transplant.
- the transplant kit further comprises apoptotic leukocytes isolated from peripheral blood.
- said apoptotic leukocytes of said tolerizing regimen are enriched for B cells by positive or negative selection.
- said apoptotic leukocytes of said tolerizing regimen are expanded at least 200 fold relative to a starting population prior to contacting with said crosslinking agent.
- a method of inhibiting immune response to a therapeutic agent in a subject comprising administering to the subject the pharmaceutical composition of any one of aspects above.
- the therapeutic agent is a biologic.
- the biologic is a protein, vaccine, antibody, aptamer, nucleic acids, DNA, RNA, antisense oligonucleotide, virus and bacteria.
- a method of abrogating or reducing an undesired immune response to a therapy in a subject comprising administering to the subject the pharmaceutical composition of any one of aspects above prior to, in conjugation with or after said therapy.
- FIG. 1 shows binding of CD40 targeting peptides to PBMC. Binding of the CD40 targeting peptides was analyzed by incubating 504 of various fluorochrome tagged CD40 interacting peptides with PBMC from rhesus monkeys for 30 minutes at 4° C. and analyzed by flow cytometry. Peptides 1 and 3 showed high binding affinity.
- FIG. 2 demonstrates identified peptides bind specifically to CD40. Binding of the identified peptides to CD40 was analyzed by ELISA. Briefly, plates were coated with CD40 and then incubated with identified peptides and detected using Flag-tagged rhsCD40. Peptide #2, Peptide #5, and Peptide #7 showed similar binding affinity to Peptide #1.
- FIGS. 3A-3E shows newly identified peptides demonstrate robust inhibition of T and B cells proliferation.
- the ability of the CD40 targeting peptides to block CD40-CD40L signaling and inhibit T and B cell proliferation was assessed in a one-way CFSE-MLR.
- FIGS. 3A-3E show results of CFSE-MLR performed on PBMC obtained from a different Rhesus monkey.
- FACS analysis demonstrates that Peptide #2 and Peptide #3 inhibited T and B cell proliferation significantly better than the CD40 targeting peptide identified earlier.
- the levels of T cell and B cell proliferation was 35-46% in all the 5 animals and for sake clarity was capped at 25% in the figure for better visualization.
- FIG. 4 shows CD40 targeting peptides block B cell activation.
- the ability of the newly identified peptides to block B cell activation by CD40L was analyzed in a peptide blocking study. Briefly, B cells were pre-incubated with newly identified peptides for 30 minutes before stimulation with rhCD40L. Activation of the B cells was analyzed by measuring the expression levels of MHC class II molecules on the cell surface by flow cytometry. FACS analysis demonstrates that Peptide #2, Peptide #4 and Peptide #5 have a similar or higher ability to block the B cell activation when compared to peptide listed in Peptide #1.
- the term “about” or “approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which can depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, “about” can mean within 1 or more than 1 standard deviation, per the practice in the art. Alternatively, “about” can mean a range of up to 20%, up to 10%, up to 5%, or up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold, of a value. Where particular values are described in the application and claims, unless otherwise stated the term “about” meaning within an acceptable error range for the particular value should be assumed.
- the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps. It is contemplated that any embodiment discussed in this specification can be implemented with respect to any method or composition of the invention, and vice versa. Furthermore, compositions of the invention can be used to achieve methods of the invention.
- a “subject”, “patient”, “individual” and like terms are used interchangeably and refers to a vertebrate, preferably a mammal, more preferably a primate, still more preferably a human.
- Mammals include, without limitation, humans, primates, rodents, wild or domesticated animals, including feral animals, farm animals, sport animals, and pets.
- Primates include, for example, chimpanzees, cynomolgous monkeys, spider monkeys, and macaques, e.g., Rhesus.
- Rodents include, for example, mice, rats, woodchucks, ferrets, rabbits and hamsters.
- Domestic and game animals include, for example, cows, horses, pigs, deer, bison, buffalo, feline species, e.g., domestic cat, and canine species, e.g., dog, fox, wolf, avian species, e.g., chicken, emu, ostrich, and fish, e.g., trout, catfish and salmon.
- feline species e.g., domestic cat
- canine species e.g., dog, fox, wolf
- avian species e.g., chicken, emu, ostrich, and fish, e.g., trout, catfish and salmon.
- a subject can be male or female.
- the subject is a mammal.
- the mammal can be a human, non-human primate, mouse, rat, dog, cat, horse, or cow, but is not limited to these examples. Mammals other than humans can be advantageously used as subjects that represent animal models of conditions or disorders associated with CD40 activity.
- the compositions and methods described herein can be used to treat domesticated animals and/or pets.
- a subject can be one who has been previously diagnosed with or identified as suffering from or under medical supervision for a CD40 associated disorder, e.g., inflammatory disease, autoimmune disease, cancer.
- a CD40 associated disorder e.g., inflammatory disease, autoimmune disease, cancer.
- a subject can be one who is diagnosed and currently being treated for, or seeking treatment, monitoring, adjustment or modification of an existing therapeutic treatment, or is at a risk of developing a CD40 associated disorder.
- a subject can be a recipient of a transplant, for example, someone who has undergone, is undergoing, or will be undergoing transplantation.
- the term “in combination” refers to the use of more than one prophylactic and/or therapeutic agent simultaneously or sequentially and in a manner such that their respective effects are additive or synergistic.
- transplant and its grammatical equivalents as used herein encompasses any procedure that involves transplantation, implantation, or infusion of cells, tissues, or organs from a donor into a recipient.
- Non-limiting exemplary types of transplant include autotransplant, autograft, allotransplant, allograft, isotransplant, isograft, xenotransplant, xenograft, and split graft, and domino transplant.
- split graft encompasses any procedure that involves the transplantation of cells, organs, tissues, or even particular proteins from a donor is split into more than one recipient.
- autotransplantation encompasses any procedure that involves the transplantation of organs, tissues, cells or even particular proteins, or expression products from one part of the body to another in the same subject.
- the subject is a member of a Laurasiatheria super order.
- the subject is an ungulate for instance a pig, giraffe, camel, deer or bovine.
- the subject is a human or non-human primate.
- the autologous tissue also called autogenous, autogeneic, or autogenic tissue
- transplanted by such a procedure is called an autograft or autotransplant.
- Non limiting example can include autologous transplant of stem cell, induced pluripotent stem cell, cells derived from stem cells and induced pluripotent cells.
- allotransplantation encompasses any procedure that involves transplantation, implantation, or infusion of cells, tissues, or organs into a recipient, where the recipient and donor are the same species.
- the recipient and/or donor are a member of a Laurasiatheria super order.
- the recipient and/or donor are ungulates for instance pig, giraffe, camel, deer or bovine.
- the cells, tissues, or organs described herein are transplanted into humans or non-human primates.
- Allotransplantation includes but is not limited to vascularized allotransplant, partially vascularized allotransplant, unvascularized allotransplant, allodressings, allobandages, and allostructures.
- an allotransplant is an isograft or isotransplant in which organs or tissues are transplanted from a donor to a genetically identical recipient (such as an identical twin).
- xenotransplantation encompasses any procedure that involves transplantation, implantation, or infusion of cells, tissues, or organs into a recipient, where the recipient and donor are different species.
- the recipient and/or donor are a member of a Laurasiatheria super order.
- the recipient and/or donor is an ungulate, for instance a pig, giraffe, camel, deer or bovine.
- the donor is a pig, and the recipient is a human or non-human primate.
- the cells, tissues, or organs described herein are transplanted into humans or non-human primates.
- Xenotransplantation includes but is not limited to vascularized xenotransplant, partially vascularized xenotransplant, unvascularized xenotransplant, xenodressings, xenobandages, and nanostructures.
- transplant rejection and its grammatical equivalents as used herein can refer to a process or processes by which an immune response of an organ transplant recipient mounts a reaction against the transplanted material (e.g., cells, tissues, and/or organs) sufficient to impair or destroy the function of the transplanted material.
- transplanted material e.g., cells, tissues, and/or organs
- hyperacute rejection and its grammatical equivalents as used herein can refer to rejection of a transplanted material or tissue occurring or beginning within the first 24 hours after transplantation.
- hyperacute rejection can encompass but is not limited to “acute humoral rejection” and “antibody-mediated rejection”.
- improving transplantation can mean lessening hyperacute rejection, which can encompass a decrease, lessening, or diminishing of an undesirable effect or symptom.
- antibody as used herein includes IgG (including IgG1, IgG2, IgG3, and IgG4), IgA (including IgA1 and IgA2), IgD, IgE, or IgM, and IgY, and is meant to include whole antibodies, including single-chain whole antibodies, and antigen-binding (Fab) fragments thereof.
- Antigen-binding antibody fragments include, but are not limited to, Fab, Fab′ and F(ab′)2, Fd (consisting of VH and CH1), single-chain variable fragment (scFv), single-chain antibodies, disulfide-linked variable fragment (dsFv) and fragments comprising either a VL or VH domain.
- the antibodies can be from any animal origin.
- Antigen-binding antibody fragments can comprise the variable region(s) alone or in combination with the entire or partial of the following: hinge region, CH1, CH2, and CH3 domains. Also included are any combinations of variable region(s) and hinge region, CH1, CH2, and CH3 domains.
- Antibodies can be monoclonal, polyclonal, chimeric, humanized, and human monoclonal and polyclonal antibodies.
- islet refers to endocrine (e.g., hormone-producing) cells present in the pancreas of an organism, or cells that mimic one or more function of cells present in the pancreas of an organism.
- islet cells can comprise different types of cells, including, but not limited to, pancreatic ⁇ cells, pancreatic ⁇ cells, pancreatic ⁇ cells, pancreatic F cells, and/or pancreatic ⁇ cells.
- Islet cells can also refer to a group of cells, cell clusters, or the like, including cells cultured in-vitro.
- islet cells are extracted from an islet donor and implanted or transplanted at a predetermined site of an islet recipient for differentiation, expansion, and vascularization to form a therapeutic dose of ⁇ -cell mass by methods, systems, and/or reagents described herein.
- the predetermined site is a renal subcapsular space of the islet recipient.
- the islets of Langerhans are the regions of the pancreas that contain the endocrine (e.g., hormone-producing) cells (e.g., beta cells).
- NICCs or NPIs are extracted from an islet donor and implanted or transplanted at a predetermined site of an islet recipient for differentiation, expansion, and vascularization to form a therapeutic dose of ⁇ -cell mass by methods, systems, and/or reagents described herein.
- the predetermined site is a renal subcapsular space of the islet recipient.
- NICCs or NPIs extracted from the donor are implanted or transplanted to the recipient under the cover of transient immunosuppression.
- islet cells can be stem cell-derived islet cells, induced pluripotent stem cell-derived islet cells, transdifferentiated, or surrogate islet cells.
- a “donor” is meant to include any mammalian organism, human or non-human, that can serve as a source of donor tissue or cells for transplantation and/or for inducing donor cell tolerance.
- Non-human mammals include, but are not limited to, ungulates, such as an even-toed ungulate (e.g., pigs, peccaries, hippopotamuses, camels, llamas, chevrotains (mouse deer), deer, giraffes, pronghorn, antelopes, goat-antelopes (which include sheep, goats and others), or cattle) or an odd-toed ungulate (e.g., horse, tapirs, and rhinoceroses), a non-human primate (e.g., a monkey, or a chimpanzee), a Canidae (e.g., a dog) or a cat.
- ungulates such as an even-toed ungulate (e.g., pigs, peccaries, hippopotamuses, camels, llamas, chevrotains (mouse deer), deer, giraffes, prong
- a non-human animal can be a member of the Laurasiatheria superorder.
- the donor can be a living donor or a cadaveric donor. In some cases, the donor is a living donor. In some cases, the donor is a cadaveric donor.
- the cadaveric donor may be, for example, a brain dead, heart beating donor (BDD).
- BDD brain dead, heart beating donor
- NHBD non-heart beating donor
- the donor can be from any animal, for example, a human or non-human animal.
- the donor can be in any stage of development, including, but not limited to fetal, perinatal, neonatal, pre-weaning, post-weaning, juvenile, young adult, or adult.
- a donor of cells used in the preparation of a tolerizing vaccine or preparatory regimen can be fully or partially MHC (major histocompatibility complex) matched to a transplant donor (e.g., a donor of cells, tissues, or organs used for transplantation).
- the partially matched donor is haploidentical to the transplant donor.
- the partially matched donor shares one or more MHC alleles with a transplant donor.
- the partially matched donor can share one or more of a MHC class I A allele, a MHC class I B allele, a MHC class II DR allele, a MHC class II DQ allele, or a combination thereof with a transplant donor.
- the partially matched donor can share one DR allele with the transplant donors.
- a “recipient” can be a human or non-human animal that can receive, is receiving, or has received a transplant graft, a tolerizing vaccine, a preparatory regimen for transplantation, and/or other compositions provided in the present disclosure.
- a recipient can also be in need of a transplant graft, a tolerizing vaccine, a preparatory regimen for transplantation, and/or other compositions provided herein.
- the recipient can be a human or non-human animal that can receive, is receiving, or has received a transplant graft.
- the recipient can be a human or non-human animal that can receive, is receiving, or has received the presently described tolerizing vaccine or preparatory regimen for transplantation.
- non-human animal and its grammatical equivalents as used herein includes all animal species other than humans, including non-human mammals, which can be a native animal or a genetically modified non-human animal.
- a non-human mammal includes, an ungulate, such as an even-toed ungulate (e.g., pigs, peccaries, hippopotamuses, camels, llamas, chevrotains (mouse deer), deer, giraffes, pronghorn, antelopes, goat-antelopes (which include sheep, goats and others), or cattle) or an odd-toed ungulate (e.g., horse, tapirs, and rhinoceroses), a non-human primate (e.g., a monkey, or a chimpanzee), a Canidae (e.g., a dog) or a cat.
- an even-toed ungulate e.g., pigs, pecca
- a non-human animal can be a member of the Laurasiatheria superorder.
- the Laurasiatheria superorder can include a group of mammals as described in Waddell et al., Towards Resolving the Interordinal Relationships of Placental Mammals. Systematic Biology 48 (1): 1-5 (1999).
- Members of the Laurasiatheria superorder can include Eulipotyphla (hedgehogs, shrews, and moles), Perissodactyla (rhinoceroses, horses, and tapirs), Carnivora (carnivores), Cetartiodactyla (artiodactyls and cetaceans), Chiroptera (bats), and Pholidota (pangolins).
- a member of Laurasiatheria superorder can be an ungulate described herein, e.g., an odd-toed ungulate or even-toed ungulate.
- An ungulate can be a pig.
- a member can be a member of Carnivora, such as a cat, or a dog.
- a member of the Laurasiatheria superorder can be a pig.
- a pig can be a wild pig, a domestic pig, mini pigs, a Sus scrofa pig, a Sus scrofa domesticus pig, or inbred pigs.
- fetal animal and its grammatical equivalents can refer to any unborn offspring of an animal. In some cases, pancreatic cell or tissue are isolated from 6 weeks old embryonic pig for transplantation.
- perinatal animal and its grammatical equivalents can refer to an animal immediately before or after birth. For example, a perinatal period can start from 20th to 28th week of gestation and ends 1 to 4 weeks after birth.
- non-human animal and its grammatical equivalents can refer to any animal before being withdrawn from the mother's milk.
- juvenile animal and its grammatical equivalents can refer to any animal before becoming a young adult animal. For example, a juvenile stage of pigs can refer to any pigs of 2 years of age or younger.
- genetically modified refers to having one or more alterations of a nucleic acid, e.g., the nucleic acid within an organism's genome.
- genetic modification can refer to alterations, additions, and/or deletion of genes.
- a genetically modified cell can also refer to a cell with an added, deleted and/or altered gene.
- a genetically modified cell can be from a genetically modified non-human animal.
- a genetically engineered cell from a genetically engineered non-human animal can be a cell isolated from such genetically engineered non-human animal.
- a genetically modified cell from a genetically modified non-human animal can be a cell originated from such genetically modified non-human animal.
- a genetically engineered cell or a genetically engineered animal can comprise a transgene, or other foreign DNA, added or incorporated, or an endogenous gene modified, including, targeted, recombined, interrupted, deleted, disrupted, replaced, suppressed, enhanced, or otherwise altered, to mediate a genotypic or phenotypic effect in at least one cell of the animal, and typically into at least one germ line cell of the animal.
- transgene and its grammatical equivalents as used herein can refer to a gene or genetic material that can be transferred into an organism.
- a transgene can be a stretch or segment of DNA containing a gene that is introduced into an organism.
- the gene or genetic material can be from a different species.
- the gene or genetic material can be synthetic.
- a transgene can retain its ability to produce RNA or polypeptides (e.g., proteins) in a transgenic organism.
- a transgene can comprise a polynucleotide encoding a protein or a fragment (e.g., a functional fragment) thereof.
- the polynucleotide of a transgene can be an exogenous polynucleotide.
- a fragment (e.g., a functional fragment) of a protein can comprise at least or at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% of the amino acid sequence of the protein.
- a fragment of a protein can be a functional fragment of the protein.
- a functional fragment of a protein can retain part or all of the function of the protein.
- An exogenous polypeptide can encode an exogenous protein or a functional fragment thereof.
- exogenous nucleic acid sequence can refer to a gene or genetic material that was transferred into a cell or animal that originated outside of the cell or animal.
- An exogenous nucleic acid sequence can by synthetically produced.
- An exogenous nucleic acid sequence can be from a different species, or a different member of the same species.
- An exogenous nucleic acid sequence can be another copy of an endogenous nucleic acid sequence.
- gene knock-out or “knock-out” can refer to any genetic modification that reduces the expression of the gene being “knocked out.” Reduced expression can include no expression.
- the genetic modification can include a genomic disruption.
- disrupting can refer to a process of altering a gene, e.g., by deletion, insertion, mutation, rearrangement, or any combination thereof.
- a gene can be disrupted by knockout.
- Disrupting a gene can be partially reducing or completely suppressing expression (e.g., mRNA and/or protein expression) of the gene.
- Disrupting can also include inhibitory technology, such as shRNA, siRNA, microRNA, dominant negative, or any other means to inhibit functionality or expression of a gene or protein.
- gene editing and its grammatical equivalents as used herein can refer to genetic engineering in which one or more nucleotides are inserted, replaced, or removed from a genome.
- gene editing can be performed using a nuclease (e.g., a natural-existing nuclease or an artificially engineered nuclease).
- condition and its grammatical equivalents as used herein can refer to a disease, event, or change in health status.
- diabetes and its grammatical equivalents as used herein can refer to is a disease characterized by high blood sugar levels over a prolonged period.
- the term “diabetes” and its grammatical equivalents as used herein can refer to all or any type of diabetes, including, but not limited to, type 1, type 2, type 3c (pancreatogenic diabetes including cystic fibrosis-related, and surgical, and hemochromatosis-related), gestational diabetes, and monogenic diabetes (HNF1A-MODY, GCK-MODY 2, etc.), and other forms of mitochondrial diabetes.
- diabetes can be a form of hereditary diabetes.
- the term “susceptible to” refers to an individual who is “susceptible to” a disease, disorder, and/or condition has not been diagnosed with the disease, disorder, and/or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and/or condition may not exhibit symptoms of the disease, disorder, and/or condition.
- an individual who is susceptible to a disease, disorder, condition, or event may be characterized by one or more of the following: (1) a genetic mutation associated with development of the disease, disorder, and/or condition; (2) a genetic polymorphism associated with development of the disease, disorder, and/or condition; (3) increased and/or decreased expression and/or activity of a protein associated with the disease, disorder, and/or condition; (4) habits and/or lifestyles associated with development of the disease, disorder, condition, and/or event (5) having undergone, planning to undergo, or requiring a transplant.
- an individual who is susceptible to a disease, disorder, and/or condition will develop the disease, disorder, and/or condition.
- an individual who is susceptible to a disease, disorder, and/or condition will not develop the disease, disorder, and/or condition.
- phenotype and its grammatical equivalents as used herein can refer to a composite of an organism's observable characteristics or traits, such as its morphology, development, biochemical or physiological properties, phenology, behavior, and products of behavior. Depending on the context, the term “phenotype” can sometimes refer to a composite of a population's observable characteristics or traits.
- “Inflammatory disorder” means an immune-mediated inflammatory condition that affects humans and is generally characterized by dysregulated expression of one or more cytokines.
- inflammatory disorders include skin inflammatory disorders, inflammatory disorders of the joints, inflammatory disorders of the cardiovascular system, certain autoimmune diseases, lung and airway inflammatory disorders, intestinal inflammatory disorders.
- skin inflammatory disorders include dermatitis, for example atopic dermatitis and contact dermatitis, acne vulgaris, and psoriasis.
- Examples of inflammatory disorders of the joints include rheumatoid arthritis.
- Examples of inflammatory disorders of the cardiovascular system are cardiovascular disease and atherosclerosis.
- autoimmune diseases include Type 1 diabetes, Graves disease, Guillain-Barre disease, Lupus, Psoriatic arthritis, and Ulcerative colitis.
- lung and airway inflammatory disorders include asthma, cystic fibrosis, COPD, emphysema, and acute respiratory distress syndrome.
- intestinal inflammatory disorders include colitis and inflammatory bowel disease.
- Other inflammatory disorders include cancer, hay fever, periodontitis, allergies, hypersensitivity, ischemia, depression, systemic diseases, post infection inflammation and bronchitis.
- the peptides and compositions of the invention may also be employed in the non-therapeutic treatment of inflammation.
- non-therapeutic treatment of inflammation include use to relieve normal, non-pathological, inflammation, for example inflammation in the muscles and joints following exercise
- protein As used herein, the terms “protein”, “peptide” and “polypeptide” are used interchangeably to designate a series of amino acid residues connected to each other by peptide bonds between the alpha-amino and carboxy groups of adjacent residues.
- the terms “protein”, “peptide” and “polypeptide” refer to a polymer of amino acids, including modified amino acids (e.g., phosphorylated, glycated, glycosylated, etc.) and amino acid analogs, regardless of its size or function.
- Protein and “polypeptide” are often used in reference to relatively large polypeptides, whereas the term “peptide” is often used in reference to small polypeptides, but usage of these terms in the art overlaps.
- the terms “protein”, “peptide” and “polypeptide” are used interchangeably herein when referring to a gene product and fragments thereof.
- the terms “increased”/“increase”, “increasing” or “enhance” are all used herein to generally mean an increase by a statically significant amount; for the avoidance of doubt, the terms “increased”, “increase”, or “enhance”, mean an increase of at least 10% as compared to a reference level, for example an increase of at least about 10%, at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% increase or any increase between 10-100% as compared to a reference level, or at least about a 2-fold, or at least about a 3-fold, or at least about a 4-fold, or at least about a 5-fold or at least about a 10-fold increase, or any increase between 2-fold and 10-fold or greater as compared to a reference level.
- the increase can be, for example, at least 10%, at least 20%, at least 30%, at least
- “decrease”, “reduce”, “reduction”, “lower” or “lowering,” or “inhibit” are all used herein generally to mean a decrease by a statistically significant amount.
- “decrease”, “reduce”, “reduction”, or “inhibit” means a decrease by at least 10% as compared to a reference level, for example a decrease by at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% decrease (e.g., absent level or non-detectable level as compared to a reference level), or any decrease between 10-100% as compared to a reference level.
- a marker or symptom by these terms is meant a statistically significant decrease in such level.
- the decrease can be, for example, at least 10%, at least 20%, at least 30%, at least 40% or more, and is preferably down to a level accepted as within the range of normal for an individual without a given disease.
- substantially pure means sufficiently homogeneous to appear free of readily detectable impurities as determined by standard methods of analysis, such as thin layer chromatography [TLC], gel electrophoresis and high performance liquid chromatography [HPLC], used by those of skill in the art to assess such purity, or sufficiently pure such that further purification would not detectably alter the physical and chemical properties, such as enzymatic and biological activities, of the substance.
- TLC thin layer chromatography
- HPLC high performance liquid chromatography
- Methods for purification of the compounds to produce substantially chemically pure compounds are known to those of skill in the art.
- a substantially chemically pure compound may, however, be a mixture of stereoisomers. In such instances, further purification might increase the specific activity of the compound.
- the compositions of the present disclosure are substantially pure.
- detecttable agent is meant a compound that is linked to a diagnostic agent to facilitate detection. Such a “detectable agent” may be covalently or non-covalently linked to a diagnostic agent, hi addition, the linkage may be direct or indirect Examples of “detectable agents” include, protein purification tags, cytotoxins, enzymes, paramagnetic labels, enzyme substrates, co-factors, enzymatic inhibitors, dyes, radionuclides, chemiluminescent labels, fluorescent markers, growth inhibitors, cytokines, antibodies, and biotin.
- biological activity refers to the in vitro, ex vivo, or in vivo activities of a compound or physiological responses that result upon in vivo administration of a compound, composition or other mixture.
- Biological activity thus, encompasses therapeutic effects and pharmaceutical activity of such compounds, compositions and mixtures.
- peptides capable of binding CD40 and thereby inhibit activity of CD40, i.e., interaction of CD40 with CD154.
- CD40 binding peptide are used interchangeably with “anti-CD40 peptide”.
- the present disclosure provides pharmaceutical compositions comprising a peptide which comprise an amino acid sequence selected from Table 1 or a portion thereof or a functional fragment thereof.
- a peptide can be full length peptide selected from Table 1 or can be a portion thereof or a functional fragment thereof.
- a peptide of the compositions and methods disclosed herein are less than 20 amino acids in length.
- the peptide is 4, 6, 8, 10, 12, 14, or 15 amino acids in length.
- the peptide comprises an amino acid sequence selected from group consisting of SEQ ID NOs: 1-6 and 8-17. The sequences of such peptides are shown in Table 1.
- the peptide of the compositions and methods disclosed herein have an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% identical to that of SEQ ID NO:1. In some embodiments, the peptide of the compositions and methods disclosed herein have an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% identical to that of SEQ ID NO: 2.
- the peptide of the compositions and methods disclosed herein have an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% identical to that of SEQ ID NO:3. In some embodiments, the peptide of the compositions and methods disclosed herein have an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% identical to that of SEQ ID NO:4.
- the peptide of the compositions and methods disclosed herein have an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% identical to that of SEQ ID NO:5. In some embodiments, the peptide of the compositions and methods disclosed herein have an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% identical to that of SEQ ID NO: 6.
- the peptide of the compositions and methods disclosed herein have an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% identical to that of SEQ ID NO:8.
- the peptide of the compositions and methods disclosed herein have an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% identical to that of SEQ ID NO:9.
- the peptide of the compositions and methods disclosed herein have an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% identical to that of SEQ ID NO:10.
- the peptide of the compositions and methods disclosed herein have an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% identical to that of SEQ ID NO:11.
- the peptide of the compositions and methods disclosed herein have an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% identical to that of SEQ ID NO:12.
- the peptide of the compositions and methods disclosed herein have an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% identical to that of SEQ ID NO:13.
- the peptide of the compositions and methods disclosed herein have an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% identical to that of SEQ ID NO:14.
- the peptide of the compositions and methods disclosed herein have an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% identical to that of SEQ ID NO:15.
- the peptide of the compositions and methods disclosed herein have an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% identical to that of SEQ ID NO:16.
- the peptide of the compositions and methods disclosed herein have an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% identical to that of SEQ ID NO:17.
- the peptide or functional fragment thereof of the present disclosure retain, the ability at a minimum, to bind a CD40 protein.
- the peptide comprises a portion (e.g., a functional fragment of the peptide) of the wild type sequence selected from SEQ ID NOs 1-6 and 8-17.
- Percent (%) amino acid sequence identity for a given peptide sequence relative to a reference sequence is defined as the percentage of identical amino acid residues identified after aligning the two sequences and introducing gaps if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity.
- Percent (%) amino acid sequence homology for a given polypeptide sequence relative to a reference sequence is defined as the percentage of identical or strongly similar amino acid residues identified after aligning the two sequences and introducing gaps if necessary, to achieve the maximum percent homology.
- Non identities of amino acid sequences include conservative substitutions, deletions or additions that do not affect the biological activity of the peptide.
- Strongly similar amino acids can include, for example, conservative substitutions known in the art. Percent identity and/or homology can be calculated using alignment methods known in the art, for instance alignment of the sequences can be conducted using publicly available software such as BLAST, Align, ClustalW2. Those skilled in the art can determine the appropriate parameters for alignment, but the default parameters for BLAST are specifically contemplated.
- wild type peptide refers to the native, un-modified peptide comprising amino acid sequence selected from SEQ ID NOs 1-6 and 8-17.
- a “fragment” or “portion” is one that substantially retains at least one biological activity normally associated with that peptide.
- the “fragment” or “portion” substantially retains all of the activities possessed by the wild type peptide.
- substantially retains biological activity, it is meant that the fragment or portion retains at least about 50%, 60%, 75%, 85%, 90%, 95%, 97%, 98%, 99%, or more, of the biological activity of the wild type peptide (and can even have a higher level of activity than the wild type peptide).
- a fragment or portion of the protein or polypeptide described herein is at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 or more contiguous amino acids and/or less than about 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, or 4 contiguous amino acids, including any combination of the foregoing as long as the lower limit is less than the upper limit and retains at least one biological activity associated with the wild type peptide.
- non-functional peptide is one that exhibits essentially no detectable biological activity normally associated with the peptide (e.g., at most, only an insignificant amount, e.g., less than about 10% or even 5%).
- a “functional” peptide or fragment is one that retains at least one biological, activity normally associated with wild type peptide.
- a “functional” peptide or fragment thereof retains all of the activities possessed by the native, unmodified or full-length wild type peptide.
- a “non-functional” peptide is one that exhibits essentially no detectable biological activity normally associated with the wild type peptide (e.g., at most, only an insignificant amount, e.g., less than about 10% or even 5%). The functional activity can be tested by one of ordinary skill in the art by the assays described below and in the Examples herein.
- compositions and methods of the present disclosure contemplate a functional derivative, functional fragment, variant, analogue or chemical derivative of a peptide disclosed herein (e.g., a peptide selected from Table 1).
- a peptide useful in the methods and compositions described herein consists of, consists essentially of, or comprises an amino acid sequence, or is a fragment thereof derived from sequence selected from SEQ ID NOs: 1-15, provided that the peptide or functional fragment retains at least one biological activity of corresponding full length wild type peptide, the biological activity being selected from at a minimum, to bind CD40, inhibiting interaction of CD40 and CD154 or modulate immune response mediated by CD40.
- the peptides or functional fragment thereof described herein can comprise conservative amino acid substitutions at one or more amino acid residues, e.g., at essential or non-essential amino acid residues but will retain a therapeutically or physiologically relevant activity of a peptide as that term is described herein.
- a “conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain.
- Families of amino acid residues having similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).
- basic side chains e.g., lysine, arginine, histidine
- acidic side chains e.g., aspartic acid
- peptide can be a variant of wild type peptide.
- variant refers to a peptide or nucleic acid that is “substantially similar” to a wild-type peptide.
- a molecule is said to be “substantially similar” to another molecule if both molecules have substantially similar structures (i.e., they are at least 50% similar in amino acid sequence as determined by BLASTp alignment set at default parameters) and are substantially similar in at least one therapeutically or physiologically relevant biological activity.
- a variant differs from the naturally occurring polypeptide or nucleic acid by one or more amino acid or nucleic acid deletions, additions, substitutions or side-chain modifications, yet retains one or more therapeutically relevant, specific functions or desired biological activities of the wild type peptide (e.g., binding CD40).
- Amino acid substitutions include alterations in which an amino acid is replaced with a different naturally-occurring or a non-conventional amino acid residue. Some substitutions can be classified as “conservative,” in which case an amino acid residue contained in a polypeptide is replaced with another naturally occurring amino acid of similar character either in relation to polarity, side chain functionality or size.
- substitutions encompassed by variants as described herein can also be “non-conservative,” in which an amino acid residue which is present in a peptide is substituted with an amino acid having different properties (e.g., substituting a charged or hydrophobic amino acid with an uncharged or hydrophilic amino acid), or alternatively, in which a naturally-occurring amino acid is substituted with a non-conventional amino acid.
- “Conservative amino acid substitutions” result from replacing one amino acid with another having similar structural and/or chemical properties, such as the replacement of a leucine with an isoleucine or valine, an aspartate with a glutamate, or a threonine with a serine.
- a “conservative substitution” of a particular amino acid sequence refers to substitution of those amino acids that are not critical for polypeptide activity or substitution of amino acids with other amino acids having similar properties (e.g., acidic, basic, positively or negatively charged, polar or non-polar, etc.) such that the substitution of even critical amino acids does not reduce the activity of the peptide, (i.e. the ability of the peptide to penetrate the blood brain barrier (BBB)).
- BBB blood brain barrier
- Conservative substitution tables providing functionally similar amino acids are well known in the art. For example, the following six groups each contain amino acids that are conservative substitutions for one another: 1) Alanine (A), Serine (S), Threonine (T); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W). (See also Creighton, Proteins, W. H.
- insertions or “deletions” are typically in the range of about 1 to 5 amino acids. The variation allowed can be experimentally determined by producing the peptide synthetically while systematically making insertions, deletions, or substitutions of nucleotides in the sequence using recombinant DNA techniques.
- substitution when referring to a peptide, refers to a change in an amino acid for a different entity, for example another amino acid or amino-acid moiety. Substitutions can be conservative or non-conservative substitutions.
- substitutions suitable for amino acids on the exterior of a protein or peptide for example, but not limited to, the following substitutions can be used: substitution of Y with F, T with S or K, P with A, E with D or Q, N with D or G, R with K, G with N or A, T with S or K, D with N or E, I with L or V, F with Y, S with T or A, R with K, G with N or A, K with R, A with S, K or P.
- non-conservative amino acid substitutions are also encompassed within the term of variants.
- derivative refers to peptides which have been chemically modified, for example but not limited to by techniques such as ubiquitination, labeling, pegylation (derivatization with polyethylene glycol), lipidation, glycosylation, or addition of other molecules.
- a molecule also a “derivative” of another molecule when it contains additional chemical moieties not normally a part of the molecule. Such moieties can improve the molecule's solubility, absorption, biological half-life, etc.
- the moieties can alternatively decrease the toxicity of the molecule, eliminate or attenuate any undesirable side effect of the molecule, etc. Moieties capable of mediating such effects are disclosed in Remington's Pharmaceutical Sciences, 18th edition, A. R. Gennaro, Ed., MackPubl., Easton, Pa. (1990), incorporated herein, by reference, in its entirety.
- a “derivative” is defined as a molecule having the amino acid sequence of a wild-type peptide (e.g., peptide selected from Table 1) or analog thereof, but additionally having a chemical modification of one or more of its amino acid side groups, alpha-carbon atoms, terminal amino group, or terminal carboxylic acid group for example by ubiquitination, labeling, pegylation (derivatization with polyethylene glycol) or addition of other molecules.
- a chemical modification includes, but is not limited to, adding chemical moieties, creating new bonds, and removing chemical moieties.
- one or more side groups, or terminal groups may be protected by protective groups known to the ordinarily-skilled synthetic chemist.
- the term “functional” when used in conjunction with “derivative” or “variant” refers to a peptide which possesses a biological activity that is substantially similar to a biological activity of the entity or molecule of which it is a derivative or variant.
- substantially similar in this context is meant that at least 50% of the relevant or desired biological activity of a corresponding wild-type peptide is retained.
- the derivatives retains at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or more, including 100% or even more (i.e., the derivative or variant has improved activity relative to wild-type) of the wild type peptide.
- a peptide useful in the composition and methods of the present disclosure is an analog of a wild type peptide (e.g., selected from Table 1).
- An “analog” of a molecule such as a peptide refers to a molecule similar in function to either the entire molecule or to a fragment thereof.
- the term “analog” is also intended to include allelic species and induced variants. Analogs typically differ from wild type peptides at one or a few positions, often by virtue of conservative substitutions. Analogs typically exhibit at least 80 or 90% sequence identity with wild type peptides. Some analogs also include unnatural amino acids or modifications of N or C terminal amino acids.
- unnatural amino acids are, for example but not limited to; disubstituted amino acids, N-alkyl amino acids, lactic acid, 4-hydroxyproline, ⁇ -carboxyglutamate, ⁇ -N,N,N-trimethyllysine, ⁇ -N-acetyllysine, O-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, ⁇ -N-methylarginine.
- unnatural amino acids include, but are not limited to the D-amino acids, 2,4-diaminobutyric acid, a-amino isobutyric acid, 2-aminoisobutyric acid, 4-aminobutyric acid, Abu, 2-amino butyric acid, g-Abu, e-Ahx, 6-amino hexanoic acid, Aib, 2-amino isobutyric acid, 3-amino propionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, naphthalene, L-1-naphthalene, citrulline, homocitrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, b-alanine, fluoro-amino acids, designer amino acids such as b-methyl amino acids, Ca-methyl amino acids, calcium
- compositions and methods of the instant disclosure also contemplate a peptidomimetic.
- a “peptidomimetic” is a compound containing non-peptidic structural elements that is capable of mimicking the biological action of a parent peptide.
- a peptidomimetic may or may not comprise peptide bonds.
- a peptide useful for compositions and methods of the present disclosure should be of a size sufficient to interact with CD40 protein in such a manner as to inhibit interaction of CD40 with CD154.
- a peptide of the present disclosure is less than 20 amino acids in length.
- a peptide can be, for example, 4, 6, 13 or 15 amino acids in length.
- the peptide consists of an amino acid selected from the group consisting of SEQ ID NO:1-6 and 8-17. The sequences of such peptides are shown below in Table 1.
- the peptide comprises at least a portion that is capable of binding CD40 and inhibiting interaction of CD40 with CD154.
- the peptide comprises at least a portion of peptide comprising an amino acid sequence selected from SEQ ID NOs: 1-6 and 8-17.
- the peptide of the present disclosure is as short as possible, yet at a minimum, retains the biological activity or functional activity of binding to CD40.
- the peptide is a shortest region of contiguous sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, or SEQ ID NO: 17 which retain at a minimum the biological ability or functional activity of binding CD40.
- Useful peptides can also comprise additional regions of sequence from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15, SEQ ID NO: 16, or SEQ ID NO: 17 which surround the shortest possible contiguous sequence capable of binding CD40.
- the peptide or portion thereof bind to CD40 at a site which overlaps the binding site of CD154.
- the peptide or portion thereof bind CD40 at a site which is the binding site of CD154.
- the peptide or portion thereof bind the CD40 at a site which is not a site of CD154 binding.
- the peptides of the present disclosure can comprise additional sequences and modifications that do not bind a CD40 protein, so long as the additional sequence do not lower the biological activity of the peptide than that of unmodified peptide, for example, biological activity of binding CD40.
- additional sequences include sequences coding for B-galactosidase, luciferase, glutathione-s-transferase, thioredoxin, HIS-tags, biotin tags, and fluorescent proteins.
- the composition comprises at least one peptide or portion thereof or functional fragment thereof is selected from Table 1. In some embodiments, the composition can comprise for example more than one peptides or portion thereof or functional fragment thereof disclosed herein (e.g., a peptide selected from Table 1).
- the compositions disclosed herein can comprise at least 2 peptides, at least 3 peptides, at least 4 peptides, at least 5 peptides, at least 6 peptides, at least 7 peptides, at least 8 peptides, at least 9 peptides, at least 10 peptides, at least 11 peptides, at least 12 peptides, at least 13 peptides, at least 14 peptides, at least 15 peptides, or portion thereof (e.g., peptides selected from Table 1).
- the composition can comprise all the peptides or portion thereof disclosed herein (e.g., peptides in Table 1).
- biological activity refers to one having structural, regulatory, or biochemical functions of an unmodified molecule or any function related to or associated with a metabolic or physiological process.
- the biological activity can include an improved desired activity, or a decreased undesirable activity.
- an entity demonstrates biological activity when it participates in a molecular interaction with another molecule (e.g., binding CD40), when it has therapeutic value in alleviating a disease condition, when it has prophylactic value or therapeutic value in modulating an immune response, when it has diagnostic and/or prognostic value in determining the presence of a molecule (e.g., cell expressing CD40).
- the biological activity and/or biological effect of a peptide disclosed herein include at least in part binding to a CD40 protein.
- the peptides or functional fragment thereof disclosed herein are CD40 binding peptides or functional fragment thereof.
- the peptide associates with CD40 to form a complex.
- An example of complex formation is association of an antigen with an antibody.
- the binding of the peptides disclosed herein to CD40 can be covalent binding interactions between proteins include, for example, disulfide bonds, ester bonds, amide bonds and the like or non-covalent bonding interactions between proteins include, for example, hydrophobic interactions, van der Waals interactions, ionic interactions, hydrogen bonding interactions and the like.
- the binding of peptides disclosed herein with CD40 can be reversible (e.g., non-covalent binding interactions) or non-reversible (e.g., covalent bonds).
- a reversible interaction can be strong or weak, the strength of the interaction being determined by the forces (e.g., ionic charges, hydrogen binding, van der Walls interactions, etc.) exerted by each protein on the other protein in the complex.
- forces e.g., ionic charges, hydrogen binding, van der Walls interactions, etc.
- Kd dissociation constant
- dissociation constant refers to the value indicating the dissociation rate of a ligand (e.g., peptides disclosed herein) from its target protein (e.g., CD40) or separation of the ligand and protein complex (e.g., complex of peptide disclosed herein with a CD40 protein) over time into free ligand and free protein.
- a ligand e.g., peptides disclosed herein
- target protein e.g., CD40
- separation of the ligand and protein complex e.g., complex of peptide disclosed herein with a CD40 protein
- peptides of the present disclosure are those that bind to a CD40 protein with a Kd of no more than about 1 ⁇ 10 ⁇ 6 M, about 1 ⁇ 10 ⁇ 7 M, or about 1 ⁇ 10 ⁇ 8 M.
- a peptide has a Kd of less than about 1 ⁇ 10 ⁇ 9 M.
- a peptide of the present disclosure binds to a CD40 protein with a Kd of less than 100 nM, less than 50 nM, less than 25 nM, less than 10 nM, less than 5 nM, less than 3 nM, less than 2 nM, or less than 1 nM.
- Kd can be determined by measurement of the kinetics of complex formation and dissociation, e.g. by the SPR method (Biacore).
- a complex comprising a peptide disclosed herein or a portion thereof non-covalently bound to a CD40 protein.
- peptide of the present disclosure selectively binds with a CD40 protein in solution, as determined using an assay such as an immunosorbent assay, or on the surface of a cell.
- an assay such as an immunosorbent assay
- the terms selectively, selective, specific, and the like indicate the peptide has a greater affinity for a CD40 protein than it does for proteins unrelated to the CD40 protein. More specifically, the terms selectively, selective, specific, and the like indicate that the affinity of the peptide for CD40 is statistically significantly higher than its affinity for a negative control (e.g., an unrelated protein such as albumin) as measured using a standard assay (e.g., ELISA).
- a negative control e.g., an unrelated protein such as albumin
- Suitable techniques for assaying the ability of a peptide to selectively interact with a CD40 protein are known to those skilled in the art. Such assays can be in vitro or in vivo assays. Methods of determining selective binding of peptides with CD40 are described in Examples herein.
- non-limiting assays to determine selective binding include, but are not limited to, an enzyme-linked immunoassay, a competitive enzyme-linked immunoassay, a radioimmunoassay, a fluorescence immunoassay, a chemiluminescent assay, a lateral flow assay, a flow-through assay, an agglutination assay, a particulate-based assay (e.g., using particulates such as, but not limited to, magnetic particles or plastic polymers, such as latex or polystyrene beads), an immunoprecipitation assay, an immunoblot assay (e.g., a western blot), a phosphorescence assay, a flow-through assay, a chromatography assay, a polyacrylamide gel electrophoresis (PAGE)-based assay, a surface plasmon resonance assay, a spectrophotometric assay, a particulate-based assay, an electronic sensory assay and
- an assay can be performed using isolated CD40 protein, of CD40 expressed on a surface of a cell in culture, or it can be performed in a whole animal. Assays can be designed to give qualitative, quantitative or semi-quantitative results, depending on how they are used and the type of result that is desired.
- CD40 antigen is a peptide of 277 amino acids having a molecular weight of 30,600, and a 19 amino acid secretory signal peptide comprising predominantly hydrophobic amino acids.
- CD40 is a member of the tumor necrosis factor (TNF)/nerve growth factor (NGF) receptor family, which is defined by the presence of cysteine-rich motifs in the extracellular region.
- CD40 or “CD40 protein” refers to a cell surface glycoprotein that is a member of the tumor necrosis factor receptor family. Other names in the art for CD40 include: TNFRSF5, p50, CDW40, CD40 receptor and Bp50.
- CD40 protein generally refers to an CD40 polypeptide that is similar or identical in sequence to a wild-type CD40.
- CD40 protein refers to a CD40 polypeptide having an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100%, identical to that of a wild-type CD40 and that retains the ability, at a minimum, to bind CD154. Accordingly in some embodiments, “CD40” can be full length CD40. In some embodiments, “CD40” can be a functional fragment of a full length CD40, a species homologue and/or functional fragments thereof, an ortholog of CD40 and/or functional fragments thereof.
- the CD40 protein can be a mammalian CD40 protein.
- the CD40 polypeptide can also be a functional isoform of the full length CD40 or functional fragment thereof.
- CD40 is a wild-type CD40 of human origin, having the following amino acid sequence (SEQ ID NO:18), or a functional fragment thereof.
- a “functional fragment” or a “portion thereof” as it relates to CD40 refers to fragment of the full length CD40 (e.g. corresponding to SEQ ID NO: 18 of at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, at least 160, at least 170, at least 180, at least 190, at least 200 or more consecutive amino acids of full length wild-type CD40, that has at least about 70%, 80%, 90%, 100% or more than 100% of the function of wild-type CD40 of binding CD154
- the functional activity can be tested by one of ordinary skill in the art by the assays described in the examples.
- polypeptide and coding nucleic acid sequences of CD40 protein of human origin and those of a number of animals are publicly available, e.g., from the NCBI website and are contemplated for use in the methods and compositions herein. Examples include, but are not limited to, Mouse (GenBank Accession No. AAB08705.1), Rat (GenBank Accession No. AAH97949.1), monkey (Genbank Accession No. AWU67715.1), Porcine (GenBank Accession No. AWU67715.1) Bovine (GenBank Accession No. AAI34766.1).
- the CD40 can be a mammalian homolog of human CD40 or a functional fragment thereof.
- the CD40 polypeptide has an amino acid sequence at least 85%, at least 90%, at least 95%, at least 97% or at least 99% identical to the amino acid sequence of SEQ ID NO:18 and is capable of binding its cognate ligand CD154.
- the CD40 is a functional fragment or a portion of SEQ ID NO: 18 of at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 110, at least 120, at least 130, at least 140 consecutive amino acids of SEQ ID NO: 18, that has at least about 50%, 60%, 70%, 80%, 90%, 100% or more than 100% of the function of wild type CD40 of binding its ligand CD154.
- the functional activity can be tested by one of ordinary skill in the art by the assays described above.
- Percent (%) amino acid sequence identity for a given polypeptide sequence relative to a reference sequence is defined as the percentage of identical amino acid residues identified after aligning the two sequences and introducing gaps if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity.
- Percent (%) amino acid sequence homology for a given polypeptide sequence relative to a reference sequence is defined as the percentage of identical or strongly similar amino acid residues identified after aligning the two sequences and introducing gaps if necessary, to achieve the maximum percent homology.
- Non identities of amino acid sequences include conservative substitutions, deletions or additions that do not affect the biological activity of CD40. Strongly similar amino acids can include, for example, conservative substitutions known in the art.
- Percent identity and/or homology can be calculated using alignment methods known in the art, for instance alignment of the sequences can be conducted using publicly available software software such as BLAST, Align, ClustalW2. Those skilled in the art can determine the appropriate parameters for alignment, but the default parameters for BLAST are specifically contemplated.
- CD40 protein useful in the methods and compositions described herein consists of, consists essentially of, or comprises an amino acid sequence, or is a portion thereof derived from SEQ ID NO: 18, provided that the polypeptide retains at least one biological activity of full length CD40 of SEQ ID NO: 18, the biological activity being selected from at a minimum, binding to CD154.
- the CD40 described herein can comprise conservative amino acid substitutions at one or more amino acid residues, e.g., at essential or non-essential amino acid residues but will retain a therapeutically or physiologically relevant activity of an inhibitory peptide as that term is described herein.
- a “conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain.
- Families of amino acid residues having similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).
- basic side chains e.g., lysine, arginine, histidine
- acidic side chains e.g., aspartic acid
- CD40 can be a variant of wild type CD40.
- variant refers to a polypeptide or nucleic acid that is “substantially similar” to a wild-type CD40.
- a molecule is said to be “substantially similar” to another molecule if both molecules have substantially similar structures (i.e., they are at least 50% similar in amino acid sequence as determined by BLASTp alignment set at default parameters) and are substantially similar in at least one therapeutically or physiologically relevant biological activity.
- a variant differs from the naturally occurring polypeptide or nucleic acid by one or more amino acid or nucleic acid deletions, additions, substitutions or side-chain modifications, yet retains one or more therapeutically relevant, specific functions or desired biological activities of the naturally occurring molecule (e.g., maintains primitive HSCs in a quiescent state, enhances hematopoietic reconstitution in vivo).
- Amino acid substitutions include alterations in which an amino acid is replaced with a different naturally-occurring or a non-conventional amino acid residue. Some substitutions can be classified as “conservative,” in which case an amino acid residue contained in a polypeptide is replaced with another naturally occurring amino acid of similar character either in relation to polarity, side chain functionality or size.
- substitutions encompassed by variants as described herein can also be “non-conservative,” in which an amino acid residue which is present in a peptide is substituted with an amino acid having different properties (e.g., substituting a charged or hydrophobic amino acid with an uncharged or hydrophilic amino acid), or alternatively, in which a naturally-occurring amino acid is substituted with a non-conventional amino acid.
- variants when used with reference to a polynucleotide or polypeptide, are variations in primary, secondary, or tertiary structure, as compared to a reference polynucleotide or polypeptide, respectively (e.g., as compared to a wild-type polynucleotide or polypeptide).
- Polynucleotide changes can result in amino acid substitutions, additions, deletions, fusions and truncations in the polypeptide encoded by the reference sequence.
- Variants can also include insertions, deletions or substitutions of amino acids in the peptide sequence. To be therapeutically useful, such variants will retain a therapeutically or physiologically relevant activity as that term is used herein.
- CD40 can be an agonist of wild-type CD40, an analog or a derivative thereof.
- the agonist of wild-type CD40, an analog or a derivative thereof retains at least one biological activity of full length CD40 of SEQ ID NO: 18, the biological activity being selected from at a minimum, to bind CD154.
- the agonist of wild-type CD40, an analog or a derivative thereof retains at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100% or more than 100% of the biological activity of full length CD40 of SEQ ID NO: 18.
- the CD40 polypeptide can be recombinant, purified, isolated, naturally occurring or synthetically produced.
- the term “recombinant” when used in reference to a nucleic acid, protein, cell or a vector indicates that the nucleic acid, protein, vector or cell containing them have been modified by introduction of a heterologous nucleic acid or protein or the alteration of a native nucleic acid or a protein, or that the cell is derived from a cell so modified.
- heterologous refers to the fact that often the transferred protein was initially derived from a different cell type or a different species from the recipient.
- the protein itself is not transferred, but instead the genetic material coding for the protein (often the complementary DNA or cDNA) is added to the recipient cell.
- Methods of generating and isolating recombinant polypeptides are known to those skilled in the art and can be performed using routine techniques in the field of recombinant genetics and protein expression. For standard recombinant methods, see Sambrook et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY (1989); Deutscher, Methods in Enzymology 182:83-9(1990); Scopes, Protein Purification: Principles and Practice, Springer-Verlag, NY (1982).
- the CD40 polypeptide can be immobilized onto a solid surface, e.g., a bead or array plate.
- the CD40 protein or a portion thereof is expressed on the surface of a cell.
- the methods and compositions of the present disclosure contemplates a cell expressing CD40 on its surface.
- the disclosure is not limiting as it relates to the type of cell expressing CD40 on its surface.
- the cell can be an immune cell (e.g., B-cell, T-cell, neutrophil, monocyte, macrophage, dendritic cell, platelet), non-immune cell (e.g., an endothelial cell, epithelial cell, smooth muscle cell, fibroblast), or a cancer cell (e.g., B lymphoma cell, carcinoma cell, melanoma cell).
- an immune cell e.g., B-cell, T-cell, neutrophil, monocyte, macrophage, dendritic cell, platelet
- non-immune cell e.g., an endothelial cell, epithelial cell, smooth muscle cell, fibroblast
- a cancer cell e.
- the CD40 expressing cell is in vitro under cell culture conditions. In other embodiments, the cell expressing CD40 protein is in vivo.
- CD40 may be detected on the surface of a cell by any one of several means known in the art. For example, an antibody specific for CD40 may be used in a fluorescence-activated cell sorting technique to determine whether cells express CD40 and isolate cells expressing CD40. Other methods of detecting cell surface molecules are also useful in detecting and isolating cell expressing CD40.
- CD154 refers to a cell surface glycoprotein that is a member of the tumor necrosis factor ligand family.
- CD40 ligand is the cognate ligand for CD40.
- Other names in the art for CD40 ligand include: CD40L, CD40LG, CD 40 ligand, TNFSF5, TRAP, gp39, HIGM1, IGM, IMD3, and T-BAM.
- An exemplary protein sequence for human CD40L is NP_000065.1, which is encoded by nucleic acid sequence NM_000074.2.
- CD40 ligand “protein, polypeptide or peptide”, or CD154 as used herein, refers to a proteinaceous CD40 ligand component that has sufficient biological activity to be biologically effective. Accordingly, “CD154” include full-length CD154 proteins and polypeptides and also CD154 that have been subject to non-native processing or biological modification. Such modifications include truncations, extensions, active domains or fragments, fusion proteins, mutants with substantial or sufficient biological activity, peptidomimetics and the like. Any form of CD154 can be used methods disclosed herein, including those isolated and purified from natural sources.
- CD154 prepared by recombinant expression can also be used, i.e., those obtained by expressing a CD40 ligand nucleic acid in a recombinant host cell and collecting the expressed CD40 ligand protein.
- CD40 ligands prepared by automated peptide synthesis are also included.
- CD40 is well known in the art to interact with its cognate ligand CD154.
- One type of interaction is binding interaction and the said binding or interaction can result in activation of immune response at least, for example, by B-cell development, lymphocyte activation and proliferation; modulation of antigen presenting cells function; regulation of activity of dendritic cells, macrophages and B cells; induction of production of inflammatory cytokines in macrophages and dendritic cells; up-regulating antigen presentation; up-regulating T cell stimulation; and promoting immunoglobulin class switching in B cells.
- the binding can result, for example, in the inhibition of biological activity mediated by CD40.
- peptides of the present disclosure bind to CD40 in such a manner as to inhibit the interaction of CD40 with CD154.
- the biological activity of the peptides of the instant disclosure can be inhibiting the interaction of a CD40 protein with a CD154 protein. Therefore the peptides or functional fragment thereof disclosed herein are anti-CD40 peptides or functional fragments thereof.
- the peptide can interact with the CD40 protein in such a manner that the strength of the interaction between the CD40 protein and a CD154 protein is increased or decreased.
- the peptides disclosed herein can inhibit the interaction between CD40 and CD154.
- the term “interact” or “interaction”, and the like mean that two molecules come into sufficient physical proximity such that they cause a biological downstream effect e.g., activation of an immune cell.
- the interaction can be, for example, a binding interaction.
- the interaction can be covalent binding interactions between proteins include, for example, disulfide bonds, ester bonds, amide bonds and the like or non-covalent bonding interactions between proteins include, for example, hydrophobic interactions, van der Waals interactions, ionic interactions, hydrogen bonding interactions and the like.
- the interaction can be reversible (e.g., non-covalent binding interactions) or non-reversible (e.g., covalent bonds).
- a reversible interaction can be strong or weak, the strength of the interaction being determined by the forces (e.g., ionic charges, hydrogen binding, van der Walls interactions, etc.) exerted by each protein on the other protein in the complex.
- forces e.g., ionic charges, hydrogen binding, van der Walls interactions, etc.
- Factors affecting the strength of an interaction between two molecules are known to those skilled in the art.
- One useful measure of the strength of binding between two molecules, such as a peptide and a protein, is the dissociation constant (Kd).
- KD can be determined by measurement of the kinetics of complex formation and dissociation, e.g. by the SPR method (Biacore).
- “Inhibit” or “inhibiting,” as used herein, e.g., as in “inhibiting an interaction” between two binding partners such as proteins refers to a process of lowering or reducing the ability of a first protein (e.g., CD40 protein) and a second protein (e.g., CD154 protein) to bind or associate, or disrupting an interaction between a first protein and a second protein. “Inhibiting an interaction” between two proteins may involve disrupting one or more covalent or non-covalent interactions between the first protein and the second protein. Covalent bonding interactions between proteins include, for example, disulfide bonds, ester bonds, amide bonds and the like.
- Non-covalent bonding interactions between proteins include, for example, hydrophobic interactions, van der Waals interactions, ionic interactions, hydrogen bonding interactions and the like.
- inhibiting an interaction binding of one molecule (e.g., CD40) with another molecule (e.g., CD154) or the inhibition of stimulation of one cell (e.g., cell expressing a CD40 protein on its surface) by another cell (e.g., cell expressing CD154 on its surface) can be through steric hindrance, conformational alterations or other biochemical mechanism (e.g., through binding of peptides of instant disclosure to CD40 protein or cell expressing CD40 protein on its surface).
- the peptides of the present disclosure can inhibit an interaction between a CD40 protein and a CD154 protein by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%), at least 90%>, or at least 95%.
- the peptides disclosed herein can completely inhibit binding of CD40 to CD154. Complete inhibition of binding between CD40 and CD154 means that when a CD40 protein is contacted (treated or brought into proximity with) a peptide disclosed herein in the presence of a CD154 protein under conditions that would normally allow the interaction of CD40 and CD154, no such interaction occurs and activation signals are not stimulated in the CD40-expressing cell. Consequently CD40/CD154 mediated modulation of immune response does not occur. Methods to measure strength of binding between two proteins, for example, CD40 and CD154, has been discussed above and are known to those skilled in the art.
- contacting refers to bringing a disclosed agent (e.g., peptides disclosed herein or a portion thereof) and a cell, a target receptor, or other biological entity (e.g., CD40 protein or cell expressing CD40 on its surface) together in such a manner that the compound can affect the activity of the target, either directly; i.e., by interacting with the target itself, or indirectly; i.e., by interacting with another molecule, co-factor, factor, or protein on which the activity of the target is dependent.
- Contacting as used herein, e.g., as in “contacting a CD40 protein” refers to contacting directly or indirectly in vitro, ex vivo, or in vivo (i.e.
- the CD40 protein can be isolated, recombinant, synthesized, or immobilized on a solid surface.
- the CD40 protein can be expressed on a surface of cell (e.g. an immune cell or a cancer cell).
- the cell expressing CD40 can be present in an in vitro cell culture condition or in vivo in a subject.
- Contacting a CD40 protein can include contacting a cell expressing said CD40 protein.
- Contacting a CD40 protein can include addition of a peptide or a portion thereof, disclosed herein to a sample comprising a cell expressing a CD40 protein, or administration to a subject.
- Contacting encompasses administration to a solution, cell, tissue, mammal, subject, patient, or human.
- contacting a cell includes adding a peptide or portion thereof, disclosed herein to a cell culture.
- provided herein is a method of inhibiting an interaction between a CD40 protein and a CD154 protein comprising contacting the CD40 protein with a peptide disclosed herein.
- peptides disclosed herein can interact with any site on the CD40 protein. In some embodiments, peptides disclosed herein bind with the CD40 protein at a site that overlaps with the CD154 binding site. In some embodiments, a peptide disclosed herein binds the CD40 protein at the CD154 binding site.
- CD40 is well known to play an important role in activation of immune response.
- B cells are known to constitutively express the CD40 molecule on the cells membrane (van Kooten C. et al, J. Leukoc. Biol. 2000). Stimulation of CD40 by CD154-expressing activated T cells, results in B cells activation, proliferation and isotype switching.
- CD40-CD154 interaction can upregulate costimulatory molecules, activate antigen presenting cells (APCs) and influence T-cell priming and T-cell-mediated effector of CD40 activities include modulating cell survival (e.g., functions as cell survival signal), antibody production, antibody isotype switching, production of cytokines (e.g., IL-2, IL-6, IL-8, IL-12, TNF- ⁇ , IL-4, IL-5 and IL-10), metalloproteases (e.g., MMP-I/collagenase and MMP-9/gelatinase B) and establishment of immune memory.
- APCs antigen presenting cells
- CD40 activities further include modulating production of proteins involved in cell-cell contact or adhesion (e.g., E-selectin, VCAM-1 and ICAM-1). This interaction can activate macrophages, natural killer (NK) cells, and endothelial cells.
- CD40 plays a significant role in immune cell function and signaling, including B-cell and T-cell activation by antigen presenting cells, such as macrophages and dendritic cells. CD40 activation stimulates antibody production, isotype switching, and establishment of memory.
- CD40 activation stimulates production of cytokines, such as IL-2, IL-6, IL-8, IL-12, TNF- ⁇ , IL-4, IL-S and IL-10; and metalloproteases, such as MMP-I/collagenase and MMP-9/gelatinase B.
- CD40 activation stimulates production of proteins involved in cell-cell contact or adhesion, such as E-selectin, VCAM-1 and ICAM-1.
- CD40 recognition on target cells provides an activation pathway for NK cell cytotoxic activity.
- immune response encompasses but is not limited to one or both of the following responses: antibody production (e.g., humoral immunity), and induction of cell-mediated immunity (e.g., cellular immunity including helper T cell and/or cytotoxic T cell responses).
- antibody production e.g., humoral immunity
- cell-mediated immunity e.g., cellular immunity including helper T cell and/or cytotoxic T cell responses.
- Immune response generally refers to innate and acquired immune responses including, but not limited to, both humoral immune responses (mediated by B lymphocytes) and cellular immune responses (mediated by T lymphocytes).
- An immune response may be beneficial and lead to immunity against infectious pathogens, or an immune response may be pathogenic and lead to autoimmune or hypersensitivity disease. Immune responses against foreign viruses, bacteria, fungi, parasites typically represent beneficial adaptive immune responses.
- Immune responses against self-tissues, innocuous foreign objects (e.g., dust mite or pollen allergens, etc.), or tissue transplants represent examples of adverse maladaptive immune responses.
- modulate refers to enhancement (e.g., an increase) or inhibition (e.g., a reduction) in the specified activity or suppression of a specified activity.
- immunosuppression also refers to inhibition of an immune response mediated by CD40 activity or one that is mediated by interaction of CD40 with CD154
- modulation of immune response can be change in the level of an immune cell (e.g., B-cell, T-cell, antigen presenting cell, activated B-cell, activated T-cell, activated macrophage), a change in level of immunomodulatory molecules (e.g., inflammatory cytokines, chemokines), or a combination thereof.
- modulation of immune response can be suppression of immune response or immunosuppression.
- level, number, count and concentration can be used interchangeably.
- immunomodulatory molecule refers to any molecule which is capable of effecting the proliferation or activation of the cells of a subject's immune system.
- Such molecules include, without limitation, prostaglandin E2 (PGE2), transforming growth factor-0 (TGF- ⁇ ), indoleamine 2,3-dioxygenase (IDO), nitric oxide, hepatocyte growth factor (HGF), interleukin 6 (IL-6) and interleukin 10 (IL-10).
- PGE2 prostaglandin E2
- TGF- ⁇ transforming growth factor-0
- IDO indoleamine 2,3-dioxygenase
- nitric oxide hepatocyte growth factor
- HGF hepatocyte growth factor
- IL-6 interleukin 6
- IL-10 interleukin 10
- Modulation of immune response can mean increase or decrease of in the level of an immune cell.
- the peptides of the present disclosure inhibit or decrease the immune response by inhibiting the level of immune cell (e.g., T-cell, B-cell, antigen presenting cell, activated T-cell, activated B-cell, or activated macrophage), inhibiting the level of immunomodulatory molecules, or a combination thereof).
- the level of immune cell e.g., T-cell, B-cell, antigen presenting cell, activated T-cell, activated B-cell, or activated macrophage
- the biological activity of peptides disclosed herein can be inhibition of CD40 activity such as CD40 mediated immune response (e.g., B-cell activation, B-cell proliferation, T-cell activation, T-cell proliferation, macrophage activation, inhibition of inflammatory cytokine production, or a combination thereof).
- CD40 mediated immune response e.g., B-cell activation, B-cell proliferation, T-cell activation, T-cell proliferation, macrophage activation, inhibition of inflammatory cytokine production, or a combination thereof.
- both a cell culture system and the immune system of a subject comprise basal levels of immune cells and immunomodulatory molecules.
- the phrases basal level and normal level can be used interchangeably.
- the basal level of a type of immune cell, or a immunomodulatory molecule refers to the average number of that cell type, or immunomodulatory molecule, present in a population of individuals considered healthy (i.e., free of metabolic, autoimmune, or infectious disease) or the basal level of a type of immune cell, or an immunomodulatory molecule, refers to the average level of that cell type, or immunomodulatory molecule, present in a population of cells that is not-activated.
- Those skilled in the art are capable of determining if an immune cell, or a population of such cells, is activated.
- the expression of CD69, CD25 and/or CD154 proteins by a T-cell indicates that the cell has been activated.
- the expression of MHC-class II, B220 and CD3 proteins by B-cell indicates that the B-cell has been activated.
- the expression of IL-12, iNOS, Arg-1, or IL-1 proteins by macrophage indicates the macrophage has been activated.
- Methods to measure immune cells are well known in the art including methods based on identifying expression of specific surface marker proteins e.g., by flow cytometry.
- Level of immune cell can be measure, for example, by measuring proliferation by 3H-Thymidine Uptake, Bromodeoxyuridine Uptake (BrdU), ATP Luminescence, Fluorescent Dye Reduction (carboxyfluorescein succinimidyl ester (CFSE)-like dyes); cytokine measurement, for example, using Multi-Analyte ELISArray Kits, bead-based multiplex assay; measuring surface antigen expression, for example, by flow cytometry; measuring cell cytotoxicity, for example, by Two-Label Flow Cytometry, Calcein AM Dye Release, Luciferase Transduced Targets, or Annexin V.
- Methods to measure T-cell responses and B-cell responses are well known in the art, for example see Expert Rev. Vaccines 9(6), 595-600 (2010), mBio. 2015
- the reference level or basal level of a cell or molecule can be a specific amount (e.g., a specific concentration) or it can encompass a range of amounts.
- Basal levels, or ranges, of immune cells and immunoregulatory molecules are known to those in the art. For example, in a healthy individual, the normal level of CD4+ T-cells present in human blood is 500-1500 cells/ml. Basal levels of cells can also be reported as a percentage of a total cell population.
- Immune cell number and function may be monitored by assays that detect immune cells by an activity such as cytokine production, proliferation, or cytotoxicity.
- Lymphoproliferation Assay which assays the ability of T cells to proliferate in response to an antigen can be used as an indicator of the presence of antigen-specific CD4+ helper T cells.
- the specimen of purified T cells or PBMCs is mixed with various dilutions of antigen or antigen in the presence of stimulator cells (irradiated autologous or HLA matched antigen-presenting cells).
- the proliferation assay can be used to compare T-cell responses before and after treatment with peptides of the present disclosure or pharmaceutical compositions disclosed herein.
- assays that can be employed for detection of proliferation of immune cells (e.g., T-cell, B-cells) include use of intracellular fluorescent dye, 5,6-carboxyfluorescein diacetate succinimidyl ester (CFSE) in mixed lymphocyte reaction (CFSE-MLR) and determination of proliferating cells using flow cytometry.
- CFSE 5,6-carboxyfluorescein diacetate succinimidyl ester
- CFSE-MLR mixed lymphocyte reaction
- Another example of an assay that can be employed is detection of secreted cytokines by ELISA and ELISPOT Assays.
- Cytokine secretion by immune cells in a subject suffering from inflammatory disease or in response to a transplant may be detected by measuring either bulk cytokine production (by an ELISA) or enumerating individual cytokine producing immune cells (by an ELISPOT assay).
- cytokine production by an ELISA
- ELISPOT assay enumerating individual cytokine producing immune cells
- PBMC specimens are incubated with antigen (with or without antigen-presenting cells), and after a defined period of time, the supernatant from the culture is harvested and added to microtiter plates coated with antibody for cytokines of interest such as IFN- ⁇ , TNF- ⁇ , or IL-2.
- Antibodies ultimately linked to a detectable label or reporter molecule are added, and the plates are washed and read.
- cytokine secretions can be measured in samples (e.g., serum or other body fluids) obtained from a subject using ELISA or ELISPOT before and after treatment with the peptides disclosed herein or pharmaceutical compositions disclosed herein.
- samples e.g., serum or other body fluids
- ELISPOT ELISA or ELISPOT
- Other useful assays include, measurement of detection of intracellular cytokine assay by flow cytometry, measurement of cytokine mRNA levels by RT-PCR and direct cytotoxicity assays of T-cell (See: Clay et al., 2001).
- Macrophage activation can be determined, for example, by measuring levels of chemokines such as IL-8/CXCL8, IP-10/CXCL10, MIP-1 alpha/CCL3, MIP-1 beta/CCL4, and RANTES/CCL5, which are released as chemoattractants for neutrophils, immature dendritic cells, natural killer cells, and activated T cells.
- chemokines such as IL-8/CXCL8, IP-10/CXCL10, MIP-1 alpha/CCL3, MIP-1 beta/CCL4, and RANTES/CCL5, which are released as chemoattractants for neutrophils, immature dendritic cells, natural killer cells, and activated T cells.
- levels of pro-inflammatory cytokines are released including IL-1 beta/IL-1F2, IL-6, and TNF-alpha can also be measured by assays well known in the art.
- Levels of proteolytic enzymes, MMP-1, ⁇ 2, ⁇ 7, ⁇ 9, and ⁇ 12, which degrade Collagen, Elastin, Fibronectin, and other ECM components can also be measured to determine macrophage activation.
- Leukocytes are attracted by the macrophage via its release of chemokines including MDC/CCL22, PARC/CCL18, and TARC/CCL17.
- Levels of activated B-cell can be determined, for example, by measuring antigen specific antibody secretion or detecting activated B-cell specific surface markers such as CD27, CD19, CD20, CD25, CD30, CD69, CD80, CD86, CD135, by assays such as flow cytometry.
- the peptides disclosed herein can reduce the level of an immune cell to less than about 50%, less than about 45%, less than about 40%, less than about 35%, less than about 30%, less than about 27%, or equal to about 25% of the total immune cell population.
- Methods of measuring different types of T-cells in the T-cell population are known to those skilled in the art.
- binding of a peptide disclosed herein with a CD40 protein decreases the number of immune cells by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%.
- binding of a peptide disclosed herein with a CD40 protein decreases the number of immune cells by a factor of at least 10, at least 100, at least 1,000, at least 10,000.
- the level of immune cells is reduced so that immune cells comprise no more than about 20%>, about 25%, about 30%>, about 35%, or about 40%> of the total immune cell population.
- the peptides can generally be prepared following techniques known in the art.
- the peptides can also be prepared using standard genetic engineering techniques known to those skilled in the art.
- the peptide can be produced enzymatically by inserting nucleic acid encoding the peptide into an expression vector, expressing the DNA, and translating the DNA into the peptide in the presence of the required amino acids.
- the peptide is then purified using chromatographic or electrophoretic techniques, or by means of a carrier protein which can be fused to, and subsequently cleaved from, the peptide. This can be done by inserting into the expression vector in phase with the peptide encoding sequence, a nucleic acid sequence encoding the carrier protein.
- the fusion protein-peptide may be isolated using chromatographic, electrophoretic or immunological techniques (such as binding to a resin via an antibody to the carrier protein).
- the peptide can be cleaved using chemical methodology or enzymatically, as by, for example, using hydrolases.
- Peptides of the disclosure can also be prepared using solution methods, by either stepwise or fragment condensations.
- An appropriately alpha amino-protected amino acid is coupled to an appropriately alpha carboxyl protected amino acid (such protection may not be required depending on the coupling method chosen) using diimides, symmetrical or unsymmetrical anhydrides, BOP, or other coupling reagents or techniques known to those skilled in the art. These techniques may be either or enzymatic.
- the alpha amino and/or alpha carboxyl protecting groups are removed and the next suitably protected amino acid or block of amino acids are coupled to extend the growing peptide.
- Various combinations of protecting groups and of chemical and/or enzymatic techniques and assembly strategies can be used in each synthesis.
- a peptide of the instant disclosure can be synthesized by conventional techniques.
- the peptides can be synthesized by chemical synthesis using solid phase peptide synthesis. These methods employ either solid or solution phase synthesis methods (see for example, J. M. Stewart, and J. D. Young, Solid Phase Peptide Synthesis, 2nd Ed., Pierce Chemical Co., Rockford 111. (1984) and G. Barany and R. B. Merrifield, The Peptides: Analysis Synthesis, Biology editors E. Gross and J. Meienhofer Vol. 2 Academic Press, New York, 1980, pp. 3-254 for solid phase synthesis techniques; and M Bodansky, Principles of Peptide Synthesis, Springer-Verlag, Berlin 1984, and E. Gross and J. Meienhofer, Eds., The Peptides: Analysis, Synthesis, Biology, suprs, Vol 1, for classical solution synthesis.)
- the peptides can be chemically synthesized by Merrifield-type solid phase peptide synthesis. This method may be routinely performed to yield peptides up to about 60-70 residues in length, and may, in some cases, be utilized to make peptides up to about 100 amino acids long. Larger peptides may also be generated synthetically via fragment condensation or native chemical ligation (Dawson et al., 2000, Ann. Rev. Biochem. 69:923-960).
- An advantage to the utilization of a synthetic peptide route is the ability to produce large amounts of peptides, even those that rarely occur naturally, with relatively high purities, i.e., purities sufficient for research, diagnostic or therapeutic purposes.
- Solid phase peptide synthesis is described by Stewart et al. in Solid Phase Peptide Synthesis, 2nd Edition, 1984, Pierce Chemical Company, Rockford, III; and Bodanszky and Bodanszky in The Practice of Peptide Synthesis, 1984, Springer-Verlag, New York.
- a suitably protected amino acid residue is attached through its carboxyl group to a derivatized, insoluble polymeric support, such as cross-linked polystyrene or polyamide resin.
- “Suitably protected” refers to the presence of protecting groups on both the alpha-amino group of the amino acid, and on any side chain functional groups.
- Side chain protecting groups are generally stable to the solvents, reagents and reaction conditions used throughout the synthesis, and are removable under conditions which will not affect the final peptide product.
- Stepwise synthesis of the oligopeptide is carried out by the removal of the N-protecting group from the initial amino acid, and coupling thereto of the carboxyl end of the next amino acid in the sequence of the desired peptide. This amino acid is also suitably protected.
- the carboxyl of the incoming amino acid can be activated to react with the N-terminus of the support-bound amino acid by formation into a reactive group, such as formation into a carbodiimide, a symmetric acid anhydride, or an “active ester” group, such as hydroxybenzotriazole or pentafluorophenyl esters.
- a reactive group such as formation into a carbodiimide, a symmetric acid anhydride, or an “active ester” group, such as hydroxybenzotriazole or pentafluorophenyl esters.
- N-terminal acetylation on the deprotected N.sup. ⁇ -amino group of peptides synthesized using the Boc strategy can be accomplished with 10% Ac2 O and 5% DIEA in NMP, followed by washing of the peptide resin with NMP and/or CH2 Cl2.
- solid phase peptide synthesis methods include the BOC method which utilized tert-butyloxcarbonyl as the alpha-amino protecting group, and the FMOC method which utilizes 9-fluorenylmethyloxcarbonyl to protect the alpha-amino of the amino acid residues, both which methods are well-known by those of skill in the art.
- Incorporation of N- and/or C-blocking groups may also be achieved using protocols conventional to solid phase peptide synthesis methods.
- C-terminal blocking groups for example, synthesis of the desired peptide is typically performed using, as solid phase, a supporting resin that has been chemically modified so that cleavage from the resin results in a peptide having the desired C-terminal blocking group.
- synthesis is performed using a p-methylbenzhydrylamine (MBHA) resin, so that, when peptide synthesis is completed, treatment with hydrofluoric acid releases the desired C-terminally amidated peptide.
- MBHA p-methylbenzhydrylamine
- incorporation of an N-methylamine blocking group at the C-terminus is achieved using N-methylaminoethyl-derivatized DVB, resin, which upon HF treatment releases a peptide bearing an N-methylamidated C-terminus.
- Blockage of the C-terminus by esterification can also be achieved using conventional procedures.
- FMOC protecting group in combination with DVB resin derivatized with methoxyalkoxybenzyl alcohol or equivalent linker, can be used for this purpose, with cleavage from the support being effected by TFA in dicholoromethane. Esterification of the suitably activated carboxyl function, e.g. with DCC, can then proceed by addition of the desired alcohol, followed by de-protection and isolation of the esterified peptide product.
- the peptides of the present disclosure are manufactured by solid phase peptide synthesis using Fmoc chemistry.
- the Fmoc group is deprotected at the N-terminus
- the side chain protection group is deprotected, and the peptide is cleaved from the resin.
- the resin is a Cl-resin.
- the condensation reaction reagent is DIC+HOBT.
- deprotection is done using Pip.
- the synthesized peptides are purified by RP-HPLC using a solvent of acetonitrile+deionized with TFA as the buffer.
- the peptides are purified by gradient elution.
- the peptides of the disclosure may be prepared by standard chemical or biological means of peptide synthesis.
- Biological methods include, without limitation, expression of a nucleic acid encoding a peptide in a host cell or in an in vitro translation system.
- nucleic acid sequences that encode the peptide of the instant disclosure.
- the methods and compositions herein contemplate use of a nucleic acid fragment encoding a peptide disclosed herein (e.g., peptide selected from Table 1).
- subclones of a nucleic acid sequence encoding a peptide of the disclosure can be produced using conventional molecular genetic manipulation for subcloning gene fragments, such as described by Sambrook et al, Molecular Cloning: A Laboratory Manual, Cold Springs Laboratory, Cold Springs Harbor, N.Y. (2012), and Ausubel et al.
- an isolated nucleic acid fragment encoding a peptide comprising an amino acid sequence selected from group consisting of SEQ ID NOs: 1-6 and 8-17 or a portion thereof. Further, the disclosure encompasses an isolated nucleic acid encoding a peptide having substantial homology to a peptide selected from group consisting of SEQ ID NOs: 1-6 and 8-17. In some embodiments, the isolated nucleic acid fragment encodes a peptide having a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% identical to that of SEQ ID NO: 1.
- the isolated nucleic acid fragment encodes a peptide having an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% identical to that of SEQ ID NO: 2. In some embodiments, the nucleic acid fragment encodes a peptide having an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% identical to that of SEQ ID NO:3.
- the nucleic acid fragment encodes a peptide having an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% identical to that of SEQ ID NO: 4. In some embodiments, the nucleic acid fragment encodes a peptide having an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% identical to that of SEQ ID NO: 5.
- the isolated nucleic acid fragment encodes a peptide having an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% identical to that of SEQ ID NO: 6. In some embodiments, the isolated nucleic acid fragment encodes a peptide having an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% identical to that of SEQ ID NO: 8.
- the isolated nucleic acid fragment encodes a peptide having an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% identical to that of SEQ ID NO: 9. In some embodiments, the isolated nucleic acid fragment encodes a peptide having an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% identical to that of SEQ ID NO: 10.
- the isolated nucleic acid fragment encodes a peptide having an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% identical to that of SEQ ID NO: 11. In some embodiments, the isolated nucleic acid fragment encodes a peptide having an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% identical to that of SEQ ID NO: 12.
- the isolated nucleic acid fragment encodes a peptide having an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% identical to that of SEQ ID NO: 13. In some embodiments, the isolated nucleic acid fragment encodes a peptide having an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% identical to that of SEQ ID NO: 14.
- the isolated nucleic acid fragment encodes a peptide having an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% identical to that of SEQ ID NO: 15. In some embodiments, the isolated nucleic acid fragment encodes a peptide having an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% identical to that of SEQ ID NO: 16.
- the isolated nucleic acid fragment encodes a peptide having an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% identical to that of SEQ ID NO: 17.
- an “isolated nucleic acid” refers to a nucleic acid segment or fragment which has been separated from sequences which flank it in a naturally occurring state, i.e., a DNA fragment which has been removed from the sequences which are normally adjacent to the fragment, i.e., the sequences adjacent to the fragment in a genome in which it naturally occurs.
- the term also applies to nucleic acids which have been substantially purified from other components which naturally accompany the nucleic acid, i.e., RNA or DNA or proteins, which naturally accompany it in the cell.
- the term therefore includes, for example, a recombinant DNA which is incorporated into a vector, into an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (i.e., as a cDNA or a genomic or cDNA fragment produced by PCR or restriction enzyme digestion) independent of other sequences. It also includes a recombinant DNA which is part of a hybrid gene encoding additional polypeptide sequence.
- A refers to adenosine
- C refers to cytosine
- G refers to guanosine
- T refers to thymidine
- U refers to uridine.
- the present disclosure also encompasses recombinant nucleic acid encoding the peptides described herein.
- a “recombinant” nucleic acid is one that has been created using genetic engineering techniques.
- Biological preparation of a peptide disclosed herein involves expression of a nucleic acid encoding a desired peptide.
- An expression cassette comprising such a coding sequence may be used to produce a desired peptide for use in the method of the disclosure.
- the vector can be readily introduced into a host cell, e.g., mammalian, bacterial, yeast or insect cell by any method in the art. Coding sequences for a desired peptide of the disclosure can be codon optimized based on the codon usage of the intended host cell in order to improve expression efficiency as demonstrated herein. Codon usage patterns can be found in the literature (Nakamura et al, 2000, Nuc Acids Res. 28:292).
- bacterial cells such as streptococci, staphylococci, E. coli, Streptomyces and Bacillus subtilis cells
- fungal cells such as yeast cells and Aspergillus cells
- insect cells such as Drosophila S2 and Spodoptera Sf9 cells
- animal cells such as CHO, COS, HeLa, C127, 3T3, BHK, HEK 293 and Bowes melanoma cells
- plant cells include bacterial cells, such as streptococci, staphylococci, E. coli, Streptomyces and Bacillus subtilis cells
- fungal cells such as yeast cells and Aspergillus cells
- insect cells such as Drosophila S2 and Spodoptera Sf9 cells
- animal cells such as CHO, COS, HeLa, C127, 3T3, BHK, HEK 293 and Bowes melanoma cells
- vectors are known in the art including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses.
- vector includes an autonomously replicating plasmid or a virus.
- the term should also be construed to include non-plasmid and non-viral compounds which facilitate transfer of nucleic acid into cells, such as, for example, polylysine compounds, liposomes, and the like.
- viral vectors include, but are not limited to, adenoviral vectors, adeno-associated virus vectors, retroviral vectors, and the like.
- the expression vector can be transferred into a host cell by physical, biological or chemical means, discussed in detail elsewhere herein.
- analysis of the peptide composition can be conducted.
- Such amino acid composition analysis may be conducted using high resolution mass spectrometry to determine the molecular weight of the peptide.
- the amino acid content of the peptide can be confirmed by hydrolyzing the peptide in aqueous acid, and separating, identifying and quantifying the components of the mixture using HPLC, or an amino acid analyzer. Protein sequenators, which sequentially degrade the peptide and identify the amino acids in order, can also be used to determine definitely the sequence of the peptide.
- the isolated nucleic acid can comprise any type of nucleic acid, including, but not limited to DNA and RNA.
- the composition comprises an isolated DNA molecule, including for example, an isolated cDNA molecule, encoding a peptide of the disclosure, or functional fragment thereof.
- the composition comprises an isolated RNA molecule encoding a peptide of the invention, or a functional fragment thereof.
- the isolated nucleic acids can be synthesized using any method known in the art.
- the nucleic acid molecules of the present disclosure can be modified to improve stability in serum or in growth medium for cell cultures. Modifications can be added to enhance stability, functionality, and/or specificity and to minimize immunostimulatory properties of the nucleic acid molecule of the disclosure.
- the 3′-residues may be stabilized against degradation, e.g., they may be selected such that they consist of purine nucleotides, particularly adenosine or guanosine nucleotides.
- substitution of pyrimidine nucleotides by modified analogues e.g., substitution of uridine by 2′-deoxythymidine is tolerated and does not affect function of the molecule.
- the nucleic acid molecule can contain at least one modified nucleotide analogue.
- the ends may be stabilized by incorporating modified nucleotide analogues.
- nucleotide analogues include sugar- and/or backbone-modified ribonucleotides (i.e., include modifications to the phosphate-sugar backbone).
- the phosphodiester linkages of natural RNA may be modified to include at least one of a nitrogen or sulfur heteroatom.
- the phosphoester group connecting to adjacent ribonucleotides is replaced by a modified group, e.g., of phosphothioate group.
- the 2′ OH-group is replaced by a group selected from H, OR, R, halo, SH, SR, NH2, NHR, NR2 or ON, wherein R is Ci-C6 alkyl, alkenyl or alkynyl and halo is F, CI, Br or I.
- nucleobase-modified ribonucleotides i.e., ribonucleotides, containing at least one non-naturally occurring nucleobase instead of a naturally occurring nucleobase.
- Bases can be modified to block the activity of adenosine deaminase.
- modified nucleobases include, but are not limited to, uridine and/or cytidine modified at the 5-position, e.g., 5-(2-amino)propyl uridine, 5-bromo uridine; adenosine and/or guanosines modified at the 8 position, e.g., 8-bromo guanosine; deaza nucleotides, e.g., 7-deaza-adenosine; O- and N-alkylated nucleotides, e.g., N6-methyl adenosine are suitable. It should be noted that the above modifications may be combined.
- the nucleic acid molecule comprises at least one of the following chemical modifications: 2′-H, 2′-O-methyl, or 2′-OH modification of one or more nucleotides.
- a nucleic acid molecule of the invention can have enhanced resistance to nucleases.
- a nucleic acid molecule can include, for example, 2′-modified ribose units and/or phosphorothioate linkages.
- the 2′ hydroxyl group (OH) can be modified or replaced with a number of different “oxy” or “deoxy” substituents.
- the nucleic acid molecules of the invention can include 2′-O-methyl, 2′-fluorine, 2′-O-methoxyethyl, 2′-O-aminopropyl, 2′-amino, and/or phosphorothioate linkages.
- LNA locked nucleic acids
- ENA ethylene nucleic acids
- certain nucleobase modifications such as 2-amino-A, 2-thio (e.g., 2-thio-U), G-clamp modifications, can also increase binding affinity to a target.
- the nucleic acid molecule includes a 2′-modified nucleotide, e.g., a 2′-deoxy, 2′-deoxy-2′-fluoro, 2′-0-methyl, 2′-0-methoxyethyl (2′-0-MOE), 2′-0-aminopropyl (2′-0-AP), 2′-0-dimethylaminoethyl (2′-0-DMAOE), 2′-0-dimethylaminopropyl (2′-O-DMAP), 2′-0-dimethylaminoethyloxyethyl (2′-0-DMAEOE), or 2′-0-N-methylacetamido (2′-0-NMA).
- the nucleic acid molecule includes at least one 2′-0-methyl-modified nucleotide, and in some embodiments, all of the nucleotides of the nucleic acid molecule include a 2′-0-methyl modification.
- Nucleic acid fragments discussed herein include otherwise unmodified RNA and DNA as well as RNA and DNA that have been modified, e.g., to improve efficacy, and polymers of nucleoside surrogates.
- Unmodified RNA refers to a molecule in which the components of the nucleic acid, namely sugars, bases, and phosphate moieties, are the same or essentially the same as that which occur in nature, preferably as occur naturally in the human body.
- the art has referred to rare or unusual, but naturally occurring, RNAs as modified RNAs, see, e.g., Limbach et al. ⁇ Nucleic Acids Res., 1994, 22:2183-2196).
- modified RNA refers to a molecule in which one or more of the components of the nucleic acid, namely sugars, bases, and phosphate moieties, are different from that which occur in nature, preferably different from that which occurs in the human body. While they are referred to as “modified RNAs” they will of course, because of the modification, include molecules that are not, strictly speaking, RNAs.
- Nucleoside surrogates are molecules in which the ribophosphate backbone is replaced with a non-ribophosphate construct that allows the bases to be presented in the correct spatial relationship such that hybridization is substantially similar to what is seen with a ribophosphate backbone, e.g., non-charged mimics of the ribophosphate backbone.
- Modifications of the nucleic acid of the disclosure can be present at one or more of, a phosphate group, a sugar group, backbone, N-terminus, C-terminus, or nucleobase.
- a “vector” or “delivery vector” can be a viral or non-viral vector that is used to deliver a nucleic acid to a cell, tissue or subject.
- exemplary vectors include, but are not limited to, adeno-associated virus vectors, adenovirus vectors, lentivirus vectors, paramyxovirus vectors, alphavirus vectors and herpes virus vectors.
- a “recombinant” vector or delivery vector refers to a viral or non-viral vector that comprises one or more heterologous nucleotide sequences (i.e., transgenes), e.g., two, three, four, five or more heterologous nucleotide sequences.
- the recombinant vectors and delivery vectors of the invention encode a fusion polypeptide of NAg and a cytokine such as IFN- ⁇ , but can also include one or more additional heterologous nucleotide sequences, for example, sequences encoding C- or N-terminal modifications and linker moieties.
- viral vector or “viral delivery vector” can refer to a virus particle that functions as a nucleic acid delivery vehicle, and which comprises the vector genome packaged within a virion. Alternatively, these terms can be used to refer to the vector genome when used as a nucleic acid delivery vehicle in the absence of the virion.
- the art is replete with suitable vectors that are useful in the present disclosure.
- the expression of natural or synthetic nucleic acids encoding a peptide is typically achieved by operably linking a nucleic acid encoding the peptide or portions thereof to a promoter, and incorporating the construct into an expression vector.
- the vectors to be used are suitable for replication and, optionally, integration in eukaryotic cells. Typical vectors contain transcription and translation terminators, initiation sequences, and promoters useful for regulation of the expression of the desired nucleic acid sequence.
- the vectors of the present invention may also be used for nucleic acid immunization and gene therapy, using standard gene delivery protocols. Methods for gene delivery are known in the art. See, e.g., U.S. Pat. Nos. 5,399,346, 5,580,859, 5,589,466, incorporated by reference herein in their entireties.
- the disclosure provides a gene therapy vector.
- the isolated nucleic acid of the disclosure can be cloned into a number of types of vectors.
- the nucleic acid can be cloned into a vector including, but not limited to a plasmid, a phagemid, a phage derivative, an animal virus, and a cosmid.
- Vectors of particular interest include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.
- the vector may be provided to a cell in the form of a viral vector.
- Viral vector technology is well known in the art and is described, for example, in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York), and in other virology and molecular biology manuals.
- Viruses which are useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses.
- a suitable vector contains an origin of replication functional in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers, (e.g., WO 01/96584; WO 01/29058; and U.S. Pat. No. 6,326,193).
- retroviruses provide a convenient platform for gene delivery systems.
- a selected gene can be inserted into a vector and packaged in retroviral particles using techniques known in the art.
- the recombinant virus can then be isolated and delivered to cells of the subject either in vivo or ex vivo.
- retroviral systems are known in the art.
- adenovirus vectors are used.
- a number of adenovirus vectors are known in the art.
- lentivirus vectors are used.
- vectors derived from retroviruses such as the lentivirus are suitable tools to achieve long-term gene transfer since they allow long-term, stable integration of a transgene and its propagation in daughter cells.
- Lentiviral vectors have the added advantage over vectors derived from onco-retroviruses such as murine leukemia viruses in that they can transduce non-proliferating cells, such as hepatocytes. They also have the added advantage of low immunogenicity.
- the composition includes a vector derived from an adeno-associated virus (AAV).
- Adeno-associated viral (AAV) vectors have become powerful gene delivery tools for the treatment of various disorders.
- AAV vectors possess a number of features that render them ideally suited for gene therapy, including a lack of pathogenicity, minimal immunogenicity, and the ability to transduce postmitotic cells in a stable and efficient manner. Expression of a particular gene contained within an AAV vector can be specifically targeted to one or more types of cells by choosing the appropriate combination of AAV serotype, promoter, and delivery method
- the vector also includes conventional control elements which are operably linked to a regulatory control sequence in a manner which permits its transcription, translation and/or expression in a cell transfected with the plasmid vector or infected with the virus produced by the disclosure.
- operably linked sequences include both expression control sequences that are contiguous with the gene of interest and expression control sequences that act in trans or at a distance to control the gene of interest.
- Expression control sequences include appropriate transcription initiation, termination, promoter and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation (poly A) signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., Kozak consensus sequence); sequences that enhance protein stability; and when desired, sequences that enhance secretion of the encoded product.
- RNA processing signals such as splicing and polyadenylation (poly A) signals
- sequences that stabilize cytoplasmic mRNA sequences that enhance translation efficiency (i.e., Kozak consensus sequence); sequences that enhance protein stability; and when desired, sequences that enhance secretion of the encoded product.
- a great number of expression control sequences including promoters which are native, constitutive, inducible and/or tissue-specific, are known in the art and may be utilized. Additional promoter elements, e.g., enhancers, regulate the frequency of transcriptional initiation.
- promoters typically contain functional elements downstream of the start site as well.
- the spacing between promoter elements frequently is flexible, so that promoter function is preserved when elements are inverted or moved relative to one another.
- tk thymidine kinase
- the spacing between promoter elements can be increased to 50 bp apart before activity begins to decline.
- individual elements can function either cooperatively or independently to activate transcription.
- a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence.
- CMV immediate early cytomegalovirus
- This promoter sequence is a strong constitutive promoter sequence capable of driving high levels of expression of any polynucleotide sequence operative ly linked thereto.
- Another example of a suitable promoter is Elongation Growth Factor-1 (EF-1a).
- constitutive promoter sequences may also be used, including, but not limited to the simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, an avian leukemia virus promoter, an Epstein-Barr virus immediate early promoter, a Rous sarcoma virus promoter, as well as human gene promoters such as, but not limited to, the actin promoter, the myosin promoter, the hemoglobin promoter, and the creatine kinase promoter. Further, the invention should not be limited to the use of constitutive promoters.
- inducible promoters are also contemplated as part of the invention.
- the use of an inducible promoter provides a molecular switch capable of turning on expression of the polynucleotide sequence which it is operatively linked when such expression is desired, or turning off the expression when expression is not desired.
- inducible promoters include, but are not limited to a metallothionine promoter, a glucocorticoid promoter, a progesterone promoter, and a tetracycline promoter.
- Enhancer sequences found on a vector also regulates expression of the gene contained therein.
- enhancers are bound with protein factors to enhance the transcription of a gene.
- Enhancers may be located upstream or downstream of the gene it regulates. Enhancers may also be tissue-specific to enhance transcription in a specific cell or tissue type.
- the vector of the present invention comprises one or more enhancers to boost transcription of the gene present within the vector.
- the expression vector to be introduced into a cell can also contain either a selectable marker gene or a reporter gene or both to facilitate identification and selection of expressing cells from the population of cells sought to be transfected or infected through viral vectors.
- the selectable marker may be carried on a separate piece of DNA and used in a co-transfection procedure. Both selectable markers and reporter genes may be flanked with appropriate regulatory sequences to enable expression in the host cells.
- Useful selectable markers include, for example, antibiotic-resistance genes, such as neo and the like.
- Reporter genes are used for identifying potentially transfected cells and for evaluating the functionality of regulatory sequences.
- a reporter gene is a gene that is not present in or expressed by the recipient organism or tissue and that encodes a polypeptide whose expression is manifested by some easily detectable property, e.g., enzymatic activity. Expression of the reporter gene is assayed at a suitable time after the DNA has been introduced into the recipient cells.
- Suitable reporter genes may include genes encoding luciferase, beta-galactosidase, chloramphenicol acetyl transferase, secreted alkaline phosphatase, or the green fluorescent protein gene (e.g., Ui-Tei et al, 2000 FEBS Letters 479: 79-82).
- Suitable expression systems are well known and may be prepared using known techniques or obtained commercially.
- the construct with the minimal 5′ flanking region showing the highest level of expression of reporter gene is identified as the promoter.
- Such promoter regions may be linked to a reporter gene and used to evaluate agents for the ability to modulate promoter-driven transcription.
- the vector can be readily introduced into a host cell, e.g., mammalian, bacterial, yeast, or insect cell by any method in the art.
- the expression vector can be transferred into a host cell by physical, chemical, or biological means.
- Physical methods for introducing a polynucleotide into a host cell include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like.
- Methods for producing cells comprising vectors and/or exogenous nucleic acids are well-known in the art. See, for example, Sambrook et al.
- a preferred method for the introduction of a polynucleotide into a host cell is calcium phosphate transfection.
- Biological methods for introducing a polynucleotide of interest into a host cell include the use of DNA and RNA vectors.
- Viral vectors, and especially retroviral vectors have become the most widely used method for inserting genes into mammalian, e.g., human cells.
- Other viral vectors can be derived from lentivirus, poxviruses, herpes simplex virus I, adenoviruses and adeno-associated viruses, and the like. See, for example, U.S. Pat. Nos. 5,350,674 and 5,585,362.
- Chemical means for introducing a polynucleotide into a host cell include colloidal dispersion systems, such as macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes.
- An exemplary colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (e.g., an artificial membrane vesicle).
- an exemplary delivery vehicle is a liposome.
- the use of lipid formulations is contemplated for the introduction of the nucleic acids into a host cell (in vitro, ex vivo or in vivo).
- the nucleic acid may be associated with a lipid.
- the nucleic acid associated with a lipid may be encapsulated in the aqueous interior of a liposome, interspersed within the lipid bilayer of a liposome, attached to a liposome via a linking molecule that is associated with both the liposome and the oligonucleotide, entrapped in a liposome, complexed with a liposome, dispersed in a solution containing a lipid, mixed with a lipid, combined with a lipid, contained as a suspension in a lipid, contained or complexed with a micelle, or otherwise associated with a lipid.
- Lipid, lipid/DNA or lipid/expression vector associated compositions are not limited to any particular structure in solution. For example, they may be present in a bilayer structure, as micelles, or with a “collapsed” structure. They may also simply be interspersed in a solution, possibly forming aggregates that are not uniform in size or shape.
- Lipids are fatty substances which may be naturally occurring or synthetic lipids.
- lipids include the fatty droplets that naturally occur in the cytoplasm as well as the class of compounds which contain long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.
- Lipids suitable for use can be obtained from commercial sources.
- DMPC dimyristyl phosphatidylcholine
- DCP dicetyl phosphate
- Choi cholesterol
- DMPG dimyristyl phosphatidylglycerol
- Stock solutions of lipids in chloroform or chloroform/methanol can be stored at about ⁇ 20° C.
- Liposome is a generic term encompassing a variety of single and multilamellar lipid vehicles formed by the generation of enclosed lipid bilayers or aggregates. Liposomes can be characterized as having vesicular structures with a phospholipid bilayer membrane and an inner aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution.
- compositions that have different structures in solution than the normal vesicular structure are also encompassed.
- the lipids may assume a micellar structure or merely exist as nonuniform aggregates of lipid molecules.
- lipofectamine-nucleic acid complexes are also contemplated.
- assays include, for example, “molecular biological” assays well known to those of skill in the art, such as Southern and Northern blotting, RT-PCR and PCR; “biochemical” assays, such as detecting the presence or absence of a particular peptide, e.g., by immunological means (ELISAs and Western blots) or by assays described herein to identify agents falling within the scope of the disclosure.
- molecular biological assays well known to those of skill in the art, such as Southern and Northern blotting, RT-PCR and PCR
- biochemical assays, such as detecting the presence or absence of a particular peptide, e.g., by immunological means (ELISAs and Western blots) or by assays described herein to identify agents falling within the scope of the disclosure.
- the present disclosure includes a composition comprising a host cell which comprises a peptide of the disclosure, a nucleic acid encoding a peptide of the disclosure, or a combination thereof.
- the term “host cell” comprises prokaryotic cells and eukaryotic cells.
- Exemplary prokaryotic host cells include E. coli, Bacillus subtilis , etc.
- Exemplary eukaryotic cells include yeast cells, insect cells, mammal cells, etc.
- the cell is genetically modified to comprise a peptide and/or nucleic acid of the disclosure.
- genetically modified cell is autologous to a subject being treated with the composition of the invention.
- the cells can be allogeneic, syngeneic, or xenogeneic with respect to the subject.
- the cell is able to secrete or release the expressed peptide of the invention into extracellular space in order to deliver the peptide to one or more other cells.
- the genetically modified cell may be modified in vivo or ex vivo, using techniques standard in the art. Genetic modification of the cell may be carried out using an expression vector or using a naked isolated nucleic acid construct. In one embodiment, the cell is obtained and modified ex vivo, using an isolated nucleic acid encoding a peptide. In one embodiment, the cell is obtained from a subject, genetically modified to express the peptide and/or nucleic acid, and is re-administered to the subject. In certain embodiments, the cell is expanded ex vivo or in vitro to produce a population of cells, wherein at least a portion of the population is administered to a subject in need. In one embodiment, the cell is genetically modified to stably express the peptide. In another embodiment, the cell is genetically modified to transiently express the peptide.
- the present disclosure provides a scaffold or substrate composition comprising a peptide of the disclosure, an isolated nucleic acid of the disclosure, a host cell comprising the peptide or isolated nucleic acid fragment of the disclosure, or a combination thereof.
- a peptide of the disclosure, an isolated nucleic acid of the disclosure, a host cell producing the peptide of the disclosure, or a combination thereof is incorporated within a scaffold.
- a peptide of the disclosure, an isolated nucleic acid of the disclosure, a host cell producing the peptide of the disclosure, or a combination thereof is applied to the surface of a scaffold.
- the scaffold of the disclosure can be of any type known in the art. Non-limiting examples of such a scaffold includes a, hydrogel, electrospun scaffold, foam, mesh, sheet, patch, and sponge.
- compositions and methods of the present disclosure contemplate use of modified peptides, so long as the modified peptide, retains, the biological activity of the wild type unmodified peptide e.g., binding CD40.
- the peptide is altered.
- altered polypeptide refers to a peptide that includes alterations, such as deletions, additions, and substitutions (generally conservative in nature as would be known to a person in the art, such as alanines), to the wild type sequence, as long as the peptide retains the desired biological activity, i.e., binding CD40, or inhibiting interaction of CD40 and CD154, and/or modulating immune responses directed by interaction of CD40 and CD154.
- modifications can be deliberate, as through site-directed mutagenesis, or can be accidental, such as through mutations of artificial hosts, such as genetically engineered bacteria, yeast or mammalian cells, that produce the proteins, or errors due to PCR amplification or other recombinant DNA methods.
- a peptide of the instant disclosure is a “modified peptide” comprising non-naturally occurring amino acids.
- the peptides comprise a combination of naturally occurring and non-naturally occurring amino acids, and in some embodiments, the peptides comprise only non-naturally occurring amino acids.
- Modified peptide may include the incorporation of non-natural amino acids into the peptides of the disclosure, including synthetic non-native amino acids, substituted amino acids, or one or more D-amino acids into the peptides (or other components of the composition, with exception for protease recognition sequences) is desirable in certain situations.
- D-amino acid-containing peptides exhibit increased stability in vitro or in vivo compared to L-amino acid-containing forms.
- the construction of peptides incorporating D-amino acids can be particularly useful when greater in vivo or intracellular stability is desired or required.
- D-peptides are resistant to endogenous peptidases and proteases, thereby providing better oral trans-epithelial and transdermal delivery of linked drugs and conjugates, improved bioavailability of membrane-permanent complexes (see below for further discussion), and prolonged intravascular and interstitial lifetimes when such properties are desirable.
- the use of D-isomer peptides can also enhance transdermal and oral trans-epithelial delivery of linked drugs and other cargo molecules.
- D-peptides cannot be processed efficiently for major histocompatibility complex class Il-restricted presentation to T helper cells, and are therefore less likely to induce humoral immune responses in the whole organism.
- Peptide conjugates can therefore be constructed using, for example, D-isomer forms of cell penetrating peptide sequences, L-isomer forms of cleavage sites, and D-isomer forms of therapeutic peptides.
- the peptides as disclosed comprise L and D amino acids, wherein no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 D-amino acids are included.
- the peptides comprise more than 10 D-amino acids, and in certain aspects all the amino acids of the peptides are D-amino acids.
- peptides which have been modified using ordinary molecular biological techniques so as to improve their resistance to proteolytic degradation or to optimize solubility properties or to render them more suitable as a therapeutic agent.
- Such variants include those containing residues other than naturally-occurring L-amino acids, e.g., D-amino acids or non-naturally-occurring synthetic amino acids.
- the peptides disclosed herein may include unnatural amino acids formed by post-translational modification or by introducing unnatural amino acids during translation. A variety of approaches are available for introducing unnatural amino acids during protein translation.
- tRNAs which have suppressor properties
- suppressor tRNAs have been used in the process of site-directed non-native amino acid replacement (SNAAR).
- SNAAR site-directed non-native amino acid replacement
- a unique codon is required on the mRNA and the suppressor tRNA, acting to target a non-native amino acid to a unique site during the protein synthesis (described in WO90/05785).
- the suppressor tRNA must not be recognizable by the aminoacyl tRNA synthetases present in the protein translation system.
- a non-native amino acid can be formed after the tRNA molecule is aminoacylated using chemical reactions which specifically modify the native amino acid and do not significantly alter the functional activity of the aminoacylated tRNA. These reactions are referred to as post-aminoacylation modifications.
- post-aminoacylation modifications For example, the epsilon-amino group of the lysine linked to its cognate tRNA (tRNALYs), could be modified with an amine specific photoaffinity label.
- a peptide of the composition and methods disclosed herein is in a cyclic form.
- the cyclic peptide comprises a linkage between amino acids.
- Cyclic derivatives of the peptides disclosed herein are also part of the present disclosure. Cyclization may allow the peptide to assume a more favorable conformation for association with other molecules. Cyclization may be achieved using techniques known in the art. For example, disulfide bonds may be formed between two appropriately spaced components having free sulfhydryl groups, or an amide bond may be formed between an amino group of one component and a carboxyl group of another component.
- Cyclization may also be achieved using an azobenzene-containing amino acid as described by Ulysse, L., et al, J. Am. Chem. Soc. 1995, 117, 8466-8467.
- the components that form the bonds may be side chains of amino acids, non-amino acid components or a combination of the two.
- cyclic peptides may comprise a beta-turn in the right position. Beta-turns may be introduced into the peptides of the invention by adding the amino acids Pro-Gly at the right position.
- a more flexible peptide may be prepared by introducing cysteines at the right and left position of the peptide and forming a disulphide bridge between the two cysteines.
- the two cysteines are arranged so as not to deform the beta-sheet and turn.
- the peptide is more flexible as a result of the length of the disulfide linkage and the smaller number of hydrogen bonds in the beta-sheet portion.
- the relative flexibility of a cyclic peptide can be determined by molecular dynamics simulations.
- the peptides of the invention may be converted into pharmaceutical salts by reacting with inorganic acids such as hydrochloric acid, sulfuric acid, hydrobromic acid, phosphoric acid, etc., or organic acids such as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, succinic acid, malic acid, tartaric acid, citric acid, benzoic acid, salicylic acid, benezenesulfonic acid, and toluenesulfonic acids.
- inorganic acids such as hydrochloric acid, sulfuric acid, hydrobromic acid, phosphoric acid, etc.
- organic acids such as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, succinic acid, malic acid, tartaric acid, citric acid, benzoic acid, salicylic acid, benezenesulfonic acid, and toluenesulf
- the peptides or fragments or derivatives thereof can be “retro-inverso peptides.”
- a “retro-inverso peptide” refers to a peptide with a reversal of the direction of the peptide bond on at least one position, i.e., a reversal of the amino- and carboxy-termini with respect to the side chain of the amino acid.
- a retro-inverso analogue has reversed termini and reversed direction of peptide bonds while approximately maintaining the topology of the side chains as in the native peptide sequence.
- the retro-inverso peptide can contain L-amino acids or D-amino acids, or a mixture of L-amino acids and D-amino acids, up to all of the amino acids being the D-isomer.
- Partial retro-inverso peptide analogues are polypeptides in which only part of the sequence is reversed and replaced with enantiomeric amino acid residues. Since the retro-inverted portion of such an analogue has reversed amino and carboxyl termini, the amino acid residues flanking the retro-inverted portion are replaced by side-chain-analogous a-substituted geminal-diaminomethanes and malonates, respectively.
- Retro-inverso forms of cell penetrating peptides have been found to work as efficiently in translocating across a membrane as the natural forms.
- Synthesis of retro-inverso peptide analogues are described in Bonelli, F. et al., Int J Pept Protein Res. 24(6):553-6 (1984); Verdini, A and Viscomi, G. C, J. Chem. Soc. Perkin Trans. 1:697-701 (1985); and U.S. Pat. No. 6,261,569, which are incorporated herein in their entirety by reference.
- Processes for the solid-phase synthesis of partial retro-inverso peptide analogues have been described (EP 97994-B) which is also incorporated herein in its entirety by reference.
- the peptides of the disclosure comprise an N-terminal and/or C-terminal modifications. Such modification can that in certain instances improve activity.
- the peptide of the disclosure comprises a butyric acid at the N-terminus of the peptide.
- the peptide is PEGylated at the C-terminus of the peptide.
- the carboxy terminus of the peptide of the present disclosure is amidated.
- the present disclosure encompasses variants of the peptide analogs, including those with terminal modifications, without terminal modifications, or having different terminal modifications.
- a peptide of the present disclosure can have, for example, a butyric acid at the N-terminus and is PEGylated and/or amidated at the C-terminus.
- the present disclosure also encompasses analogs and derivatives of peptides disclosed herein, including peptides having different terminal modifications or no terminal modifications.
- a peptide or compositions disclosed herein can be PEGylated.
- PEGylation is considered as a gold standard for chemical modifications of peptides. It involves one or more polyethylene glycol (PEG) chains linked to a protein, peptide or non-peptide molecule. PEGylation is the term given to conjugation with monomethoxy poly (ethyleneglycol) or mPEG.
- PEG polymer, a polyether compound, or a polymer of ethylene oxide is highly soluble in water.
- the peptides of the disclosure may further be conjugated to non-amino acid moieties that are useful in their therapeutic application.
- moieties that improve the stability, biological half-life, water solubility, and/or immunologic characteristics of the peptide are useful.
- a non-limiting example of such a moiety is polyethylene glycol (PEG).
- a stable bond is formed between PEG and polypeptide.
- an active PEG derivative with a functional group—such as active ester, active carbonate, tresylate or aldehyde suitable for coupling to a given peptide Preferred positions for PEGylation are the N-terminal amino group of the polypeptide backbone and the ⁇ -amino group in the side chain of the amino acid residue lysine. Being highly labile, mild chemical reaction conditions are used by proteins and peptides for the conjugation at the hydrophobic sites.
- Polysialic acid is highly hydrophilic in nature which increases the systemic circulation. It has the advantage of being biodegradable and its catabolic products are not toxic (e.g. NeuNAc). It markedly reduces proteolysis, prolongs the half life in systemic circulation (up to 40 h) and retains the activity of protein and peptide in-vivo. It also reduces immunogenicity and antigenicity.
- Polysialic acids are linear polymers of N-acetylneuraminic acid (sialic acid) abundantly present on the surface of cells and many proteins. Polysialylation has been tested and are used in therapeutics for the treatment of various diseases. It includes preservation of stability and function, optimal pharmacokinetics and pharmacodynamics, and
- Covalent attachment of biologically active compounds to water-soluble polymers is one method for alteration and control of biodistribution, pharmacokinetics, and often, toxicity for these compounds (Duncan et al, 1984, Adv. Polym. Sci. 57:53-101).
- PEG-conjugated or “PEGylated” protein therapeutics containing single or multiple chains of polyethylene glycol on the protein, have been described in the scientific literature (Clark et al., 1996, J. Biol. Chem. 271: 21969-21977; Hershfield, 1997, Biochemistry and immunology of poly(ethylene glycol)-modified adenosine deaminase (PEG-ADA). In J. M. Harris and S.
- the peptides or modified peptides further comprise co-translational and post-translational (C-terminal peptide cleavage) modifications, such as, for example, disulfide-bond formation, glycosylation, acetylation, phosphorylation, proteolytic cleavage (e.g., cleavage by furins or metalloproteases), and the like.
- co-translational and post-translational (C-terminal peptide cleavage) modifications such as, for example, disulfide-bond formation, glycosylation, acetylation, phosphorylation, proteolytic cleavage (e.g., cleavage by furins or metalloproteases), and the like.
- the main post-translational modifications associated with peptides are: Acetylation, acylation, ADP-ribosylation, Amidation, ⁇ -Carboxylation and ⁇ -hydroxylation, Disulfide bond formation, Glycosylation, Phosphorylation, Proteolytic processing, Sulfation, Methylation, Acyl Lipidation.
- a peptide modification includes phosphorylation (e.g., on a Tyr, Ser or Thr residue), N-terminal acetylation, C-terminal amidation, C-terminal hydrazide, C-terminal methyl ester, fatty acid attachment, sulfonation (tyrosine), N-terminal dansylation, N-terminal succinylation, tripalmitoyl-S-Glyceryl Cysteine (PAMb 3 Cys-OH) as well as famesylation of a Cys residue.
- Systematic chemical modification of a peptide can, for example, be performed in the process of peptide optimalization.
- post-translational modifications that fall within the scope of the present disclosure include signal peptide cleavage, glycosylation, acetylation, isoprenylation, proteolysis, myristoylation, protein folding and proteolytic processing, etc.
- Some modifications or processing events require introduction of additional biological machinery.
- processing events such as signal peptide cleavage and core glycosylation, are examined by adding canine microsomal membranes or Xenopus egg extracts (U.S. Pat. No. 6,103,489) to a standard translation reaction.
- Modifications (which do not normally alter primary sequence) include in vivo, or in vitro chemical derivatization of polypeptides, e.g., acetylation, or carboxylation.
- glycosylation e.g., those made by modifying the glycosylation patterns of a polypeptide during its synthesis and processing or in further processing steps; e.g., by exposing the polypeptide to enzymes which affect glycosylation, e.g., mammalian glycosylating or deglycosylating enzymes.
- enzymes which affect glycosylation e.g., mammalian glycosylating or deglycosylating enzymes.
- sequences which have phosphorylated amino acid residues e.g., phosphotyrosine, phosphoserine, or phosphothreonine.
- fusion protein refers to a recombinant protein of two or more proteins. Fusion proteins can be produced, for example, by a nucleic acid sequence encoding one protein is joined to the nucleic acid encoding another protein such that they constitute a single open-reading frame that can be translated in the cells into a single polypeptide harboring all the intended proteins. The order of arrangement of the proteins can vary.
- Fusion proteins can include an epitope tag or a half-life extender.
- Epitope tags include biotin, FLAG tag, c-myc, hemaglutinin, His6, digoxigenin, FITC, Cy3, Cy5, green fluorescent protein, V5 epitope tags, GST, ⁇ -galactosidase, AU1, AUS, and avidin.
- Half-life extenders include Fc domain and serum albumin. Accordingly, in some embodiments, the peptides disclosed herein can be conjugated to a peptide tag.
- a peptide of the present disclosure further comprises a peptide tag.
- Tolbert et al used a betaine moiety, a small molecule, containing a positively charged quaternary ammonium group (MW-100) is introduced as a small solubility enhancement tag onto the N-terminus of polypeptides by chemical ligation or expressed protein ligation.
- the modified forms of polypeptides are more water soluble.
- Modification with small ubiquitin related modifier (SUMO) has the ability to enhance protein expression and solubility.
- a peptide of the present disclosure can be conjugated to a detectable agent.
- detectable agent as used herein that is conjugated directly or indirectly to a probe (e.g., peptide) to generate a “labeled” probe.
- the label may be detectable by itself (e.g. radioisotope labels, fluorescent agent or chemiluminescent agent) or, in the case of an enzymatic label, may catalyze chemical alteration of a substrate compound or composition that is detectable (e.g., avidin-biotin).
- primers can be labeled to detect a PCR product.
- the peptides of the compositions and methods disclosed herein is modified by linkage to a carrier peptide.
- conjugated are used to refer to any method known in the art for functionally connecting moieties (such as detectable agent, carrier peptide), including, without limitation, recombinant fusion, covalent bonding, disulfide bonding, ionic bonding, hydrogen bonding, and electrostatic bonding.
- carrier peptide indicates peptides that transport a specific substance or group of substances (e.g., peptides of the present disclosure) through intracellular compartments or in extracellular fluids (e.g. in the blood) or else across the cell membrane.
- Exemplary carrier proteins comprise subunit B of cholera toxin, Avidin, BTG protein, Bovine G globulin, Bovine Immunoglobulin G, Bovine Thyroglobulin, Bovine Serum Albumin (BSA), Conalbumin, Edestein, Exoprotein A from Pseudomonas aeruginosa , HC (Hemocyanin from crab Paralithodes camtschatica), Helix Promatia Haemocyanin (HPH), Human Serum Albumin (HSA), KTI (Kunits trypsin inhibitor from soybeans), Keyhole Limpet Heamocyanin (KLH), LPH (Haemocyanin from Limulus polyphemus ) Ovalbumin, Pam3Cys-Th, Polylysine, porcine Thyroglobulin (PTG), Purified Protein Derivative (PPD), Rabbit Serum Albumin (RSA), Soybeab Trypsin Inhibitor (STI) (Sunflower Globulin (SF
- a peptide of the compositions and methods disclosed herein is fused to a carrier peptide.
- attachment of the carrier is performed at the C-terminus or N-terminus of the fragment.
- the fusion protein i.e., peptides of the present disclosure and a carrier peptide
- the fusion protein can be provided as a single polypeptide through recombinant DNA technology and related processes, such as cloning, chimeric constructs, Polymerase Chain Reaction and additional procedures identifiable by a skilled person.
- attachment can be performed through chemical linkage of the fragment to the carrier using methods also identifiable by a skilled person.
- Non-limiting examples of such transportable peptides, or vectors, suitable for coupling to the pharmaceutical agent include transferrin, insulin-like growth factors I and II, basic albumin and prolactin.
- the conjugation can be carried out using bifunctional reagents which are capable of reacting with each of the polypeptides and forming a bridge between the two.
- the preferred method of conjugation involves polypeptide thiolation, wherein the two polypeptides are treated with a reagent such as N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP) to form a disulfide bridge between the two polypeptides.
- SPDP N-succinimidyl 3-(2-pyridyldithio)propionate
- conjugation agents can be used, so long as they provide linkage of the two polypeptides (i.e. therapeutic polypeptide drug and the transportable peptide) together without denaturing them. Preferably, the linkage can be easily broken once the chimeric polypeptide has entered the region of interest.
- the peptides of the present disclosure can be linked to short cell penetrating peptides, which have the ability to cross cell membrane bilayers. Non limiting examples of such peptides include TAT (HIV-1 transactivating transcriptor) SynB3, Tat, transportan.
- Variants of suitable peptides of the invention can also be expressed. Variants may be made by, for example, the deletion, addition, or alteration of amino acids that have either (i) minimal influence on certain properties, secondary structure, and hydropathic nature of the polypeptide or (ii) substantial effect on one or more properties of the peptide mimetics of the invention. Variants may also include, for example, a peptide conjugated to a linker or other sequence for ease of synthesis, purification, identification, or therapeutic use (i.e., delivery) of the peptide.
- the variants of the peptides according to the present invention may be (i) one in which one or more of the amino acid residues are substituted with a conserved or non-conserved amino acid residue (preferably a conserved amino acid residue) and such substituted amino acid residue may or may not be one encoded by the genetic code, (ii) one in which there are one or more modified amino acid residues, e.g., residues that are modified by the attachment of substituent groups, (iii) one in which the peptide is an alternative splice variant of the peptide of the present invention, (iv) fragments of the peptides and/or (v) one in which the peptide is fused with another peptide, such as a leader or secretory sequence or a sequence which is employed for purification (for example, His-tag) or for detection (for example, Sv5 epitope tag).
- a conserved or non-conserved amino acid residue preferably a conserved amino acid residue
- the fragments include peptides generated via proteolytic cleavage (including multi-site proteolysis) of an original sequence. Variants may be post-translationally, or chemically modified. Such variants are deemed to be within the scope of those skilled in the art from the teaching herein.
- the peptides disclosed herein can comprise a lipophilic molecular group.
- lipophilic molecular group refers to a lipid moiety, such as a fatty acid, glyceride or phospholipid which when coupled to a therapeutic molecule, increases its lipophilicity.
- the lipophilic molecular group can be attached to the therapeutic molecule through an ester bond.
- Lipophilic molecular groups can comprise lipid moieties such as fatty acid, glyceride or phospholipids.
- Lipidization can involve acylation of the ⁇ -amino group of lysine residues or acylation of N-terminal ⁇ -amino groups.
- the presence of a lipid group in peptides changes their secondary structures, hydrophobicity and self-assembling propensities but retains its ability to bind to target receptors.
- Reversible Aqueous lipidization technology This is a new method for developing fatty acids-peptide conjugates which is carried out in aqueous solution and can regenerate the original active peptides in tissues or the blood and thus called REAL Technology. Lipidization can be carried out at cysteine residues following cleavage of intra-disulfide bonds with a reducing agent. This type of lipidization is typically reversible. Methods of REAL are described in, for example, Shen et al. In some embodiments, a modification combining site specific modification and lipidization technique is carried out, e.g., Chang et. al. L Pharm Sci 2002.
- modification of a peptide disclosed herein is a modification by adding an opposite charged surfactant that binds to peptide to obtain neutral hydrophobicity entity.
- Solubility of ionic compounds is high due to ease of solvation of the counter ions with ionic detergents. Positively charged peptides and negatively charged surfactants should be employed, since cationic surfactants might have toxic side effects.
- Replacement of counter ions decreases the aqueous solubility and increases the lipophilicity. Thus, it changes the partition coefficient by orders of magnitude. HIP method is inexpensive and reversible. The process involves replacement of counter ions (e.g. chloride, acetate, nitrate) with an ionic detergent of similar charge.
- counter ions e.g. chloride, acetate, nitrate
- ion-pairing is a useful method for increasing the bioavailability of drugs and enhancing permeation of certain drugs.
- Dai et al. (Int. J. Pharm. 2007; 336: 58-66), Sun et al. (Int. J. Nanomed, 2011) prepared insulin hydrophobic ion paired complex using different anionic surfactants like sodium lauryl sulphate, surfoplex, sodium deoxycholate and sodium oleate respectively in 1:6 ratio.
- peptides disclosed herein can be modified by attaching one or more amphiphilic oligomers, for example, Nobex Technology. Creation of amphiphilic oligomers is accomplished by binding a lipophilic alkyl unit to a hydrophilic polyethylene glycol (PEG) unit. The resulting oligomer is then covalently bound to a specific site or sites on the target molecule to facilitate the desired changes in properties of the peptide.
- PEG polyethylene glycol
- the two types of bonds are involved in this technology are hydrolyzable bond at the site of attachment, and non-hydrolyzable bond to create a ‘micro-pegylated’ peptide. Methods for this modification are described in, for example, Still J G. et al. Diabetes Metab. Res. Rev.
- the peptide disclosed herein can be modified by contacting with small hydrophobic organic compounds non-covalently.
- Emisphere'seligenTM technology See Singh B et al. Oral Delivery of Therapeutic Macromolecules: A Perspective Using the EligenTM Technology; 2014.
- compositions comprising peptides or portion thereof capable of biding CD40 and methods of their use.
- the compositions disclosed herein are formulated for administration via intracerebroventricular, intravenous, intradermal, intraperitoneal, oral, intramuscular, subcutaneous, intranasal, intracranial, intracelial, intracerebellar, intrathecal, transdermal, pulmonary, or topical administration route.
- compositions provided herein can be prepared in a variety of ways depending on the intended use of the compositions.
- a composition useful in practicing the technology herein may be a liquid comprising a peptide disclosed herein in solution, in suspension, or both (solution/suspension).
- solution/suspension refers to a liquid composition where a first portion of the active agent is present in solution and a second portion of the active agent is present in particulate form, in suspension in a liquid matrix.
- a liquid composition also includes a gel.
- the liquid composition may be aqueous or in the form of an ointment, salve, cream, or the like.
- An aqueous suspension or solution/suspension useful for practicing the methods disclosed herein may contain one or more polymers as suspending agents.
- Useful polymers include water-soluble polymers such as cellulosic polymers and water-insoluble polymers such as cross-linked carboxyl-containing polymers.
- An aqueous suspension or solution/suspension of the present disclosure can be viscous or muco-adhesive, or both viscous and muco-adhesive.
- the pharmaceutical compositions herein comprise a pharmaceutically acceptable carrier.
- pharmaceutically acceptable means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly, in humans.
- phrases “pharmaceutically acceptable carrier” as used herein means a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent, media, encapsulating material, manufacturing aid (e.g., lubricant, talc magnesium, calcium or zinc stearate, or steric acid), or solvent encapsulating material, involved in maintaining the stability, solubility, or activity of, active agents in the compositions.
- manufacturing aid e.g., lubricant, talc magnesium, calcium or zinc stearate, or steric acid
- solvent encapsulating material involved in maintaining the stability, solubility, or activity of, active agents in the compositions.
- Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient.
- materials which can serve as pharmaceutically-acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, methylcellulose, ethyl cellulose, microcrystalline cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) excipients, such as cocoa butter and suppository waxes; (8) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (9) glycols, such as propylene glycol; (10) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol (PEG); (11) esters, such as ethyl oleate and ethyl laurate; (12) agar; (13) buffering agents, such as magnesium hydro
- Release agents, coating agents, preservatives, and antioxidants can also be present in the formulation.
- excipient carrier
- pharmaceutically acceptable carrier or the like are used interchangeably herein. Examples of suitable pharmaceutical carriers are described in “Remington's Pharmaceutical Sciences” by E. W. Martin, and still others are familiar to skilled artisans.
- compositions of the present disclosure can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like, including those adapted for the following: (1) parenteral administration, for example, by subcutaneous, intramuscular, intravenous or epidural injection as, for example, a sterile solution or suspension, or sustained-release formulation; (2) topical application, for example, as a cream, ointment, or a controlled-release patch or spray applied to the skin; (3) intravaginally or intrarectally, for example, as a pessary, cream or foam; (4) ocularly; (5) transdermally; (6) transmucosally; or (7) nasally.
- parenteral administration for example, by subcutaneous, intramuscular, intravenous or epidural injection as, for example, a sterile solution or suspension, or sustained-release formulation
- topical application for example, as a cream, ointment, or a controlled-release patch or spray applied to the
- compositions of the invention can be formulated as neutral or salt forms.
- Pharmaceutically acceptable salts include those formed with free amino groups such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with free carboxyl groups such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc.
- compositions disclosed herein can comprise a preservative.
- a “preservative” is a compound which can be added to the diluent to essentially reduce bacterial action in the reconstituted formulation, thus facilitating the production of a multi-use reconstituted formulation, for example.
- potential preservatives include octadecyldimethylbenzyl ammonium chloride, hexamethonium chloride, benzalkoniurn chloride (a mixture of alkylbenzyldimethylammonium chlorides in which the alkyl groups are long-chain compounds), and benzethonium chloride.
- preservatives include aromatic alcohols such as phenol, butyl and benzyl alcohol, alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol.
- aromatic alcohols such as phenol, butyl and benzyl alcohol
- alkyl parabens such as methyl or propyl paraben
- catechol resorcinol
- cyclohexanol 3-pentanol
- m-cresol m-cresol
- the most preferred preservative herein is benzyl alcohol.
- One important purpose of the present formulation is to stabilize these peptide or analogue against spontaneous dimerization. This is preferably accomplished by formulating the peptides at low pH that will prevent disulfide bond formation between two Cys residues.
- acid buffers are preferably biocompatible.
- Examples include citrate, acetate, 2-(N-morpholino)ethanesulfonic acid (MES), or any similar buffer with a pK of about 5, wherein in the presence of the buffer, the pH of the solution is ⁇ 7.5 but preferably not below 3.0.
- a preferred acidic formulation comprises citrate, preferably at about 25 mM.
- the buffer is preferably supplemented with glycine (GIy) as an excipient and bulking agent.
- GIy glycine
- a preferred concentration is about 50 mg/ml GIy.
- Another advantage of GIy is that it is an accepted excipient for intravenous infusion or injection in humans.
- Other amino acids or compounds can be used in place of GIy. Examples of desirable acids or combinations are citrate+acetate, acetate and Tris, and the like.
- a pharmaceutical composition of the disclosure is formulated to be compatible with its intended route of administration.
- routes of administration include, but are not limited to, parenteral, e.g., intravenous, intradermal, subcutaneous, oral, intranasal (e.g., inhalation), transdermal (e.g., topical), transmucosal, and rectal administration.
- the composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous, subcutaneous, intramuscular, oral, intranasal, or topical administration to human beings.
- compositions for intravenous administration are solutions in sterile isotonic aqueous buffer.
- the composition may also include a solubilizing agent and a local anesthetic such as lignocaine to ease pain at the site of the injection.
- compositions of the disclosure are to be administered topically, the compositions can be formulated in the form of an ointment, cream, transdermal patch, lotion, gel, shampoo, spray, aerosol, solution, emulsion, or other form well-known to one of skill in the art. See, e.g., Remington's Pharmaceutical Sciences and Introduction to Pharmaceutical Dosage Forms, 19th ed., Mack Pub. Co., Easton, Pa. (1995).
- viscous to semi-solid or solid forms comprising a carrier or one or more excipients compatible with topical application and having a dynamic viscosity preferably greater than water are typically employed.
- Suitable formulations include, without limitation, solutions, suspensions, emulsions, creams, ointments, powders, liniments, salves, and the like, which are, if desired, sterilized or mixed with auxiliary agents (e.g., preservatives, stabilizers, wetting agents, buffers, or salts) for influencing various properties, such as, for example, osmotic pressure.
- auxiliary agents e.g., preservatives, stabilizers, wetting agents, buffers, or salts
- Other suitable topical dosage forms include sprayable aerosol preparations wherein the active ingredient, preferably in combination with a solid or liquid inert carrier, is packaged in a mixture with a pressurized volatile (e.g., a gaseous propellant, such as freon) or in a squeeze bottle.
- a pressurized volatile e.g., a gaseous propellant, such as freon
- humectants can also be added to pharmaceutical composition
- compositions disclosed herein are to be formulated for intranasal administration of a composition
- the composition can be formulated in an aerosol form, spray, mist or in the form of drops.
- prophylactic or therapeutic agents for use according to the present invention can be conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable propellant (e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas).
- a suitable propellant e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
- the dosage unit may be determined by providing a valve to deliver a metered amount.
- Capsules and cartridges for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.
- compositions can be formulated orally in the form of tablets, capsules, cachets, gel caps, solutions, suspensions, and the like.
- Tablets or capsules can be prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g., pregelatinized maize starch, polyvinylpyrrolidone, or hydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystalline cellulose, or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc, or silica); disintegrants (e.g., potato starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulphate).
- binding agents e.g., pregelatinized maize starch, polyvinylpyrrolidone, or hydroxypropyl methylcellulose
- fillers e.g., lactose, microcrystalline cellulose, or calcium hydrogen phosphate
- lubricants e.
- Liquid preparations for oral administration may take the form of, but not limited to, solutions, syrups or suspensions, or they may be presented as a dry product for constitution with water or other suitable vehicle before use.
- Such liquid preparations may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives, or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters, ethyl alcohol, or fractionated vegetable oils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid).
- the preparations may also contain buffer salts, flavoring, coloring, and sweetening agents as appropriate.
- Preparations for oral administration may be suitably formulated for slow release, controlled release, or sustained release of a prophylactic or therapeutic agent(s).
- the method of the disclosure may comprise pulmonary administration, e.g., by use of an inhaler or nebulizer, of a composition formulated with an aerosolizing agent.
- pulmonary administration e.g., by use of an inhaler or nebulizer
- a composition formulated with an aerosolizing agent See, e.g., U.S. Pat. Nos. 6,019,968, 5,985,320, 5,985,309, 5,934,272, 5,874,064, 5,855,913, 5,290,540, and 4,880,078; and PCT Publication Nos. WO 92/19244, WO 97/32572, WO 97/44013, WO 98/31346, and WO 99/66903, each of which is incorporated herein by reference their entireties.
- an antibody of the invention, combination therapy, and/or composition of the invention is administered using Alkermes AIR pulmonary drug delivery technology (Alkermes, Inc., Cambridge, Mass.).
- the method of the disclosure may comprise administration of a composition formulated for parenteral administration by injection (e.g., by bolus injection or continuous infusion).
- Formulations for injection may be presented in unit dosage form (e.g., in ampoules or in multi-dose containers) with an added preservative.
- the compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
- the active ingredient may be in powder form for constitution with a suitable vehicle (e.g., sterile pyrogen-free water) before use.
- compositions formulated as depot preparations may additionally comprise of administration of compositions formulated as depot preparations.
- long acting formulations may be administered by implantation (e.g., subcutaneously or intramuscularly) or by intramuscular injection.
- the compositions may be formulated with suitable polymeric or hydrophobic materials (e.g., as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives (e.g., as a sparingly soluble salt).
- compositions formulated as neutral or salt forms include those formed with anions such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with cations such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc.
- compositions are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent.
- a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent.
- composition can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline.
- an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.
- the disclosure also provides that one or more of the peptides or pharmaceutical compositions of the disclosure is packaged in a hermetically sealed container such as an ampoule or sachette indicating the quantity of the agent.
- a hermetically sealed container such as an ampoule or sachette indicating the quantity of the agent.
- one or more of the peptides, or pharmaceutical compositions of the disclosure is supplied as a dry sterilized lyophilized powder or water free concentrate in a hermetically sealed container and can be reconstituted (e.g., with water or saline) to the appropriate concentration for administration to a subject.
- one or more of the peptides or pharmaceutical compositions of the disclosure is supplied as a dry sterile lyophilized powder in a hermetically sealed container at a unit dosage of at least 5 mg, more preferably at least 10 mg, at least 15 mg, at least 25 mg, at least 35 mg, at least 45 mg, at least 50 mg, at least 75 mg, or at least 100 mg.
- the lyophilized peptides or pharmaceutical compositions of the invention should be stored at between 2° C. and 8° C.
- the peptides, or pharmaceutical compositions of the disclosure should be administered within 1 week, preferably within 5 days, within 72 hours, within 48 hours, within 24 hours, within 12 hours, within 6 hours, within 5 hours, within 3 hours, or within 1 hour after being reconstituted.
- one or more of the peptides or pharmaceutical compositions of the disclosure is supplied in liquid form in a hermetically sealed container indicating the quantity and concentration of the agent.
- the liquid form of the administered composition is supplied in a hermetically sealed container at least 0.25 mg/ml, more preferably at least 0.5 mg/ml, at least 1 mg/ml, at least 2.5 mg/ml, at least 5 mg/ml, at least 8 mg/ml, at least 10 mg/ml, at least 15 mg/ml, at least 25 mg/ml, at least 50 mg/ml, at least 75 mg/ml or at least 100 mg/ml.
- the liquid form should be stored at between 2° C. and 8° C. in its original container.
- compositions can be administered in various ways, depending on the preference for local or systemic treatment, and on the area to be treated. Administration may be done topically (including ophthalmically, vaginally, rectally, intranasally), orally, by inhalation, or parenterally, for example by intravenous drip or intraperitoneal, subcutaneous, subdural, intramuscular or intravenous injection, or via an implantable delivery device.
- Formulations for topical administration may include, but are not limited to, lotions, ointments, gels, creams, suppositories, drops, liquids, sprays and powders Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable.
- compositions for oral administration include powders or granules, suspensions or solutions in water or nonaqueous media, sachets, capsules or tablets. Thickeners, diluents, flavorings, dispersing aids, emulsifiers or binders may be desirable.
- Formulations for parenteral administration may include, but are not limited to, sterile solutions, which may also contain buffers, diluents and other suitable additives.
- Formulations for implantable delivery devices may similarly include, but are not limited to, sterile solutions, which may also contain buffers, diluents and other suitable additives.
- compositions as described above is in a pharmaceutically acceptable medium suitable for administration to a recipient subject.
- Pharmaceutically acceptable mediums suitable for administration to a subject are known in the art.
- compositions disclosed herein can be conveniently provided as sterile liquid preparations, e.g., isotonic aqueous solutions, suspensions, emulsions, dispersions, or viscous compositions, which may be buffered to a selected pH. Liquid preparations are normally easier to prepare than gels, other viscous compositions, and solid compositions. Additionally, liquid compositions are somewhat more convenient to administer, especially by injection (e.g., intravenous injection).
- Viscous compositions can be formulated within the appropriate viscosity range to provide longer contact periods with specific tissues.
- Liquid or viscous compositions can comprise carriers, which can be a solvent or dispersing medium containing, for example, water, saline, phosphate buffered saline, polyol (for example, glycerol, propylene, glycol, liquid polyethylene glycol, and the like) and suitable mixtures thereof.
- Sterile injectable solutions can be prepared by incorporating the compositions disclosed herein in the required amount of the appropriate solvent with various amounts of the other ingredients, as desired.
- Such compositions may be in admixture with a suitable carrier, diluent, or excipient such as sterile water, physiological saline, glucose, dextrose, or the like.
- the compositions can also be lyophilized.
- the compositions can contain auxiliary substances such as wetting, dispersing, or emulsifying agents (e.g., methylcellulose), pH buffering agents, gelling or viscosity enhancing additives, preservatives, flavoring agents, colors, and the like, depending upon the route of administration and the preparation desired. Standard texts, such as “Remington's Pharmaceutical Science”, 17th edition, 1985, incorporated herein by reference, may be consulted to prepare suitable preparations, without undue experimentation.
- compositions which enhance the stability and sterility of the compositions, including antimicrobial preservatives, antioxidants, chelating agents, and buffers, may be added. Prevention of the action of microorganisms can be ensured by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, and the like.
- the compositions can be isotonic, i.e., they can have the same osmotic pressure as blood and lacrimal fluid.
- the desired isotonicity of the compositions herein may be accomplished using sodium chloride, or other pharmaceutically acceptable agents such as dextrose, boric acid, sodium tartrate, propylene glycol or other inorganic or organic solutes. Sodium chloride is preferred particularly for buffers containing sodium ions.
- Parenteral dosage forms of the compositions can also be administered to a subject by various routes, including, but not limited to subcutaneous, intravenous (including bolus injection), intramuscular, and intraarterial. Since administration of parenteral dosage forms typically bypasses the subject's natural defenses against contaminants, parenteral dosage forms are preferably sterile or capable of being sterilized prior to administration to a subject. Examples of parenteral dosage forms include, but are not limited to solutions ready for injection, dry products ready to be dissolved or suspended in a pharmaceutically acceptable vehicle for injection, suspensions ready for injection, controlled-release parenteral dosage forms, and emulsions. Suitable vehicles that can be used to provide parenteral dosage forms of the disclosure are well known to those skilled in the art.
- Examples include, without limitation: sterile water; water for injection USP; saline solution; glucose solution; aqueous vehicles such as but not limited to sodium chloride injection, Ringer's injection, dextrose Injection, dextrose and sodium chloride injection, and lactated Ringer's injection; water-miscible vehicles such as, but not limited to ethyl alcohol, polyethylene glycol, and propylene glycol; and non-aqueous vehicles such as, but not limited to corn oil, cottonseed oil, peanut oil, sesame oil, ethyl oleate, isopropyl myristate, and benzyl benzoate.
- aqueous vehicles such as but not limited to sodium chloride injection, Ringer's injection, dextrose Injection, dextrose and sodium chloride injection, and lactated Ringer's injection
- water-miscible vehicles such as, but not limited to ethyl alcohol, polyethylene glycol, and propylene glycol
- compositions provided herein can be packaged in a pressurized aerosol container together with suitable propellants, for example, hydrocarbon propellants like propane, butane, or isobutane with conventional adjuvants.
- suitable propellants for example, hydrocarbon propellants like propane, butane, or isobutane with conventional adjuvants.
- Compositions can also be administered in a non-pressurized form such as in a nebulizer or atomizer.
- Compositions can also be administered directly to the airways in the form of a dry powder, for example, by use of an inhaler.
- suitable powder compositions include, by way of illustration, powdered preparations of a composition comprising a peptide disclosed herein thoroughly intermixed with lactose, or other inert powders acceptable for intrabronchial administration.
- the powder compositions can be administered via an aerosol dispenser or encased in a breakable capsule which can be inserted by the subject into a device that punctures the capsule and blows the powder out in a steady stream suitable for inhalation.
- the compositions can include propellants, surfactants, and co-solvents and can be filled into conventional aerosol containers that are closed by a suitable metering valve.
- Aerosols for the delivery to the respiratory tract are known in the art. See for example, Adjei, A. and Garren, J. Pharm. Res., 1: 565-569 (1990); Zanen, P. and Lamm, J.-W. J. Int. J. Pharm., 114: 111-115 (1995); Gonda, I. “Aerosols for delivery of therapeutic and diagnostic agents to the respiratory tract,” in Critical Reviews in Therapeutic Drug Carrier Systems, 6:273-313 (1990); Anderson et al, Am. Rev. Respir.
- compositions disclosed herein further encompass anhydrous pharmaceutical compositions and dosage forms comprising the disclosed compounds as active ingredients, since water can facilitate the degradation of some compounds.
- water e.g., 5%
- Anhydrous pharmaceutical compositions and dosage forms of the disclosure can be prepared using anhydrous or low moisture containing ingredients and low moisture or low humidity conditions.
- compositions and dosage forms that comprise lactose and at least one active ingredient that comprises a primary or secondary amine are preferably anhydrous if substantial contact with moisture and/or humidity during manufacturing, packaging, and/or storage is expected.
- Anhydrous compositions are preferably packaged using materials known to prevent exposure to water such that they can be included in suitable formulary kits. Examples of suitable packaging include, but are not limited to hermetically sealed foils, plastics, unit dose containers (e.g., vials) with or without desiccants, blister packs, and strip packs.
- the compositions can be administered to a subject by controlled- or delayed-release means.
- the use of an optimally designed controlled-release preparation in medical treatment is characterized by a minimum of drug substance being employed to cure or control the condition in a minimum amount of time.
- Advantages of controlled-release formulations include: 1) extended activity of the active agent; 2) reduced dosage frequency; 3) increased patient compliance; 4) usage of less total drug; 5) reduction in local or systemic side effects; 6) minimization of drug accumulation; 7) reduction in blood level fluctuations; 8) improvement in efficacy of treatment; 9) reduction of potentiation or loss of drug activity; and 10) improvement in speed of control of diseases or conditions.
- Controlled-release formulations can be used to control a compound of formula (I)'s onset of action, duration of action, plasma levels within the therapeutic window, and peak blood levels.
- controlled- or extended-release dosage forms or formulations can be used to ensure that the maximum effectiveness of a compound of formula (I) is achieved while minimizing potential adverse effects and safety concerns, which can occur both from under-dosing a drug (i.e., going below the minimum therapeutic levels) as well as exceeding the toxicity level for the drug.
- a variety of known controlled- or extended-release dosage forms, formulations, and devices can be adapted for use with the compositions described herein. Examples include, but are not limited to those described in U.S. Pat. Nos. 3,845,770; 3,916,899; 3,536,809; 3,598, 123; 4,008,719; 5674,533; 5,059,595; 5,591,767; 5, 120,548; 5,073,543; 5,639,476; 5,354,556; 5,733,566; and 6,365,185 B 1, each of which is incorporated herein by reference in their entireties.
- dosage forms can be used to provide slow or controlled-release of one or more active ingredients using, for example, hydroxypropylmethyl cellulose, other polymer matrices, gels, permeable membranes, osmotic systems (such as OROS). (Alza Corporation, Mountain View, Calif. USA)), multilayer coatings, microparticles, liposomes, or microspheres or a combination thereof to provide the desired release profile in varying proportions.
- ion exchange materials can be used to prepare immobilized, adsorbed salt forms of the disclosed compounds and thus effect controlled delivery of the drug. Examples of specific anion exchangers include, but are not limited to Duolite. A568 and Duolite. AP143 (Rohm&Haas, Spring House, Pa.
- compositions described herein can be administered to a subject by sustained release or in pulses.
- Pulse therapy is not a form of discontinuous administration of the same amount of a composition over time, but comprises administration of the same dose of the composition at a reduced frequency or administration of reduced doses.
- Sustained release or pulse administrations are particularly preferred when the disorder occurs continuously in the subject, for example where the subject has continuous or chronic symptoms of an inflammatory disease.
- Each pulse dose can be reduced and the total amount of a peptide disclosed herein can be administered over the course of treatment to the subject is minimized.
- the interval between pulses when necessary, can be determined by one of ordinary skill in the art. Often, the interval between pulses can be calculated by administering another dose of the composition when the composition or the active component of the composition is no longer detectable in the subject prior to delivery of the next pulse. Intervals can also be calculated from the in vivo half-life of the composition. Intervals can be calculated as greater than the in vivo half-life, or 2, 3, 4, 5 and even 10 times greater the composition half-life.
- Various methods and apparatus for pulsing compositions by infusion or other forms of delivery to the patient are disclosed in U.S. Pat. Nos. 4,747,825; 4,723,958; 4,948,592; 4,965,251 and 5,403,590.
- Formulations suitable for parenteral administration such as, for example, by intraarticular (in the joints), intravenous, intramuscular, intradermal, intraperitoneal, and subcutaneous routes, and carriers include aqueous isotonic sterile injection solutions, which can contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, preservatives, liposomes, microspheres and emulsions.
- the compositions described herein include, but are not limited to therapeutic compositions useful for practicing the therapeutic methods described herein.
- compositions contain a physiologically tolerable carrier together with an active agent as described herein, dissolved or dispersed therein as an active ingredient.
- the therapeutic composition is not immunogenic (e.g., allergenic) when administered to a mammal or human patient for therapeutic purposes.
- pharmaceutically acceptable e.g., physiologically tolerable and grammatical variations thereof, as they refer to compositions, carriers, diluents and reagents, are used interchangeably and represent that the materials are capable of administration to or upon a mammal without the production of undesirable physiological effects such as nausea, dizziness, gastric upset and the like.
- a pharmaceutically acceptable carrier will not promote the raising of an immune response to an agent with which it is admixed, unless so desired.
- the preparation of a pharmacological composition that contains active ingredients dissolved or dispersed therein is well understood in the art and need not be limited based on formulation.
- the active ingredient can be mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredient and in amounts suitable for use in the therapeutic methods described herein. Suitable excipients include, for example, water, saline, dextrose, glycerol, ethanol or the like and combinations thereof.
- the composition can contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents and the like which enhance the effectiveness of the active ingredient.
- the compositions described herein can include pharmaceutically acceptable salts of the components therein.
- Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the polypeptide) that are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, tartaric, mandelic and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine and the like. Physiologically tolerable carriers are well known in the art.
- Exemplary liquid carriers are sterile aqueous solutions that contain no materials in addition to the active ingredients and water, or contain a buffer such as sodium phosphate at physiological pH value, physiological saline or both, such as phosphate-buffered saline. Still further, aqueous carriers can contain more than one buffer salt, as well as salts such as sodium and potassium chlorides, dextrose, polyethylene glycol and other solutes. Liquid compositions can also contain liquid phases in addition to and to the exclusion of water. Exemplary of such additional liquid phases are glycerin, vegetable oils such as cottonseed oil, and water-oil emulsions. The amount of an active agent used in the methods described herein that will be effective in the treatment of a particular disorder or condition will depend on the nature of the disorder or condition, and can be determined by standard clinical techniques.
- compositions can be formulated for any appropriate manner of administration, including for example, topical, oral, nasal, intravenous, intracranial, intraperitoneal, subcutaneous or intramuscular administration.
- parenteral administration such as subcutaneous injection
- the carrier preferably comprises water, saline, alcohol, a fat, a wax or a buffer.
- any of the above carriers or a solid carrier such as mannitol, lactose, starch, magnesium stearate, sodium saccharine, talcum, cellulose, glucose, sucrose, and magnesium carbonate, may be employed.
- Biodegradable microspheres can also be employed as carriers for the pharmaceutical compositions of this invention. Suitable biodegradable microspheres are disclosed, for example, in U.S. Pat. Nos. 4,897,268 and 5,075,109.
- Such compositions can also comprise buffers (e.g., neutral buffered saline or phosphate buffered saline), carbohydrates (e.g., glucose, mannose, sucrose or dextrans), mannitol, proteins, polypeptides or amino acids such as glycine, antioxidants, chelating agents such as EDTA or glutathione, adjuvants (e.g., aluminum hydroxide) and/or preservatives.
- buffers e.g., neutral buffered saline or phosphate buffered saline
- carbohydrates e.g., glucose, mannose, sucrose or dextrans
- mannitol e.g., proteins, polypeptides or amino acids
- glycine
- compositions as described herein can be formulated as a lyophilizate.
- Compounds can also be encapsulated within liposomes using well known technology.
- the compositions described herein can be administered as part of a sustained release formulation (i.e., a formulation such as a capsule or sponge that effects a slow release of compound following administration).
- sustained release formulations can generally be prepared using well known technology and administered by, for example, oral, rectal or subcutaneous implantation, or by implantation at the desired target site.
- Sustained-release formulations can contain a polypeptide, polynucleotide dispersed in a carrier matrix and/or contained within a reservoir surrounded by a rate controlling membrane.
- Carriers for use within such formulations are biocompatible, and can also be biodegradable; preferably the formulation provides a relatively constant level of active component release.
- the amount of active compound contained within a sustained release formulation depends upon the site of implantation, the rate and expected duration of release and the nature of the condition to be treated or prevented.
- the pharmaceutical composition is contained in an implantable pump.
- the pump can be designed to deliver to the intended site of action, at the required rate of administration, and in the proper therapeutic dose.
- One example of such pump includes but is not limited to the commercially available, the Alzet osmotic mini pump to delivering drugs at a controlled rate and dose over extended periods.
- the peptides of the compositions disclosed herein can be contained in a continuous flow pump.
- the delivery mechanism of one such pump is based on the expansion of Freon gas at 37° C. that pushes a diaphragm “plunger/pusher” plate.
- the pump reservoir is implanted subcutaneously and is connected to a catheter to deliver the therapeutic molecules.
- the peptides can be contained in a programmable pump.
- Programmable pumps include electromechanical pumps of the peristaltic type, powered by batteries. Their built-in electronics can be remotely controlled from an external programming unit. An example is the SynchroMed system (Medtronic Inc.).
- the infusion can be programmed in various modes: continuous hourly infusions, repeated bolus infusions with a specified delay, multiple doses over a programmed interval, or a single bolus infusion.
- the pharmaceutical composition comprising peptides disclosed herein is contained in a syringe, including a blunt tip syringe for injection to the target site. Microspheres can be implanted stereotactically in the target site.
- the compositions disclosed herein can be formulated in liposomes, for example to improve stability and promote delivery.
- liposome refers to a vesicular structure having lipid-containing membranes enclosing an aqueous interior.
- a vesicular structure is a hollow, lamellar, spherical structure, and provides a small and enclosed compartment, separated from the cytosol by at least one lipid bilayer.
- Liposomes can have one or more lipid membranes.
- Oligolamellar large vesicles and multilamellar vesicles have multiple, usually concentric, membrane layers and are typically larger than 100 nm.
- Liposomes with several nonconcentric membranes i.e., several smaller vesicles contained within a larger vesicle, are termed multivesicular vesicles.
- Liposomes can further comprise one or more additional lipids and/or other components such as sterols, e.g., cholesterol. Additional lipids can be included in the liposome compositions for a variety of purposes, such as to prevent lipid oxidation, to stabilize the bilayer, to reduce aggregation during formation or to attach ligands onto the liposome surface. Any of a number of additional lipids and/or other components can be present, including amphipathic, neutral, cationic, anionic lipids, and programmable fusion lipids. Such lipids and/or components can be used alone or in combination.
- One or more components of the liposome can comprise a ligand, e.g., a targeting ligand.
- Liposome compositions can be prepared by a variety of methods that are known in the art. See e.g., U.S. Pat. No. 6,339,069B1.
- Niosomes are non-phospholipid based synthetic vesicles that have properties and function like liposomes. Liposomes are vesicular structures with an aqueous core surrounded by a hydrophobic lipid membrane created by extrusion of phospholipids and known in the art to be used for drug delivery purposes.
- the peptide is encapsulated in a liposome.
- Liposomes can vary in size from 15 nm to 100 ⁇ and are contemplated to have either a single layer (uni-lamellar), or multiple phospholipid bilayer membranes (mutilamellar structure).
- the peptide can be encapsulated in a niosome, a non-phospholipid-based synthetic vesicle
- compositions disclosed herein are formulated using micelles formed from lipid-associated peptides disclosed herein, e.g., peptides of the present disclosure conjugated to at least one amphiphilic carrier, in which the micelles have an average diameter of less than about 100 nm, preferably. More preferred embodiments provide micelles having an average diameter less than about 50 nm, and even more preferred embodiments provide micelles having an average diameter less than about 100 nm, or even less than about 20 nm.
- micelles are a particular type of molecular assembly in which amphipathic molecules are arranged in a spherical structure such that all hydrophobic portions on the molecules are directed inward, leaving the hydrophilic portions in contact with the surrounding aqueous phase. The converse arrangement exists if the environment is hydrophobic.
- the peptides of the present disclosure are encapsulated in a micelle.
- Micelles are spherical aggregates of amphiphilic molecules dispersing in water with their hydrophilic head groups on the surface of the sphere, and their hydrophobic tails collected inside.
- An important property of micelles is their ability to increase the solubility and bioavailability of poorly soluble pharmaceuticals.
- the amphiphilic molecules in micelles are in constant exchange with those in the bulk solution.
- polymeric micelles also known as polymersomes, are self-assembled polymer shells composed of block copolymer amphiphiles such as polyethylene glycol-polylactic acid (PEG-PLA) and PEG-polycaprolactone (PEG-PCL).
- Polymeric micelles differ from nanoparticles that are either more solid or monolithic (nanospheres) or contain an oily or aqueous core and are surrounded by a polymer shell (nanocapsules). However, in practice, polymeric micelles also be referred to as nanoparticle or nanocarriers because of their particle size.
- the peptides of the compositions disclosed herein is encapsulated in a nanoparticle.
- the peptide disclosed herein can be encapsulated in a microcapsule or a microsphere, which are free flowing powders consisting of spherical particles of 2 millimeters or less in diameter, usually 500 microns or less in diameter.
- nanoparticle refers to a particle having a size between 1 and 1000 nm which can be manufactured from artificial or natural macromolecular substances.
- To such nanoparticles can be bound drugs or other biologically active materials by covalent, ionic or adsorptive linkage, or the latter can be incorporated into the material of the nanoparticles.
- Nanoparticles may or may not exhibit size-related properties that differ significantly from those observed in fine particles or bulk materials. Nanoparticles provide improved bioavailability by enhancing aqueous solubility, increasing resistance time in the body (increasing half-life for clearance/increasing specificity for its cognate receptors and targeting drug to specific location in the body (its site of action). This results in concomitant reduction in quantity of the drug required and dosage toxicity, enabling the safe delivery of toxic therapeutic drugs and protection of non-target tissues and cells from severe side effects.
- Non-limiting examples of nanoparticles include solid lipid nanoparticles (comprise lipids that are in solid phase at room temperature and surfactants for emulsification, the mean diameters of which range from 50 nm to 1000 nm for colloid drug delivery applications), liposomes, nanoemulsions (oil-in-water emulsions done on a nano-scale), albumin nanoparticles, and polymeric nanoparticles. Nanoparticles can be surface coated to modulate their stability, solubility, and targeting. A coating that is multivalent or polymeric confers high stability.
- a non-limiting example includes coating with hydrophilic polymer such as polyethylene glycol or ploysorbate-80.
- Nanoparticles are solid matrix colloidal particles with diameters ranging from 1-100 nm formed using various polymers like degradable starch, dextran, chitosan, microcrystalline cellulose (MCC), hydroxypropyl cellulose (HPC), hydroxypropyl ethylcellulose (HPMC), carbomer, and wax-like starch, gelatin polymers.
- MCC microcrystalline cellulose
- HPMC hydroxypropyl cellulose
- carbomer hydroxypropyl ethylcellulose
- the drug can be loaded via either incorporation with the system or its adsorption on the particulate system.
- the encapsulating nanoparticle can be, for example, solid-lipid nanoparticles (SLNs), polymeric nanoparticles, or oil-in-water nanoemulsions.
- Solid-lipid nanoparticles are surfactant-stabilized aqueous colloidal dispersions of lipid nanoparticles that solidify upon cooling. They contain a lipid phase dispersed in an aqueous environment.
- Polymeric nanoparticles are solid colloidal particles created from polymeric systems. These nanoparticles are made from biocompatible polymers that encapsulate or adsorb drugs for prolonged release.
- Nanoemulsions are oil-in-water (OAV) or water-in-oil (W/O) formulations made with edible or otherwise pharmaceutically acceptable oils, surface-active agents (surfactants), and water, where the diameter of the inner phase is reduced to nanometer length scale.
- OAV oil-in-water
- W/O water-in-oil
- the versatility of nanoemulsions is based on the different types of oils and surface modifiers that can be used. For instance, oils that are rich in omega-3 polyunsaturated fatty acids (PUFA) can play a very important role in overcoming biological barriers, including the blood brain barrier.
- PUFA omega-3 polyunsaturated fatty acids
- the liposomes and nanoparticles encapsulating the peptides disclosed herein can be further linked with and/or coated with other agents.
- an agent can be an antibody binding fragment such as Fab, F(ab′)2, Fab′ or a single antibody chain polypeptide which binds to a receptor molecule present on a target cell of interest, (e.g., immune cell expressing CD40 or a neoplastic cell expressing CD40 on its surface).
- the nanoparticles encapsulating the peptides of the compositions and methods disclosed herein can be coated with poly(ethylene glycol) or polysorbate 80 or albumin or its functional groups.
- PEG-containing surfactants poly(oxy-ethylene)-poly(oxy-propylene) can also be used for coating nanoparticles.
- Polysorbate 80-coated poly(n-butylcyanoacrilate) nanoparticles have been formulated by emulsion polymerization method to target selectively rivastigmine or tacrine. Nanoparticles and liposomes can also be linked to carrier peptides for examples TAT, to improve their lipophilicity.
- the peptides of the compositions and methods disclosed herein can be fused to a carrier peptide, for example for transdermal administration. See for example, US20080305989A1, EP2857033A2
- compositions of the present disclosure are formulated for intranasal administration. Therefore, in order to enhance the absorption of a therapeutic drug or agent into the olfactory neurons, the drug or agent should be capable of at least partially dissolving in the fluids that are secreted by the mucous membrane that surround the cilia of the olfactory receptor cells of the olfactory epithelium. Therefore the therapeutic peptide can be linked to a carrier that increases its dissolution within nasal secretions.
- Non-limiting examples of such carriers include GM-1 ganglioside, phosphotidylserine (PS), and emulsifiers such as polysorbate 80.
- compositions of the present disclosure comprises a ganglioside or a phosphatidylserine (e.g., composition comprising a peptide disclosed herein formulated for intranasal administration).
- the peptides of the present disclosure can be encapsulated in nanoparticles, liposomes, micelles, microspheres, niosomes, cyclodextrin-inclusion complexes, or nanoemulsions.
- the nanoparticles can be coated with polymers such as polyethylene glycol-polylactic acid (PEG-PLA) or Chitosan (CS). Methods for preparation of (PEG-PLA) nanoparticles are well known in the art.
- the chitosan nanoparticles can be complexed with cyclodextrins.
- the compositions of the present disclosure further comprise cyclodextrin.
- cyclodextrins refers to cyclic oligosaccharides, like ⁇ -, ⁇ - and ⁇ -cyclodextrin and their derivatives, preferably ⁇ -cyclodextrin and its derivatives, preferably methylated ⁇ -cyclodextrin, with a degree of CH3-substitution between 0.5 and 3.0, more preferably between 1.7 and 2.1.
- saccharides refers to disaccharides, like lactose, maltose, saccharose and also refers to polysaccharides, like dextrans, with an average molecular weight between 10,000 and 100,000, preferably 40,000 and 70,000.
- sugar alcohols refers to mannitol and sorbitol.
- the compositions of the present disclosure further comprises saccharides selected from the group consisting of cyclodextrins, disaccharides, polysaccharides and combinations thereof. Peptides can be encapsulated in carriers, like cyclodextrins inclusion complexes containing a hydrophobic core and a hydrophilic shell which can help improve upon the solubility problems.
- compositions can be dispensed intranasally as a powdered or liquid nasal spray, nose drops, a gel or ointment, injection or infusion contained in a tube or catheter, by syringe, by pledge, or by submucosal infusion.
- the composition can made viscous using vehicles such as natural gums, methylcellulose and derivatives, acrylic polymers (carbopol) and vinyl polymers (polyvinylpyrrolidone).
- Many other excipients known in the pharmaceutical literature, can be added, such as preservatives, surfactants, co-solvents, adhesives, antioxidants, buffers, viscosity enhancing agents, and agents to adjust the pH or the osmolarity.
- Nasal powder compositions can be made by mixing the active agent and the excipient, both possessing the desired particle size.
- Other methods to make a suitable powder formulation can be selected. Firstly, a solution of the active agent and the cyclodextrin and/or the other saccharide and/or sugar alcohol is made, followed by precipitation, filtration and pulverization. It is also possible to remove the solvent by freeze drying, followed by pulverization of the powder in the desired particle size by using conventional techniques, known from the pharmaceutical literature. The final step is size classification for instance by sieving, to get particles that are less than 100 microns in diameter, preferably between 50 and 100 microns in diameter. Powders can be administered using a nasal insufflator.
- Powders may also be administered in such a manner that they are placed in a capsule.
- the capsule is set in an inhalation or insufflation device.
- a needle is penetrated through the capsule to make pores at the top and the bottom of the capsule and air is sent to blow out the powder particles.
- Powder formulation can also be administered in a jet-spray of an inert gas or suspended in liquid organic fluids.
- the composition for intranasal administration can be adapted for aerosolization and inhalation.
- the composition can be administered nasally via pressurized aerosol, aqueous pump spray or other standard methods known to those skilled in the art.
- compositions of present disclosure can be administered in the form of spray in a non-pressurized aerosol device, for example a Pfeiffer pump.
- the compositions can be contained in a syringe, catheter, an inhaler, a nebulizer, a nasal spray pump, a nasal irrigation pump, or a nasal lavage pump.
- Non-limiting example for intranasal administration of liquid formulation can include (a) delivering drops with a drop pipette, (b) rhinyle catheter and squirt tube which involves inserting the tip of a fine catheter or micropipette to the desired area under visual control and squirt the liquid into the desired location, (c) squeeze bottles which involve squeezing a partly air-filled plastic bottle, to deliver the atomized drug from a jet outlet, (d) metered-dose spray pumps which deliver a unit dose of drug per use, (e) single- and duo-dose spray devices which is used for a single administration of a unit dose of drug, (f) nasal pressurized metered-dose inhalers delivers a nasal aerosol preparation, and (g) powered nebulizers and atomizers to administer drug in the form of a mist.
- Non-limiting examples for intranasal administration of liquid formulations can include nasal powder inhalers (e.g., Rhinocort Turbuhaler®; BiDoseTM/ProhalerTM) from Pfeiffer/Aptar), nasal powder sprayers (e.g., Fit-lizerTM device, Unidose-DPTM), and nasal powder insufflators (e.g., Bi-DirectionalTM nasal delivery, Optinose).
- nasal powder inhalers e.g., Rhinocort Turbuhaler®; BiDoseTM/ProhalerTM
- nasal powder sprayers e.g., Fit-lizerTM device, Unidose-DPTM
- nasal powder insufflators e.g., Bi-DirectionalTM nasal delivery, Optinose.
- compositions of the present disclosure can be provided in a lyophilized form.
- the composition in a lyophilized form can further comprise a lyoprotectant.
- a “lyoprotectant” is a molecule which, when combined with a protein or peptide of interest, significantly prevents or reduces chemical and/or physical instability of the protein or peptide upon lyophilization and subsequent storage.
- Exemplary lyoprotectants include sugars such as sucrose or trehalose; an amino acid such as monosodium glutamate or histidine; a methylamine such as betaine; a lyotropic salt such as magnesium sulfate; a polyol such as trihydric or higher sugar alcohols, e.g., glycerin, erythritol, glycerol, arabitol, xylitol, sorbitol, and mannitol; propylene glycol; polyethylene glycol; pluronics; and combinations thereof.
- the preferred lyoprotectant is a non-reducing sugar, such as trehalose or sucrose.
- the lyoprotectant is added to the pre-lyophilized formulation in a “lyoprotecting amount” which means that, following lyophilization of the peptide in the presence of the lyoprotecting amount of the lyoprotectant, the peptide essentially retains its physical and chemical stability and integrity upon lyophilization and storage.
- the composition in a lyophilized form can comprise a bulking agent.
- a “bulking agent” is a compound which adds mass to the lyophilized mixture and contributes to the physical structure of the lyophilized cake (e.g., facilitates the production of an essentially uniform lyophilized cake which maintains an open pore structure).
- Exemplary bulking agents include mannitol, glycine, polyethylene glycol and xorbitol.
- the lyophilized formulation may be reconstituted with a diluent such that the peptide concentration in the reconstituted formulation is at least 10 mg/mL, for example from about 10 mg/mL to about 1000 mg/mL, more preferably from about 50 mg/mL to about 500 mg/mL, and most preferably from about 100 mg/mL to about 500 mg/mL.
- a diluent such that the peptide concentration in the reconstituted formulation is at least 10 mg/mL, for example from about 10 mg/mL to about 1000 mg/mL, more preferably from about 50 mg/mL to about 500 mg/mL, and most preferably from about 100 mg/mL to about 500 mg/mL.
- the peptide concentration in the reconstituted formulation is significantly higher than that in the pre-lyophilized formulation.
- the peptide concentration in the reconstituted formulation may be about 2-40 times, preferably 3-10 times and most preferably 3-6 times (e.g., at least three fold or at least four fold) that of the pre-lyophilized formulation.
- Reconstitution generally takes place at a temperature of about 25° C. to ensure complete hydration, although other temperatures may be employed as desired.
- the time required for reconstitution will depend, for example, on the type of diluent, amount of excipient(s) and peptide.
- the “diluent” of interest is one which is pharmaceutically acceptable (safe and non-toxic for administration to a human) and is useful for diluting a peptide in solution or the preparation of a reconstituted formulation.
- Exemplary diluents include sterile water, bacteriostatic water for injection (BWFI), a pH buffered solution (e.g.
- the diluent optionally contains a preservative.
- a preservative Exemplary preservatives have been described above, with aromatic alcohols such as benzyl or phenol alcohol being the preferred preservatives.
- the amount of preservative employed is determined by assessing different preservative concentrations for compatibility with the peptide and preservative efficacy testing. For example, if the preservative is an aromatic alcohol (such as benzyl alcohol), it can be present in an amount from about 0.1-2.0% and preferably from about 0.5-1.5%, but most preferably about 1.0-1.2%.
- the reconstituted formulation has less than 6000 particles per vial which are >10 ⁇ m in size.
- the composition comprising the peptides
- the composition has a shelf life in packaged form of at least 2, 3 or 4 days, up to 1, 2 or 3 weeks or one month, or in some embodiments a shelf life in packaged form of at least one or two months up to 6 months or one to two years, or more, at a temperature of 37° C., or at room temperature.
- Shelf life has its conventional meaning in the art and refers to the time period during which the active agent retains sufficient activity for accurate and effective administration thereof, and the formulation retains sufficient stability (e.g., a single phase solution remains as a single phase solution) for accurate and effective administration thereof at the recited temperature conditions.
- the peptides of the present disclosure selectively bind CD40 protein.
- the CD40 protein is expressed on a surface of a cell (e.g., an immune cell, a non-immune cell, a cancer cell). It will be well appreciated by those skilled in the art that the peptides or portion thereof disclosed herein can bind CD40 protein in vitro, (for example when used in a cell culture system), ex vivo, and in vivo. In some embodiments, peptides of the present disclosure can be used to detect CD40 expressing cells.
- the peptide is conjugated to a detectable agent, for example, fluorescent agent, chemiluminescent agent, radioistope, or enzyme-substrate agent such as alkaline phosphatase.
- a detectable agent for example, fluorescent agent, chemiluminescent agent, radioistope, or enzyme-substrate agent such as alkaline phosphatase.
- An assay for detecting a cell expressing CD40 in a sample comprises, for example, a) contacting the sample with a peptide of the present disclosure under condition suitable to allow binding of the peptide with CD40 on the cell surface; b) washing the unbound peptide; and c) detecting the peptide bound to cells.
- the peptide can be detected, for example, by screening for the detectable agent conjugated to the peptide. The presence of detectable agent indicates presence of CD40 expressing cell.
- a secondary detection agent e.g., an antibody binding the peptide disclosed herein
- a secondary detection agent e.g., an antibody binding the peptide disclosed herein
- the peptide useful for such assays can be immobilized onto a solid surface.
- the solid surface can be in the form of tubes, beads, discs, silicon chips, microplates, polyvinylidene difluoride (PVDF) membrane, nitrocellulose membrane, nylon membrane, other porous membrane, non-porous membrane, e.g., plastic, polymer, perspex, silicon, amongst others, a plurality of polymeric pins, or a plurality of microtitre wells, or any other surface suitable for immobilizing polypeptide or polynucleotides.
- PVDF polyvinylidene difluoride
- Activated immune cells expressing CD40 are associated with risk of developing inflammatory diseases, autoimmune disease or cancer. Accordingly, in some embodiments, the peptides disclosed herein can be used to identify patients at risk of developing inflammatory diseases, autoimmune disease or a cancer. A sample obtained from candidate patients can be assayed for presence of CD40 expressing cells using, for example, an assay described above. In some embodiments, the sample can be screened for a specific type of cell expressing CD40 alone or in combination with other surface markers specific for that cell type.
- a sample can be assayed for a population of activated T-cell by using the peptides disclosed herein to detect expression of CD40 alone or using the peptides disclosed herein in combination with molecules detecting activated T-cell specific surface proteins such as CD25, CD38, CD69 and the like.
- a sample can be assayed to identify a CD40 expressing cancer cell using the peptides disclosed herein alone or by using the peptides disclosed herein to detect the CD40 in combination with molecules detecting cancer markers on cell surface.
- An increase in number of a specific type of cell (e.g., activated T-cell) relative to the total cell population in a sample can be indicative of a disease condition (e.g. inflammatory diseases, autoimmune disease or a cancer).
- a disease condition e.g. inflammatory diseases, autoimmune disease or a cancer.
- the peptides disclosed herein can be used to screen for molecules (e.g., small molecule, proteins, peptides) that bind CD40 and modulate CD40 activity.
- the assay can be designed, for example, as a competitive binding assay.
- the assay can be designed, to test the ability of a test molecule to compete with the peptides disclosed herein for binding to a CD40 protein.
- the peptides disclosed herein useful for such an assay can be one that is fused to a detectable agent.
- the method comprises: a) contacting a cell or biological sample with a peptide disclosed herein in the presence or absence of a test agent under conditions which allow for their binding to CD40; b) washing the sample to remove any unbound peptides; and c) screening the sample for presence of detectable agent.
- absence of a detectable agent indicates the test molecule is able to compete with the peptide for binding to a CD40 protein.
- the assay can be used, for example, to screen for molecules that partially inhibit binding of peptides to the CD40 protein. Methods of competitive binding assay are well known in the art.
- test agents can be obtained using any of the numerous approaches in combinatorial-library methods known in the art, including: biological libraries; spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; the “one-bead one-compound” library method; and synthetic library methods using affinity chromatography selection.
- biological library approach is limited to peptide libraries, while the other four approaches are applicable to peptide, non-peptide oligomer or small molecule libraries of compounds (Lam et al., 1997, Anticancer Drug Des.12:45). Examples of methods for the synthesis of molecular libraries can be found in the art, for example, in: DeWitt et al, 1993, Proc. Natl. Acad.
- high throughput screening methods involve providing a library containing a large number of compounds (candidate compounds) potentially having the desired activity. Such “combinatorial chemical libraries” are then screened in one or more assays, as described herein, to identify those library members (particular chemical species or subclasses) that display a desired characteristic activity.
- the compounds thus identified can serve as conventional “lead compounds” or can themselves be used as potential or actual therapeutics.
- Assays disclosed herein are meant as examples of useful assays and it is understood that other assays or modifications of assays described above using the peptides disclosed herein can be employed. Suitable assay techniques are known to those skilled in the art and are disclosed for example in Molecular Cloning: A Laboratory Manual, Sambrook, J., Fritsch, E. F., and Maniatis, T, Cold Spring Harbor Laboratory Press; 2nd Edition (December 1989). All referenced cited herein are incorporated herein in their entirety.
- immunosuppression as used herein relates to an inhibition in the activation and/or maintenance of an immune response (for example, immune response directed towards a transplant).
- immune response refers to the alteration in the reactivity of an organism's immune system upon exposure to an antigen.
- immuno response encompasses but is not limited to one or both of the following responses: antibody production (e.g., humoral immunity), and induction of cell-mediated immunity (e.g., cellular immunity including helper T cell and/or cytotoxic T cell responses).
- Immune response generally refers to innate and acquired immune responses including, but not limited to, both humoral immune responses (mediated by B lymphocytes) and cellular immune responses (mediated by T lymphocytes).
- An immune response may be beneficial and lead to immunity against infectious pathogens, or an immune response may be pathogenic and lead to autoimmune or hypersensitivity disease.
- Immune responses against foreign viruses, bacteria, fungi, parasites typically represent beneficial adaptive immune responses.
- Immune responses against self tissues, innocuous foreign objects (e.g., dust mite or pollen allergens, etc.), or tissue transplants represent examples of adverse maladaptive immune responses.
- modulate refers to enhancement (e.g., an increase) or inhibition (e.g., a reduction) in the specified activity or suppression thereof.
- immunosuppression also refers to inhibition of an immune response mediated by CD40 activity or one that is mediated by interaction of CD40 with CD154.
- CD40 is well known to play an important role in activation of immune response.
- B cells are known to constitutively express the CD40 molecule on the cells membrane (van Kooten C. et al, J. Leukoc. Biol. 2000). Stimulation of CD40 by CD154-expressing activated T cells, results in B cells activation, proliferation and isotype switching.
- CD40-CD154 interaction can upregulate costimulatory molecules, activate APCs, and influence T-cell priming and T-cell-mediated effector CD40 activities include modulating cell survival (e.g., functions as cell survival signal), antibody production, antibody isotype switching, production of cytokines (e.g., IL-2, IL-6, IL-8, IL-12, TNF- ⁇ , IL-4, IL-5 and IL-10), metalloproteases (e.g., MMP-I/collagenase and MMP-9/gelatinase B) and establishment of immune memory.
- cell survival e.g., functions as cell survival signal
- cytokines e.g., IL-2, IL-6, IL-8, IL-12, TNF- ⁇ , IL-4, IL-5 and IL-10
- metalloproteases e.g., MMP-I/collagenase and MMP-9/gelatinase B
- CD40 activities further include modulating production of proteins involved in cell-cell contact or adhesion (e.g., E-selectin, VCAM-1 and ICAM-1). This interaction can activate macrophages, natural killer (NK) cells, and endothelial cells.
- CD40 plays a significant role in immune cell function and signaling, including B-cell and T-cell activation by antigen presenting cells, such as macrophages and dendritic cells. CD40 activity stimulates antibody production, isotype switching, and establishment of memory.
- CD40 activity stimulates production of cytokines, such as IL-2, IL-6, IL-8, IL-12, TNF- ⁇ , IL-4, IL-S and IL-10; and metalloproteases, such as MMP-I/collagenase and MMP-9/gelatinase B.
- CD40 activity stimulates production of proteins involved in cell-cell contact or adhesion, such as E-selectin, VCAM-1 and ICAM-1.
- CD40 recognition on target cells provides an activation pathway for NK cell cytotoxic activity.
- the disclosure further provides methods of modulating cell survival, antibody production, isotype switching, establishment of memory, production of cytokines, metalloproteases or proteins involved in cell-cell contact or adhesion, NK cell cytotoxic activity, B-cell activation, B-cell proliferation, T-cell activation, T-cell proliferation, macrophage activation, or migration of an immune cell, comprising contacting a cell expressing CD40 with an effective amount of a peptide disclosed herein.
- a cell expressing CD40 herein is intended any normal or malignant cells that express detectable levels of the CD40 antigen.
- the cell can be an immune cell, a nonimmune cell, or a cancer cell, for example, lymphoma cell.
- CD40-expressing cells are cells that express detectable levels of cell-surface CD40 antigen.
- the cell to be contacted can be informed by the immune response to be modulated.
- the immune response to be modulated for example, in embodiments related to B-cell mediated immune response, such as B-cell proliferation, B-cell activation, the cell contacted with the peptides or compositions disclosed herein can be a B-cell.
- the cell contacted with the peptides or compositions disclosed herein can be a macrophage.
- the cell to be contacted can be easily determined by one of skill in the art.
- the disclosure provides for a method for inducing immunosuppression in a subject in need thereof, the method comprising administering to the subject an effective amount of a peptide of the present disclosure or a pharmaceutical composition disclosed herein.
- compositions, methods, kits, and systems provided herein can be utilized to prevent and/or treat an autoimmune disorder.
- autoimmune disorder autoimmune disease
- autoimmune condition autoimmune condition
- the tolerance vaccine provided herein can be crosslinked to autoantigenic peptides, autoantigens, or other cellular carriers and used as a tolerance therapy for an autoimmune disorder.
- the cellular carrier is an apoptotic cellular carrier.
- the cellular carrier is a syngeneic apoptotic cellular carrier.
- the tolerizing vaccine as described herein can be used with a cellular carrier (e.g., autoantigens, autoantigenic peptides, apoptotic cellular carriers) to induce antigen-specific T cell tolerance for treatment of an autoimmune condition.
- a cellular carrier e.g., autoantigens, autoantigenic peptides, apoptotic cellular carriers
- the tolerizing vaccine with or without the carrier can be taken up, processed, and presented in a tolerogenic manner by host splenic antigen presenting cells, thereby inducing regulatory T cells, and the secretion of immune suppressive cytokines (e.g., IL-4, IL-10, IL-13, TGF- ⁇ ).
- CD40 associated disorder means any undesirable physiological condition or pathological disorder in which modulating a CD40 activity (e.g., binding CD154 or activation of immune response mediated by CD40 may improve or reduce one or more undesirable symptoms of the condition or disorder.
- a peptide disclosed herein or a pharmaceutical composition disclosed herein can be administered to a subject having, or at risk of having autoimmunity, hypersensitivity, inflammation or transplant rejection in order to induce immunosuppression and inhibit or prevent autoimmunity, hypersensitivity, inflammation or transplant rejection in the subject.
- the method of inducing immunosuppression disclosed herein can be useful to a subject who is suffering from or is at a risk of developing an inflammatory disease.
- “Inflammatory disease” means an immune-mediated inflammatory condition, generally characterized by dysregulated expression of one or more cytokines.
- Examples of inflammatory disease include skin inflammatory disorders, inflammatory disorders of the joints, and inflammatory disorders of the cardiovascular system, autoimmune diseases, lung and airway inflammatory disorders, intestinal inflammatory disorders.
- skin inflammatory disorders include dermatitis, for example atopic dermatitis and contact dermatitis, acne vulgaris, and psoriasis.
- Examples of inflammatory disorders of the joints include rheumatoid arthritis.
- Examples of inflammatory disorders of the cardiovascular system are cardiovascular disease and atherosclerosis.
- Examples of autoimmune diseases include Type 1 diabetes, Graves' disease, Guillain-Barre disease, Lupus, Psoriatic arthritis, and Ulcerative colitis.
- Examples of lung and airway inflammatory disorders include asthma, cystic fibrosis, COPD, emphysema, and acute respiratory distress syndrome.
- Examples of intestinal inflammatory disorders include colitis and inflammatory bowel disease.
- Other inflammatory disorders include cancer, hay fever, periodontitis, allergies, hypersensitivity, ischemia, depression, systemic diseases, post infection inflammation, amyotrophic lateral sclerosis and bronchitis.
- autoimmune disease refers generally to those diseases characterized by the failure of one or more B- and/or T-cell populations, or gene products thereof, to distinguish between self and non-self antigenic determinants.
- Autoimmune diseases are often characterized by the infiltration of the target cells with inflammatory lymphoid cells, for example, mononuclear phagocytes, lymphocytes and plasma cells as well as secondary lymphoid follicles.
- Exemplary autoimmune diseases include, but are not limited to, organ specific disorders such as Hashimoto's thyroiditis, primary myxoedema thyrotoxicosis, pernicious anemia, Addison's disease, and insulin-dependent diabetes mellitus as well as non-organ specific disorders such as systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), multiple sclerosis, dermatomyositis, scleroderma and psoriasis.
- organ specific disorders such as Hashimoto's thyroiditis, primary myxoedema thyrotoxicosis, pernicious anemia, Addison's disease, and insulin-dependent diabetes mellitus as well as non-organ specific disorders such as systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), multiple sclerosis, dermatomyositis, scleroderma and psoria
- Non-limiting examples of autoimmune disorders treatable with a peptide or pharmaceutical composition of the disclosure include rheumatoid arthritis, lupus (e.g., SLE, lupus nephritis), production of auto-antibodies which, in the case of antibodies against myelin basic protein contribute to multiple sclerosis and in the case of antibodies against insulin contribute to diabetes, and Crohn's disease.
- Non-limiting examples of hypersensitivity treatable with a peptide disclosed herein or pharmaceutical compositions of the disclosure include allergic reactions to antigens, antibiotics, etc.
- Non-limiting examples of inflammation treatable with a peptide or pharmaceutical composition of the present disclosure include vascular inflammatory disease (e.g., artherosclerotic lesions, plaque disruption and thrombus formation), production of inflammatory cytokines (e.g., LIF, GM-CSF, and IL-6), lung fibrosis and inflammation associated with multiple sclerosis or a tissue or organ transplant.
- vascular inflammatory disease e.g., artherosclerotic lesions, plaque disruption and thrombus formation
- production of inflammatory cytokines e.g., LIF, GM-CSF, and IL-6
- lung fibrosis e.g., fibrosis and inflammation associated with multiple sclerosis or a tissue or organ transplant.
- CD40 activity can be associated with inflammation caused by viral myocarditis and, as such, a peptide or pharmaceutical composition disclosed herein can be used to inhibit inflammation associated with viral infection.
- a method of treating a subject suffering from an inflammatory disease comprising, administering to the subject an effective amount of a pharmaceutical composition disclosed herein.
- CD40 is also present in various other tissues and cells.
- CD40 is present in epithelial cells, vascular endothelium and smooth muscle cells, and CD40 ligand (CD 154) was expressed by thrombin-activated platelets.
- CD40 ligand CD 154 was expressed by thrombin-activated platelets.
- CD40 is also expressed in human renal tubules, thymic epithelia and neural cells. Interestingly, CD40 activity appears to induce apoptosis in neural cells, in contrast to its cell survival role in the immune system. Thus, a peptide or pharmaceutical composition herein can be used to inhibit apoptosis in neural cells. Accordingly, CD40 can be used to treat neural disorders characterized by cell degeneration or undesirable or excessive cell death, such as Parkinson's disease, Alzheimer's, Huntington disease, spinocerebellar ataxias/atrophies, etc.
- CD40 is also expressed in carcinomas, such as melanoma, Kaposi's sarcoma, osteosarcoma and Ewing' sarcoma.
- CD40 in malignant melanoma appears to be predictive of a negative prognosis.
- CD40 in human bladder carcinoma cells inhibits fas-mediated apoptosis.
- CD40 has been detected in tumor vasculature in a renal carcinoma mass. Stimulation of CD40 in B-cell lymphomas stimulates growth.
- CD40 can therefore function as a cell survival or growth factor in some tumors, and may promote angiogenesis. Other data indicate that CD40 may induce cell death in transformed cells.
- a peptide or compositions disclosed herein that decreases a CD40 activity associated with tumor survival will be useful in treating cell proliferative disorders (e.g., tumors) in which CD40 functions as a cell survival signal or growth promoter, either directly (e.g., in the tumor cell) or indirectly (i.e., through stimulation of angiogenesis within a tumor mass).
- cell proliferative disorders e.g., tumors
- CD40 functions as a cell survival signal or growth promoter either directly (e.g., in the tumor cell) or indirectly (i.e., through stimulation of angiogenesis within a tumor mass).
- Other biological pathways and physiological conditions that CD40 participates in are described in Biancone, et al. (Int. J. Mol. Med. 3:343 (1999)), Laman et al. (Dev. Immunol. 6:215 (1998)), Kooten and Bachereau (J. Leukoc. Biol. 67:2 (2000)), Noelle et al
- the peptides or compositions disclosed can find use in treatment of a neoplastic disease.
- a method of treating a subject suffering from or is at risk of developing a neoplastic disease characterized by a neoplastic cell that expresses CD40 comprising administering to the subject an effective amount of peptides disclosed herein or a composition comprising the peptides disclosed herein.
- CD40 has been shown to be expressed not only on immune cells, but also on tumor cells, including B-cell malignancies and solid tumors.
- the term “neoplastic cells that expresses CD40,” refers to a cell (e.g., an immune cell such as B-cell, T-cell, or an non-immune cell, such as epithelial cell, endothelial cell, smooth muscle cell) that expresses a CD40 protein on its cell surface and exhibits abnormal cell growth, cell division and proliferation not undergoing apoptosis, or both, under inappropriate conditions.
- a “neoplastic cell” may undergo cell division when a corresponding non-neoplastic cell does not undergo cell division, or, alternatively, a “neoplastic cell” may not respond to normal cellcycle checkpoint controls.
- a neoplastic cell is one that exhibits abnormal proliferation of cells.
- the growth of neoplastic cells exceeds that of normal tissue around it and it is not coordinated with that of the normal tissue around it resulting in neoplasms.
- Neoplasms may be benign (e.g., benign tumor and atypical hyperplasia), pre-malignant (e.g., carcinoma in situ and pre-cancer) or malignant (e.g., cancer).
- cancer refers to any of the various malignant neoplasms characterized by the proliferation of cells that have the capability to invade surrounding tissue and/or metastasize to new colonization sites, including but not limited to leukemias, lymphomas, carcinomas, melanomas, sarcomas, germ cell tumors and blastomas.
- exemplary cancers include cancers of the brain, bladder, breast, cervix, colon, head and neck, kidney, lung, non-small cell lung, mesothelioma, ovary, prostate, stomach and uterus, leukemia and medulloblastoma.
- Neoplastic tissues can originate from any cell type or tissue found in a mammal, including, but not limited to hepatic, skin, breast, prostate, neural, optic, intestinal, cardiac, vasculature, lymph, spleen, renal, bladder, lung, muscle, connective, tissue, pancreatic, pituitary, endocrine, reproductive organs, bone, and blood.
- the neoplastic tissue for analysis may include any type of solid tumor or hematological cancer.
- the neoplastic tissue is a breast cancer tissue.
- the neoplastic tissue is a breast tissue with atypical hyperplasia.
- leukemia refers to broadly progressive, malignant diseases of the blood-forming organs and is generally characterized by a distorted proliferation and development of leukocytes and their precursors in the blood and bone marrow.
- Leukemia diseases include, for example, acute nonlymphocytic leukemia, chronic lymphocytic leukemia, acute granulocytic leukemia, chronic granulocytic leukemia, acute promyelocytic leukemia, adult T-cell leukemia, aleukemic leukemia, a leukocythemic leukemia, basophylic leukemia, blast cell leukemia, bovine leukemia, chronic myelocytic leukemia, leukemia cutis, embryonal leukemia, eosinophilic leukemia, Gross' leukemia, hairy-cell leukemia, hemoblastic leukemia, hemocytoblastic leukemia, histiocytic leukemia, stem cell leukemia, acute monocytic leukemia,
- carcinoma refers to a malignant new growth made up of epithelial cells tending to infiltrate the surrounding tissues and give rise to metastases.
- exemplary carcinomas include, for example, acinar carcinoma, acinous carcinoma, adenocystic carcinoma, adenoid cystic carcinoma, carcinoma adenomatosum, carcinoma of adrenal cortex, alveolar carcinoma, alveolar cell carcinoma, basal cell carcinoma, carcinoma basocellulare, basaloid carcinoma, basosquamous cell carcinoma, bronchioalveolar carcinoma, bronchiolar carcinoma, bronchogenic carcinoma, cerebriform carcinoma, cholangiocellular carcinoma, chorionic carcinoma, colloid carcinoma, comedo carcinoma, corpus carcinoma, cribriform carcinoma, carcinoma en cuirasse, carcinoma cutaneum, cylindrical carcinoma, cylindrical cell carcinoma, duct carcinoma, carcinoma durum, embryonal carcinoma, encephaloid carcinoma, epiennoid carcinoma, carcinoma epitheliale adenoides, exophytic carcinoma, carcinoma ex ulcere, carcinoma fibro
- sarcoma generally refers to a tumor which arises from transformed cells of mesenchymal origin. Sarcomas are malignant tumors of the connective tissue and are generally composed of closely packed cells embedded in a fibrillar or homogeneous substance.
- Sarcomas include, for example, chondrosarcoma, fibrosarcoma, lymphosarcoma, melanosarcoma, myxosarcoma, osteosarcoma, Abemethy's sarcoma, adipose sarcoma, liposarcoma, alveolar soft part sarcoma, ameloblastic sarcoma, botryoid sarcoma, chloroma sarcoma, chorio carcinoma, embryonal sarcoma, Wilms' tumor sarcoma, endometrial sarcoma, stromal sarcoma, Ewing's sarcoma, fascial sarcoma, fibroblastic sarcoma, giant cell sarcoma, granulocytic sarcoma, Hodgkin's sarcoma, idiopathic multiple pigmented hemorrhagic sarcoma,
- melanoma is taken to mean a tumor arising from the melanocytic system of the skin and other organs.
- Melanomas include, for example, acral-lentiginous melanoma, amelanotic melanoma, benign juvenile melanoma, Cloudman's melanoma, S91 melanoma, Harding-Passey melanoma, juvenile melanoma, lentigo maligna melanoma, malignant melanoma, nodular melanoma subungal melanoma, and superficial spreading melanoma.
- the Examples herein show that the CD40 binding peptides disclosed herein can bind CD40 expressed on B-cell and inhibit, for example, B-cell proliferation and B-cell activation.
- the peptides of the present disclosure can find use, for example, in the treatment of B-cell lymphomas, prevention of EBV-induced B-cell lymphoma that can occur after transplantation or in other instances of immunosuppression, such as AIDS, and which present a significant risk in such patient populations.
- the methods disclosed herein can be useful in prevention of immunoblastic B-cell lymphomas that frequently arise in immunocompromised individuals. In such preventative methods, a mammal at risk of developing an immunoblastic B-cell lymphoma is administered an effective amount of peptides disclosed herein.
- the peptides can be administered for as long as the state of immunocompromise that places the individual at risk exists.
- peptides of the present disclosure or compositions comprising the peptides can be useful for treatment of cancer characterized by “neoplastic B-cell growth” is intended any disease or condition (including pre-malignant conditions) involving uncontrolled growth of cells of B-cell lineage.
- Such diseases and conditions include, but are not limited to, acute lymphoblastic leukemia (ALL), acute myelogenous leukemia (AML), chronic myelogenous leukemia (CIVIL), chronic lymphocytic leukemia (CLL), prolymphocytic leukemia (PLL), small lymphocytic leukemia (SLL), diffuse small lymphocytic leukemia (DSLL), diffuse large B-cell lymphoma (DLBCL), hairy cell leukemia, non-Hodgkin's lymphomas, Hodgkin's disease, Epstein-Barr Virus (EBV) induced lymphomas, myelomas such as multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, mucosal associated lymphoid tissue lymphoma, monocytoid B cell lymphoma, splenic lymphoma, lymphomatoid granulomatosis, intravascular lymphomatosis, immunoblastic lymphomas, AIDS-
- compositions and methods used to prevent occurrence (or reoccurrence) or treatment of neoplastic disease characterized by other types of malignant cells that express CD40 in individuals at risk for such disease include those with family history or other genetic characteristics indicating predisposition to cancers in which the neoplastic cells express CD40, and individuals that develop drug-resistant neoplastic disease as a result of chemotherapy, in which the drug-resistant neoplastic cells express CD40.
- Treatment refers to initiation of therapy after clinical symptoms or signs of disease have been observed.
- the methods may be used in conjunction with other therapies appropriate for afflicted individuals, including chemotherapy, radiation therapy, and immunotherapy.
- the “effective amount” to be administered will be governed by such considerations, and refers to the minimum amount necessary to ameliorate, treat, or stabilize, the cancer; to increase the time until progression (duration of progression free survival) or to treat or prevent the occurrence or recurrence of a tumor, a dormant tumor, or a micrometastases.
- the peptides, disclosed herein can be optionally formulated with one or more additional therapeutic agents currently used to prevent or treat cancer or a risk of developing a cancer.
- the effective amount of such other agents depends on the amount of peptides present in the formulation, the type of disorder or treatment, and other factors such age, weight etc. These are generally used in the same dosages and with administration routes as used herein before or about from 1 to 99% of the heretofore employed dosages.
- the efficacy of the treatment methods for cancer comprising administering the peptides disclosed herein or pharmaceutical compositions of the present disclosure can be measured by various endpoints commonly used in evaluating cancer treatments, including but not limited to, tumor regression, tumor weight or size shrinkage, time to progression, duration of survival, progression free survival, overall response rate, duration of response, and quality of life.
- the peptides disclosed herein can require unique measures and definitions of clinical responses to drugs.
- the therapeutically effective amount of the peptides disclosed herein or compositions comprising the same can reduce the number of cancer cells; reduce the tumor size; inhibit (i.e., slow to some extent and preferably stop) cancer cell infiltration into peripheral organs; inhibit (i.e., slow to some extent and preferably stop) tumor metastasis; inhibit, to some extent, tumor growth; and/or relieve to some extent one or more of the symptoms associated with the disorder.
- the peptides disclosed herein act to prevent growth and/or kill existing cancer cells; it can be cytostatic and/or cytotoxic.
- efficacy in vivo can, for example, be measured by assessing the duration of survival, duration of progression free survival (PFS), the response rates (RR), duration of response, and/or quality of life.
- described herein are methods for increasing progression free survival of a human subject susceptible to or diagnosed with a cancer, for example, skin cutaneous melanoma.
- Time to disease progression is defined as the time from administration of the drug until disease progression or death.
- the combination treatment of the disclosure using a peptide or compositions disclosed herein, and one or more chemotherapeutic agents may significantly increase progression free survival by at least about 1 month, 1.2 months, 2 months, 2.4 months, 2.9 months, 3.5 months, such as by about 1 to about 5 months, when compared to a treatment with chemotherapy alone.
- the methods described herein may significantly increase response rates in a group of human subjects susceptible to or diagnosed with a cancer that are treated with various therapeutics.
- Response rate is defined as the percentage of treated subjects who responded to the treatment.
- the combination treatment described herein using a peptide or compositions disclosed herein, and one or more chemotherapeutic agents significantly increases response rate in the treated subject group compared to the group treated with chemotherapy alone.
- the methods described herein comprise administering an effective amount of the peptides of the instant disclosure or compositions comprising said peptides, to a subject in order to alleviate a symptom of a cancer.
- “alleviating a symptom of a cancer” is ameliorating or reducing any condition or symptom associated with the cancer.
- reduction or degree of prevention is at least 5%, 10%, 20%, 40%, 50%, 60%, 80%, 90%, 95%, or 100% as measured by any standard technique.
- the cancer is completely cleared as detected by any standard method known in the art, in which case the cancer is considered to have been treated.
- a patient who is being treated for a cancer is one who a medical practitioner has diagnosed as having such a condition.
- Diagnosis can be by any suitable means. Diagnosis and monitoring can involve, for example, detecting the level of cancer cells in a biological sample (for example, a tissue or lymph node biopsy, blood test, or urine test), detecting the level of a surrogate marker of the cancer in a biological sample, detecting symptoms associated with the specific cancer, or detecting immune cells involved in the immune response typical of such a cancer.
- a biological sample for example, a tissue or lymph node biopsy, blood test, or urine test
- the treatment and/or prevention of cancer includes, but is not limited to, alleviating symptoms associated with cancer, the inhibition of the progression of cancer, the promotion of the regression of cancer, the promotion of the immune response, inhibition of tumor growth, inhibition of tumor size, inhibition of metastasis, inhibition of cancer cell growth, inhibition of cancer cell proliferation, or cause cancer cell death.
- compositions, systems, and methods for inducing graft tolerance relate to administering a tolerizing vaccine or a preparatory regimen before, during, and/or after administration of donor transplant cells, tissue(s), or organ(s).
- the tolerizing vaccine or preparatory regimen can induce tolerance to the allograft or xenograft in the graft recipient.
- An organ, tissue, or cell can be differentiated from stem cells, grown de novo, or isolated from an animal (e.g., a human or non-human animal) and can be transplanted into a recipient in need of a transplant from the same species (an allotransplant) or a different species (a xenotransplant).
- the donor of the organ, tissue, or cell can be referred to herein as a transplant donor.
- the transplanted organ, tissue, or cell can be referred to herein as a transplant or a graft.
- the donor of transplant or graft can be any animal, including human and non-human animals. In some cases the donor is a human. In some cases, the transplant donor is a non-human animal.
- the transplant donor e.g., a non-human animal donor
- the donor of a transplant or graft can be a living donor or a cadaveric donor.
- the transplant donor is a living donor.
- the transplant donor is a cadaveric donor.
- the cadaveric donor may be a brain dead, heart beating donor (BDD).
- the cadaveric donor may be a non-heart beating donor (NHBD).
- BDD brain dead, heart beating donor
- NHBD non-heart beating donor
- the donor can be from any animal, for example, a human or non-human animal.
- the donor of a transplant or graft can be at any age or stage of development.
- the transplant donor can be a fetal, perinatal, neonatal, pre-weaning, post-weaning, juvenile, young adult, or adult human or animal.
- Transplants or grafts can be used to treat diseases or disorders in recipients in need thereof.
- Suitable diseases that can be treated are any in which an organ, tissue, or cell of a recipient is defective or injured, and the recipient can be treated by transplantation of an organ, tissue, or cell (e.g., a kidney, heart, lung, liver, vein, skin, endocrine pancreas, pancreatic islet cell, or a combination thereof).
- the transplant comprises a kidney, liver, heart, lung, pancreas, endocrine pancreas, islet cell, small bowel, bone marrow, hematopoietic stem cell, embryonic or induced pluripotent stem cell-derived cells such as islet beta cells or hepatocytes, embryonic or induced pluripotent stem cell-derived islet, embryonic or induced pluripotent stem cell-derived hepatocyte, or a combination thereof.
- methods for inducing immunosuppression can be useful to prevent graft rejection in a subject who has undergone, is undergoing or will be undergoing a cell, organ or tissue transplant.
- the peptides and compositions disclosed herein can find use in preventing transplant rejection by inhibiting a CD40 mediated immune response in a recipient, towards an antigen on a transplanted material (e.g., cells, tissues, an/or organs).
- a transplanted material e.g., cells, tissues, an/or organs.
- a method of inhibiting an immune response to a donor cell or tissue transplantation in a recipient comprising administering to the recipient a peptide or compositions disclosed herein.
- transplant rejection acute or chronic
- Transplantation includes grafting of tissues or organ from the body of an individual to a different place within the same individual, or a different individual.
- Transplantation also involves grafting of tissues or organs from one area of the body to another. Transplantation of tissues or organs between genetically dissimilar animals of the same species is termed as allogeneic transplantation. Transplantation of animal organs into humans is termed xenotransplants.
- Transplant/graft rejection can involve recognition of donor-specific antigens, for example, recognition of donor-specific antigens presented to T cells by host antigen presenting cells (indirect) or donor antigen presenting cells (direct). T cell activation in response to donor-specific antigens can lead to a pro-inflammatory response and transplant rejection. Transplant rejection (e.g., T cell-mediated transplant rejection) can be prevented by chronic immunosuppression with one or more immunomodulatory molecules. However, immunosuppression is costly and associated with the risk of serious side effects.
- a method described herein to prevent transplantation rejection or prolong the time to transplantation rejection without or with minimal immunosuppressive drug use involves using a genetically modified animal as a cell, organ, or tissue donor.
- the cells, organs, and/or tissues of the altered donor animal e.g., a donor non-human animal, can be harvested and used in allografts or xenografts.
- a cell, organ, or tissue can be extracted from an animal, and used to generate a genetically-altered cell, organ or tissue.
- primary cells can be extracted from an animal, and used to make genetically altered cells.
- a cell, organ, or tissue derived from an animal e.g., a cell line
- Transplant rejection can also be reduced or eliminated by inducing tolerance to a transplant or graft using a tolerizing vaccine or preparatory regimen.
- a tolerizing vaccine or preparatory regimen of the disclosure can be used to prevent transplant rejection or delay rejection, for example, by reducing a pro-inflammatory immune response to the transplant, and/or enhancing a tolerance-promoting immune response.
- a tolerizing vaccine or preparatory regimen of the disclosure can circumvent the need for long-term immunosuppression of the recipient.
- an effective amount is the minimum amount necessary to inhibit an immune response in a recipient that would result in rejection of the transplant, but as much as necessary to achieve a longer transplant survival time. Such amount is preferably below the amount that is toxic to the recipient or renders the recipient significantly more susceptible to infections.
- the amount of peptides or compositions disclosed herein used for inhibiting immune response directed to a transplant can be lower than the amount of an immunosuppressive agent normally required for transplanted grafts and depends on the individual circumstances surrounding the transplant.
- a treatment with the peptides or compositions disclosed herein can also lessen or prevent the side effects frequently observed in transplant recipients who undergo immune suppressive therapy,
- the present disclosure therefore provides a method for inhibiting transplant rejection and a method to increase transplant survival.
- the peptides or compositions disclosed herein can administered as a part of a transplantation protocol.
- a method for transplantation of a cell, organ, or tissue from a mammalian donor in a recipient comprising administering an effective amount of the peptides or pharmaceutical compositions disclosed herein.
- the method of transplantation further comprises transplanting the donor cell tissue or organ in a recipient.
- An organ, tissue, or cell isolated from an animal can be transplanted into a recipient in need of a transplant from the same species (an allotransplant) or a different species (a xenotransplant).
- Transplants or grafts can be used to treat diseases or disorders in a recipient in need thereof.
- Suitable diseases that can be treated are any in which an organ, tissue, or cell of a recipient is defective or injured, (e.g., a kidney, heart, lung, liver, vein, skin, pancreatic islet cell, or a combination thereof) and the recipient can be treated by transplantation of an organ, tissue, or cell.
- the transplant comprises a kidney, liver, heart, lung, pancreas, islet cell, small bowel, bone marrow, hematopoietic stem cell, embryonic or induced pluripotent stem cell-derived cells, embryonic or induced pluripotent stem cell-derived islet, embryonic or induced pluripotent stem cell-derived hepatocyte, or a combination thereof.
- the methods herein can comprise administering one or more dose of a peptide or compositions disclosed herein to a recipient before, after, and/or during transplant of donor cells, organs, and/or tissues to inhibit donor-specific immune response in a recipient.
- a first dose of a peptide or compositions disclosed herein can be given on or on about day ⁇ 100, day ⁇ 90, day ⁇ 80, day ⁇ 70, day ⁇ 60, day ⁇ 50, day ⁇ 40, day ⁇ 30, day ⁇ 20, day ⁇ 15, day ⁇ 14, day ⁇ 13, day ⁇ 12, day ⁇ 11, day ⁇ 10, day ⁇ 9, day ⁇ 8, day ⁇ 7, day ⁇ 6, day ⁇ 5, day ⁇ 4, day ⁇ 3, day ⁇ 2 or day ⁇ 1, relative to transplant of donor cells, organs, and/or tissues on day 0.
- a first dose of a peptide or compositions disclosed herein can be given on or on about day ⁇ 100 to ⁇ 50; ⁇ 50 to ⁇ 40; ⁇ 40 to ⁇ 30; ⁇ 30 to ⁇ 20; ⁇ 20 to ⁇ 10; ⁇ 10 to ⁇ 5; ⁇ 7 to ⁇ 1, relative to transplant of donor cells, organs, and/or tissues on day 0.
- a first dose of a peptide or compositions disclosed herein can be given 8 days (e.g., day ⁇ 8) before transplant of donor cells, organs, and/or tissues.
- a peptide or compositions of the present disclosure can be administered on the same day (e.g., day 0) as transplant of donor cells, organs, and/or tissues.
- a peptide or a composition disclosed herein can be administered on or on about day 300, day 200, day 100, day 90, day 80, day 70, day 60, day 50, day 40, day 30, day 20, day 15, day 14, day 13, day 12, day 11, day 10, day 9, day 8, day 7, day 6, day 5, day 4, day 3, day 2 or day 1, relative to transplant of donor cells, organs, and/or tissues on day 0.
- a peptide or compositions disclosed herein can be administered on or on about day 400 to 350; 350 to 300; 300 to 250; 250 to 200; 200 to 150; 150 to 100; 100 to 50; 50 to 40; 40 to 30; 30 to 20; 20 to 10; 10 to 5; 7 to 1, relative to transplant of donor cells, organs, and/or tissues on day 0.
- a peptide or compositions disclosed herein can be administered on 1 day after (e.g., day 1) transplant of donor cells, organs, and/or tissues.
- a peptide or compositions disclosed herein can be administered of day 14 after transplant of donor cells, organs, and/or tissues.
- a peptide or compositions disclosed herein can be administered for a lifetime of the recipient.
- a recipient e.g., a human or a non-human animal
- a recipient e.g., a human or a non-human animal
- a recipient e.g., a human or a non-human animal
- a recipient e.g., a human or a non-human animal
- a recipient e.g., a human or a non-human animal
- a recipient e.g., a human or a non-human animal
- the transplantation method of the present disclosure can comprise multiple doses of a peptide or a composition disclosed herein before, and/or during and/or after transplantation of a graft cell, tissue, or organ.
- the multiple doses can be referred to as comprising an initial dose and one or more booster doses.
- the initial dose occurs prior to or concurrently with the transplant of the graft cell tissue or organ.
- the booster dose(s), when administered, occur after the initial dose.
- a booster dose is administered to achieve and maintain a particular serum/blood trough level of the peptides disclosed herein.
- one or more booster doses can be administered before, and/or concurrently with, and/or after transplant of the graft cell, tissue, or organ.
- Subsequent (e.g., booster) dose(s) of the peptide or compositions disclosed herein can be administered in any interval of time following a preceding dose (e.g., an initial dose).
- the subsequent dose can be administered 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 31 days, 32 days, 33 days, 34 days, 35 days, 36 days, 37 days, 38 days, 39 days, 40 days, 41 days, 42 days, 43 days, 44 days, 45 days, 46 days, 47 days, 48 days, 49 days, 50 days, 51 days, 52 days, 53 days, 54 days, 55 days, 56 days, 57 days, 58 days, 59 days, 60 days, 90 days, 120 days, 150 days, or 180 days
- subsequent (booster) dose(s) can be administered before, concurrently with, or after transplantation of the graft cell, tissue, or organ.
- the methods of the present disclosure comprise at least one dose of the peptide or compositions disclosed herein prior to transplantation.
- the methods of the present disclosure comprises at least two doses of a peptide or compositions disclosed herein prior to transplantation (e.g., an initial dose and a booster dose).
- the methods herein comprises at least three doses of the peptide or compositions disclosed herein prior to transplantation (e.g., an initial dose and two booster doses).
- the preparatory regimen comprises an initial dose of the peptide or compositions disclosed herein prior to transplantation and at least one dose of booster concurrently with or after transplantation of the graft cell, tissue, or organ.
- two doses of a peptide or compositions thereof can be administered.
- the first dose can be administered on day ⁇ 8 relative to transplant of donor cells, organs, and/or tissues on day 0.
- the second dose can be administered on day ⁇ 1 relative to transplant of donor cells, organs, and/or tissues on day 0.
- the first dose can be administered on day ⁇ 8, ⁇ 9, ⁇ 10, ⁇ 11, ⁇ 12, ⁇ 13, or ⁇ 14 relative to transplant of donor cells, organs, and/or tissues on day 0.
- the second dose can be administered on day ⁇ 1, ⁇ 2, ⁇ 3, ⁇ 4, ⁇ 5, or ⁇ 6 relative to transplant of donor cells, organs, and/or tissues on day 0.
- a second dose (e.g., a booster dose) can be administered on day 100, day 90, day 80, day 70, day 60, day 50, day 40, day 30, day 29, day 28, day 27, day 26, day 25, day 24, day 23, day 22, day 21, day 20, day 19, day 18, day 17, day 16, day 15, day 14, day 13, day 12, day 11, day 10, day 9, day 8, day 7, day 6, day 5, day 4, day 3, day 2 or day 1, relative to transplant of donor cells, organs, and/or tissues on day 0.
- the second dose (e.g., a booster dose) can be administered on 1 day after (e.g., day 1) transplant of donor cells, organs, and/or tissues.
- a third dose of a peptide or compositions disclosed herein can be administered on day 300, day 200, day 100, day 90, day 80, day 70, day 60, day 50, day 40, day 30, day 29, day 28, day 27, day 26, day 25, day 24, day 23, day 22, day 21, day 20, day 19, day 18, day 17, day 16, day 15, day 14, day 13, day 12, day 11, day 10, day 9, day 8, day 7, day 6, day 5, day 4, day 3, day 2 or day 1, relative to transplant of donor cells, organs, and/or tissues on day 0.
- the peptide or compositions disclosed herein can be administered on or on about day 300 to 200; 200 to 100; 100 to 50; 50 to 40; 40 to 30; 30 to 20; 20 to 10; 10 to 5; 7 to 1, relative to transplant of donor cells, organs, and/or tissues on day 0.
- a fourth dose of a peptide or compositions disclosed herein can be administered on day 600, day 500, day 400, day 300, day 200, 100, day 90, day 80, day 70, day 60, day 50, day 40, day 30, day 29, day 28, day 27, day 26, day 25, day 24, day 23, day 22, day 21, day 20, day 19, day 18, day 17, day 16, day 15, day 14, day 13, day 12, day 11, day 10, day 9, day 8, day 7, day 6, day 5, day 4, day 3, day 2 or day 1, relative to transplant of donor cells, organs, and/or tissues on day 0.
- the peptide or compositions disclosed herein can be administered on or on about day 600 to 500; 500 to 400; 400 to 300; 300 to 200; 200 to 100; 100 to 50; 50 to 40; 40 to 30; 30 to 20; 20 to 10; 10 to 5; 7 to 1, relative to transplant of donor cells, organs, and/or tissues on day 0.
- a fifth dose of a peptide or compositions disclosed herein can be administered on day 1,000, day 900, day 800, day 700, day 600, day 500, day 400, day 300, day 200, 100, day 90, day 80, day 70, day 60, day 50, day 40, day 30, day 29, day 28, day 27, day 26, day 25, day 24, day 23, day 22, day 21, day 20, day 19, day 18, day 17, day 16, day 15, day 14, day 13, day 12, day 11, day 10, day 9, day 8, day 7, day 6, day 5, day 4, day 3, day 2 or day 1, relative to transplant of donor cells, organs, and/or tissues on day 0.
- a booster dose can be administered on day 1,000, day 900, day 800, day 700, day 600, day 500, day 400, day 300, day 200, 100, day 90, day 80, day 70, day 60, day 50, day 40, day 30, day 29, day 28, day 27, day 26, day 25, day 24, day 23, day 22, day 21, day 20, day 19, day 18, day 17, day 16, day 15, day 14, day 13, day 12, day 11, day 10, day 9, day 8, day 7, day 6,
- the peptide or compositions disclosed herein can be administered on or on about day 1,000 to 900; 900 to 800; 800 to 700; 700 to 600; 600 to 500; 500 to 400; 400 to 300; 300 to 200; 200 to 100; 100 to 50; 50 to 40; 40 to 30; 30 to 20; 20 to 10; 10 to 5; 7 to 1, relative to transplant of donor cells, organs, and/or tissues on day 0.
- a second dose of a booster is not required.
- a second dose of a booster is given concomitantly on day 0 with transplant donor cells, organs, and/or tissues.
- Booster doses of a peptide or compositions disclosed herein can be lower dose than the initial or preceding dose of the peptide or compositions disclosed herein.
- a booster or subsequent dose can be about: 1%, 2%, 3%, 4%, 5%, 7.5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, or 75% lower than the initial or preceding dose.
- the transplantation method comprises administering a tolerizing agent in the subject.
- a tolerizing agent or a “toleragen” as used herein means any donor antigen (such as a protein, nucleic acid, carbohydrate, lipid, or combination of any thereof) that mediates host unresponsiveness.
- a tolerizing agent works by inducing the tolerized host not to produce antibodies or cell-mediated immune responses specific for the toleragen. Additional discussion of tolerizing may be found, for instance, in PCT publication WO 2006/052668, which is incorporated herein in its entirety.
- the tolerizing agent can be a tolerizing vaccine comprising inactivated or apoptotic donor cells that upon intravenous injection can result in tolerance to transplanted donor cells or tissues or compositions comprising said cells with similar marks.
- the tolerizing agent can be an anti-CD40 agent (e.g., agent comprising one or more amino acid sequences listed in any one of SEQ ID 1-6 and 8-17).
- the tolerizing agent can be mammalian leukocytes fixed with a crosslinking agent.
- the tolerizing agent can be mammalian leukocytes fixed with a crosslinking agent, administered together with an anti-CD40 binding agent (e.g., agent comprising one or more amino acid sequences listed in any one of SEQ ID 1-6 and 8-17).
- an anti-CD40 binding agent e.g., agent comprising one or more amino acid sequences listed in any one of SEQ ID 1-6 and 8-17.
- the tolerizing agent can be mammalian leukocytes fixed with a crosslinking agent, surface-conjugated with an anti-CD40 binding agent (e.g., agent comprising one or more amino acid sequences listed in any one of SEQ ID 1-6 and 8-17).
- the preparatory regimen can be inactivated or apoptotic donor cells that upon intravenous injection can result in tolerance to transplanted donor cells or tissues or compositions comprising said cells with similar marks.
- the preparatory regimen can be an anti-CD40 agent (e.g., agent comprising one or more amino acid sequences listed in any one of SEQ ID 1-6 and 8-17).
- the preparatory regimen can be mammalian leukocytes fixed with a crosslinking agent.
- the preparatory regimen can be mammalian leukocytes fixed with a crosslinking agent, administered together with an anti-CD40 binding agent (e.g., agent comprising one or more amino acid sequences listed in any one of SEQ ID 1-6 and 8-17).
- an anti-CD40 binding agent e.g., agent comprising one or more amino acid sequences listed in any one of SEQ ID 1-6 and 8-17.
- the preparatory regimen can be fixed mammalian leukocytes surface-conjugated with an anti-CD40 binding agent (e.g., agent comprising one or more amino acid sequences listed in any one of SEQ ID 1-6 and 8-17).
- a tolerizing vaccine or preparatory regimen of the disclosure can comprise administering an anti-CD40 agent or an anti-CD40 ligand agent, e.g., any one or more of the peptides comprising an amino acid sequence with at least 80%, at least 85%, at least 90% or at least 95% sequence identity to the sequences set forth in any one of SEQ ID NOs. 1-6 and 8-17.
- an anti-CD40 agent or an anti-CD40 ligand agent e.g., any one or more of the peptides comprising an amino acid sequence with at least 80%, at least 85%, at least 90% or at least 95% sequence identity to the sequences set forth in any one of SEQ ID NOs. 1-6 and 8-17.
- the tolerizing vaccine or preparatory regimen can further comprise immunosuppression agents such as (i) an mTOR inhibitor, (ii) an anti-tumor necrosis factor agent or an anti-tumor necrosis factor receptor agent, or (iii) an anti-interleukin 6 agent or an anti-interleukin 6 receptor agent.
- immunosuppression agents such as (i) an mTOR inhibitor, (ii) an anti-tumor necrosis factor agent or an anti-tumor necrosis factor receptor agent, or (iii) an anti-interleukin 6 agent or an anti-interleukin 6 receptor agent.
- a tolerizing vaccine or preparatory regimen of the disclosure can comprise administering apoptotic cells (e.g., apoptotic donor leukocytes) to a recipient conjugated to an anti-CD40 agent or an anti-CD40 ligand agent, e.g., any one or more of the peptides comprising an amino acid sequence with at least 80%, at least 85%, at least 90% or at least 95% sequence identity to the sequences set forth in any one of SEQ ID NOs. 1-6 and 8-17.
- apoptotic cells e.g., apoptotic donor leukocytes
- an anti-CD40 agent or an anti-CD40 ligand agent e.g., any one or more of the peptides comprising an amino acid sequence with at least 80%, at least 85%, at least 90% or at least 95% sequence identity to the sequences set forth in any one of SEQ ID NOs. 1-6 and 8-17.
- Cells for the preparation of a tolerizing vaccine or preparatory regimen can be obtained from any source, including animals, cells lines, and/or lab-generated cells.
- the cells can be obtained from a human or non-human animal.
- the cells can be from a cell line (e.g., a human or non-human cell line).
- the cells are human cells.
- the cells are non-human cells.
- the cells are of a non-human primate.
- the cells are of a member of the Laurasiatheria superorder.
- the cells are of an ungulate, for instance a camelid or a pig.
- the cells are of a pig.
- the cells are from the same species as the transplant donor. In some cases, the cells are from the same species as the transplant recipient. In some cases, the cells are from the same species as the transplant donor and the transplant recipient. In some cases, the cells are from a different species than the transplant donor. In some cases, the cells are from a different species than the transplant recipient. In some cases, the cells are from a different species than the transplant donor and the transplant recipient.
- Cells for the preparation of a tolerizing vaccine or preparatory regimen can be obtained from living donors or cadaveric donors.
- the donor is a living donor.
- the donor is a cadaveric donor.
- the cadaveric donor may be, for example, a brain dead, heart beating donor (BDD).
- the cadaveric donor may be, for example, a non-heart beating donor (NHBD).
- the donor can be from any animal, for example, a human or non-human animal.
- cells for the preparation of a tolerizing vaccine can be from the same donor as a graft or transplant.
- cells for the preparation of a tolerizing vaccine can be from a different donor than the graft or transplant.
- Cells for the preparation of a tolerizing vaccine or preparatory regimen can be obtained from a donor animal of any age or stage of development.
- the donor animal can be a fetal, perinatal, neonatal, pre-weaning, post-weaning, juvenile, young adult, or adult animal.
- non-human animals can be past weaning age.
- donor animals can be at least or at least about six months old.
- donor animals can be at least or at least about 18 months old.
- cells for the preparation of a tolerizing vaccine or preparatory regimen can be obtained (for example, differentiated) from stem cells (e.g., embryonic stem cells, induced pluripotent stem cells, and/or mesenchymal stem cells).
- stem cells e.g., embryonic stem cells, induced pluripotent stem cells, and/or mesenchymal stem cells.
- the cells used to make a tolerizing vaccine or preparatory regimen can include one or more cells from tissues, organs, or bodily fluids.
- the cells can be from tissues, organs, or bodily fluids including, but not limited to: brain, lung, liver, heart, spleen, pancreas, small intestine, large intestine, skeletal muscle, smooth muscle, skin, bones, adipose tissues, hairs, thyroid, trachea, gall bladder, kidney, ureter, bladder, aorta, vein, esophagus, diaphragm, stomach, rectum, adrenal glands, bronchi, ears, eyes, retina, genitals, hypothalamus, larynx, nose, tongue, spinal cord, or ureters, uterus, ovary, testis, blood, spinal fluid, lymph fluid, or a combination thereof.
- the cells used to make a tolerizing vaccine or preparatory regimen can include one or more types of cells.
- the cells can include, but are not limited to: trichocytes, keratinocytes, gonadotropes, corticotropes, thyrotropes, somatotropes, lactotrophs, chromaffin cells, parafollicular cells, glomus cells melanocytes, nevus cells, Merkel cells, odontoblasts, cementoblasts corneal keratocytes, retina Muller cells, retinal pigment epithelium cells, neurons, glias (e.g., oligodendrocyte astrocytes), ependymocytes, pinealocytes, pneumocytes (e.g., type I pneumocytes, and type II pneumocytes), clara cells, goblet cells, G cells, D cells, ECL cells, gastric chief cells, parietal cells, foveolar cells, K cells, D cells, I cells, goble
- the cells used to make a tolerizing vaccine or preparatory regimen comprise a cell type that expresses MHC class II. In some cases, the cells used to make a tolerizing vaccine or preparatory regimen comprise a cell type that does not expresses MHC class II.
- a tolerizing vaccine or preparatory regimen can comprise leukocytes.
- Leukocytes can include, for example, neutrophils, eosinophils, basophils, lymphocytes, monocytes, or a combination thereof.
- Lymphocytes can include, for example, B lymphocytes (B cells), T lymphocytes (T cells), natural killer (NK) cells, or a combination thereof.
- Leukocytes in a tolerizing vaccine or preparatory regimen can be obtained from any source, including, for example, a donor, a cell line, or a differentiated stem cell.
- Leukocytes obtained from a donor can include leukocytes obtained from a spleen (e.g., splenocytes, splenic B cells); a liver; peripheral blood (including peripheral blood B cells); a lymph node; a thymus; bone marrow; or any other organ, tissue, or bodily fluid; or any combination thereof.
- the tolerizing vaccine or preparatory regimen comprises splenic B cells, peripheral blood B cells, or a combination thereof.
- the tolerizing vaccine or preparatory regimen comprises cells mobilized from the bone marrow to peripheral blood with a mobilization agent, e.g., cells mobilized with granulocyte colony-stimulating factor (G-CSF), granulocyte macrophage colony-stimulating factor (GM-CSF), mozobil, or a combination thereof.
- a mobilization agent e.g., cells mobilized with granulocyte colony-stimulating factor (G-CSF), granulocyte macrophage colony-stimulating factor (GM-CSF), mozobil, or a combination thereof.
- G-CSF granulocyte colony-stimulating factor
- GM-CSF granulocyte macrophage colony-stimulating factor
- mozobil or a combination thereof.
- a donor of cells used in the preparation of a tolerizing vaccine or preparatory regimen can be genetically modified.
- cells obtained from a donor can be genetically modified ex vivo.
- cell lines are genetically modified to produce cells for use in a tolerizing vaccine or preparatory regimen.
- the genetically modified donors and/or cells can be produced using any method known in the art, including those described herein. Regardless of whether the genetically modified cells are isolated from a genetically modified animal, produced in culture, or a combination thereof, the genetically modified cells can be of any animal species, including human and non-human animals.
- Genetically modified cells used in a tolerizing vaccine or preparatory regimen can comprise one or more genetic modifications that reduce or eliminate expression or a gene or gene product (e.g., a protein).
- the genetic modification(s) can be modifications to the gene whose expression is reduced or eliminated. Such genes can be referred to as disrupted genes.
- the genetic modification(s) can also be to areas of the genome separate from the gene whose expression is reduced or eliminated (for example, modification to a promoter, enhancer, silencer, transcription factor, etc.).
- the genetically modified cells used in the tolerizing vaccine or preparatory regimen can comprise, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, or more genes whose expression is reduced or eliminated by genetic modification.
- Non-limiting examples of genes whose expression can be reduced or eliminated by genetic modification in the cells used in a tolerizing vaccine or preparatory regimen include, but are not limited to: alpha 1,3 galactosyltransferase (GGTA1); putative cytidine monophosphatase-N-acetylneuraminic acid hydroxylase-like protein (CMAH); ⁇ 1,4N-acetylgalactosaminyltransferase (B4GALNT2); a component of a major histocompatibility complex (MHC) I-specific enhanceosome (e.g., a NOD-like receptor family CARD domain containing 5 (NLRC5)); a transporter of an MHC I-binding peptide (e.g., transporter associated with antigen processing 1 (TAP1)); complement component 3 (C3); a CXC chemokine receptor 3 ligand (CXCL3); a CXC motif chemokine ligand10 (CXCL10)
- the genetically modified cells used in a tolerizing vaccine can comprise disruptions in one or more genes comprising GGTA1, CMAH, B4GALNT2, B2M, NLRC5 or any combination thereof.
- the genetically modified cells used to make a tolerizing vaccine or preparatory regimen can have disrupted GGTA1 only, or disrupted CMAH only, or disrupted B4GALNT2, B2M or NLRC5 only.
- the genetically modified cells used to make a tolerizing vaccine or preparatory regimen can also have disrupted GGTA1 and CMAH, disrupted GGTA1 and B4GALNT2, or disrupted CMAH and B4GALNT2, or disrupted NLRC5 and B2M.
- the genetically modified cells used to make a tolerizing vaccine or preparatory regimen can have disrupted GGTA1, CMAH, and B4GALNT2. Without wishing to be bound by any particular theory, such disruptions can minimize or eliminate cell-mediated immunity, antibody-mediated immunity, antibody-dependent cell-mediated immunity, and/or cell-dependent antibody-mediated immunity to organ, tissue, cell, and cell line grafts (e.g., xenografts or allografts comprising the same genetic modification(s) as the cells used in the tolerizing vaccine).
- organ, tissue, cell, and cell line grafts e.g., xenografts or allografts comprising the same genetic modification(s) as the cells used in the tolerizing vaccine.
- Genetically modified cells used in a tolerizing vaccine or preparatory regimen can comprise, or further comprise, one or more genetic modifications that increase expression of one or more genes or gene products.
- the increased expression can be from zero expression, e.g., the increased expression can be of a gene or gene product that is not normally expressed in the cell without genetic modification.
- the increased expression can be compared to a threshold level, e.g., a level normally expressed in the cell without genetic modification.
- the genetic modification(s) can comprise one or more exogenous polynucleotides encoding a polypeptide (e.g., an endogenous or exogenous polypeptide).
- exogenous polynucleotides include, but are not limited to, polynucleotides encoding one or more of an MHC I formation suppressor (e.g., an infected cell protein 47 (ICP47)); a regulator of complement activation (e.g., CD46, CD55, or CD59); an inhibitory ligand for NK cells; a B7 family member (e.g., a programmed death ligand such as PD-L1 or PD-L2); a serine protease inhibitor (e.g., Spi9); a galectin; an interleukin (e.g., IL-37); a CD40:CD40L blocking agent (e.g., a CD40 antagonist polypeptide, an anti-CD40 ligand polypeptide); a Fas ligand (FasL); any functional fragment thereof; or any combination thereof.
- an MHC I formation suppressor e.g., an infected cell protein 47 (ICP47)
- an inhibitory ligand for NK cells is a human leukocyte antigen (HLA), such as human leukocyte antigen E (HLA-E), human leukocyte antigen G (HLA-G), ⁇ -2-microglobulin (B2M) or any combination thereof.
- HLA-G is HLA-G1, HLA-G2, HLA-G3, HLA-G4, HLA-G5, HLA-G6, HLA-G7, or any combination thereof.
- galectins is galectin-1, galectin-2, galectin-3, galectin-4, galectin-5, galectin-6, galectin-7, galectin-8, galectin-9, galectin-10, galectin-11, galectin-12, galectin-13, galectin-14, or galectin-15.
- a galectin can be galectin-9.
- Donor leukocytes can be retrieved from a living donor's spleen.
- B lymphocytes can be taken from the donor in one or more blood draws and/or apheresis procedures and optionally expanded ex vivo, or a separate cell donor can be identified that is a suitable match or partial match to the transplant donor.
- separate donors can be used for the tolerogenic leukocytes and the allograft.
- splenocytes from a cadaveric donor who is fully matched or partially matched (e.g., haploidentical) with the prospective living transplant donor are a clinically viable source of tolerogenic leukocytes.
- a tolerization protocol can be initiated with the infusion of splenocytes on e.g., day ⁇ 7 followed by the living donor transplant (e.g., a kidney transplant) on e.g., day 0 and the infusion of ex vivo expanded splenic B cells on e.g., on day +1.
- the living donor transplant e.g., a kidney transplant
- the infusion of ex vivo expanded splenic B cells e.g., on day +1.
- Cells used in preparing a tolerizing vaccine/regimen or preparatory regimen can be expanded ex vivo.
- leukocyte cells can be expanded in vitro in the presence of one or more reagents for a predetermined amount of time prior to use as a tolerizing vaccine.
- the cells can be contacted with at least one cytokine for about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 160, 165, 170, 175, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 390, or 300 hours.
- the cells can be contacted with at least one cytokine for about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more days.
- the cells can be contacted with at least one cytokine for about 1 to 2, 2 to 3, 3 to 4, 1 to 4, 1 to 3, or 2 to 4 weeks.
- the cytokine is one or more interleukin.
- the interleukin (IL) is at least one of IL-2, IL-4, IL-21, BAFF, multimer CD40L, IL-10, IL-12, and IL-15.
- the cells are contacted with IL-2, IL-4, IL-21, BAFF, and multimer CD40L.
- Leukocytes used in a tolerizing vaccine/regimen or preparatory regimen can comprise at least or at least about 10%, e.g., 25%, CD19 positive, CD20 positive, or CD21 positive MHC Class II positive B cells.
- donor splenocytes can comprise at least or at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or 80% CD19, CD20, and/or CD21 positive MHC Class II positive B cells, e.g., about, at least, or at least about 10 to 20; 20 to 30; 30 to 40; 40 to 50; 50 to 60%, or 60 to 70%.
- splenic B cells or leukocytes used in a tolerizing vaccine/regimen or preparatory regimen can comprise at least or at least about 60%, e.g., 90%, CD19, CD20, or CD21 positive MHC Class II positive B cells.
- splenic B cells or leukocytes used in a tolerizing vaccine/regimen or preparatory regimen can comprise about, at least, or at least about 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% CD19, CD20, or CD21 positive MEW Class II positive B cells e.g., at least or at least about 60 to 70; 70 to 80; 80 to 90; or 90 to 95%.
- donor splenocytes or leukocytes used in a tolerizing vaccine/regimen or preparatory regimen can comprise from or from about 50% to 100%, e.g., from or from about 60% to 100% or 80% to 100%, CD19, CD20, or CD21 positive MEW Class II positive B cells.
- the MEW class II is swine leukocyte antigen (SLA) Class II.
- the MEW class II is human leukocyte antigen (HLA) Class II.
- a tolerizing agent of the methods herein can comprise apoptotic cells, non-apoptotic cells, or a combination thereof.
- Cells for a tolerizing vaccine or preparatory regimen can be made apoptotic a number of different ways.
- the cells can be contacted with a chemical (e.g., a fixative or cross-linking agent, a cellular damaging agent, or a combination thereof), to make some or all of the cells apoptotic.
- the cells can be irradiated (e.g., with ultraviolet radiation, gamma radiation, etc.) to make some or all of the cells apoptotic.
- Cells for a tolerizing vaccine or preparatory regimen can be contacted with a chemical, such as a fixative or cross-linking agent, a carbodiimide, or a carbodiimide derivative, a diimidoester, a sulfhydryl-to-sulfhydryl crosslinker, an amine-to-sulfhydryl crosslinker, a sulfhydryl-to-carbohydrate crosslinker, a photoreactive crosslinker, an in vivo crosslinker, a cellular damaging agent or an apoptosis inducer.
- the contacting can make some or all of the cells apoptotic.
- Suitable fixatives or cross-linking agents include, but are not limited to: an amine-to-amine crosslinker, a sulfhydryl-to-sulfhydryl crosslinker, an amine-to-sulfhydryl crosslinker, an in vivo crosslinker, a sulfhydryl-to-carbohydrate crosslinker, a photoreactive crosslinker, a chemoselective ligation crosslinking agent, a carboxyl-to-amine crosslinker, a carbodiimide, genipin, acrylic aldehyde, diformyl, osmium tetroxide, a diimidoester, mercuric chloride, zinc sulphate, zinc chloride, trinitrophenol (picric acid), potassium dichromate, ethanol, methanol, acetone, acetic acid, or a combination thereof.
- the carbodiimide can comprise ethylcarbodiimide; ethylene carbodiimide; N,N′-diisopropylcarbodiimide (DIC); N,N′-dicyclohexylcarbodiimide (DCC); 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDCI, EDC, ECDI, or EDAC); or a combination thereof.
- the carbodiimide comprises ethylcarbodiimide.
- the carbodiimide comprises ethylene carbodiimide.
- the carbodiimide comprises N,N′-diisopropylcarbodiimide (DIC).
- the carbodiimide comprises N,N-dicyclohexylcarbodiimide (DCC). In some cases, the carbodiimide comprises 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDCI, EDC, ECDI, or EDAC). In some cases, the tolerizing vaccine comprises cells treated with EDCI derivatives and/or functionalized EDCI. Methods of preparation, administration and use of tolerizing agent e.g., a tolerizing vaccine is well known in the art, for example, see U.S. Pat. No. 8,734,786B2, U.S. Pat. No. 9,888,673B2.
- Cells for a tolerizing vaccine or preparatory regimen can be contacted with a diimidoester.
- the contacting can be for a pre-determined time.
- the contacting can make some or all of the cells apoptotic.
- the diimidoester can comprise cyanuric chloride; diisocyanate; diethylpyrocarbonate (DEPC) or diethyl dicarbonate; a maleimide; benzoquinone; or a combination thereof.
- Cells for a tolerizing vaccine or preparatory regimen can be contacted with an amine-to-amine crosslinker.
- the contacting can be for a pre-determined time.
- the contacting can make some or all of the cells apoptotic.
- the amine-to-amine-crosslinker comprises disuccinimidyl glutarate (DSG); disuccinimidyl suberate (DSS); bis(sulfosuccinimidyl)suberate (BS3); tris-(succinimidyl) aminotriacetate (TSAT); BS(PEG)5; BS(PEG)9; dithiobis(succinimidyl propionate) (DSP); 3,3′-dithiobis(sulfosuccinimidyl propionate) (DTSSP); disuccinimidyl tartrate (DST); bis(2-(succinimidooxycarbonyloxy)ethyl)sulfone (BSO
- the amine-to-amine crosslinker comprises an imidoester, such as dimethyl adipimidate (DMA); dimethyl pimelimidate (DMP); dimethyl suberimidate (DMS); dimethyl 3,3′-dithiobispropionimidate (DTBP); or any combination thereof.
- the amine-to-amine crosslinker comprises a difluoro, such as 1,5-difluoro-2,4-dinitrobenzene (DFDNB).
- Cells for a tolerizing vaccine or preparatory regimen can be contacted with a sulfhydryl-to-sulfhydryl crosslinker.
- the contacting can be for a pre-determined time.
- the contacting can make some or all of the cells apoptotic.
- the sulfhydryl-to-sulfhydryl crosslinker comprises a maleimide, such as bismaleimidoethane (BMOE); 1,4-bismaleimidobutane (BMB); bismaleimidohexane (BMH); tris(2-maleimidoethyl)amine (TMEA); BM(PEG)2 (such as 1,8-bismaleimido-diethyleneglycol); BM(PEG)3 (such as 1,11-bismaleimido-triethyleneglycol), dithiobismaleimidoethane (DTME); or any combination thereof.
- BMOE bismaleimidoethane
- BMB 1,4-bismaleimidobutane
- BMH bismaleimidohexane
- TMEA tris(2-maleimidoethyl)amine
- BM(PEG)2 such as 1,8-bismaleimido-diethyleneglycol
- BM(PEG)3
- Cells for a tolerizing vaccine or preparatory regimen can be contacted with an amine-to-sulfhydryl crosslinker.
- the contacting can be for a pre-determined time.
- the contacting can make some or all of the cells apoptotic.
- the amine-to-sulfhydryl crosslinker comprises a NHS-haloacetyl crosslinker, a NHS-maleimide, a NHS-pyridyldithiol crosslinker, a sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) crosslinker, or any combination thereof.
- SMCC sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate
- the NHS-haloacetyl crosslinkers can comprise succinimidyl iodoacetate (SIA); succinimidyl 3-(bromoacetamido)propionate (SBAP); succinimidyl (4-iodoacetyl)aminobenzoate (SIAB); sulfo-SIAB; or a combination thereof.
- SIA succinimidyl iodoacetate
- SBAP succinimidyl 3-(bromoacetamido)propionate
- SIAB succinimidyl (4-iodoacetyl)aminobenzoate
- sulfo-SIAB sulfo-SIAB
- the NHS-maleimide can comprise N- ⁇ -maleimidoacet-oxysuccinimide ester (AMAS); N- ⁇ -maleimidopropyl-oxysuccinimide ester (BMPS); N- ⁇ -maleimidobutyryl-oxysuccinimide ester (GMBS); sulfo-GMBS; m-maleimidobenzoyl-N-hydrosuccinimide ester (MBS); sulfo-MBS; SMCC; sulfo-SMCC; N- ⁇ -malemidocaproyl-oxysuccinimide ester (EMCS); sulfo-EMCS; succinimidyl 4-(p-maleimidophenyl)butyrate (SMPB); sulfo-SMPB; succinimidyl 6-((beta-maleimidopropionamido)hexanoate) (SMPH); sulfosuccinimidyl 4-(N-maleimido
- the NHS-pyridyldithiol crosslinker can comprise succinimidyl 3-(2-pyridyldithio)propionate (SPDP), succinimidyl 6-(3(2-pyridyldithio)propionamido)hexanoate (LC-SPDP), sulfo-LC-SPDP, or 4-succinimidyloxycarbonyl-alpha-methyl- ⁇ (2-pyridyldithio)tolune (SMPT).
- SPDP succinimidyl 3-(2-pyridyldithio)propionate
- LC-SPDP succinimidyl 6-(3(2-pyridyldithio)propionamido)hexanoate
- sulfo-LC-SPDP sulfo-LC-SPDP
- Cells for a tolerizing vaccine or preparatory regimen can be contacted with a sulfhydryl-to-carbohydrate crosslinker.
- the contacting can be for a pre-determined time.
- the contacting can make some or all of the cells apoptotic.
- the sulfhydryl-to-carbohydrate crosslinker comprises (N- ⁇ -maleimidopropionic acid hydrazide (BMPH), N- ⁇ -maleimidocaproic acid hydrazide (EMCH), 4-(4-N-maleimidophenyl)butyric acid hydrazide (MPBH), N- ⁇ -maleimidoundecanoic acid hydrazide (KMUH), 3-(2-pyridyldithio)propionyl hydrazide (PDPH), or any combination thereof.
- BMPH N- ⁇ -maleimidopropionic acid hydrazide
- EMCH N- ⁇ -maleimidocaproic acid hydrazide
- MPBH 4-(4-N-maleimidophenyl)butyric acid hydrazide
- KMUH N- ⁇ -maleimidoundecanoic acid hydrazide
- PDPH 3-(2-pyridyldithio
- the carboxyl-to-amine crosslinker is dicyclohexylcarbodiimide (DCC), 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDCI, EDC, or EDAC), N-hydroxysuccinimide (NHS), sulfo-NHS, or any combination thereof.
- DCC dicyclohexylcarbodiimide
- EDCI 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide
- EDC 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide
- NHS N-hydroxysuccinimide
- sulfo-NHS sulfo-NHS
- Cells for a tolerizing vaccine or preparatory regimen can be contacted with a photoreactive crosslinker.
- the contacting can be for a pre-determined time.
- the contacting can make some or all of the cells apoptotic.
- the photoreactive crosslinker comprises a NHS ester/aryl azide, a NHS ester/diazirine, or a combination thereof.
- the NHS ester/aryl azide can comprise N-5-azido-2-nitrobenzoyloxysuccinimide (ANB-NOS), sulfo-SANPAH, or a combination thereof.
- the NHS ester/diazirine can comprise SDA (NHS-diazirine/succinimidyl 4,4′-azipentanoate), sulfo-SDA, LC-SDA (NHS-LC-diazirine/succinimidyl 6-(4,4′-azipentanamido)hexanoate), sulfo-LC-SDA, SDAD (NHS—SS-diazirine/succinimidyl 2-((4,4′-azipentanamido)ethyl)1,3′-dithiopropionate), sulfo-SDAD, or a combination thereof.
- SDA NHS-diazirine/succinimidyl 4,4′-azipentanoate
- LC-SDA NHS-LC-diazirine/succinimidyl 6-(4,4′-azipentanamido)hexanoate
- SDAD NHS—SS-
- Cells for a tolerizing vaccine or preparatory regimen can be contacted with an in vivo crosslinker.
- the contacting can be for a pre-determined time.
- the contacting can make some or all of the cells apoptotic.
- the in vivo crosslinker can comprise BS3, DTSSP, sulfo-EGS, DSG, DSP, DSS, EGS, sulfo-SDA, sulfo-LC-SDA, sulfo-SDAD, SDA, LC-SDA, SDAD, NHS-ester diazirine, or any combination thereof.
- the cells for use in a tolerizing vaccine or preparatory regimen are treated with a cellular damaging agent or an apoptosis inducer.
- the cellular damaging agent induces apoptosis in some or all of the contacted cells.
- Non-limiting exemplary cellular damaging agents include doxorubicin, staurosporine, etoposide, comptothecin, paclitaxel, vinblastine, or any combination thereof.
- Non-limiting exemplary apoptosis inducers include marinopyrrole A, maritoclax, (E)-3,4,5,4′-tetramethoxystilbene, 17-(Allylamino)-17-demethoxygeldanamycin, 2,4,3′,5′-tetramethoxystilbene, 2OHOA, 6,8-bis(benzylthio)-octanoic acid, AT101, apoptolidin, FU 40A, ara-G hydrate, arylquin 1, BAD, BAM7, BAX activator molecule 7, BH3I-1, BID, BMS-906024, BV02, bendamustine, borrelidin, borrelidine, cyclopentanecarboxylic acid, NSC 216128, treponemycin, brassinin, brassinine, brefeldin A, ascotoxin, BFA, cyanein, decumbin, bufalin, CCF642, CCT007093
- Cells for a tolerizing vaccine or preparatory regimen can be made apoptotic by contacting the cells with a chemical (e.g., a fixative or cross-linking agent, a cellular damaging agent, or a combination thereof) for a predetermined amount of time.
- a chemical e.g., a fixative or cross-linking agent, a cellular damaging agent, or a combination thereof
- the cells in the tolerizing vaccine or the preparatory regimen are made apoptotic by fixing for a predetermined amount time with the crosslinking agent (e.g., ECDI).
- the predetermined amount of time is about 30 minutes, about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 12 hours, about 18 hours, about 24 hours, about 36 hours, about 48 hours, about 60 hours, or about 72 hours.
- the predetermined amount of time is less than an hour. In some cases, the predetermined time is at least about 1 minute, 5 minutes, 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 60 minutes, 75, minutes, 90 minutes, 120 minutes, 150 minutes, 180 minutes, 210 minutes or 240 minutes. In some cases, the predetermined time is at most about 30 minutes, 40 minutes, 50 minutes, 60 minutes, 75, minutes, 90 minutes, 120 minutes, 150 minutes, 180 minutes, 210 minutes or 240 minutes.
- the predetermined amount of time is about 1 minute to about 240 minutes, 1 minute to about 10 minutes, 10 minutes to about 240 minutes, about 10 minutes to about 180 minutes, about 10 minutes to about 120 minutes, about 10 minutes to about 90 minutes, about 10 minutes to about 60 minutes, about 10 minutes to about 30 minutes, about 30 minutes to about 240 minutes, about 30 minutes to about 180 minutes, about 30 minutes to about 120 minutes, about 30 minutes to about 90 minutes, about 30 minutes to about 60 minutes, about 50 minutes to about 240 minutes, about 50 minutes to about 180 minutes, about 50 minutes to about 120 minutes, about 50 minutes to about 90 minutes, about 50 minutes to about 60 minutes, about 10 minutes to about 20 minutes, about 20 minutes to about 30 minutes, about 30 minutes to about 40 minutes, about 40 minutes to about 50 minutes, about 50 minutes to about 60 minutes, about 60 minutes to about 70 minutes, about 70 minutes to about 80 minutes, about 80 minutes to about 90 minutes, about 90 minutes to about 100 minutes, about 100 minutes to about 110 minutes, about 110 minutes to about 120 minutes, about 10 minutes to about 30 minutes, about 30 minutes, about
- the contacting can be at any temperature. In some cases the contacting is performed on ice (e.g., at 4° C.). In other cases, the contacting is performed at room temperature. In some cases, the contacting is performed at a temperature of at least about 0° C., 2° C., 4° C., 8° C., 15° C., 20° C., 25, 30° C., 35° C., or 37° C. In some cases, the contacting is performed at a temperature of at most about 4° C., 8° C., 15° C., 20° C., 25, 30° C., 35° C., 37° C., or 40° C. In some cases, the contacting is performed at a temperature of about 0° C.
- Cells in a tolerizing vaccine or preparatory regimen can aggregate as a result of the method of making some or all of the cells apoptotic.
- cells can aggregate after contacting with a chemical, such as a fixative or crosslinking agent.
- the predetermined amount of time that the cells are contacted with the chemical can be selected to minimize the amount of aggregation in the tolerizing vaccine or preparatory regimen.
- aggregates can be removed, for example, by washing and/or filtration.
- a tolerizing vaccine or preparatory regimen can comprise from or from about 0.01 to 10 aggregates, per ⁇ l.
- the tolerizing vaccine or preparatory regimen can comprise from or from about 0.01 to 1, 0.1 to 1, 0.25 to 1, 0.5 to 1, 1 to 5; or 1 to 10 aggregate per ⁇ l.
- the tolerizing vaccine or preparatory regimen can comprise less than about 0.1, 0.5, 0.75, 1, 5, or 10 aggregates per ⁇ L.
- the tolerizing vaccine or preparatory regimen can comprise less than 5 aggregates per ⁇ L.
- the tolerizing vaccine or preparatory regimen can comprise less than about: 5, 4, 3, 2, 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.05, or 0.01 aggregates per ⁇ L.
- the tolerizing vaccine or preparatory regimen comprises 1 or fewer aggregates per ⁇ L.
- the tolerizing vaccine or preparatory regimen can comprise about 0.01, about 0.05, about 0.1, about 0.2, about 0.3, about 0.4, about 0.5 about 0.6, about 0.7, about 0.8, about 0.9, or about 1.0 aggregates per ⁇ L.
- the tolerizing vaccine or preparatory regimen can include from or from about 0.01% to 10%, e.g., from or from about 0.01% to 2%, necrotic cells.
- cells of a tolerizing vaccine or preparatory regimen can comprise from or from about 0.01% to 10%; 0.01% to 7.5%, 0.01% to 5%; 0.01% to 2.5%; or 0.01% to 1% necrotic cells.
- the cells of a tolerizing vaccine or preparatory regimen of the disclosure can comprise at most about 0.01%, 0.1%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% necrotic cells.
- conjugate or “conjugated to,” and the like refer to molecular entities (.e.g, peptides of the present disclosure and an apoptotic leucocyte) being linked together through covalent or non-covalent bonds. Conjugation may be accomplished by directly coupling the two molecular entities, e.g., creating an ester or amide from an hydroxyl group, amino group, and a carboxylic acid. Conjugation may be accomplished by indirectly coupling the two molecular entities, e.g., instituting a linking group such as a polyethylene glycol.
- Conjugation may be accomplished by modifying the molecular entities with chemical groups that react with one another, e.g., alkyne-functionalized entity with an azide-functionalized entity or the reduction of thiol groups on individual entities to form a disulfide bond.
- Conjugates such as ethylene carbodiimide (ECDI), hexamethylene diisocyanate, propyleneglycol di-glycidylether which contain 2 epoxy residues, and epichlorohydrin can be used for fixation of peptides or proteins to the apoptotic leucocyte surface.
- Reactive carboxyl groups on the surface of an apoptotic leukocyte can be joined to free amines (e.g., from Lys residues) on the peptide or protein, by reacting them with, for example, 1-ethyl-3-[3,9-dimethyl aminopropyl] carbodiimide hydrochloride (EDC) or N-hydroxysuccinimide ester (NHS).
- EDC 1-ethyl-3-[3,9-dimethyl aminopropyl] carbodiimide hydrochloride
- NHS N-hydroxysuccinimide ester
- the same chemistry may be used to conjugate free amines on the surface of an apoptotic leukocyte with free carboxyls (e.g., from the C-terminus, or from Asp or GIu residues) on the peptide or protein.
- free amine groups on the surface of an apoptotic leuckocyte may be covalently bound to peptides and proteins, or peptide or protein fusion proteins, using sulfo-SIAB chemistry, essentially as described by Arano et al. (1991) Chem. 2:71-6.
- a great variety of means, well known in the art, may be used to conjugate the peptides to surface of apoptotic leuckocytes. These methods include any standard chemistry which do not destroy or severely limit the biological activity of the peptides and that of the apoptotic leuckocytes, and which allow for a sufficient number of peptides to be conjugated to the surface in an orientation which allows for inducing tolerance.
- the C-terminal regions of a peptide are conjugated.
- the N-terminus of a peptide can be conjugated onto the surface of the apoptotic leucocyte.
- the present disclosure provides preparatory regimen and/or tolerizing vaccines and regimen comprising apoptotic cells such as leucocytes or mesenchymal stromal cells comprising one or more peptides conjugated to the surface of the apoptotic cell.
- apoptotic cells such as leucocytes or mesenchymal stromal cells
- the one or more peptides can comprise an amino acid sequence set forth in any one of SEQ ID NOs. 1-6 and 8-17.
- the peptide conjugated to the surface of an apoptotic cell such as a leukocyte can vary in length.
- the one or more peptides can comprise a sequence set forth in any one of SEQ ID NOs.
- the one or more peptides can comprise a sequence from 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, 99.9%, 99.95% or 100% identical to the sequences set forth in any of SEQ ID NOs. 1-6 and 8-17.
- the one or more peptides can comprise a sequence set forth in any one of SEQ ID NOs. 1-6 and 8-17 and can be about 9, about 10, about 1, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 75, about 80, about 90, about 100, about 120, about 140, about 150, about 160, about 170, about 180, about 200, about 220, about 250, about 270, about 300, about 330, about 350, about 400, about 440, about 500, about 600, about 700, about 800, about 900 or about 1000 amino acids in length.
- the one or more peptides can comprise a sequence set forth in any one of SEQ ID NOs.
- the one or more peptides can comprise a sequence set forth in any one of SEQ ID NOs.
- 1-6 and 8-17 can be about 5-250, 10-250, 20-250, 30-250, 50-250, 100-250, 5-100, 10-100, 20-100, 30-100, 50-100, 5-50, 10-50, 20-50, 30-50, 5-30, 10-30, 15-30, or 20-30 amino acids in length.
- the peptides conjugated on the surface of apoptotic leukocytes are derived from a MHC molecule.
- MHC molecule refers to a molecule comprising Major Histocompatibility Complex (MHC) glycoprotein protein sequences.
- MHC Major Histocompatibility Complex
- HLA human leukocyte antigen
- HLA Human Leukocyte Antigens
- HLA is the human form of “MHC” and therefore can be used interchangeably.
- HLA proteins that can be utilized in accordance with the presently disclosed and claimed inventive concept(s) include, but are not limited to, an HLA class I ⁇ chain, an HLA class II ⁇ chain and an HLA class II ⁇ chain.
- Specific examples of HLA class II ⁇ and/or ⁇ proteins that may be utilized in accordance with the presently disclosed and claimed inventive concept(s) include, but are not limited to, those encoded at the following gene loci: HLA-DRA; HLA-DRB1; HLA-DRB3,4,5; HLA-DQA; HLA-DQB; HLA-DPA; and HLA-DPB.
- the apoptotic cells further comprise one or more peptides derived from a MHC class I molecule.
- the sequences of amino acid residues in the peptide can be substantially similar or functionally comparable to a polypeptide sequence in the MHC molecule.
- a peptide derived from a MHC class II molecule refers to a peptide that has a sequence “from a region in an MHC class II molecule” (e.g., the hypervariable region), and is a peptide that has a sequence 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, 99.9%, 99.95% or 100% identical to the naturally occurring MHC amino acid sequence of the region.
- the peptide derived from a MHC class II molecule can comprise a sequence from 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, 99.9%, 99.95% or 100% identical to the hypervariable region of the WIC class II molecule.
- a peptide derived from a MHC class I molecule refers to a peptide that has a sequence “from a region in an WIC class I molecule” (e.g., the hypervariable region), and is a peptide that has a sequence 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, 99.9%, 99.95% or 100% identical to the naturally occurring WIC amino acid sequence of the region.
- the peptide derived from a MHC class I molecule will comprise a sequence from the hypervariable region of the MHC class I molecule.
- the peptides derived from a recipient's MHC class II molecule may comprise an entire WIC class II molecule.
- the peptides derived from a recipient's WIC class II molecule may comprise an entire ⁇ chain of DR, DQ or DP.
- the peptides derived from a recipient's MHC class II molecule may comprise entire ⁇ chain of DR, DQ, or DP.
- the peptides derived from a recipient's WIC class II molecule may comprise entire ⁇ 1 and/or ⁇ 2 domains of DR, DQ or DP.
- the peptides derived from a recipient's WIC class II molecule may comprise entire ⁇ 1 and/or ⁇ 2 domains of DR, DQ, or DP.
- the peptides derived from a recipient's WIC class II molecule may comprise MHC-DR1, MHC-DR2, MHC-DR3, MHC-DR4, and/or MHC-DRS.
- the peptides derived from a recipient's MHC class II molecule may comprise a fragment of an ⁇ 1 and/or ⁇ 2 domain of DR, DQ or DP.
- the peptides derived from a recipient's WIC class II molecule may comprise a fragment of a ⁇ 1 and/or ⁇ 2 domain of DR, DQ, or DP.
- the peptides derived from a recipient's WIC class II molecule may comprise a fragment of MHC-DR1, MHC-DR2, MHC-DR3, MHC-DR4, and/or MHC-DRS.
- the peptides derived from a recipient's MHC class II molecule may comprise a sequence from a hypervariable region.
- the peptides derived from a recipient's WIC class II molecule can comprise an in silico-identified high, medium, or low affinity peptides from the hypervariable region of the DRB molecule (e.g., a 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid peptide).
- the peptides derived from a recipient's MHC class II molecule may be synthesized or recombinant. In some cases, the peptides derived from a recipient's MHC class II molecule may between about 10 and 30 amino acids in length. The peptides derived from a recipient's MHC class II molecule may be at least 10 to 30 amino acids in length. In some embodiments, the peptides derived from a recipient's MHC class II molecule can be at least about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 275, 200, or 250 amino acids in length.
- the peptides derived from a recipient's MHC class II molecule can be at most about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 275, 200, or 250 amino acids in length. In some embodiments, the peptides derived from a recipient's MHC class II molecule can be about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 275, 200, or 250 amino acids in length.
- the peptides derived from a recipient's MHC class II molecule can be about 5-250, 10-250, 20-250, 30-250, 50-250, 100-250, 5-100, 10-100, 20-100, 30-100, 50-100, 5-50, 10-50, 20-50, 30-50, 5-30, 10-30, 15-30, or 20-30 amino acids in length.
- Cells for a tolerizing vaccine or preparatory regimen can be treated with a fixative or crosslinking agent (e.g., a carbodiimide such as ECDI) in the presence of one or more antigens and/or epitopes.
- a fixative or crosslinking agent e.g., a carbodiimide such as ECDI
- the epitopes can comprise a peptide containing an amino acid sequence set forth in any one of SEQ ID NOs. 1-6 and 8-17.
- the antigens and/or epitopes can comprise antigens and/or epitopes from a transplant donor, a transplant recipient, a third party, or a combination thereof.
- the cells in the tolerizing vaccine or preparatory regimen are coupled to recipient antigens and/or epitopes. In some cases, the cells in the tolerizing vaccine or preparatory regimen are coupled to third party antigens and/or epitopes. In some cases, the cells in the tolerizing vaccine or preparatory regimen are coupled to transplant donor antigens and/or epitopes.
- a “cocktail” of peptides can be conjugated on the surface of the apoptotic leucocytes.
- the apoptotic cells such as leucocytes can comprise one or more peptides derived from MHC class II molecule.
- the apoptotic cells such as leucocytes can comprise, for example, at least 2, 3, 5, 7, 10, 15, 20, 30, 40, 50, 100 or more peptides derived from a MHC class II molecule.
- the apoptotic leucocytes can comprise one or more peptides derived from a MHC class I molecule.
- the apoptotic cells such as leucocytes can comprise, for example, at least 2, 3, 5, 7, 10, 15, 20, 30, 40, 50, 100 or more peptides derived from a MHC class I molecule.
- a mixture of more than one peptide derived from a MHC class II molecule has the advantage of inducing increased immune tolerance response in the recipient.
- the increased tolerance can be, for example, through a mechanism called linked supersession.
- the mechanism of linked suppression will be known to an artisan skilled in the art. For instance, peptides comprising sequences from hypervariable regions of ⁇ and ⁇ chains may be used in combination.
- the size of a protein or a peptide that is conjugated to the surface of the apoptotic leukocyte may comprise, but is not limited to 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 110, 120, 130, 140, 150, 160, 170, 180
- Cells e.g., leukocytes
- a fixative or crosslinking agent e.g., a carbodiimide such as ECDI
- the one or more immunomodulatory molecules can comprise one or more peptides comprising an amino acid sequence set forth in any one of SEQ ID NOs. 1-6 and 8-17.
- the one or more immunomodulatory molecules can comprise all or a portion of: IFN- ⁇ , an NF-kB inhibitor, vitamin D3, siCD40, anti-CD40 antibody, cobalt protoporphyrin, insulin B9-23, ⁇ 1-antitrypsin, a cluster of differentiation protein, a gp39 antagonist, ⁇ 1-antitrypsin, CD47, PD-L1, PD-L2, CTLA-4, rapamycin, compstatin, abatacept, ipilimumab, or a combination thereof.
- the NF-kB inhibitor can comprise dehydroxymethylepoxyquinomicin (DHMEQ), curcumin, triptolide, Bay-117085, or a combination thereof.
- the cluster of differentiation protein can comprise CD4, CD46, CD47, CD55, CD59, or a portion thereof, or a combination thereof.
- the one or more immunomodulatory molecules can comprise all or a portion of a calcineurin inhibitor (e.g., cyclosporine or tacrolimus), a costimulatory signal blockade, an IL-2 signaling inhibitor (e.g., daclizumab or basiliximab), a cell cycle blocker (e.g., mycophenolate mofetil (MMF) or azathioprine), a T cell recirculation inhibitor (e.g., FTY720 or another sphingosine 1-phosphate (S1P) receptor agonist), a nitrogen mustard alkylating agent (e.g., cyclophosphamide), a complement C3 or C5 inhibitor, or any combination thereof.
- a calcineurin inhibitor e.g., cyclosporine or tacrolimus
- a costimulatory signal blockade e.g., daclizumab or basiliximab
- a cell cycle blocker e.g.,
- the immunomodulatory molecules can target T cell receptor (TCR), CD3e, FK506-binding protein 12 (FKBP12), cytotoxic T lymphocyte associated protein 4 (CTLA-4), programmed cell death protein 1 (PD-1, e.g., pembrolizumab), programmed death ligand 1 (PD-L1, e.g., MPDL3280A), CD40L (CD154), CD40, inducible costimulatory (ICOS), IL-2, TNF- ⁇ (e.g., infliximab), IL-6 or IL-6R (e.g., tocilizumab, actemra, clazakizumab, ALD518, siltuximab, elsilimomab, sirukumab, sarilumab, olokizumab), IL-7, CD2, CD20, CD52, ⁇ -4 integrin, mTOR (e.g., rapamycin or everolimus), T cell receptor
- Cells for tolerizing vaccine or preparatory regimen can be treated with a fixative or crosslinking agent (e.g., a carbodiimide such as ECDI) in the presence of an agent that increases expression of anti-inflammatory cytokines in a recipient.
- a fixative or crosslinking agent e.g., a carbodiimide such as ECDI
- the agent can comprise one or more peptides comprising an amino acid sequence set forth in any one of SEQ ID NOs. 1-6 and 8-17.
- the anti-inflammatory cytokines may include, for example, TGF- ⁇ , IL-10, IL-13, or a combination thereof.
- the agent that increases expression of anti-inflammatory cytokines in the recipient comprises ⁇ 1-antitrypsin.
- ADLs from all the doses can all be conjugated with the same immunomodulatory molecules, agents that increases expression of anti-inflammatory cytokines, and/or antigens or epitopes.
- ADLs from one or more doses can be conjugated with a first set of immunomodulatory molecules, agents that increases expression of anti-inflammatory cytokines, and/or antigens or epitopes, and ADLs from other dose(s) can be conjugated with a different set of immunomodulatory molecules, agents that increases expression of anti-inflammatory cytokines, and/or antigens or epitopes.
- a preparatory regimen or tolerizing vaccine of the disclosure can comprise administering one or more immunosuppression agents and/or anti-inflammatory agents to a transplant recipient.
- the regimen may include administering one or more immunosuppression agents and/or anti-inflammatory agents in addition to apoptotic cells (such as apoptotic donor leukocytes or mesenchymal stromal cells).
- the regimen may include administering one or more immunosuppression agents and/or anti-inflammatory agents in addition to an anti-CD40 agent, for e.g., a peptide comprising an amino acid sequence with at least 80%, at least 85%, at least 90% or at least 95% sequence identity to the sequences set forth in any one of SEQ ID NOs. 1-6 and 8-17.
- an anti-CD40 agent for e.g., a peptide comprising an amino acid sequence with at least 80%, at least 85%, at least 90% or at least 95% sequence identity to the sequences set forth in any one of SEQ ID NOs. 1-6 and 8-17.
- the short term immunosuppression comprises administering an antagonistic anti-CD40 agent.
- An anti-CD40 agent or an anti-CD40 ligand agent can be, for example, a peptide comprising an amino acid sequence with at least 80%, at least 85%, at least 90% or at least 95% sequence identity to the sequences set forth in any one of SEQ ID NOs. 1-6 and 8-17.
- the anti-CD40 agent or anti-CD40 ligand agent can be, for example, a peptide comprising an amino with at least 80%, 85%, 88%, 90%, 93%95% or 99% sequence identity to the sequences set forth in any one of SEQ ID NOs. 1-6 and 8-17.
- an anti-CD40 agent or an anti-CD40 ligand agent can be, for example, an antagonistic anti-CD40 antibody, and antagonistic anti-CD40 ligand antibody, or an antigen binding fragmented thereof.
- An anti-CD40 agent or an anti-CD40 ligand agent can be an antagonistic anti-CD40 antibody or antigen-binding fragment thereof.
- Non-limiting examples of antagonistic anti-CD40 antibodies include 2C10, 2C10R4, ASKP1240, 4D11, bleselumab, BI-655064, HCD122, CFZ533, ch5D12, CDP7657, and FFP104.
- the anti-CD40 agent is ASKP1240.
- An anti-CD40 agent or an anti-CD40 ligand agent can be an antagonistic anti-CD40 ligand antibody or antigen-binding fragment thereof.
- antagonistic anti-CD40 ligand antibodies include BG9588, ruplizumab, toralizumab, IDEC-131, dapirolizumab, letolizumab, BMS-986004, VIB4920, and MEDI4920.
- an immunosuppression agent used for short term immunosuppression can be any one or more of MMF (mycophenolate mofetil (Cellcept)), ATG (anti-thymocyte globulin), anti-CD154 (CD40L), alemtuzumab (Campath), CTLA4-Ig (Abatacept/Orencia), belatacept (LEA29Y), daclizumab (Ze-napax), basiliximab (Simulect), infliximab (Remicade), cyclosporin, deoxyspergualin, soluble complement receptor 1, cobra venom factor, compstatin, anti C5 antibody (eculizumab/Soliris), methylprednisolone, FTY720, everolimus, anti-CD154-Ab, leflunomide, anti-IL-2R-Ab, anti-CXCR3 antibody, anti-ICOS antibody, anti-OX40 antibody, and anti-CD122 antibody, human anti-CD154 mono
- an immunosuppression agent used for short term immunosuppression can target T cell receptor (TCR), CD3e, FK506-binding protein 12 (FKBP12), cytotoxic T lymphocyte associated protein 4 (CTLA-4), programmed cell death protein 1 (PD-1), programmed death ligand 1 (PD-L1), CD40L (CD154), CD40, inducible costimulatory (ICOS), IL-2, TNF- ⁇ , IL-6, IL-7, CD2, CD20, CD52, ⁇ -4 integrin, mTOR (mechanistic target of rapamycin, everolimus, serolimus), DNA synthesis, or any combination thereof.
- TCR T cell receptor
- CD3e FK506-binding protein 12
- FKBP12 cytotoxic T lymphocyte associated protein 4
- CTLA-4 cytotoxic T lymphocyte associated protein 4
- PD-1 programmed cell death protein 1
- PD-L1 programmed death ligand 1
- CD40L CD154
- CD40 inducible costim
- an immunosuppression agent used for short term immunosuppression can be a MHC/TCR interaction blockade, a nonselective depleting agent, calcineurin inhibitor, costimulatory signal blockade, cytokine blockade, lymphocyte depleting agent, cell adhesion inhibitor, IL-2 signaling inhibitor, cell cycle blocker, or any combination thereof.
- the MHC/TCR interaction blockade can be anti-abTCR mAb T10B9.
- the nonselective depleting agent can be anti-CD3 mAb (OKT3) or antithymocyte globulin (ATG).
- the calcineurin inhibitor can be cyclosporine or tacrolimus.
- the costimulatory signal blockade can be anti-CTLA-4 mAb, abatacept, ipilimumab, anti-PD-1 (such as pembrolizumab), anti-PD-L1 (such as MPDL3280A), anti-CD154 mAb, anti-CD40 mAb, or anti-ICOS mAb.
- the cytokine blockade can be anti-CD25 mAb (such as daclizumab or basiliximab), anti-TNF (etanercept, infliximab, adalimumab, certolizumab pegol, and golimumab), anti-IL-6/IL-6R mAb (such as clazakizumab, ALD518, siltuximab, elsilimomab, sirukumab, olokizumab, sarilumab, tocilizumab, actemra), or anti-IL-7 mAb.
- anti-CD25 mAb such as daclizumab or basiliximab
- anti-TNF etanercept, infliximab, adalimumab, certolizumab pegol, and golimumab
- anti-IL-6/IL-6R mAb such as clazakizumab, ALD518, si
- the lymphocyte depleting agent can be anti-CD2 mAb, fusion protein with IgG1 (such as alefacept), anti-CD20 mAb (such as rituximab), or anti-CD52 mAb (such as alemtuzumab).
- the cell adhesion inhibitor can be anti-very large antigen 4 (VLA4) (such as natalizumab).
- VLA4 very large antigen 4
- the IL-2 signaling inhibitor can be sirolimus (rapamycin) or everolimus.
- the cell cycle blocker can be mycophenolate mofetil (MMF) or azathioprine.
- Short term immunosuppression can comprise administering one or more immunosuppression agents and/or anti-inflammatory agents of the disclosure for at most about 100 days after a transplant.
- Short term immunosuppression can comprise administering one or more immunosuppression agents and/or anti-inflammatory agents of the disclosure, for example, for at most about 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 25, 28, 29, 30, 31, 32, 33, 34, 35, 40, 42, 49, 50, 55, 60, 70, 80, 90, or 100 days after a transplant.
- short term immunosuppression can conclude within 28 days after a transplant.
- short term immunosuppression can conclude about 21 days after a transplant.
- short term immunosuppression can begin prior to transplantation.
- short term immunosuppression can commence about or at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 25, 28, 29, 30, 35, or 50 days prior to a transplant.
- short term immunosuppression can commence at most about 10 days prior to a transplant.
- short term immunosuppression can commence about 7 days prior to a transplant.
- the duration of short term immunosuppression (e.g., the length of time between administering a first dose and a final dose of an immunosuppression agents and/or anti-inflammatory agents can be about or at most about 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 25, 28, 29, 30, 31, 32, 33, 34, 35, 40, 42, 49, 50, 55, 60, 70, 80, 90, or 100 days.
- the duration of short term immunosuppression can be about or at most about 30 days. In some cases, the duration of short term immunosuppression can be about 28 days.
- a peptide or compositions disclosed herein can be administered as a tolerizing agent.
- a peptide or compositions disclosed herein can be administered before, after, and/or during the administration of a tolerizing agent.
- the peptide of compositions disclosed herein can be administered between day ⁇ 100 and day 0, e.g., on day ⁇ 90, day ⁇ 80, day ⁇ 70, day ⁇ 60, day ⁇ 50, day ⁇ 40, day ⁇ 30, day ⁇ 20, day ⁇ 15, day ⁇ 14, day ⁇ 13, day ⁇ 12, day ⁇ 11, day ⁇ 10, day ⁇ 9, day ⁇ 8, day ⁇ 7, day ⁇ 6, day ⁇ 5, day ⁇ 4, day ⁇ 3, day ⁇ 2, day ⁇ 1, or day 0 relative to the administration of a tolerizing agent.
- the peptide or compositions disclosed herein can be administered concomitantly with the tolerizing agent. In some cases, the peptide or compositions disclosed herein can be administered on or on about day ⁇ 100 to ⁇ 50; ⁇ 50 to ⁇ 40; ⁇ 40 to ⁇ 30; ⁇ 30 to ⁇ 20; ⁇ 20 to ⁇ 10; ⁇ 10 to ⁇ 5; ⁇ 7 to ⁇ 1, relative to the administration of a tolerizing agent.
- the peptide or composition disclosed herein can be administered between day 0 and day 100, e.g., on day 100, day 90, day 80, day 70, day 60, day 50, day 40, day 30, day 20, day 15, day 14, day 13, day 12, day 11, day 10, day 9, day 8, day 7, day 6, day 5, day 4, day 3, day 2 or day 1 relative to the administration of a tolerizing agent.
- the peptide or compositions disclosed herein can be administered on or on about day 100 to 50; 50 to 40; 40 to 30; 30 to 20; 20 to 10; 10 to 5; 7 to 1, relative to the administration of a tolerizing agent.
- the peptide or compositions disclosed herein can be administered between day 0 and day 300, e.g., on day 300, day 200, day 100, day 90, day 80, day 70, day 60, day 50, day 40, day 30, day 20, day 15, day 14, day 13, day 12, day 11, day 10, day 9, day 8, day 7, day 6, day 5, day 4, day 3, day 2 or day 1 relative to the administration of a tolerizing agent.
- the peptide or compositions disclosed herein can be administered on or on about day 300 to 200; 200 to 100; 100 to 50; 50 to 40; 40 to 30; 30 to 20; 20 to 10; 10 to 5; 7 to 1, relative to the administration of a tolerizing agent.
- a peptide or compositions disclosed herein can be administered between day 0 and day 600, e.g., on day 600, day 500, day 400, day 300, day 200, day 100, day 90, day 80, day 70, day 60, day 50, day 40, day 30, day 20, day 15, day 14, day 13, day 12, day 11, day 10, day 9, day 8, day 7, day 6, day 5, day 4, day 3, day 2 or day 1 relative to the administration of a tolerizing agent.
- a peptide or composition disclosed herein can be administered on or on about day 600 to 500; day 500 to 400; day 400 to 300; 300 to 200; 200 to 100; 100 to 50; 50 to 40; 40 to 30; 30 to 20; 20 to 10; 10 to 5; 7 to 1, relative to the administration of a tolerizing agent.
- a peptide or compositions disclosed herein can be administered between day 0 and day 1,000, e.g., on day 1,000, day 900, day 800, day 700, day 600, day 500, day 400, day 300, day 200, day 100, day 90, day 80, day 70, day 60, day 50, day 40, day 30, day 20, day 15, day 14, day 13, day 12, day 11, day 10, day 9, day 8, day 7, day 6, day 5, day 4, day 3, day 2 or day 1 relative to the administration of a tolerizing agent.
- a peptide or compositions disclosed herein can be administered on or on about day 1,000 to 900; 900 to 800; 800 to 700; 700 to 600; 600 to 500; day 500 to 400; day 400 to 300; 300 to 200; 200 to 100; 100 to 50; 50 to 40; 40 to 30; 30 to 20; 20 to 10; 10 to 5; 7 to 1, relative to the administration of a tolerizing agent.
- a peptide or compositions disclosed herein can be administered on the day when a tolerizing agent is administered. In other cases, a peptide or can be administered before and after the administration of the tolerizing vaccine or preparatory regimen.
- a peptide or compositions disclosed herein can be administered before, after, and/or during the day of the transplantation of the graft cell, tissue, or organ.
- the peptide of compositions disclosed herein can be administered between day ⁇ 100 and day 0, e.g., on day ⁇ 90, day ⁇ 80, day ⁇ 70, day ⁇ 60, day ⁇ 50, day ⁇ 40, day ⁇ 30, day ⁇ 20, day ⁇ 15, day ⁇ 14, day ⁇ 13, day ⁇ 12, day ⁇ 11, day ⁇ 10, day ⁇ 9, day ⁇ 8, day ⁇ 7, day ⁇ 6, day ⁇ 5, day ⁇ 4, day ⁇ 3, day ⁇ 2, day ⁇ 1, or day 0 relative to the day of the transplantation of the graft cell, tissue, or organ.
- the peptide or compositions disclosed herein can be administered concomitantly with the day of the transplantation of the graft cell, tissue, or organ. In some cases, the peptide or compositions disclosed herein can be administered on or on about day ⁇ 100 to ⁇ 50; ⁇ 50 to ⁇ 40; ⁇ 40 to ⁇ 30; ⁇ 30 to ⁇ 20; ⁇ 20 to ⁇ 10; ⁇ 10 to ⁇ 5; ⁇ 7 to ⁇ 1, relative to the day of the transplantation of the graft cell, tissue, or organ.
- the peptide or composition disclosed herein can be administered between day 0 and day 100, e.g., on day 100, day 90, day 80, day 70, day 60, day 50, day 40, day 30, day 20, day 15, day 14, day 13, day 12, day 11, day 10, day 9, day 8, day 7, day 6, day 5, day 4, day 3, day 2 or day 1 relative to the day of the transplantation of the graft cell, tissue, or organ.
- the peptide or compositions disclosed herein can be administered on or on about day 100 to 50; 50 to 40; 40 to 30; 30 to 20; 20 to 10; 10 to 5; 7 to 1, relative to the day of the transplantation of the graft cell, tissue, or organ.
- the peptide or compositions disclosed herein can be administered between day 0 and day 300, e.g., on day 300, day 200, day 100, day 90, day 80, day 70, day 60, day 50, day 40, day 30, day 20, day 15, day 14, day 13, day 12, day 11, day 10, day 9, day 8, day 7, day 6, day 5, day 4, day 3, day 2 or day 1 relative to the day of the transplantation of the graft cell, tissue, or organ.
- the peptide or compositions disclosed herein can be administered on or on about day 300 to 200; 200 to 100; 100 to 50; 50 to 40; 40 to 30; 30 to 20; 20 to 10; 10 to 5; 7 to 1, relative to the day of the transplantation of the graft cell, tissue, or organ.
- a peptide or compositions disclosed herein can be administered between day 0 and day 600, e.g., on day 600, day 500, day 400, day 300, day 200, day 100, day 90, day 80, day 70, day 60, day 50, day 40, day 30, day 20, day 15, day 14, day 13, day 12, day 11, day 10, day 9, day 8, day 7, day 6, day 5, day 4, day 3, day 2 or day 1 relative to the day of the transplantation of the graft cell, tissue, or organ.
- a peptide or composition disclosed herein can be administered on or on about day 600 to 500; day 500 to 400; day 400 to 300; 300 to 200; 200 to 100; 100 to 50; 50 to 40; 40 to 30; 30 to 20; 20 to 10; 10 to 5; 7 to 1, relative to the day of the transplantation of the graft cell, tissue, or organ.
- a peptide or compositions disclosed herein can be administered between day 0 and day 1,000, e.g., on day 1,000, day 900, day 800, day 700, day 600, day 500, day 400, day 300, day 200, day 100, day 90, day 80, day 70, day 60, day 50, day 40, day 30, day 20, day 15, day 14, day 13, day 12, day 11, day 10, day 9, day 8, day 7, day 6, day 5, day 4, day 3, day 2 or day 1 relative to the day of the transplantation of the graft cell, tissue, or organ.
- the a peptide or compositions disclosed herein can be administered on or on about day 1,000 to 900; 900 to 800; 800 to 700; 700 to 600; 600 to 500; day 500 to 400; day 400 to 300; 300 to 200; 200 to 100; 100 to 50; 50 to 40; 40 to 30; 30 to 20; 20 to 10; 10 to 5; 7 to 1, relative to the day of the transplantation of the graft cell, tissue, or organ.
- a peptide or compositions disclosed herein can be administered on the day of the transplantation of the graft cell, tissue, or organ. In other cases, a peptide or can be administered before and after the day of the transplantation of the graft cell, tissue, or organ.
- a preparatory regimen can comprise multiple doses of a peptide or composition described herein, before, and/or during and/or after transplantation of a graft cell, tissue, or organ.
- a preparatory regimen can comprise multiple doses of a peptide comprising a sequence with at least 80%, 85% 90% or 95% sequence identity set forth in any one of SEQ ID NOs. 1-6 and 8-17, before, and/or during and/or after transplantation of a graft cell, tissue, or organ.
- a tolerizing regimen can comprise multiple doses of a peptide or composition described herein, before, and/or during and/or after transplantation of a graft cell, tissue, or organ.
- a tolerizing regimen can comprise multiple doses of a peptide comprising a sequence with at least 80%, 85% 90% or 95% sequence identity set forth in any one of SEQ ID NOs. 1-6 and 8-17, before, and/or during and/or after transplantation of a graft cell, tissue, or organ.
- the multiple doses can be referred to as comprising an initial dose and one or more booster doses.
- the initial dose can occur prior to or concurrently with the transplant of the graft cell tissue or organ.
- the booster dose(s), when administered, occur after the initial dose.
- one or more booster doses can be administered before, and/or concurrently with, and/or after transplant of the graft cell, tissue, or organ.
- Subsequent (e.g., booster) dose(s) of a preparatory regimen or tolerizing vaccine can be administered in any interval of time following a preceding dose (e.g., an initial dose).
- the subsequent dose can be administered about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 31 days, 32 days, 33 days, 34 days, 35 days, 36 days, 37 days, 38 days, 39 days, 40 days, 41 days, 42 days, 43 days, 44 days, 45 days, 46 days, 47 days, 48 days, 49 days, 50 days, 51 days, 52 days, 53 days, 54 days, 55 days, 56 days, 57 days, 58 days, 59 days, 60 days, 90 days, 120 days, 150 days, or
- subsequent (booster) dose(s) can be administered before, concurrently with, or after transplantation of the graft cell, tissue, or organ.
- the preparatory regimen comprises at least one dose, at least two doses, at least three doses of a preparatory regimen or tolerizing vaccine prior to transplantation.
- two doses of the tolerizing vaccine or preparatory regimen can be administered.
- the first dose can be administered, for example, on day ⁇ 14, ⁇ 10, ⁇ 9, ⁇ 8, ⁇ 7, ⁇ 6, ⁇ 5, ⁇ 4, ⁇ 3, ⁇ 2, ⁇ 1, 0, 1, 2, 3, 4, 5, 6, or 7 relative to transplant of donor cells, organs, and/or tissues on day 0.
- the second dose can be administered, for example, on day ⁇ 3, ⁇ 2, ⁇ 1, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 relative to transplant of donor cells, organs, and/or tissues on day 0.
- the first dose is administered on day ⁇ 8 and the second dose is administered on day ⁇ 1.
- the first dose is administered on day ⁇ 8 and the second dose is administered on day 0. In some embodiments, the first dose is administered on day ⁇ 8 and the second dose is administered on day 1. In some embodiments, the first dose is administered on day ⁇ 7 and the second dose is administered on day ⁇ 1. In some embodiments, the first dose is administered on day ⁇ 7 and the second dose is administered on day 0. In some embodiments, the first dose is administered on day ⁇ 7 and the second dose is administered on day 1. In some embodiments, the first dose is administered on day ⁇ 12 and the second dose is administered on day ⁇ 4. In some embodiments, the first dose is administered on day ⁇ 11, ⁇ 12, ⁇ 13, or ⁇ 14 and the second dose is administered on day ⁇ 3, ⁇ 4, ⁇ 5, or ⁇ 6.
- a second dose of the tolerizing vaccine/preparatory regimen can be administered on day 100, day 90, day 80, day 70, day 60, day 50, day 40, day 30, day 29, day 28, day 27, day 26, day 25, day 24, day 23, day 22, day 21, day 20, day 19, day 18, day 17, day 16, day 15, day 14, day 13, day 12, day 11, day 10, day 9, day 8, day 7, day 6, day 5, day 4, day 3, day 2 or day 1, relative to transplant of donor cells, organs, and/or tissues on day 0.
- the second dose of the tolerizing vaccine e.g., a booster vaccine
- a second dose of a tolerizing vaccine is given concomitantly on day 0 with transplant donor cells, organs, and/or tissues.
- a second dose of a tolerizing vaccine is not required.
- three doses of the tolerizing vaccine/preparatory regimen or preparatory regimen can be administered.
- the first dose can be administered, for example, on day ⁇ 14, ⁇ 10, ⁇ 9, ⁇ 8, ⁇ 7, ⁇ 6, ⁇ 5, ⁇ 4, ⁇ 3, ⁇ 2, ⁇ 1, 0, 1, 2, 3, 4, 5, 6, or 7 relative to transplant of donor cells, organs, and/or tissues on day 0.
- the second dose can be administered, for example, on day ⁇ 3, ⁇ 2, ⁇ 1, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 relative to transplant of donor cells, organs, and/or tissues on day 0.
- the third dose can be administered, for example, on day 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, 100, 120, 150, 180, 200, 250, 300, 350, or 365 relative to transplant of donor cells, organs, and/or tissues on day 0.
- the first dose is administered on day ⁇ 8, the second dose is administered on day 1, and the third dose is administered on day 7.
- the first dose is administered on day ⁇ 8, the second dose is administered on day 1, and the third dose is administered on day 14.
- the first dose is administered on day ⁇ 8, the second dose is administered on day 1, and the third dose is administered on day 21.
- the first dose is administered on day ⁇ 7, the second dose is administered on day 1, and the third dose is administered on day 7. In some embodiments, the first dose is administered on day ⁇ 7, the second dose is administered on day 1, and the third dose is administered on day 14. In some embodiments, the first dose is administered on day ⁇ 7, the second dose is administered on day 1, and the third dose is administered on day 21.
- a third dose of the tolerizing vaccine/preparatory regimen can be administered on day 300, day 200, day 100, day 90, day 80, day 70, day 60, day 50, day 40, day 30, day 29, day 28, day 27, day 26, day 25, day 24, day 23, day 22, day 21, day 20, day 19, day 18, day 17, day 16, day 15, day 14, day 13, day 12, day 11, day 10, day 9, day 8, day 7, day 6, day 5, day 4, day 3, day 2 or day 1, relative to transplant of donor cells, organs, and/or tissues on day 0.
- the tolerizing vaccine can be administered on or on about day 300 to 200; 200 to 100; 100 to 50; 50 to 40; 40 to 30; 30 to 20; 20 to 10; 10 to 5; or 7 to 1, relative to transplant of donor cells, organs, and/or tissues on day 0.
- a fourth dose of the tolerizing vaccine/preparatory regimen can be administered on day 600, day 500, day 400, day 300, day 200, 100, day 90, day 80, day 70, day 60, day 50, day 40, day 30, day 29, day 28, day 27, day 26, day 25, day 24, day 23, day 22, day 21, day 20, day 19, day 18, day 17, day 16, day 15, day 14, day 13, day 12, day 11, day 10, day 9, day 8, day 7, day 6, day 5, day 4, day 3, day 2 or day 1, relative to transplant of donor cells, organs, and/or tissues on day 0.
- a booster vaccine e.g., a booster vaccine
- the tolerizing vaccine can be administered on or on about day 600 to 500; 500 to 400; 400 to 300; 300 to 200; 200 to 100; 100 to 50; 50 to 40; 40 to 30; 30 to 20; 20 to 10; 10 to 5; 7 to 1, relative to transplant of donor cells, organs, and/or tissues on day 0.
- a fifth dose of the tolerizing vaccine or preparatory regimen can be administered on day 1,000, day 900, day 800, day 700, day 600, day 500, day 400, day 300, day 200, 100, day 90, day 80, day 70, day 60, day 50, day 40, day 30, day 29, day 28, day 27, day 26, day 25, day 24, day 23, day 22, day 21, day 20, day 19, day 18, day 17, day 16, day 15, day 14, day 13, day 12, day 11, day 10, day 9, day 8, day 7, day 6, day 5, day 4, day 3, day 2 or day 1, relative to transplant of donor cells, organs, and/or tissues on day 0.
- a booster vaccine e.g., a booster vaccine
- the tolerizing vaccine can be administered on or on about day 1,000 to 900; 900 to 800; 800 to 700; 700 to 600; 600 to 500; 500 to 400; 400 to 300; 300 to 200; 200 to 100; 100 to 50; 50 to 40; 40 to 30; 30 to 20; 20 to 10; 10 to 5; 7 to 1, relative to transplant of donor cells, organs, and/or tissues on day 0.
- the long term tolerance is for a period of at least one month, at least two months, at least three months, at least four months, at least five months, at least six months, at least seven months, at least eight months, at least nine months, at least ten months, at least eleven months, at least twelve months, at least thirteen months, at least fourteen months, at least fifteen months, at least sixteen months, at least seventeen months, at least eighteen months, at least nineteen months, at least twenty months, at least twenty-one months, at least twenty-two months, at least twenty-three months, or at least twenty-four months.
- the long term tolerance is for a period of at least 1 year, at least 2 years, at least 3 years, at least 4 years, at least 5 years, at least 6 years, at least 7 years, at least 8 years, at least 9 years, or at least 10 years.
- the long term tolerance is achieved in the absence of a booster vaccine or booster regimen.
- the long term tolerance is achieved with an administration of a booster vaccine or booster regimen in one or multiple doses.
- one or more booster vaccine doses are administered on the day of, or at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 31 days, 32 days, 33 days, 34 days, 35 days, 36 days, 37 days, 38 days, 39 days, 40 days, 41 days, 42 days, 43 days, 44 days, 45 days, 46 days, 47 days, 48 days, 49 days, 50 days, 51 days, 52 days, 53 days, 54 days, 55 days, 56 days, 57 days, 58 days, 59 days, 60 days, 65 days, 70 days, 75 days, 80 days, 85 days, 90 days, 95 days, 100 days, 105 days, 110 days, 115 days, 120 days, 125 days, 130 days, 135 days, 140 days, 85 days
- a tolerizing vaccine or preparatory regimen can be administered with or without an adjuvant (e.g., one or more immunomodulatory molecules).
- an adjuvant e.g., one or more immunomodulatory molecules.
- the adjuvant enhances the tolerogenic properties of the tolerizing vaccine by inhibiting activation and maturation of antigen presenting cells.
- a dose of a tolerizing vaccine or preparatory regimen can vary based upon the weight of a recipient of a tolerizing vaccine.
- the dose of the tolerizing vaccine or preparatory regimen can comprise about: 1 ⁇ 101 cells/kg, 1 ⁇ 102 cells/kg, 1 ⁇ 103 cells/kg, 1 ⁇ 104 cells/kg, 1 ⁇ 105 cells/kg, 1 ⁇ 106 cells/kg, 1 ⁇ 107 cells/kg, 1 ⁇ 108 cells/kg, 1 ⁇ 109 cells/kg, 1 ⁇ 1010 cells/kg, 1 ⁇ 1011 cells/kg, 1 ⁇ 1012 cells/kg, or more.
- a dose of the tolerizing vaccine or preparatory regimen can comprises about: 1 ⁇ 101 to 1 ⁇ 102 cells/kg; 1 ⁇ 102 to 1 ⁇ 103 cells/kg; 1 ⁇ 103 to 1 ⁇ 104 cells/kg; 1 ⁇ 104 to 1 ⁇ 105 cells/kg; 1 ⁇ 105 to 1 ⁇ 106 cells/kg; 1 ⁇ 106 to 1 ⁇ 107 cells/kg; 1 ⁇ 107 to 1 ⁇ 108 cells/kg; 1 ⁇ 108 to 1 ⁇ 109 cells/kg; 1 ⁇ 109 to 1 ⁇ 1010 cells/kg, 1 ⁇ 1010 to 1 ⁇ 1011 cells/kg, 1 ⁇ 1011 to 1 ⁇ 1012 cells/kg.
- a dose of the tolerizing vaccine or preparatory regimen for administration can be about 0.01 ⁇ 109 cells/kg, 0.02 ⁇ 109 cells/kg, 0.03 ⁇ 109 cells/kg, 0.04 ⁇ 109 cells/kg, 0.05 ⁇ 109 cells/kg, 0.06 ⁇ 109 cells/kg, 0.07 ⁇ 109 cells/kg, 0.08 ⁇ 109 cells/kg, 0.09 ⁇ 109 cells/kg, 0.1 ⁇ 109 cells/kg, 0.2 ⁇ 109 cells/kg, 0.21 ⁇ 109 cells/kg, 0.22 ⁇ 109 cells/kg, 0.23 ⁇ 109 cells/kg, 0.24 ⁇ 109 cells/kg, 0.25 ⁇ 109 cells/kg, 0.26 ⁇ 109 cells/kg, 0.27 ⁇ 109 cells/kg, 0.28 ⁇ 109 cells/kg, 0.29 ⁇ 109 cells/kg, 0.3 ⁇ 109 cells/kg, 0.4 ⁇ 109 cells/kg, 0.5 ⁇ 109 cells/kg, 0.6 ⁇ 109 cells/kg, 0.7 ⁇ 109 cells/kg, 0.8 ⁇ 109 cells/kg, 0.9 ⁇ 109 cells/kg, 1.0 ⁇ 109 cells/kg, 1.5 ⁇ 109 cells/kg, 2.0 ⁇
- a dose of the tolerizing vaccine or preparatory regimen can comprise at least about: 1 ⁇ 104 cells/kg, 5 ⁇ 104 cells/kg, 1 ⁇ 105 cells/kg, 5 ⁇ 105 cells/kg, 1 ⁇ 106 cells/kg, 5 ⁇ 106 cells/kg, 1 ⁇ 107 cells/kg, 5 ⁇ 107 cells/kg, 1 ⁇ 108 cells/kg, 2 ⁇ 108 cells/kg, 3 ⁇ 108 cells/kg, 4 ⁇ 108 cells/kg, 5 ⁇ 108 cells/kg, 6 ⁇ 108 cells/kg, 7 ⁇ 108 cells/kg, 8 ⁇ 108 cells/kg, 9 ⁇ 108 cells/kg, 1 ⁇ 109 cells/kg, 1 ⁇ 1010 cells/kg, or more.
- the methods herein can comprise administering at least or at least about 0.25 ⁇ 109 cells (e.g., apoptotic donor leukocytes (ADLs), such as ECDI-treated cells, e.g., ECDI-treated leukocytes, or apoptotic mesenchymal stromal cells) per kg recipient body weight.
- ADLs apoptotic donor leukocytes
- ECDI-treated cells e.g., ECDI-treated leukocytes, or apoptotic mesenchymal stromal cells
- at least or at least about 1 ⁇ 107, 1 ⁇ 108, 0.25 ⁇ 109, 0.50 ⁇ 109, 0.75 ⁇ 109, 1.00 ⁇ 109, 1.25 ⁇ 109, 1.50 ⁇ 109, 1.75 ⁇ 109 or 2 ⁇ 109 cells (e.g., ECDI-treated cells, e.g., ECDI-treated leukocytes) per kg recipient body weight ECDI-treated cells can be administered.
- the cells can comprise leukocytes, e.g., splenocytes, peripheral blood mononuclear cells (PBMCs), stem-cell derived leukocytes, or a combination thereof.
- splenocytes, PBMCs, stem-cell derived leukocytes, or the combination thereof can comprise B cells or B lymphocytes.
- the cells can comprise primary cells, cells expanded ex vivo, cells of a cell line, or a combination thereof.
- the cells can comprise mesenchymal stromal cells.
- Cells of tolerizing vaccine/regimen or preparatory regimen for each dose of administration can be suspended in a volume suitable for transfusion.
- the cells can be suspended in a volume of about: 0.1 ml, 0.2 ml, 0.3 ml, 0.4 ml, 0.5 ml, 0.6 ml, 0.7 ml, 0.8 ml, 0.9 ml, 1 ml, 2 ml, 3 ml, 4 ml, 5 ml, 6 ml, 7 ml, 8 ml, 9 ml, 10 ml, 11 ml, 12 ml, 13 ml, 14 ml, 15 ml, 16 ml, 17 ml, 18 ml, 19 ml, 20 ml, 21 ml, 22 ml, 23 ml, 24 ml, 25 ml, 26 ml, 27 ml, 28 ml, 29 ml, 30 ml, 31 m
- the cells of tolerizing vaccine or preparatory regimen for each dose of administration can be suspended in a volume of about: 0.1 ml to 1 ml; 1 ml to 10 ml; 10 ml to 50 ml; 50 ml to 100 ml; 100 ml to 200 ml; 200 ml to 300 ml; 300 ml to 400 ml; or 400 ml to 500 ml.
- 75 ⁇ 106 cells of tolerizing vaccine or preparatory regimen can be suspended in a volume of 0.5 ml.
- Tolerizing vaccines/regimen or preparatory regimens can be administered (e.g., by intravenous infusion) in a volume that varies depending upon the weight of the recipient.
- the tolerizing vaccine or preparatory regimen can be given intravenously in a volume of at least or at least about 0.01 ml, 0.1 ml, 0.5 ml, 1 ml, 2 ml, 3 ml, 4 ml, 5 ml, 10 ml, 20 ml, 30 ml, 40 ml or 50 ml per kg recipient body weight, e.g., at least or at least about 0.01 to 0.1, 0.1 to 1, 1 to 2; 2 to 3; 3 to 4; 4 to 5; 1 to 5; 5 to 10; 10 to 20; 20 to 30; 30 to 40; or 40 to 50 ml per kg recipient body weight.
- the tolerizing vaccine e.g., comprising ECDI-treated cells
- Booster doses of a tolerizing vaccine or preparatory regimen can comprise fewer cells than an initial dose of the tolerizing vaccine or preparatory regimen.
- a booster or subsequent dose of the tolerizing vaccine or preparatory regimen can comprise about: 1%, 2%, 3%, 4%, 5%, 7.5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, or 75% fewer cells, or less than the initial or preceding dose of the tolerizing vaccine or preparatory regimen.
- a cell of a tolerizing vaccine or a preparatory regimen can have a circulation half-life after it is administered to a subject.
- a tolerizing vaccine or preparatory regimen described herein can have a circulation half-life of at least or at least about 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 18, 24, 36, 48, 60, or 72 hours.
- the circulation half-life of the tolerizing vaccine or preparatory regimen cells can be from or from about 0.1 to 0.5; 0.5 to 1.0; 1.0 to 2.0; 1.0 to 3.0; 1.0 to 4.0; 1.0 to 5.0; 5 to 10; 10 to 15; 15 to 24; 24 to 36; 36 to 48; 48 to 60; or 60 to 72 hours.
- a tolerizing vaccine or preparatory regimen described herein can have a circulation half-life of at least or at least about 3 hours.
- the cells of the tolerizing vaccine or preparatory regimen can be treated to enhance their circulation half-life.
- Such treatment can include coating the cell with a protein, e.g., CD47.
- the cell treated to enhance its circulation half-life can be a non-apoptotic cell.
- the cell treated to enhance its circulation half-life can be an apoptotic cell.
- the cell in a tolerizing vaccine or preparatory regimen can be genetically modified (e.g., insertion of a transgene such as CD47 in its genome) to enhance its circulation half-life.
- the cell genetically modified to enhance its circulation half-life can be a non-apoptotic cell.
- the cell genetically modified to enhance its circulation half-life can be an apoptotic cell.
- the tolerizing vaccine or the preparatory regimen can be advantageous in transplantation, for example, in xenotransplantation or in allotransplantation, by tolerizing a graft recipient and preventing or delaying graft rejection.
- the tolerization or the preparatory regimen can be conferred to a graft recipient without the use of immunosuppressive therapies (e.g., one or more immunomodulatory molecules).
- immunosuppressive therapies e.g., one or more immunomodulatory molecules.
- other immunosuppressive therapies can be used in combination with tolerizing vaccines to prevent, decrease, or delay transplantation rejection.
- the dose of a peptide or composition thereof required in presence of a tolerizing agent can be lower than the dose of a peptide or composition disclosed herein required in its absence by at least or at least about 5%.
- dose of a peptide or composition disclosed herein can be lower by at least or at least about 5%, 10%, 20%, 30%, 40%, 50%, or more.
- the term “lower” and its grammatical equivalents as used herein can refer to using less peptide or compositions thereof compared to a required dose when one or more wild-type cells, organs, and/or tissues is transplanted into a recipient (e.g., a human or a non-human animal).
- the methods disclosed herein can increase the duration of survival of a transplant (e.g., a xenograft or an allograft transplant) in a recipient for a period of at least one month, at least two months, at least three months, at least four months, at least five months, at least six months, at least 1 year, at least 2 years, at least 3 years, at least 4 years, at least 5 years, at least 6 years, at least 7 years, at least 8 years, at least 9 years, or at least 10 years.
- a transplant e.g., a xenograft or an allograft transplant
- the transplant survives for a period of at least one month, at least two months, at least three months, at least four months, at least five months, at least six months, at least seven months, at least eight months, at least nine months, at least ten months, at least eleven months, at least twelve months, at least thirteen months, at least fourteen months, at least fifteen months, at least sixteen months, at least seventeen months, at least eighteen months, at least nineteen months, at least twenty months, at least twenty-one months, at least twenty-two months, at least twenty-three months, or at least twenty-four months.
- the transplant survives for a period of at least 1 year, at least 2 years, at least 3 years, at least 4 years, at least 5 years, at least 6 years, at least 7 years, at least 8 years, at least 9 years, or at least 10 years.
- the survival is achieved in the absence of a booster vaccine or booster regimen.
- the transplant survival is achieved with an administration of a booster dose in one or multiple doses.
- a first dose of a peptide or compositions disclosed herein can comprise at least or at least about 1 mg, 5 mg, 10 mg, 20 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, 100 mg, or 200 mg of the a peptide or compositions disclosed herein per kg recipient body weight.
- a first dose of a peptide or compositions disclosed herein can comprise at least or at least about 30 mg, 40 mg, 50 mg, 60 mg, 70 mg of a peptide or compositions disclosed herein per kg recipient body weight.
- a first dose of a peptide or compositions disclosed herein can comprise from or from about 1 mg to 200 mg, e.g., from or from about 20 mg to 100 mg; 30 mg to 80 mg; 30 mg to 70 mg; 40 mg to 70 mg; 40 mg to 60 mg; 50 mg to 70 mg; or 60 mg to 80 mg of the a peptide or compositions disclosed herein per kg recipient body weight.
- Methods of the present disclosure further comprises administering an immunomodulatory agent to the recipient.
- the immunomodulatory agent can be for example, an immunosuppressive agent.
- immunosuppressive agent refers to substances that act to suppress or mask the immune system of the host into which the graft is being transplanted. This would include substances that suppress cytokine production, down-regulate or suppress self-antigen expression, or mask the MHC antigens.
- peptides or compositions of the disclosure can be used alone or in some embodiments in combination with immunosuppressive agent that inhibits rejection of a transplant.
- the immunosuppressive agent can be administered simultaneously or at a separate time than the peptide or compositions herein.
- Non-limiting examples of immunosuppressive agents are azathioprine, corticosteroids, cyclosporine, 2-amino-6-aryl-5-substituted pyrimidines; azathioprine or cyclophosphamide; bromocryptine; glutaraldehyde; antiidiotypic antibodies for MHC antigens; cyclosporin A; one or more steroids, preferably corticosteroids and glucocorticosteroids such as prednisone, methyl prednisolone, and dexamethasone; anti-interferon-gamma antibodies; anti-tumor necrosis factor-alpha antibodies; anti-tumor necrosis factor-beta antibodies; anti-interleukin-2 antibodies; anticytokine receptor antibodies such as anti-IL-2 receptor antibodies; heterologous anti-lymphocyte globulin; pan-T antibodies, preferably OKT-3 monoclonal antibodies; antibodies to CD4; streptokinase; strept
- the immunosuppressive agent inhibits T cell activation.
- the immunosuppressive agent that inhibits T cell activation can be an anti-CD40 agent or anti-CD40L (CD154) agent.
- the anti-CD40 agent can be an anti-CD40 antibody.
- the anti-CD40 antibody can be an antagonistic antibody.
- the anti-CD40 antibody can be a Fab′ anti-CD40L monoclonal antibody fragment CDP7657.
- the anti-CD40 antibody can be a FcR-engineered, Fc silent anti-CD40L monoclonal domain antibody.
- the peptide or compositions disclosed herein is administered with one or more additional immunosuppression agent described herein, such as further comprising providing to the recipient one or more of an anti-CD40 agent or anti-CD40L (CD154) agent (e.g., an anti-CD40 antibody), a B-cell targeting agent (e.g., B cell depleting biologics, for example, a biologic targeting CD20, CD19, or CD22, and/or B cell modulating biologics, for example, a biologic targeting BAFF, BAFF/APRIL, CD40, IgG4, ICOS, IL-21, B7RP1), an mTOR inhibitor, a TNF-alpha inhibitor, a IL-6 inhibitor, a nitrogen mustard alkylating agent (e.g., cyclophosphamide), a complement C3 or C5 inhibitor, IFN ⁇ , an NF ⁇ B inhibitor, ⁇ 1-antitrypsin, vitamin D3, siCD40, cobalt protoporphyrin, insulin B9
- the NF ⁇ B inhibitor is dehydroxymethylepoxyquinomicin (DHMEQ), curcumin, triptolide, Bay-117085, or a combination thereof.
- B-cell targeting biologic can be Rituximab, anti-CD20 antibody.
- immunosuppressive agent can be MMF (mycophenolate mofetil (Cellcept)), ATG (anti-thymocyte globulin), anti-CD154 (CD40L), alemtuzumab (Campath), B-cell targeting agent (e.g., B cell depleting biologics, for example, a biologic targeting CD20, CD19, or CD22, and/or B cell modulating biologics, for example, a biologic targeting BAFF, BAFF/APRIL, CD40, IgG4, ICOS, IL-21, B7RP1), anti-IL-6R antibody (tocilizumab, Actemra), anti-IL-6 antibody (sarilumab, olokizumab), CTLA4-Ig (Abatacept/Orencia), belatacept (LEA29Y), mTOR inhibitor (sirolimus (e.g., Rapamune), rapamycin, everolimus), tacrolimus (Prograf), daclizumab (
- Non-limiting examples of B-cell targeting biologics include antagonistic anti-CD40 mAb antibody, Fc-engineered anti-CD40L antibodies, Rituximab, anti-CD20 antibody.
- One or more than one immunosuppressive agents/drugs can be used together or sequentially.
- One or more than one immunosuppressive agents/drugs can be used for induction therapy or for maintenance therapy.
- the same or different drugs can be used during induction and maintenance stages.
- daclizumab Zaclizumab (Zenapax) is used for induction therapy and tacrolimus (Prograf) and sirolimus (Rapamune), or everolimus, or any other mTOR inhibitor is used for maintenance therapy.
- daclizumab (Zenapax) is used for induction therapy and low dose tacrolimus (Prograf) and low dose sirolimus (Rapamune) is used for maintenance therapy.
- Immunosuppression can also be achieved using non-drug regimens including, but not limited to, whole body irradiation, thymic irradiation, and full and/or partial splenectomy. These techniques can also be used in combination with one or more immunosuppressive drug.
- one or more immunomodulatory molecules can target T cell receptor (TCR), CD3e, FK506-binding protein 12 (FKBP12), cytotoxic T lymphocyte associated protein 4 (CTLA-4), programmed cell death protein 1 (PD-1), programmed death ligand 1 (PD-L1), CD40L (CD154), CD40, inducible costimulatory (ICOS), IL-2, TNF- ⁇ , IL-6, IL-7, CD2, CD20, CD52, ⁇ -4 integrin, mTOR (mechanistic target of rapamycin, everolimus, serolimus), DNA synthesis, or any combination thereof.
- TCR T cell receptor
- CD3e FK506-binding protein 12
- FKBP12 cytotoxic T lymphocyte associated protein 4
- CTLA-4 cytotoxic T lymphocyte associated protein 4
- PD-1 programmed cell death protein 1
- PD-L1 programmed death ligand 1
- CD40L CD154
- CD40 inducible costimulatory
- IL-2
- the one or more immunomodulatory molecule can target B cell, (e.g., B cell depleting biologics, for example, a biologic targeting CD20, CD19, or CD22, and/or B cell modulating biologics, for example, a biologic targeting BAFF, BAFF/APRIL, CD40, IgG4, ICOS, IL-21, B7RP1).
- B cell targeting agent can be anti-CD20 mAb (such as rituximab) or other B-cell depleting antibody.
- the immunosuppressive drugs can be a MHC/TCR interaction blockade, a nonselective depleting agent, calcineurin inhibitor, costimulatory signal blockade, cytokine blockade, lymphocyte depleting agent, cell adhesion inhibitor, IL-2 signaling inhibitor, cell cycle blocker, or any combination thereof.
- the MHC/TCR interaction blockade can be anti-abTCR mAb T10B9.
- the nonselective depleting agent can be anti-CD3 mAb (OKT3) or antithymocyte globulin (ATG).
- the calcineurin inhibitor can be cyclosporine or tacrolimus.
- the costimulatory signal blockade can be anti-CELA-4 mAb, abatacept, ipilimumab, anti-PD-1 (such as pembrolizumab), anti-PD-L1 (such as MPDL3280A), anti-CD154 mAb, anti-CD40 mAb, or anti-ICOS mAb.
- the cytokine blockade can be anti-CD25 mAb (such as daclizumab or basiliximab), anti-TNF (infliximab), anti-IL-6 mAb (such as ALD518), or anti-IL-7 mAb.
- the lymphocyte depleting agent can be anti-CD2 mAb, fusion protein with IgG1 (such as alefacept), anti-CD20 mAb (such as rituximab), or anti-CD52 mAb (such as alemtuzumab).
- the cell adhesion inhibitor can be anti-very large antigen 4 (VLA4) (such as natalizumab).
- VLA4 very large antigen 4
- the IL-2 signaling inhibitor can be sirolimus (rapamycin) or everolimus.
- the cell cycle blocker can be mycophenolate mofetil (MMF) or azathioprine.
- a peptide or compositions disclosed herein or an immunomodulatory agent can be easily determined by one of skill in the art.
- the key factor in selecting an appropriate dose and scheduling is the result obtained, i.e., graft survival long-term.
- a relatively high dose may be needed either initially for the treatment of hyperacute graft rejection, which can be attributed to antibody-mediated graft destruction, or at a later stage characterized by a sudden decline in graft function.
- an immunosuppressive agent When used, it may be administered by any suitable means, including parenteral, and, if desired for local immunosuppressive treatment, intralesionally.
- Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, and subcutaneous administration.
- an immunosuppressive agent when used, it is suitably administered by pulse infusion, particularly with declining doses, or by continuous infusion.
- compositions disclosed herein can further comprise an immunomodulatory agent, for example, an immunosuppressive agent.
- an immunosuppressive agent for example, an immunosuppressive agent.
- An “effective amount” of an immunosuppressive agent and peptides disclosed herein is an amount which achieves a degree of immunosuppression sufficient to delay, inhibit, suppress or moderate tissue transplant rejection and/or delay, inhibit, suppress or moderate one or more symptoms of an inflammatory disease e.g., autoimmune disease described herein.
- the peptides of the present disclosure in combination with an immunomodulatory agent are used at dosages suitable for inducing transplant survival or, alternatively, at dosages at which the immunosuppressive activities of the two agents synergize.
- sub-therapeutic dosages can be used in the disclosed methods, i.e., dosages which are lower than the amounts that would be effective when the peptide or immunomodulatory agent is used alone.
- Suitable sub-therapeutic dosages of one or both agents of the disclosed combination therapy are those which are sufficient, when the two agents are used in combination, to delay, inhibit, suppress or moderate an undesired immune response to an antigen, or inhibit transplant rejection as a result as described above.
- the skilled artisan will be able to determine such dosages using ordinary experimentation, such as by using animal models.
- One advantage of the disclosed combined therapy therefore, is that the subject being treated can in some instances be spared the side-effects of higher levels of immunosuppression resulting from, for example, corticosteroids and inhibitors of calcineurin.
- compositions and methods of the present disclosure can comprise an additional therapeutic agent.
- the therapeutic agent can be, for example, anti-inflammatory agent, immunosuppressive agent, anti-tumor agent as relevant for the condition to be treated. Immunosuppressive agents have been described above.
- Non-limiting example of anti-tumor agents include antibiotics and analogs (e.g., aclacinomycins, actinomycin f1, anthramycin, azaserine, bleomycins, cactinomycin, carubicin, carzinophilin, chromomycins, dactinomycin, daunorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, idarubicin, menogaril, mitomycins, mycophenolic acid, nogalamycin, olivomycines, peplomycin, pirarubicin, plicamycin, porfiromycin, puromycin, streptonigrin, streptozocin, tubercidin, zinostatin, zorubicin), antimetabolites (e.g., antibiotics and analogs (e.g., aclacinomycins, actinomycin f1, anthramycin,
- folic acid analogs e.g., denopterin, edatrexate, methotrexate, piritrexim, pteropterin, Tomudex®, trimetrexate
- purine analogs e.g., cladribine, fludarabine, 6-mercaptopurine, thiamiprine, thioguanine
- pyrimidine analogs e.g., ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, doxifluridine, emitefur, enocitabine, floxuridine, fluorouracil, gemcitabine, tagafur).
- Anti-inflammatory agent can be Steroidal Anti-inflammatory Agents, such as 21-acetoxypregnenolone, alclometasone, algestone, amcinonide, beclomethasone, betamethasone, budesonide, chloroprednisone, clobetasol, clobetasone, clocortolone, cloprednol, corticosterone, cortisone, cortivazol, deflazacort, desonide, desoximetasone, dexamethasone, diflorasone, diflucortolone, difluprednate, enoxolone, fluazacort, flucloronide, flumethasone, flunisolide, fluocinolone acetonide, fluocinonide, fluocortin butyl, fluocortolone, fluorometholone, fluperolone acetate, fluprednidene acetate, flupredn
- Anti-inflamatory agent can be Non-Steroidal antiinflammatory agents such as Aminoarylcarboxylic acid derivatives (e.g., enfenamic acid, etofenamate, flufenamic acid, isonixin, meclofenamic acid, mefenamic acid, niflumic acid, talniflumate, terofenamate, tolfenamic acid), arylacetic acid derivatives (e.g., aceclofenac, acemetacin, alclofenac, amfenac, amtolmetin guacil, bromfenac, bufexamac, cinmetacin, clopirac, diclofenac sodium, etodolac, felbinac, fenclozic acid, fentiazac, glucametacin, ibufenac, indomethacin, isofezolac, isoxepac, lonazolac, met
- the therapeutic agent can be a biologic.
- a biologic refers to any biological material suitable for a therapeutic or in vivo diagnostic purpose.
- Biologic therapeutics include peptides, proteins, vaccines, antibodies, aptamers, nucleic acids, DNA, RNA, antisense oligonucleotides, viruses and bacteria.
- the terms “treat,” “treatment,” “treating,” or “amelioration” refer to therapeutic treatments, wherein the object is to reverse, alleviate, ameliorate, inhibit, slow down or stop the progression or severity of a condition associated with, a disease or disorder.
- the term “treating” includes reducing or alleviating at least one adverse effect or symptom of a condition, disease or disorder associated with a CD40 activity, such as, but not limited to, a inflammation, autoimmune disease or a cancer.
- Treatment is generally “effective” if one or more symptoms or clinical markers are reduced. Alternatively, treatment is “effective” if the progression of a disease is reduced or halted.
- treatment includes not just the improvement of symptoms or markers, but also a cessation of at least slowing of progress or worsening of symptoms that would be expected in absence of treatment.
- Beneficial or desired clinical results include, but are not limited to, alleviation of one or more symptom(s), diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable.
- treatment also includes providing relief from the symptoms or side-effects of the disease (including palliative treatment).
- the methods of the disclosure including treating a CD40 associated disorder of a subject, likely results in an improvement in the subjects' condition, a reduction of symptoms or decreasing the subject's risk for developing symptoms associated with a CD40 associated disorder. Improvements therefore include one or more decreased symptoms associated with autoimmunity, allergy, cancer, etc. An improvement may also be reducing the frequency or amount of a drug used for treating a subject having or at risk of having a CD40 associated disorder. For example, autoimmune patients treated with steroids may require less steroid when treated in combination with a peptide or compositions disclosed herein. An improvement therefore would include reducing the dosage frequency or amount of steroid that the subject was administered in comparison to the dosage frequency or amount administered prior to treatment with a peptide or compositions disclosed herein. An improvement may be, for example, longer transplant survival, or reduction of side effects associated with conventional immunosuppressive regiments for transplantation.
- An improvement may be relatively short in duration, e.g., several hours, days or weeks, or extend over a longer period of time, e.g., months or years.
- the improvement need not be a complete ablation of any or all symptoms of the disorder. For example, reducing severe rheumatoid arthritis to a less severe form is an improvement.
- a satisfactory clinical endpoint is achieved when there is an incremental improvement or a partial reduction in the subject's condition or associated symptoms, over a short or long duration.
- the term “effective amount” as used herein refers to the amount needed to alleviate at least one or more symptom of the disease or disorder, and relates to a sufficient amount of pharmacological composition to provide the desired effect (e.g., binding to CD40 and/or inhibition of the interaction of CD40 and CD154).
- the term “therapeutically effective amount” therefore refers to an amount that is sufficient to effect a particular effect when administered to a typical subject.
- An effective amount as used herein would also include an amount sufficient to delay the development of a symptom of the disease, alter the course of a symptom disease (for example but not limited to, slow the progression of a symptom of the disease), or reverse a symptom of the disease. Thus, it is not possible to specify the exact “effective amount”. For any given case, however, an appropriate “effective amount” can be determined by one of ordinary skill in the art using only routine experimentation.
- Effective amounts, toxicity, and therapeutic efficacy can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population).
- the dosage can vary depending upon the dosage form employed and the route of administration utilized.
- the dose ratio between toxic and therapeutic effects is the therapeutic index and can be expressed as the ratio LD50/ED50.
- Compositions and methods that exhibit large therapeutic indices are preferred.
- a therapeutically effective dose can be estimated initially from cell culture assays.
- a dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the antibody or antigen binding fragment thereof), which achieves a half-maximal inhibition of symptoms as determined in cell culture, or in an appropriate animal model.
- IC50 i.e., the concentration of the antibody or antigen binding fragment thereof
- Levels in plasma can be measured, for example, by high performance liquid chromatography.
- the effects of any particular dosage can be monitored by a suitable bioassay. The dosage can be determined by a physician and adjusted, as necessary, to suit observed effects of the treatment.
- compositions of the disclosure are administered in effective amounts.
- An “effective amount” is that amount of a composition that alone, or together with further doses, produces the desired response.
- a desired response is inhibition of transplant rejection or increasing transplant survival.
- the desired response is inhibiting the progression of the disease. This may involve only slowing the progression of the disease temporarily, although more preferably, it involves halting the progression of the disease permanently. This can be monitored by routine methods.
- Such amounts will depend, of course, on the particular condition being treated, the severity of the disorder, the activity of the specific compound, the route of administration, the rate of clearance of the composition, the duration of treatment, the drugs used in combination or coincident with the compositions, the age, body weight, sex, diet, and general health of the subject, and like factors well known in the medical arts and sciences.
- compositions used in the foregoing methods preferably are sterile and contain an effective amount of the active agents for producing the desired response in a unit of weight or volume suitable for administration to a patient.
- an article of manufacture which contains the pharmaceutical composition in a solution form or in a lyophilized form or a kit comprising an article of manufacture.
- the kit can comprise instructions for diluting the composition or for its reconstitution and/or use.
- the article of manufacture comprises a container. Suitable containers include, for example, bottles, vials (e.g. dual chamber vials), syringes (such as dual chamber syringes) and test tubes.
- the container may be formed from a variety of materials such as glass or plastic.
- the container holds the lyophilized formulation and a label on, or associated with, the container may indicate directions for reconstitution and/or use.
- the label may indicate that the lyophilized formulation is reconstituted to peptide concentrations as described above.
- the label may further indicate that the formulation is useful or intended for subcutaneous administration.
- the container holding the formulation may be a multi-use vial, which allows for repeat administrations (e.g., from 2-6 administrations) of the reconstituted formulation.
- the article of manufacture may further comprise a second container comprising a suitable diluent (e.g., BWFI). Upon mixing of the diluent and the lyophilized formulation, the final protein concentration in the reconstituted formulation will generally be at least 50 mg/mL.
- the article of manufacture may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use.
- Therapeutic kits may have a single container which contains the formulation of the peptide pharmaceutical compositions with or without other components (e.g., other compounds or pharmaceutical compositions of these other compounds) or may have distinct container for each component.
- therapeutic kits of the disclosure include a formulation of peptide disclosed herein or compositions comprising said peptides or an acid addition salt thereof as disclosed herein packaged for use in combination with the co-administration of a second compound (such as an anti-inflammatory agent, immunomodulating agent, anti-tumor agent, a natural product, a hormone or antagonist, a anti-angiogenesis agent or inhibitor, a apoptosis-inducing agent or a chelator) or a pharmaceutical composition thereof.
- a second compound such as an anti-inflammatory agent, immunomodulating agent, anti-tumor agent, a natural product, a hormone or antagonist, a anti-angiogenesis agent or inhibitor, a apoptosis-inducing agent or a chelator
- the components of the kit may be pre-complexed or each component may be in a separate distinct container prior to administration to a patient.
- the components of the kit may be provided in one or more liquid solutions, preferably, an aqueous solution, more preferably, a sterile aqueous solution.
- the components of the kit may also be provided as solids, which may be converted into liquids by addition of suitable solvents, which are preferably provided in another distinct container.
- the container of a therapeutic kit may be a vial, test tube, flask, bottle, syringe, or any other means of enclosing a solid or liquid.
- the kit will contain a second vial or other container, which allows for separate dosing.
- the kit may also contain another container for a pharmaceutically acceptable liquid.
- a therapeutic kit will contain apparatus (e.g., one or more needles, syringes, eye droppers, pipette, etc.), which enables administration of the peptides of the disclosure which are components of the present kit.
- an article of manufacture which contains compositions comprising apoptotic leukocytes and an anti-CD40 agent comprising one or more peptides with an amino sequence with at least 85%, at least 90% or at least 95% sequence identity to the sequences set forth in any one of SEQ ID NOs. 1-6 and 8-17, in a solution form or in a lyophilized form or a kit comprising an article of manufacture.
- the kits of the instant disclosure can be for use in transplantation of a transplant in a recipient or reducing or inhibiting occurrence of GVHD in a recipient. In some embodiments, the kit is useful as a preparatory regimen prior to the transplantation.
- the kit is useful as a tolerizing regimen post-transplantation.
- the kit can comprise instructions for diluting the composition or for its reconstitution and/or use.
- the article of manufacture comprises one or more containers. Suitable containers include, for example, bottles, vials (e.g. dual chamber vials), syringes (such as dual chamber syringes) and test tubes.
- the container may be formed from a variety of materials such as glass or plastic.
- the container holds the compositions e.g., in a lyophilized or solution form and a label on, or associated with, the container may indicate directions for reconstitution and/or use.
- the label may indicate that the lyophilized composition is reconstituted to an effective amount as described above.
- the label may further indicate that the composition is useful or intended for subcutaneous administration or intravenous administration.
- the container holding the composition may be a multi-use vial, which allows for repeat administrations (e.g., from 2-6 administrations) of the composition.
- the article of manufacture may further comprise a container comprising a suitable diluent (e.g., BWFI). Upon mixing of the diluent and the composition, the final concentration in the composition can be, for example, an effective amount suitable for administration.
- the article of manufacture may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use.
- Kits may have a single container which contains, for example apoptotic leukocytes fixed in a crosslinking agent with or without other components (e.g., and an anti-CD40 agent or an anti-CD40 ligand agent).
- the anti-CD40 agent conjugated to the apoptotic leukocytes may comprise a peptide comprising an amino acid sequence with at least 85%, at least 90% or at least 95% sequence identity to the sequences set forth in any one of SEQ ID NOs. 1-6 and 8-17.
- the components of the kit may be pre-complexed or each component may be in a separate distinct container prior to administration to a patient.
- kits of the disclosure include the components disclosed herein packaged for use in combination with the co-administration of an additional component (such as an anti-inflammatory agent, immunomodulating agent, anti-tumor agent, a natural product, a hormone or antagonist, an anti-angiogenesis agent or inhibitor, a apoptosis-inducing agent, or a chelator).
- an additional component such as an anti-inflammatory agent, immunomodulating agent, anti-tumor agent, a natural product, a hormone or antagonist, an anti-angiogenesis agent or inhibitor, a apoptosis-inducing agent, or a chelator.
- the container of a kit may be a vial, test tube, flask, bottle, syringe, or any other means of enclosing a solid or liquid.
- the kit will contain a second vial or other container, which allows for separate dosing.
- the kit may also contain another container for a pharmaceutically acceptable liquid.
- a kit will contain apparatus (e.g., one or more needles, syringes, eye droppers, pipette, etc.), which enables administration of the compositions of the disclosure which are components of the present kit.
- the kit disclosed herein further comprises the transplant.
- the transplant is a kidney, liver, heart, lung, pancreas, islet cell, small bowel, bone marrow, hematopoietic stem cell, embryonic or induced pluripotent stem cell-derived islet beta cell, embryonic or induced pluripotent stem cell-derived islet, embryonic or induced pluripotent stem cell-derived hepatocyte or a combination thereof.
- the transplant can be autologous, allograft, or a xenograft.
- Table 1 lists the amino acid sequences of CD40 binding peptides.
- a control CD40-binding peptide, Peptide 1 was included in the studies described below for comparison.
- Peptide 1 has the amino acid sequence of VLQWAEKGYYTMSNN (SEQ ID NO: 7).
- Amino Acid Complement Wave method was used to identify peptides 2-7 that exhibited high affinity to the human CD40 epitope.
- Peptides that can bind CD40 and block the CD40-CD40L signaling cascade were identified. Sequences of the peptides are presented in the table below.
- Peptides 8-17 are exemplary fragments of peptide 2-7 expected to exhibit high affinity to the human CD40 epitope.
- PBMC peripheral blood mononuclear cells
- RM Rhesus macaques
- the plates were washed and the enzymatic activity was determined using 3,3′,5,5′-tetramethyl-benzidine substrate for 15 minutes and the reaction was stopped by the addition of 2 M H2SO4, and optical density was measured at 450 nm, with the correction wavelength set at 620 nm. Similar to the mononuclear cell binding studies, the Peptide #1 showed high binding affinity to recombinant human CD40, whereas Peptide 4 had the lowest binding affinity. These binding studies indicate Peptide 2, Peptide 3, and Peptide 7 showed specific binding to human CD40, and can therefore block CD40-CD154 interaction.
- MLR mixed lymphocyte reactions
- PBMC peripheral blood mononuclear cells
- proliferating cells were quantitated by determining the frequency of CF SE-low CD4, CD8 and CD20 cells.
- Stained cells were acquired on a 3-laser BD Canto II instrument (BD Bioscience).
- a minimum of 200,000 events were acquired with FACSDIVA 6.1.3.
- FACS analysis was performed with FlowJo Version 10 software. Analysis of the results demonstrated that all the peptides 1-7 showed significant suppression of CD4, CD8 and CD20 cell proliferation in 4/5 donor recipient combinations. More significantly, Peptide 2 and Peptide 3 suppressed the proliferation of CD4, CD8, and CD20 cells at a higher efficacy compared to the other peptides.
- CD40-binding peptides 1-7 as listed in Table 1 was assessed by analyzing the expression of MHC class II molecules on the B cell when subsequently incubated in the presence of recombinant hCD40L. Briefly, B cells were purified from 3 RM PBMC by magnetic sorting. B cells were incubated with peptides at 30 ⁇ M for 15 minutes at 37° C., then activated for 30 minutes with recombinant hCD40L, and then analyzed for expression of MHC class II by staining the cells with specific antibody. Stained cells were acquired on a 3-laser BD Canto II instrument (BD Bioscience). A minimum of 200,000 events were acquired with FACSDIVA 6.1.3. FACS analysis was performed with FlowJo Version 10 software. FACS analysis of the B cells demonstrated that pre-incubation with the peptides resulted in a 50% reduction in MHC class II expression on B cells following activation with CD40L.
- This study identified new peptides that can bind to CD40 and inhibit CD40 activity (i.e., CD40 interaction with CD40L).
- the identified peptides bind specifically to CD40.
- Peptide 2, Peptide 3, and Peptide 5 listed in Table 1 exhibited a higher affinity to bind to CD40 when compared to the control Peptide 1.
- Peptide 2, Peptide 3 and Peptide 5 show an enhanced ability to bind CD40 and inhibit CD40-CD40L interaction and down-stream signaling as evidenced by complete abrogation of T and B cell proliferation as well as activation of B cells.
- This example examines effect of anti-CD40 peptides and the tolerogenic effect of ECDI-treated donor cells for islet transplantation in vivo.
- the effect of an exemplary CD40 binding peptide, peptide 3 is illustrated.
- ECDI-treated splenocytes from a xenogeneic or allogeneic source are administered to a human or a non-human primate transplanted with islets, thereby minimizing the possibility of graft rejection in the human or non-human primate.
- the preparatory regimen of this example can readily be adapted to allotransplantation or xenotransplantation in human recipients (for example, transplant of islets, kidneys, or other cells, tissues, or organs).
- a streptozotocin-induced model of diabetes is utilized with non-human primate subjects. Diabetes is induced by intravenous treatment with streptozotocin. Recipient subjects are transplanted with islets from a MHC-I disparate, one MHC-II DRB-allele matched donor.
- Recipients are treated with a short term immunosuppressive regimen comprising: (i) an anti-CD40 peptide (Peptide 3), given intravenously at a dose of 50 mg/kg on day ⁇ 8, ⁇ 1, 7, and 14 relative to transplant; (ii) rapamycin, given orally from day ⁇ 7 to day 21 relative to transplant with a target trough level of 5-12 ng/mL; (iii) soluble TNF receptor, given intravenously at a dose of 1 mg/kg on days ⁇ 7 and 0 relative to transplant and subcutaneously at a dose of 0.5 mg/kg on days 3, 7, 10, 14, and 21 relative to transplant; and (iv) antagonistic anti-IL-6 receptor antibody, given intravenously at a dose of 10 mg/kg on days ⁇ 7, 0, 7, 14, and 21 relative to transplant.
- a short term immunosuppressive regimen comprising: (i) an anti-CD40 peptide (Peptide 3), given intravenously at a dose of 50 mg/kg on day
- Transplant recipients in experimental groups receive an intravenous infusion of ECDI-treated apoptotic donor leukocytes (ADLs) together with anti-CD40 peptide, peptide 3 (or any one or more peptide selected from peptides 1-17) before and optionally after transplantation.
- ADLs together with an anti-CD40 peptide, peptide 3 are administered about 8 days before transplantation.
- ADLs are administered about 7 days before transplantation.
- ADLs together with an anti-CD40 peptide, peptide 3 are administered about 1 day after transplantation.
- ADLs together with an anti-CD40 peptide are administered about 7 days after transplantation. In some cases, ADLs together with an anti-CD40 peptide are administered about 14 days after transplantation. In some cases, ADLs together with an anti-CD40 peptide are administered about 7 or 8 days before transplantation, and about 1, 7, and/or 14 days after transplantation.
- ADLs can be from the same donor as the islets or a different donor as disclosed herein.
- Transplant recipients in a control group do not receive ADLs. Small doses of exogenous insulin can be administered through day 21 after transplantation. Transplant recipients that receive ADLs together with an anti-CD40 peptide are expected to exhibit improved transplant survival compared to the group that do not receive ADLs. Transplant recipients that receive ADLs together with an anti-CD40 peptide are expected to exhibit improved rejection-free transplant survival compared to the group that do not receive ADLs. Transplant recipients that receive ADLs together with an anti-CD40 peptide are expected to exhibit long-term functional survival of islet allografts.
- transplant recipients that receive ADLs together with an anti-CD40 peptide are expected to exhibit improved blood glucose control after transplant (e.g., become normoglycemic), including after they stop receiving exogenous insulin (e.g., past day 100 or day 365 post-transplant).
- Blood glucose control can be evaluated, for example, by intravenous glucose tolerance test (IVGTT), a mixed meal tolerance test (MMTT), or any other metabolic test established for monitoring pancreatic islet beta cell function.
- IVGTT intravenous glucose tolerance test
- MMTT mixed meal tolerance test
- IVGTT exogenous glucose is injected intravenously, and the blood glucose level is measured over time after the injection.
- Transplant recipients that receive ADLs together with an anti-CD40 peptide are expected to exhibit rapid decreases in blood glucose levels and reduced area under the glucose concentration curve after IVGTT (e.g., comparable levels to those prior to streptozotocin treatment).
- Islet transplant recipients that receive ADLs together with an anti-CD40 peptide are expected to exhibit decreased hemoglobin A1C levels after transplant.
- Transplant recipients that receive ADLs together with an anti-CD40 peptide are expected to exhibit increased C-peptide levels after transplant that are maintained over time, indicating the transplanted islets are functional (e.g., fasted C-peptide levels, glucose-stimulated C-peptide levels, and/or increase in C-peptide levels upon glucose stimulation).
- conjugating anti-CD40 peptides e.g., Peptides 1-17 listed in table 1
- ADLs apoptotic donor leukocytes
- conjugating recipient-type WIC class II molecules to ADLs can enhance ADL efficacy in inducing tolerance to a transplanted cell, tissue, or organ.
- Coupling one or more peptides that can abrogate CD40-CD154 binding (e.g., Peptides 1-17 listed in table 1) to the surface of ADLs can enhance tolerance to a transplanted cell, tissue or organ, after uptake of ADLs (for example, by recipient spleen marginal zone antigen presenting cells or liven sinusoidal endothelial cells). Additionally, coupling recipient-type WIC class II peptides for presentation after uptake of ADLs, derived from one (or more) of the transplant recipient's WIC class II molecules can promote tolerance in the recipient, for example, via regulatory T cell subsets.
- apoptotic donor leukocytes coupled via ECDI with anti-CD40 peptides e.g. peptides listed in Table 1
- recipient-type DRB peptide e.g. peptides listed in Table 1
- MLR mixed lymphocyte reaction
- Recipient PBMCs are Mauritian Cynomolgus Monkey (Mafa M4A, M4A, M4B, M4B, M4DR, M1DR).
- Apoptotic donor leukocytes are Mauritian Cynomolgus Monkey (M3A, M3A, M3B, M3B, M3DR, M3DR), with 14 ⁇ g of synthetic peptides (e.g., Peptides 1-17 listed in table 1) conjugated to the cell surface via ECDI.
- 3 ⁇ 106 apoptotic donor leukocytes with and without conjugated anti-CD40peptide are used to stimulate 3 ⁇ 106 recipient PBMCs at 37° C. in a CO2 incubator.
- Serial samples collected post stimulation on day 1 and day 3 are analyzed for the induction of DC-10 (CD141+CD163+ of CD14+CD16+), Treg cells (CD25hi CD127 ⁇ FoxP3+ of CD4+) and Tr1 cells (CD49b+LAG-3+ of CD4+) by flow cytometry.
- Stimulated cells show that in comparison to PBLs stimulated with control apoptotic donor leukocytes alone, there is an increase in the frequency of Tregs on day 1 and further on day 3 following stimulation with anti-CD40 peptide-conjugated apoptotic donor leukocytes. Similarly, a gradual increase in the frequency of Tr1 cells (on day 1 and day 3), is observed following stimulation with anti-CD40 peptide-conjugated versus control apoptotic donor leukocytes.
- conjugating anti-CD40 peptides e.g., Peptides 1-17 listed in table 1
- the preparatory regimen of this example can readily be adapted to allotransplantation or xenotransplantation in mammalian recipients (for example, transplant of islets, kidneys, or other cells, tissues, or organs, such as embryonic stem cell, induced pluripotent stem cell (iPS)-derived, or mesenchymal stem cell-derived cells, tissues and organs).
- mammalian recipients for example, transplant of islets, kidneys, or other cells, tissues, or organs, such as embryonic stem cell, induced pluripotent stem cell (iPS)-derived, or mesenchymal stem cell-derived cells, tissues and organs.
- recipient type MHC peptides are conjugated to ADLs, and the ADLs are used to induce tolerance to a human islet transplant recipient.
- Splenocytes are obtained from a fully MHC-I and MHC-II mismatched xenogeneic or allogeneic source and are conjugated during ECDI treatment to anti-CD40 peptides (e.g., Peptides 1-17 listed in table 1). Following that, MHC class II chains, domains, and/or peptides are conjugated during ECDI treatment to generate ADLs conjugated to anti-CD40 peptides and peptides derived from MHC-II. The ADLs conjugated to anti-CD40 peptides and peptides derived from MHC-II are administered to human subjects that receive islet transplants, thereby reducing the possibility of graft rejection.
- anti-CD40 peptides e.g., Peptides 1-17 listed in table 1
- MHC class II chains, domains, and/or peptides are conjugated during ECDI treatment to generate ADLs conjugated to anti-CD40 peptides and peptides derived from M
- Transplant recipients receive an intravenous infusion of ADLs conjugated to anti-CD40 peptides and peptides derived from MHC-II before and optionally after transplantation, for example, on day ⁇ 7 and day +1 relative to transplantation.
- ADLs can be from the same donor as the islets or a different donor as disclosed herein.
- Recipient subjects are transplanted with islets from the fully MHC class I and MHC class II mismatched donor.
- Recipients are treated with a short term immunosuppressive regimen comprising: (i) an antagonistic anti-CD40 peptide (e.g., Peptides 1-17 listed in table 1), given intravenously at a dose of 50 mg/kg on day ⁇ 8, ⁇ 1, 7, and 14 relative to transplant; (ii) rapamycin, given orally from day ⁇ 7 to day 21 relative to transplant with a target trough level of 5-12 ng/mL; (iii) soluble TNF receptor, given intravenously at a dose of 1 mg/kg on days ⁇ 7 and 0 relative to transplant and subcutaneously at a dose of 0.5 mg/kg on days 3, 7, 10, 14, and 21 relative to transplant; and (iv) antagonistic anti-IL-6 receptor antibody, given intravenously at a dose of 10 mg/kg on days ⁇ 7, 0, 7, 14, and 21 relative to transplant.
- Transplant recipients in control groups do not receive ADLs, or receive ADLs without anti-CD40 peptide and MHC class II peptides conjugated.
- Transplant recipients that receive anti-CD40 peptide and MHC-II peptides conjugated ADLs are expected to exhibit improved survival compared to recipients that do not receive anti-CD40 peptide and MHC-II-conjugated ADLs.
- Transplant recipients that receive ADLs conjugated with anti-CD40 peptide and peptides derived from MHC-II are expected to exhibit improved rejection-free survival compared to recipients that do not receive anti-CD40 peptide and MHC-II-conjugated ADLs.
- Transplant recipients that receive ADLs conjugated to anti-CD40 peptides and peptides derived from MHC-II are expected to exhibit long-term functional survival of islet allografts compared to recipients that do not receive ADLs conjugated to anti-CD40 peptides and peptides derived from MHC-II.
- transplant recipients that receive ADLs conjugated to anti-CD40 peptides and peptides derived from MHC-II are expected to exhibit improved blood glucose control after transplant (e.g., become normoglycemic), including after they stop receiving exogenous insulin (e.g., past day 100 or day 365 post-transplant).
- Blood glucose control can be evaluated, for example, by intravenous glucose tolerance test (IVGTT), a mixed meal tolerance test (MMTT), or any other metabolic test established for monitoring pancreatic islet beta cell function.
- IVGTT intravenous glucose tolerance test
- MMTT mixed meal tolerance test
- Transplant recipients that receive ADLs conjugated to anti-CD40 peptides and peptides derived from MHC-II are expected to exhibit rapid decreases in blood glucose levels and reduced area under the glucose concentration curve after IVGTT (e.g., comparable levels to a healthy subject).
- Transplant recipients that receive ADLs conjugated to anti-CD40 peptides and peptides derived from MHC-II are expected to exhibit decreased hemoglobin A1C levels after transplant.
- Transplant recipients that receive anti-CD40L and MHC-II conjugated ADLs are expected to exhibit increased C-peptide levels after transplant that are maintained over time, indicating the transplanted islets are functional (e.g., fasted C-peptide levels, glucose-stimulated C-peptide levels, and/or increase in C-peptide levels upon glucose stimulation).
- Stem cells from one donor can be differentiated into a first population of cells for use as apoptotic donor leukocytes (ADLs), and separately differentiated into a second population of cells for transplant.
- ADLs apoptotic donor leukocytes
- This technique can be used to induce tolerance to any universal cell-derived cell, tissue, or organ transplant.
- the stem cells can be embryonic stems cells, induced pluripotent stem cells (iPSCs), and/or mesenchymal stem cells.
- the stem cells are differentiated into a first population of cells that express both MHC class I and MHC class II antigens.
- iPSCs from a transplant donor are differentiated into B lymphocytes that express both MHC class I and II antigens. Methods of differentiating iPSCs into B cells are described, for example, in French A et al. (2015), Stem Cells and Development 24(9):1082-95.
- the donor stem cell-derived B cells share one MHC class II antigen with the recipient (e.g., at least one MHC class II DR allele, MHC class II DQ allele, or MHC class II DP allele).
- the iPSCs are differentiated into a second population of cells to be transplanted.
- the stem-cell derived B cells are treated with ECDI in the presence of an anti-CD40 peptide (e.g., Peptides 1-17 listed in table 1) to generate anti-CD40 conjugated apoptotic donor leukocytes (anti-CD40 peptide conjugated ADLs).
- an anti-CD40 peptide e.g., Peptides 1-17 listed in table 1
- the stem cell-derived, anti-CD40 peptide conjugated ADLs are administered to a subject that receives a transplant of cells, tissues, or organs derived from the same stem cell donor, thereby reducing the possibility of transplant rejection.
- the transplant recipient receives an intravenous infusion of stem cell-derived anti-CD40 peptide conjugated ADLs before and optionally after transplantation, for example, on day ⁇ 7 and day +1 relative to transplantation.
- the recipients can optionally be treated with a short term immunosuppressive regimen comprising: (i) an antagonistic anti-CD40 peptide (e.g., Peptides 1-17 listed in table 1), given intravenously at a dose of 50 mg/kg on day ⁇ 8, ⁇ 1, 7, and 14 relative to transplant; (ii) rapamycin, given orally from day ⁇ 7 to day 21 relative to transplant with a target trough level of 5-12 ng/mL; (iii) soluble TNF receptor, given intravenously at a dose of 1 mg/kg on days ⁇ 7 and 0 relative to transplant and subcutaneously at a dose of 0.5 mg/kg on days 3, 7, 10, 14, and 21 relative to transplant; and (iv) antagonistic anti-IL-6 receptor antibody, given intravenously at a dose of 10 mg/kg on days ⁇ 7, 0, 7, 14, and 21 relative to transplant.
- an antagonistic anti-CD40 peptide e.g., Peptides 1-17 listed in table 1
- rapamycin given
- the recipient is expected to exhibit improved rejection-free survival compared to the recipients that do not receive the stem-cell derived, anti-CD40 peptide conjugated ADLs.
- This technique can be used to induce tolerance to any universal cell-derived cell, tissue, or organ transplant.
- ECDI-fixed B cell products conjugated with CD40 binding peptide (e.g., Peptides 1-17 listed in table 1) generated under different experimental conditions are compared for their ability to induce maturation-arrest in dendritic cells (DC).
- DC dendritic cells
- Human monocyte-derived DC are generated with IL-4 and GCSF. These DC are incubated in the presence or absence of various ECDI-fixed B cell products coupled to inhibitors of DC maturation (e.g., rapamycin, curcumin, vitamin D3, Bay-117085, siCD40, cobalt protoporphyrin, and ⁇ 1-antitrypsin).
- Readouts of DC maturation arrest include i) expression of DC phenotypic markers (CD83, CD80, CD86, MHC class II, CD40), ii) STAT-6 phosphorylation, iii) RELP nuclear translocation, iv) IL-12p70 production, v) allostimulatory capacity, and vi) priming of T cells with regulatory phenotypes (CD4+CD25hiCD127lowFoxp3+ Tregs and CD4+CD49b+Lag-3+CD45RA ⁇ Tr1 cells). ECDI-treated B cell products are identified that induce maturation-arrest in DC.
- ECDI-fixed donor B lymphocytes are generated, including B cells with various anti-CD40 peptides conjugated to their surface by ECDI (e.g., Peptides 1-17 listed in table 1).
- ECDI e.g., Peptides 1-17 listed in table 1.
- MLRs One-way mixed lymphocyte reactions
- Cells are phenotyped and proliferation evaluated at various time points by multi-parametric flow cytometry (e.g., including CFSE dilution and staining for markers that differentiate cell subsets of interest). Blocking antibodies are added and distinct cell subsets are depleted to dissect underlying mechanisms and to determine how the enhanced ECDI-fixed B cell products influence T cell immunity (e.g., B cell products with or without coupled anti-CD40 peptides).
- multi-parametric flow cytometry e.g., including CFSE dilution and staining for markers that differentiate cell subsets of interest.
- Blocking antibodies are added and distinct cell subsets are depleted to dissect underlying mechanisms and to determine how the enhanced ECDI-fixed B cell products influence T cell immunity (e.g., B cell products with or without coupled anti-CD40 peptides).
- Readouts include i) fold-proliferation of CD4+ and CD8+ T cells with effector phenotypes as determined by surface markers, intracellular cytokines, and/or transcription factors; ii) fold-proliferation of CD4+ and CD8+ T cells with regulatory phenotypes as determined by surface markers, intracellular cytokines, and/or transcription factors; and iii) CD8+ T cell-mediated cytotoxicity against target cells in the MLR.
- the efficacy of peri-transplantation infusions of ADL products coupled to anti-CD40 peptides (e.g., Peptides 1-17 listed in table 1), for inducing stable renal allograft tolerance is evaluated in one DRB-matched, SI non-human primates.
- An experimental group receives ADL products comprising ex vivo expanded donor B cells coupled to anti-CD40 peptides (e.g., Peptides 1-17 listed in table 1), while a control group receives saline infusions.
- SI mammalian recipients are identical in both groups and long-term maintenance drugs are not given to any recipient. Renal allograft failure is defined by serum creatinine>2.5 mg/dL and confirmed by graft histology.
- Purpose-bred, qualified non-human primate donors and recipients are selected from qualified vendors.
- the donors and recipients have a defined MHC disparity (MHC class I-disparate and one MHC class II DRB allele-matched donor-recipient pairs, based on high-resolution MHC class I and II genotyping using Fluidigm Access Arrays to generate amplicons for deep sequencing).
- Eligibility criteria for recipients can include ABO compatibility, low memory alloreactivity as defined by negative panel reactive antibodies (PRA; OneLambda Bead assay), negative donor-specific antibodies (DSA) by flow, and IFN- ⁇ ELISPOT ⁇ 12 SFC/106 PBMC (B cell ELISPOT) against donor non-human primate.
- PRA negative panel reactive antibodies
- DSA negative donor-specific antibodies
- IFN- ⁇ ELISPOT ⁇ 12 SFC/106 PBMC B cell ELISPOT
- Kidney transplantation in non-human primates follows established procedures. Briefly, following systemic heparinization of both donor and recipient, the donor organ is excised and the vessels are anastomosed to the recipient's infrarenal aorta and vena cava. Typically, this is performed in a left-to-right fashion owing to the longer length of the left renal vessels.
- the donor ureter is tunneled through the retroperitoneum and a primary ureteroneocystostomy is formed typically on the posterior wall of the bladder using a modified Leadbetter-Politano approach. Particular attention is paid to avoid urine leakage and ureteral stenosis. Bilateral native nephrectomy is completed prior to closure.
- Ex vivo expanded and ECDI-fixed donor B cells coupled to anti-CD40 peptides are infused IV into experimental recipients on days ⁇ 7 and +1 at a dose of 0.25 ⁇ 109/kg recipient body weight.
- Approximately 60 ml of blood (corresponding to 1% of body weight) is drawn from donors on day ⁇ 21 or ⁇ 22 ( ⁇ 2 days) relative to planned renal transplant, and B cells are purified by magnetic sorting using non-human primate CD20 beads.
- B cells can be enriched from leukapheresis products.
- a B cell expansion protocol is adapted from the culture system reported by Su et al (J Immunol 2016, 197:4163-76).
- Purified B cells (approximately 24 ⁇ 106 B cells from 60 ml of blood) are expanded ex vivo in a GREX100M flask (Wilson Wolf) until day ⁇ 7 in RPMI 1640 medium with added 5% rhesus macaque serum, 55 ⁇ M 2-ME, 2 mM L-glutamine, 100 U/ml penicillin, 100 ⁇ g/ml streptomycin, 10 mM HEPES, 1 mM sodium pyruvate, and 1% MEM nonessential amino acids.
- the culture medium is supplemented with anti-CD40 peptides (e.g., Peptides 1-17 listed in table 1), IL-2 (50 ng/ml), IL-4 (10 ng/ml), IL-21 (10 ng/ml), and BAFF (10 ng/ml).
- anti-CD40 peptides e.g., Peptides 1-17 listed in table 1
- IL-2 50 ng/ml
- IL-4 10 ng/ml
- IL-21 10 ng/ml
- BAFF 10 ng/ml
- Input cell numbers, medium volume, and the concentration of CD40L-multimeric are optimized in feasibility studies. Cells are counted after 7 and 14 days, split after 14 days (day ⁇ 7), and the cells not infused on day ⁇ 7 are expanded for another 8 days for infusion on day +1.
- ECDI electrospray induced cell proliferation
- DPBS DPBS
- CD40 binding peptides e.g., Peptides 1-17 listed in table 1
- ECDI-fixed B cells coupled to CD40 binding peptides, meeting all release criteria, are loaded into cold syringes for IV infusion with a maximum concentration of 20 ⁇ 106 cells/mL; the cells remain on ice until recipient administration.
- Induction of apoptosis is monitored in vitro by incubating an aliquot of ECDI-fixed cells coupled to CD40 binding peptides at 37° C. for 4-6 hours, labelling with Annexin V/PI, and analyzing via fluorescent microscopy or flow cytometry.
- Identical short-term immunosuppression and anti-inflammatory therapies are administered to control and experimental subjects.
- the first dose of each drug is given on day ⁇ 8 or ⁇ 7 relative to transplantation on day 0.
- the antagonistic anti-CD40 peptides are given intravenously at 50 mg/kg on days ⁇ 8, ⁇ 1, 7, and 14.
- Rapamycin (Rapamune®) is given orally (PO) from day ⁇ 7 through day 21 post-transplant; the target trough level is 5 to 12 ng/ml.
- Concomitant anti-inflammatory therapy is with i) ⁇ -IL-6R (tocilizumab, Actemra®) at 10 mg/kg IV on days ⁇ 7, 0, 7, 14, and 21, and ii) sTNFR (etanercept, Enbrel®) at 1 mg/kg IV on days ⁇ 7 and 0 and 0.5 mg/kg subcutaneously on days 3, 7, 10, 14, and 21.
- ⁇ -IL-6R tocilizumab, Actemra®
- sTNFR etanercept, Enbrel®
- the primary efficacy outcome is the proportion of transplanted non-human primate with rejection-free allograft survival (confirmed by histopathology) at day 365 post-transplant. Accordingly, follow-up is to day 365 or graft failure, whichever occurs first.
- the group experimental group that received ADLs coupled to CD40 binding peptides is expected to exhibit enhanced rejection-free allograft survival compared to the control group that received only the short-term immunosuppression and anti-inflammatory therapies.
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Animal Behavior & Ethology (AREA)
- Pharmacology & Pharmacy (AREA)
- Immunology (AREA)
- Epidemiology (AREA)
- Engineering & Computer Science (AREA)
- Cell Biology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Organic Chemistry (AREA)
- Hematology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Microbiology (AREA)
- Mycology (AREA)
- Virology (AREA)
- Biomedical Technology (AREA)
- Zoology (AREA)
- Molecular Biology (AREA)
- Biochemistry (AREA)
- Developmental Biology & Embryology (AREA)
- Genetics & Genomics (AREA)
- Biotechnology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- General Chemical & Material Sciences (AREA)
- Biophysics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Diabetes (AREA)
- Gastroenterology & Hepatology (AREA)
- Transplantation (AREA)
- Optics & Photonics (AREA)
- Dispersion Chemistry (AREA)
- Physics & Mathematics (AREA)
- Nanotechnology (AREA)
- Obesity (AREA)
- Endocrinology (AREA)
Abstract
Description
- This application is a continuation of U.S. application Ser. No. 17/089,463, filed Nov. 4, 2020, which is a continuation of International Patent Application PCT/US2019/061325, filed Nov. 13, 2019, which claims benefit to U.S. Provisional Patent Application No. 62/760,796, filed Nov. 13, 2018, each of which is incorporated herein by reference in their entirety.
- The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Jul. 13, 2021, is named 199830-718302-Squence_Listing.txt and is 8,192 bytes in size.
- CD40 ligand (CD40L), or CD154, binds to i) its classical receptor CD40 on B cells and other antigen presenting cells, ii) αMβ2, also known as Mac-1, on neutrophils, monocytes, and macrophages, iii) α5β1 on monocytes, and αIIbβ3 on platelets. The interaction of CD154 with CD40 is implicated in the activation of CD40 expressing immune cells such as B-cell, T-cell, neutrophil, monocyte, macrophage, dendritic cell, and platelet.
- 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. Absent any indication otherwise, publications, patents, and patent applications mentioned in this specification are incorporated herein by reference in their entireties.
- In one aspect provided herein is a pharmaceutical composition comprising a peptide or a portion thereof, wherein the peptide comprises an amino acid sequence with at least 85%, or at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-6 and 8-17, and a pharmaceutically acceptable carrier, wherein said peptide binds a CD40 protein or portion thereof. In one aspect, provided herein is a pharmaceutical composition comprising a peptide, wherein the peptide comprises an amino acid sequence with at least 90% sequence identity to the sequences set forth in any one of SEQ ID NOs: 1-6 and 8-17, and a pharmaceutically acceptable carrier, wherein said peptide binds a CD40 protein or a portion thereof.
- In some embodiments, the peptide comprises the amino acid sequence of SEQ ID NO: 1. In some embodiments, the peptide comprises the amino acid sequence of SEQ ID NO: 2. In some embodiments, the peptide comprises the amino acid sequence of SEQ ID NO: 3. In some embodiments, the peptide comprises the amino acid sequence of SEQ ID NO: 4. In some embodiments, the peptide comprises the amino acid sequence of SEQ ID NO: 5. In some embodiments, the peptide comprises the amino acid sequence of SEQ ID NO: 6. In some embodiments, the peptide comprises the amino acid sequence of SEQ ID NO: 8. In some embodiments, the peptide comprises the amino acid sequence of SEQ ID NO: 9. In some embodiments, the peptide comprises the amino acid sequence of SEQ ID NO: 10. In some embodiments, the peptide comprises the amino acid sequence of SEQ ID NO: 11. In some embodiments, the peptide comprises the amino acid sequence of SEQ ID NO: 12. In some embodiments, the peptide comprises the amino acid sequence of SEQ ID NO: 13. In some embodiments, the peptide comprises the amino acid sequence of SEQ ID NO: 14. In some embodiments, the peptide comprises the amino acid sequence of SEQ ID NO: 15. In some embodiments, the peptide comprises the amino acid sequence of SEQ ID NO: 16. In some embodiments, the peptide comprises the amino acid sequence of SEQ ID NO: 17.
- In some embodiments, the peptide is from 4 to 20 amino acids in length. In some embodiments, the peptide comprises at least one non-naturally occurring amino acid. In some embodiments, the peptide comprises a carboxy terminus that is amidated. In some embodiments, the peptide is in a cyclic form. In some embodiments, the peptide further comprises PEG. In some embodiments, the peptide is comprises a lipophilic molecular group. In some embodiments, the peptide is recombinant or synthesized. In some embodiments, the peptide is fused to a carrier polypeptide. In some embodiments, the peptide is further conjugated to a detectable agent, a peptide tag, a magnetic particle, a diagnostic agent, a therapeutic agent, or a combination thereof. In some embodiments, the pharmaceutical composition disclosed herein, comprises a detectable agent that is an enzyme—substrate agent, a fluorescent agent, a chemiluminescent agent, or a radioisotope. In some embodiments, the pharmaceutical composition disclosed herein, comprises a therapeutic agent that is an anti-inflammatory, immunosuppressive, immunomodulatory, or an anti-tumor agent. In some embodiments, the peptide is in a soluble form.
- In some embodiments, the peptide is formulated in a liposome or a nanoparticle delivery system. In some embodiments, the peptide is formulated with a biocompatible polymer. In some embodiments, the pharmaceutical composition of the aspects above further comprises a pharmaceutically acceptable diluent, additive or excipient. In some embodiments, the pharmaceutical composition of the aspects disclosed above is formulated for administration via a subcutaneous, intravenous, intradermal, intraperitoneal, oral, intramuscular, intracerebroventricular, intranasal, intracranial, intracelial, intracerebellar, intrathecal, transdermal, pulmonary, or topical administration route. In some embodiments, the pharmaceutical composition of the aspects disclosed above is formulated for administration via intravenous administration route. In some embodiments, the pharmaceutical composition further comprises a ganglioside and/or a phosphotidylserine.
- In some embodiments, the pharmaceutical composition further comprises saccharides selected from the group consisting of cyclodextrins, disaccharides, polysaccharides, and a combination thereof. In some embodiments, the pharmaceutical composition of the aspects disclosed above is in a solution form or in a lyophilized form. In some embodiments, the pharmaceutical composition is contained in a delivery device selected from the group consisting of a syringe, a blunt tip syringe, a catheter, an inhaler, a nebulizer, a nasal spray pump, a nasal irrigation pump or nasal lavage pump, and an implantable pump. In some embodiments, the pharmaceutical composition has a shelf life of at least 2 days, 2 weeks, 1 month to 2 years at room temperature. In some embodiments, the pharmaceutical composition has a shelf life of at least 2 days, 2 weeks, 1 month to 2 years at 4° C. In some embodiments, the peptide or portion thereof binds a CD40 protein or a portion of the CD40 protein. In some embodiments, the peptide or portion thereof, wherein the peptide or the functional thereof is in an amount sufficient to inhibit binding of the CD40 protein to a CD154.
- In one aspect, the technology herein relates to a complex comprising a peptide or a portion thereof non-covalently bound to a CD40 protein or a portion of the CD40 protein, wherein the peptide comprises an amino acid sequence with at least 85% or at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-6 and 8-17. In some embodiments, the CD40 protein is mammalian. In some embodiments, the CD40 protein is human. In some embodiments, the CD40 protein is recombinant. In one aspect provided herein is an isolated nucleic acid fragment encoding a peptide or a portion thereof, wherein the peptide comprises an amino acid sequence with at least 85% or at least 90% sequence identity to an amino acid sequence set forth in any one of SEQ ID NOs: 1-6 and 8-17. In some embodiments, the isolated nucleic acid fragment of the aspects disclosed above further comprising a nucleotide sequence encoding an agent, wherein the agent is a targeting agent, a detectable agent, a diagnostic agent, or a therapeutic agent. In one aspect provided herein is a vector comprising the isolated nucleic acid fragment of any one of aspects disclosed above. In some embodiments, the isolated nucleic acid fragment is operably linked to a regulatory control sequence. In one aspect provided herein is a host cell comprising the isolated nucleic acid fragment of aspects above or the vector of aspects above.
- In one aspect, provided herein is a preparatory regimen for transplantation of a cell, tissue or organ from a recipient to a donor, comprising an anti-CD40 agent; wherein the anti-CD40 agent comprises a peptide containing an amino acid sequence with at least 85% or at least 90% sequence identity to the sequences set forth in any one of SEQ ID NOs. 1-6 and 8-17. In another aspect, provided herein is a preparatory regimen for transplantation of a cell, tissue or organ from a recipient to a donor, comprising an anti-CD40 agent; wherein the anti-CD40 agent comprises a peptide containing an amino acid sequence with at least 90% sequence identity to the sequences set forth in any one of SEQ ID NOs. 8-17. In one embodiment, the preparatory regimen can further comprise instructions for administration of said anti-CD40 agent to said recipient. In one embodiment, said anti-CD40 agent is administered to said recipient between about 10 days before and about 30 days after said transplantation. In another embodiment, said anti-CD40 agent is administered to said recipient at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days before said transplantation. In another embodiment, said anti-CD40 agent is administered to said recipient at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 15, 17, 18, 19, 20, 21, 22, 23, 24, or 25 days before said transplantation. In another embodiment, said anti-CD40 agent is administered to said recipient subcutaneously, intravenously, intradermally, intraperitoneally, orally, intramuscularly, intracerebroventricularly, intranasally, intracranially, intracelially, intracerebellarly, intrathecally, or transdermally, or topically. In another embodiment, said anti-CD40 agent is administered to said recipient at a dose of between about 1 mg/kg and 100 mg/kg. In one embodiment, the anti-CD40 agent is administered to said recipient at a dose of 50 mg/kg.
- In one embodiment, the preparatory regimen provides long term tolerance to said transplant cell, tissue, or organ. In one embodiment, the preparatory regimen further comprises administering an effective amount of apoptotic donor leukocytes. In one embodiment, the donor leukocytes are fixed in a cross-linking agent. In one embodiment, the apoptotic leukocytes are mammalian leukocytes. In one embodiment, the apoptotic leukocytes are porcine leukocytes. In one embodiment, the apoptotic leukocytes are human leukocytes. In one embodiment, the apoptotic leukocytes are from a cadaveric donor, a brain dead donor, a non-heart beating donor, or a living donor. In one embodiment, the apoptotic leukocytes are ex vivo expanded leukocytes. In one embodiment, the apoptotic leukocytes are isolated from a spleen, or peripheral blood. In one embodiment, the apoptotic leukocytes comprise B-lymphocytes. In one embodiment, the apoptotic leukocytes comprise cells that have been differentiated from stem cells or induced pluripotent stem cells ex vivo. In some embodiments, the stem cells are derived from the donor of said transplant cell, tissue, or organ. In one embodiment, the apoptotic leukocytes and the recipient are matched for at least one of MHC class I A allele, MHC class I B allele, MHC class II DR allele, MHC class II DQ allele, or MHC class II DP allele. In one embodiment, the apoptotic leukocytes and the transplant are matched for at least one of MHC class I A allele, MHC class I B allele, MHC class II DR allele, MHC class II DQ allele, or MHC class II DP allele. In one embodiment, the apoptotic leucocytes and the transplant are haploidentical. the apoptotic leucocytes are from the donor of the transplant.
- In one embodiment, transplant cell, tissue, or organ is a kidney, liver, heart, lung, pancreas, islet cell, small bowel, bone marrow, hematopoietic stem cell, embryonic stem cell-derived islet beta cell, induced pluripotent stem cell-derived islet beta cell, embryonic stem cell-derived islet, induced pluripotent stem cell-derived islet, a stem cell derived cell, tissue or organ, or a combination thereof. In one embodiment, recipient and the donor of said transplant cell, tissue, or organ are matched for at least one MEW class I A allele, MEW class I B allele, MHC class II DR allele, MEW class II DQ allele, or MHC class II DP allele.
- In one aspect, provided herein is a preparatory regimen for transplanting a cell, tissue or organ transplant to a recipient, comprising: one or more anti-CD40 peptides conjugated to the surface of an apoptotic leukocyte, wherein the one or more anti-CD40 peptides, comprise an amino acid sequence with at least 85% or at least 90% sequence identity to a sequence selected from SEQ ID NOs: 1-6 and 8-17. In another aspect, provided herein is a preparatory regimen for transplanting a cell, tissue or organ transplant to a recipient, comprising: one or more anti-CD40 peptides conjugated to the surface of an apoptotic leukocyte, wherein the one or more anti-CD40 peptides, comprises an amino acid sequence with at least 90% sequence identity to a sequence selected from SEQ ID NOs: 8-17. the apoptotic leukocyte is further conjugated to one or more peptides derived from a MEW class II molecule of the recipient. In one embodiment, the apoptotic leukocyte is further conjugated to one or more peptides derived from a MEW class II molecule of the recipient. In one embodiment, the one or more peptides derived from the MHC class II molecule comprises peptides derived from a DR β-chain, a DQ β-chain, or a DP β-chain, or a combination thereof. In one embodiment, the one or more peptides derived from the MHC class II molecule comprise a sequence from a hypervariable region. In one embodiment, the one or more peptides conjugated to the surface of the leukocyte are synthesized in-vitro or recombinantly produced. In one embodiment, the apoptotic leukocyte is MHC class II matched to the donor and MEW class II mismatched to the recipient. In one embodiment, the apoptotic leukocyte is conjugated to one or more peptides derived from a MEW class I molecule of the donor of the cell, tissue or organ transplant. In one embodiment, the MEW class I molecule is expressed in the donor of the transplant. In one embodiment, the MHC class I molecule is HLA-A1, HLA-A3, HLA-B7, or HLAB8. In one embodiment, the MEW class I molecule is encoded by HLA-A*02, 24, 01 or HLA-B*35, 44, 51. In one embodiment, the apoptotic leukocyte is MEW class I matched to the recipient, or MHC class II matched to the recipient, or both.
- In one embodiment, the preparatory regimen further comprises administering one or more agents for short-term immunosuppression of said recipient. In one embodiment, the one or more agents for short term immunosuppression is an anti-CD40 agent, wherein the anti-CD40 agent comprises a peptide containing an amino acid sequence with at least 85% or at least 90% sequence identity to an amino acid sequence set forth in any one of SEQ ID NOs: 1-6 and 8-17. In one embodiment, the one or more agents for short term immunosuppression is an anti-CD40 agent, wherein the anti-CD40 agent comprises a peptide containing an amino acid sequence with at least 90% sequence identity to an amino acid sequence set forth in any one of SEQ ID NOs: 8-17. In one embodiment, the one or more agents for short term immunosuppression comprise an mTOR inhibitor, an anti-tumor necrosis factor agent or an anti-tumor necrosis factor receptor agent, an
anti-interleukin 6 agent or an anti-interleukin 6 receptor agent, an anti-CD40 agent, or a combination thereof. In one embodiment, the preparatory regimen further comprises instructions for administration of said one or more agents for short term immunosuppression to said transplant recipient. In one embodiment, the apoptotic leukocytes have further been contacted with an amount of one or more immunomodulatory molecules. In one embodiment, said amount of one or more immunomodulatory molecules is sufficient to modify a function of antigen-presenting cells in said recipient. In one embodiment, said one or more immunomodulatory molecules are selected from IFN-γ, an NF-kB inhibitor, vitamin D3, siCD40, cobalt protoporphyrin, insulin B9-23, all or a portion of a cluster of differentiation protein, or a combination thereof. In one embodiment, said NF-kB inhibitor is curcumin, triptolide, Bay-117085, or a combination thereof. - In one embodiment, the one or more peptides are conjugated to the surface of the apoptotic leukocytes by treatment with the crosslinking agent. In one embodiment, said crosslinking agent comprises a carbodiimide. In one embodiment, said carbodiimide comprises 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (ECDI); N,N′-diisopropylcarbodiimide (DIC); N,N′-dicyclohexylcarbodiimide (DCC); or a combination thereof. In one embodiment, said crosslinking agent does not comprise a carbodiimide. In one embodiment, said crosslinking agent comprises genipin, acrylic aldehyde, diformyl, osmium tetroxide, a diimidoester, mercuric chloride, zinc sulphate, zinc chloride, trinitrophenol (picric acid), potassium dichromate, ethanol, methanol, acetone, acetic acid, or a combination thereof. IN one embodiment, said diimidoester is selected from cyanuric chloride, diisocyanate, diethylpyrocarbonate (DEPC), a maleimide, benzoquinone, or a combination thereof. In one embodiment, said apoptotic leukocytes are fixed for a predetermined amount of time. In one embodiment, said predetermined time is at least about 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 60 minutes, 75, minutes, 90 minutes, 120 minutes, 150 minutes, 180 minutes, 210 minutes, or 240 minutes. In one embodiment, said apoptotic leukocytes are contacted with an amount of one or more immunomodulatory molecules, before fixation.
- In one aspect, provided herein is a tolerizing regimen for post-transplant stabilization of a cell, tissue or organ transplant in a recipient, comprising administering to the recipient an effective amount of an anti-CD40 peptide, wherein the anti-CD40 peptide comprises an amino acid sequence with at least 85%. or at least 90% sequence identity to a sequence selected from SEQ ID NOs. 1-6 and 8-17. In another aspect, provided herein is a tolerizing regimen for post-transplant stabilization of cell, tissue or organ transplant in a recipient, comprising administering to the recipient an effective amount of an anti-CD40 peptide, wherein the anti-CD40 peptide comprises an amino acid sequence with at least 90% sequence identity to a sequence selected from SEQ ID NOs. 8-17. In one embodiment, the tolerizing regimen further comprises apoptotic leukocytes. In one embodiment, the apoptotic leukocytes are expanded at least about 3 fold, 5 fold, 10 fold, 50 fold, 100 fold, 150 fold, 200 fold or 250 fold relative to the starting population prior to contacting with said crosslinking agent. In one embodiment, the apoptotic leukocytes and the anti-CD40 peptide are administered separately. In one embodiment, the apoptotic leukocytes and the anti-CD40 peptide are administered simultaneously.
- In one aspect, provided herein is a method of inhibiting an interaction between a CD40 protein and a CD154 protein, the method comprising contacting the CD40 protein with an anti-CD40 agent, wherein the anti-CD40 agent comprises a peptide containing an amino acid sequence with at least 85% or at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-6 and 8-17, thereby inhibiting the interaction between the CD40 protein and the CD154 protein. In another aspect, provided herein is a method of inhibiting an interaction between a CD40 protein and a CD154 protein, the method comprising contacting the CD40 protein with an anti-CD40 agent, wherein the anti-CD40 agent comprises a peptide containing an amino acid sequence with at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 8-17, thereby inhibiting the interaction between the CD40 protein and the CD154 protein. In one embodiment, the peptide or the functional fragment thereof binds the CD40 protein. In one embodiment, the CD40 protein is mammalian. In one embodiment, the inhibition is greater than that exhibited by contacting a peptide or a functional fragment thereof comprising a sequence set forth in SEQ ID NO: 7.
- In one embodiment, the CD40 protein is human. In one embodiment, the CD154 protein is mammalian. In one embodiment, the CD154 protein is human. In one embodiment, the CD40 protein is expressed by an immune cell. In one embodiment, the immune cell is selected from the group consisting of B-cell, T-cell, neutrophil, monocyte, macrophage, dendritic cell, and platelet. In one embodiment, the contacting inhibits B-cell activation, T-cell proliferation, T-cell activation, B-cell proliferation, macrophage activation, cytokine production, or a combination thereof. In one embodiment, the CD40 protein is expressed by an endothelial cell, an epithelial cell, a fibroblast cell, a smooth muscle cell, a B lymphoma cell, a melanoma cell, or a carcinoma cell. In one embodiment, the contacting comprises administering the peptide or a functional fragment thereof to a subject. In one embodiment, the subject is a human.
- In one aspect, provided herein is a method of transplantation of a cell or tissue transplant from a mammalian donor in a recipient, the method comprising administering an effective amount of the pharmaceutical composition of any one the aspects described above. In another aspect, provided herein is a method of transplantation of a cell, tissue or organ from a donor to a recipient, the method comprising administering an effective amount of an anti-CD40 agent; wherein the anti-CD40 agent comprises a peptide containing an amino acid sequence with at least 85% or at least 90% sequence identity to the sequences set forth in any one of SEQ ID NOs. 1-6 and 8-17 to the recipient. In another aspect, provided herein is a method of transplantation of a cell, tissue or organ from a donor to a recipient, the method comprising administering an effective amount of an anti-CD40 agent; wherein the anti-CD40 agent comprises a peptide containing an amino acid sequence with at least 90% sequence identity to the sequences set forth in any one of SEQ ID NOs. 8-17 to the recipient.
- In one embodiment, the peptide is conjugated to apoptotic leukocytes. In one embodiment, the method further comprises administering apoptotic leukocytes. In one embodiment, the method further comprises administering apoptotic leukocytes fixed with a cross-linking agent. In one embodiment, the method further comprises administering the apoptotic leukocytes and the peptide separately. In one embodiment, the method further comprises administering the apoptotic leukocytes and the peptide simultaneously. In one embodiment, the method further comprises the method further comprises transplanting the cell, tissue or organ in the recipient. In one embodiment, the cell or tissue transplant is a kidney, liver, heart, lung, pancreas, islet cell, small bowel, bone marrow, hematopoietic stem cell, embryonic or induced pluripotent stem cell-derived islet beta cell, embryonic or induced pluripotent stem cell-derived islet, embryonic or pluripotent stem-cell derived hepatocyte or a combination thereof. In one embodiment, the cell or tissue transplant is a xenotransplant. In one embodiment, the cell or tissue transplant is an allotransplant. In one embodiment, the administering is subcutaneous, intravenous, intradermal, intraperitoneal, oral, intramuscular, intracerebroventricular, intranasal, intracranial, intracelial, intracerebellar, intrathecal, transdermal, pulmonary, or topical administration. In one embodiment, the administering is intravenous administration. In one embodiment, the administering is performed prior to, during, and after the transplantation. In one embodiment, the administering is performed at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more days prior to the transplantation. In one embodiment, the administering is performed during the transplantation. In one embodiment, the administering is performed at least 1, 2, 3, 4, 5, 6, 7, 10, or more days after the transplantation. In one embodiment, administering a tolerizing agent to the recipient. In one embodiment, the tolerizing agent comprises mammalian leukocytes fixed with a crosslinking agent. In one embodiment, the mammalian leukocytes are from the mammalian donor or from a donor that is MEW matched to the mammalian donor. In one embodiment, the mammalian donor is genetically modified. In one embodiment, administering an immunomodulatory agent to the recipient. In one embodiment, the immunomodulatory agent is an anti-CD40 agent, anti-CD40L agent, a B-cell depleting or modulating agent, an mTOR inhibitor, a TNF-alpha inhibitor, a IL-6 inhibitor, alpha 1antitrypsin inhibitor, dehydroxymethylepoxyquinomycin (DHMEQ), a nitrogen mustard alkylating agent, a complement C3 or C5 inhibitor, IFNγ, an NFκB inhibitor, vitamin D3, siCD40, cobalt protoporphyrin, insulin B9-23, a cluster of differentiation protein, or any combination thereof. In one embodiment, the cell or tissue transplant survives for at least 30 days, 50 days, 100 days, 1 year or more in the recipient. In one embodiment, the recipient is a mammal. In one embodiment, the recipient is a human.
- In one aspect, provided herein is a method for inducing immunosuppression in a subject in need thereof, the method comprising administering to the subject an effective amount of the pharmaceutical composition in any one of the aspects described above. In one aspect, provided herein is a method of inducing immunosuppression in a subject in need thereof, the method comprising; administering to the recipient an effective amount of an anti-CD40 agent, wherein the agent is a peptide comprising an amino acid sequence with at least 85% or at least 90% sequence identity to the sequences set forth in any one of SEQ ID NOs. 1-6 and 8-17. In one aspect provided herein is a method of inducing immunosuppression in a subject in need thereof, the method comprising; administering to the recipient an effective amount of an anti-CD40 agent, wherein the agent is a peptide comprising an amino acid sequence with at least 90% sequence identity to the sequences set forth in any one of SEQ ID NOs. 8-17. In one embodiment, the method comprises further administering apoptotic leukocytes with the anti-CD40 agent. In one embodiment, the peptide is conjugated to apoptotic leukocytes. In one embodiment, the apoptotic leukocytes fixed with a cross-linking agent. In one embodiment, the apoptotic leukocytes and the peptides are administered separately. In one embodiment, the apoptotic leukocytes and the peptides are administered simultaneously. In one embodiment, the method further comprises transplanting a cell, tissue or organ in the subject. In one embodiment, the immune response comprises B-cell activation, T-cell proliferation, B-cell proliferation, macrophage activation, cytokine production, or a combination thereof. In one embodiment, the subject has undergone, is undergoing or will be undergoing an allotransplant. In one embodiment, the subject has undergone, is undergoing or will be undergoing a xenotransplant. In one embodiment, the subject is a human. In one embodiment, inhibits B-cell activation, T-cell proliferation, T-cell activation, B-cell proliferation, macrophage activation, cytokine production, or a combination thereof, thereby inducing immunosuppression. In one embodiment, the subject is suffering from or is at a risk of developing an inflammatory disease. In one embodiment, the inflammatory disease is an autoimmune disease.
- In one aspect, provided herein is a method of inducing immune tolerance in a subject in need thereof, the method comprising administering to the subject an effective amount of the pharmaceutical composition of any one of the aspects described above. In another aspect, provided herein is a method of inducing tolerance to a cell, tissue or organ transplant in a recipient, the method comprising, administering to the recipient an effective amount of a composition comprising an anti-CD40 agent, wherein the agent comprises a peptide containing an amino acid sequence with at least 85% or at least 90% sequence identity to any one of the sequences set forth in SEQ ID NOs. 1-6 and 8-17. In another aspect, provided herein is a method of inducing tolerance to a cell, tissue or organ transplant in a recipient, the method comprising, administering to the recipient an effective amount of a composition comprising an anti-CD40 agent, wherein the anti-CD40 agent comprises a peptide containing an amino acid sequence with at least 90% sequence identity to the sequences set forth any one of SEQ ID NOs. 8-17.
- In one embodiment, the anti-CD40 agent is conjugated to apoptotic leukocytes. In one embodiment, the method further comprises administering apoptotic leukocytes. In one embodiment, the method further comprises administering apoptotic leukocytes fixed with a cross-linking agent. In one embodiment, the method further comprises administering the apoptotic leukocytes and the anti-CD40 agent separately. In one embodiment, the method further comprises administering the apoptotic leukocytes and the anti-CD40 agent simultaneously. In one embodiment, the method further comprises apoptotic leukocytes conjugated to one or more peptides derived from a MHC class II molecule of the recipient. In one embodiment, the apoptotic leukocytes comprise cells that have been differentiated from stem cells ex vivo, wherein the stem cells are derived from a donor of the cell, tissue, or organ transplant. In one embodiment, the apoptotic leukocytes are MHC matched to the recipient. In one embodiment, the apoptotic leukocytes are conjugated to one or more peptides derived from a MHC class I molecule of a donor of the cell, tissue or organ transplant. In one embodiment, the method further comprises transplanting the cell, tissue or organ transplant. In one embodiment, the transplanting is performed at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more days after administering the composition. In one embodiment, the method further comprises administering at least one booster dose of the composition. In one embodiment, the booster dose is administered at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 15, 17, 20, 21, or 24 days after the transplanting. In one embodiment, said tolerance is for a period of at least one month. In one embodiment, said tolerance is for a period of at least 100 days. In one embodiment, said tolerance is for a period of at least one year.
- In one aspect, provided herein is a method of post-transplant immune tolerizing a subject comprising administering to said subject: an anti-CD40 agent, wherein the agent comprises a peptide comprising an amino acid sequence with at least 85% or at least 90% sequence identity to the sequences set forth in any one of SEQ ID NOs: 1-6 and 8-17. In another aspect, provided herein is a method of post-transplant immune tolerizing a subject comprising administering to said subject: an anti-CD40 agent, wherein the agent comprises a peptide comprising an amino acid sequence with at least 90% sequence identity to the sequences set forth in any one of SEQ ID NOs: 8-17. In another embodiment, the anti-CD40 agent is conjugated to apoptotic leukocytes.
- In one embodiment, the anti-CD40 agent is conjugated to apoptotic leukocytes. In one embodiment, the method further comprises administering apoptotic leukocytes. In one embodiment, the method further comprises administering apoptotic leukocytes fixed with a cross-linking agent. In one embodiment, the method further comprises administering the apoptotic leukocytes and the anti-CD40 agent separately. In one embodiment, the method further comprises administering the apoptotic leukocytes and the anti-CD40 agent simultaneously. In one embodiment, the apoptotic leukocytes are further conjugated to one or more peptides derived from a MHC class II molecule of the recipient. In one embodiment, the apoptotic leukocytes comprise cells that have been differentiated from stem cells ex vivo, wherein the stem cells are derived from a donor of the cell, tissue, or organ transplant. In one embodiment, the apoptotic leukocytes are MHC matched to the recipient. In one embodiment, the apoptotic leukocytes are conjugated to one or more peptides derived from a MHC class I molecule of a donor of the cell, tissue or organ transplant. In one embodiment, the method further comprises transplanting the cell, tissue or organ transplant. In one embodiment, the method further comprises apoptotic leukocytes isolated from peripheral blood. In one embodiment, the method further comprises apoptotic leukocytes enriched for B cells by positive or negative selection. In one embodiment, said apoptotic leukocytes are expanded at least 200 fold relative to a starting population prior to said modulating with said carbodiimide crosslinking agent.
- In one aspect, provided herein is a method of treating a subject suffering from an inflammatory disease, the method comprising administering to the subject an effective amount of the pharmaceutical composition of any one of the aspects described above. In one aspect, provided herein is a method of treating a subject suffering from or is at a risk of developing a neoplastic disease characterized by a neoplastic cell that expresses CD40, the method comprising administering to the subject an effective amount of the pharmaceutical composition of any one of the aspects described above. In one embodiment, the inflammatory disease is an autoimmune disease. In one embodiment, the inflammatory disease is selected from the group consisting of type I diabetes, multiple sclerosis, rheumatoid arthritis, systemic lupus, erythematosa, chronic obstructive pulmonary disease, atherosclerosis, Crohn's colitis, ulcerative gastritis, primary biliary cirrhosis, Guillain-Barre syndrome, Psoriasis, Graves' disease, Hashimoto's Thyroiditis, Myasthenia Gravis, Vasculitis, acute lung injury, bronchial asthma and acute respiratory distress syndrome.
- In one aspect, provided herein is a method of treating a subject suffering from or is at a risk of developing a neoplastic disease characterized by a neoplastic cell that expresses CD40, the method comprising administering to the subject an effective amount of the pharmaceutical composition of any of the aspects described above. In one embodiment, the neoplastic disease is a lymphoma, a myeloma or a carcinoma. In one embodiment, the lymphoma is a B cell lymphoma. In one embodiment, the subject is a mammal. In one embodiment, the subject is human.
- In one aspect, provided herein is a method of reducing the number of donor cells required for a transplantation in a recipient, the method comprising administering an effective amount of the pharmaceutical composition of any one of the aspects described above. In one embodiment, the donor cells are islet cells, stem cell, induced pluripotent stem cell-derived islet cells or a combination thereof. In one embodiment, the donor cells are autologous, allogeneic, and xenogeneic.
- In one aspect, provided herein is a kit for transplantation of a cell, tissue or organ transplant in a recipient comprising; (a) a first container comprising a first composition comprising apoptotic leukocytes fixed in a crosslinking agent, an optionally a container comprising a composition comprising an anti-CD40 agent, wherein the agent is a peptide comprising an amino acid sequence with at least 85% or at least 90% sequence identity to the sequences set forth in any one of SEQ ID NOs:1-6 and 8-17. In another aspect, provided herein is a kit for transplantation of a cell, tissue or organ transplant in a recipient comprising; (a) a first container comprising a first composition comprising apoptotic leukocytes fixed in a crosslinking agent, an optionally a container comprising a composition comprising an anti-CD40 agent, wherein the agent is a peptide comprising an amino acid sequence with at least 90% sequence identity to the sequences set forth in any one of SEQ ID NOs:8-17. The kit of any one of claims Error! Reference source not found.-227, wherein the anti-CD40 agent is conjugated to apoptotic leukocytes. In one embodiment, the apoptotic leukocytes is further conjugated to one or more peptides derived from a MHC class II molecule of the recipient. In one embodiment, the apoptotic leukocytes are MHC matched to the recipient. In one embodiment, the apoptotic leukocytes are further conjugated to one or more peptides derived from a MHC class I molecule of a donor of the cell, tissue or organ transplant. In one embodiment, the kit further comprises the cell, tissue or organ transplant. In one embodiment, the first container and the optional second container is a bottle, a vial, a syringe, or a test tube. In one embodiment, the first container and the optional second container is a multi-use container
- In one aspect, provided herein is a transplant kit comprising a preparatory regimen and a tolerizing regimen: said preparatory regimen comprising an anti-CD40 agent, wherein the agent comprises one or more peptides comprising an amino acid sequence with at least 85% or at least 90% sequence identity to the sequences set forth in any one of SEQ ID NOs: 8-17; said tolerizing regimen comprising: an anti-CD40 agent, wherein the agent comprises one or more peptides comprising an amino acid sequence with at least 85% or at least 90% sequence identity to the sequences set forth in any one of the sequences set forth in SEQ ID NOs: 8-17; wherein said preparatory regimen is administered to a subject prior to transplantation, and said tolerizing regimen is administered post-transplantation to said subject. In another aspect, provided herein is a transplant kit comprising a preparatory regimen and a tolerizing regimen: said preparatory regimen comprising an anti-CD40 agent, wherein the agent comprises one or more peptides comprising an amino acid sequence with at least 85% or at least 90% sequence identity to the sequences set forth in any one of SEQ ID NOs: 1-6; said tolerizing regimen comprising: an anti-CD40 agent, wherein the agent comprises one or more peptides comprising an amino acid sequence with at least 85% or at least 90% sequence identity to the sequences set forth in any one of the sequences set forth in SEQ ID NOs: 1-6; wherein said preparatory regimen is administered to a subject prior to transplantation, and said tolerizing regimen is administered post-transplantation to said subject
- In one embodiment, the transplant kit further comprises the transplant kit further comprises a cell, tissue or organ transplant. In one embodiment, the anti-CD40 agent is conjugated to apoptotic leukocytes. In one embodiment, the transplant kit further comprises apoptotic leukocytes. In one embodiment, the transplant kit further comprises apoptotic leukocytes fixed with a cross-linking agent. In one embodiment, the transplant kit further comprises apoptotic leukocytes conjugated with one or more peptides derived from a MHC class II molecule of the recipient. In one embodiment, the transplant kit further comprises apoptotic leukocytes conjugated one or more peptides derived from a MHC class I molecule of a donor of the cell, tissue or organ transplant. In one embodiment, the transplant kit further comprises apoptotic leukocytes that have been differentiated from stem cells ex vivo, wherein the stem cells are derived from a donor of the cell, tissue, or organ transplant. In one embodiment, the transplant kit further comprises apoptotic leukocytes that are MHC matched to the recipient. In one embodiment, the transplant kit further comprises instructions for transplanting the cell, tissue or organ transplant. In one embodiment, the transplant kit further comprises apoptotic leukocytes isolated from peripheral blood. In one embodiment, said apoptotic leukocytes of said tolerizing regimen are enriched for B cells by positive or negative selection. In one embodiment, said apoptotic leukocytes of said tolerizing regimen are expanded at least 200 fold relative to a starting population prior to contacting with said crosslinking agent.
- In one aspect provided herein is a method of inhibiting immune response to a therapeutic agent in a subject, the method comprising administering to the subject the pharmaceutical composition of any one of aspects above.
- In some embodiments, the therapeutic agent is a biologic. In some embodiments, the biologic is a protein, vaccine, antibody, aptamer, nucleic acids, DNA, RNA, antisense oligonucleotide, virus and bacteria.
- In one aspect provided herein is a method of abrogating or reducing an undesired immune response to a therapy in a subject, the method comprising administering to the subject the pharmaceutical composition of any one of aspects above prior to, in conjugation with or after said therapy.
- The features of the present disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure can be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the disclosure are utilized, and the accompanying drawings of which:
-
FIG. 1 shows binding of CD40 targeting peptides to PBMC. Binding of the CD40 targeting peptides was analyzed by incubating 504 of various fluorochrome tagged CD40 interacting peptides with PBMC from rhesus monkeys for 30 minutes at 4° C. and analyzed by flow cytometry.Peptides -
FIG. 2 demonstrates identified peptides bind specifically to CD40. Binding of the identified peptides to CD40 was analyzed by ELISA. Briefly, plates were coated with CD40 and then incubated with identified peptides and detected using Flag-tagged rhsCD40.Peptide # 2,Peptide # 5, andPeptide # 7 showed similar binding affinity toPeptide # 1. -
FIGS. 3A-3E shows newly identified peptides demonstrate robust inhibition of T and B cells proliferation. The ability of the CD40 targeting peptides to block CD40-CD40L signaling and inhibit T and B cell proliferation was assessed in a one-way CFSE-MLR. Each of theFIGS. 3A-3E show results of CFSE-MLR performed on PBMC obtained from a different Rhesus monkey. FACS analysis demonstrates thatPeptide # 2 andPeptide # 3 inhibited T and B cell proliferation significantly better than the CD40 targeting peptide identified earlier. The levels of T cell and B cell proliferation was 35-46% in all the 5 animals and for sake clarity was capped at 25% in the figure for better visualization. -
FIG. 4 shows CD40 targeting peptides block B cell activation. The ability of the newly identified peptides to block B cell activation by CD40L was analyzed in a peptide blocking study. Briefly, B cells were pre-incubated with newly identified peptides for 30 minutes before stimulation with rhCD40L. Activation of the B cells was analyzed by measuring the expression levels of MHC class II molecules on the cell surface by flow cytometry. FACS analysis demonstrates thatPeptide # 2,Peptide # 4 andPeptide # 5 have a similar or higher ability to block the B cell activation when compared to peptide listed inPeptide # 1. - The following discussion of the present disclosure has been presented for purposes of illustration and description. The following is not intended to limit the invention to the form or forms disclosed herein. Although the description of the present disclosure has included description of one or more embodiments and certain variations and modifications, other variations and modifications are within the scope of the present disclosure, e.g., as may be within the skill and knowledge of those in the art, after understanding the present disclosure. It is intended to obtain rights which include alternative embodiments to the extent permitted, including alternate, interchangeable and/or equivalent structures, functions, ranges or steps to those claimed, whether or not such alternate, interchangeable and/or equivalent structures, functions, ranges or steps are disclosed herein, and without intending to publicly dedicate any patentable subject matter.
- The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.
- Although various features of the disclosure may be described in the context of a single embodiment, the features can also be provided separately or in any suitable combination. Conversely, although the disclosure may be described herein in the context of separate embodiments for clarity, various aspects and embodiments can be implemented in a single embodiment.
- The practice of some embodiments disclosed herein employ, unless otherwise indicated, conventional techniques of immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics and recombinant DNA, which are within the skill of the art. See for example Sambrook and Green, Molecular Cloning: A Laboratory Manual, 4th Edition (2012); the series Current Protocols in Molecular Biology (F. M. Ausubel, et al. eds.); the series Methods In Enzymology (Academic Press, Inc.), PCR 2: A Practical Approach (M. J. MacPherson, B. D. Hames and G. R. Taylor eds. (1995)), Harlow and Lane, eds. (1988) Antibodies, A Laboratory Manual, and Culture of Animal Cells: A Manual of Basic Technique and Specialized Applications, 6th Edition (R. I. Freshney, ed. (2010)).
- The following definitions supplement those in the art and are directed to the current application and are not to be imputed to any related or unrelated case, e.g., to any commonly owned patent or application. Although any methods and materials similar or equivalent to those described herein can be used in the practice for testing of the present disclosure, the preferred materials and methods are described herein. Accordingly, the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.
- In this application, the use of the singular includes the plural unless specifically stated otherwise. It must be noted that, as used in the specification, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise. Furthermore, use of the term “including” as well as other forms, such as “include”, “includes,” and “included,” is not limiting.
- The terms “and/or” and “any combination thereof” and their grammatical equivalents as used herein, can be used interchangeably. These terms can convey that any combination is specifically contemplated. Solely for illustrative purposes, the following phrases “A, B, and/or C” or “A, B, C, or any combination thereof” can mean “A individually; B individually; C individually; A and B; B and C; A and C; and A, B, and C.”
- The term “or” can be used conjunctively or disjunctively, unless the context specifically refers to a disjunctive use.
- The term “about” or “approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which can depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, “about” can mean within 1 or more than 1 standard deviation, per the practice in the art. Alternatively, “about” can mean a range of up to 20%, up to 10%, up to 5%, or up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold, of a value. Where particular values are described in the application and claims, unless otherwise stated the term “about” meaning within an acceptable error range for the particular value should be assumed.
- As used in this specification and claim(s), the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps. It is contemplated that any embodiment discussed in this specification can be implemented with respect to any method or composition of the invention, and vice versa. Furthermore, compositions of the invention can be used to achieve methods of the invention.
- Reference in the specification to “some embodiments,” “an embodiment,” “one embodiment” or “other embodiments” means that a particular feature, structure, or characteristic described in connection with the embodiments is included in at least some embodiments, but not necessarily all embodiments, of the inventions.
- As used herein, a “subject”, “patient”, “individual” and like terms are used interchangeably and refers to a vertebrate, preferably a mammal, more preferably a primate, still more preferably a human. Mammals include, without limitation, humans, primates, rodents, wild or domesticated animals, including feral animals, farm animals, sport animals, and pets. Primates include, for example, chimpanzees, cynomolgous monkeys, spider monkeys, and macaques, e.g., Rhesus. Rodents include, for example, mice, rats, woodchucks, ferrets, rabbits and hamsters. Domestic and game animals include, for example, cows, horses, pigs, deer, bison, buffalo, feline species, e.g., domestic cat, and canine species, e.g., dog, fox, wolf, avian species, e.g., chicken, emu, ostrich, and fish, e.g., trout, catfish and salmon. The terms, “individual,” “patient” and “subject” are used interchangeably herein. A subject can be male or female.
- Preferably, the subject is a mammal. The mammal can be a human, non-human primate, mouse, rat, dog, cat, horse, or cow, but is not limited to these examples. Mammals other than humans can be advantageously used as subjects that represent animal models of conditions or disorders associated with CD40 activity. In addition, the compositions and methods described herein can be used to treat domesticated animals and/or pets.
- A subject can be one who has been previously diagnosed with or identified as suffering from or under medical supervision for a CD40 associated disorder, e.g., inflammatory disease, autoimmune disease, cancer. A subject can be one who is diagnosed and currently being treated for, or seeking treatment, monitoring, adjustment or modification of an existing therapeutic treatment, or is at a risk of developing a CD40 associated disorder. A subject can be a recipient of a transplant, for example, someone who has undergone, is undergoing, or will be undergoing transplantation.
- As used herein, the term “in combination” refers to the use of more than one prophylactic and/or therapeutic agent simultaneously or sequentially and in a manner such that their respective effects are additive or synergistic.
- The term “transplant” and its grammatical equivalents as used herein encompasses any procedure that involves transplantation, implantation, or infusion of cells, tissues, or organs from a donor into a recipient. Non-limiting exemplary types of transplant include autotransplant, autograft, allotransplant, allograft, isotransplant, isograft, xenotransplant, xenograft, and split graft, and domino transplant. In an embodiment, the term “split graft” encompasses any procedure that involves the transplantation of cells, organs, tissues, or even particular proteins from a donor is split into more than one recipient.
- The term “autotransplantation”, “autotransplant”, “autograft”, “autologous transplantation” or grammatical equivalents as used herein encompasses any procedure that involves the transplantation of organs, tissues, cells or even particular proteins, or expression products from one part of the body to another in the same subject. In an embodiment, the subject is a member of a Laurasiatheria super order. In an embodiment, the subject is an ungulate for instance a pig, giraffe, camel, deer or bovine. In an embodiment, the subject is a human or non-human primate. The autologous tissue (also called autogenous, autogeneic, or autogenic tissue) transplanted by such a procedure is called an autograft or autotransplant. Non limiting example can include autologous transplant of stem cell, induced pluripotent stem cell, cells derived from stem cells and induced pluripotent cells.
- The term “allotransplantation”, “allotransplant”, “allograft” or their grammatical equivalents as used herein encompasses any procedure that involves transplantation, implantation, or infusion of cells, tissues, or organs into a recipient, where the recipient and donor are the same species. In an embodiment, the recipient and/or donor are a member of a Laurasiatheria super order. In an embodiment, the recipient and/or donor are ungulates for instance pig, giraffe, camel, deer or bovine. In an embodiment, the cells, tissues, or organs described herein are transplanted into humans or non-human primates. Allotransplantation includes but is not limited to vascularized allotransplant, partially vascularized allotransplant, unvascularized allotransplant, allodressings, allobandages, and allostructures. In some cases, an allotransplant is an isograft or isotransplant in which organs or tissues are transplanted from a donor to a genetically identical recipient (such as an identical twin).
- The term “xenotransplantation”, “xenotransplant”, “xenograft” or its grammatical equivalents as used herein encompasses any procedure that involves transplantation, implantation, or infusion of cells, tissues, or organs into a recipient, where the recipient and donor are different species. In an embodiment, the recipient and/or donor are a member of a Laurasiatheria super order. In an embodiment, the recipient and/or donor is an ungulate, for instance a pig, giraffe, camel, deer or bovine. In an embodiment, the donor is a pig, and the recipient is a human or non-human primate. In an embodiment, the cells, tissues, or organs described herein are transplanted into humans or non-human primates. Xenotransplantation includes but is not limited to vascularized xenotransplant, partially vascularized xenotransplant, unvascularized xenotransplant, xenodressings, xenobandages, and nanostructures.
- The term “transplant rejection” and its grammatical equivalents as used herein can refer to a process or processes by which an immune response of an organ transplant recipient mounts a reaction against the transplanted material (e.g., cells, tissues, and/or organs) sufficient to impair or destroy the function of the transplanted material.
- The term “hyperacute rejection” and its grammatical equivalents as used herein can refer to rejection of a transplanted material or tissue occurring or beginning within the first 24 hours after transplantation. For example, hyperacute rejection can encompass but is not limited to “acute humoral rejection” and “antibody-mediated rejection”.
- “Improving,” “enhancing,” “bettering,” and its grammatical equivalents as used herein can mean any improvement recognized by one of skill in the art. For example, improving transplantation can mean lessening hyperacute rejection, which can encompass a decrease, lessening, or diminishing of an undesirable effect or symptom.
- The term “antibody” as used herein includes IgG (including IgG1, IgG2, IgG3, and IgG4), IgA (including IgA1 and IgA2), IgD, IgE, or IgM, and IgY, and is meant to include whole antibodies, including single-chain whole antibodies, and antigen-binding (Fab) fragments thereof. Antigen-binding antibody fragments include, but are not limited to, Fab, Fab′ and F(ab′)2, Fd (consisting of VH and CH1), single-chain variable fragment (scFv), single-chain antibodies, disulfide-linked variable fragment (dsFv) and fragments comprising either a VL or VH domain. The antibodies can be from any animal origin. Antigen-binding antibody fragments, including single-chain antibodies, can comprise the variable region(s) alone or in combination with the entire or partial of the following: hinge region, CH1, CH2, and CH3 domains. Also included are any combinations of variable region(s) and hinge region, CH1, CH2, and CH3 domains. Antibodies can be monoclonal, polyclonal, chimeric, humanized, and human monoclonal and polyclonal antibodies.
- The term “islet”, “islet cells”, “islet equivalent”, “islet-like cells”, “pancreatic islets,” “native islet cells,” “non-native islet cells” and their grammatical equivalents as used herein refers to endocrine (e.g., hormone-producing) cells present in the pancreas of an organism, or cells that mimic one or more function of cells present in the pancreas of an organism. For example, islet cells can comprise different types of cells, including, but not limited to, pancreatic α cells, pancreatic β cells, pancreatic δ cells, pancreatic F cells, and/or pancreatic ε cells. Islet cells can also refer to a group of cells, cell clusters, or the like, including cells cultured in-vitro. In some embodiments, islet cells are extracted from an islet donor and implanted or transplanted at a predetermined site of an islet recipient for differentiation, expansion, and vascularization to form a therapeutic dose of β-cell mass by methods, systems, and/or reagents described herein. In an embodiment, the predetermined site is a renal subcapsular space of the islet recipient. The islets of Langerhans are the regions of the pancreas that contain the endocrine (e.g., hormone-producing) cells (e.g., beta cells). In some embodiments, provided herein are neonatal islet cluster (NICC) or neonatal porcine islet (NPI) comprising pancreas lineage cells (e.g., beta-like cells or a cell population comprising beta-like cells) isolated from a donor by methods, systems, and/or reagents described herein. In some embodiments, NICCs or NPIs are extracted from an islet donor and implanted or transplanted at a predetermined site of an islet recipient for differentiation, expansion, and vascularization to form a therapeutic dose of β-cell mass by methods, systems, and/or reagents described herein. In an embodiment, the predetermined site is a renal subcapsular space of the islet recipient. In some embodiments, NICCs or NPIs extracted from the donor are implanted or transplanted to the recipient under the cover of transient immunosuppression. In some embodiments, islet cells can be stem cell-derived islet cells, induced pluripotent stem cell-derived islet cells, transdifferentiated, or surrogate islet cells. A “donor” is meant to include any mammalian organism, human or non-human, that can serve as a source of donor tissue or cells for transplantation and/or for inducing donor cell tolerance. Non-human mammals include, but are not limited to, ungulates, such as an even-toed ungulate (e.g., pigs, peccaries, hippopotamuses, camels, llamas, chevrotains (mouse deer), deer, giraffes, pronghorn, antelopes, goat-antelopes (which include sheep, goats and others), or cattle) or an odd-toed ungulate (e.g., horse, tapirs, and rhinoceroses), a non-human primate (e.g., a monkey, or a chimpanzee), a Canidae (e.g., a dog) or a cat. A non-human animal can be a member of the Laurasiatheria superorder. The donor can be a living donor or a cadaveric donor. In some cases, the donor is a living donor. In some cases, the donor is a cadaveric donor. The cadaveric donor may be, for example, a brain dead, heart beating donor (BDD). The cadaveric donor may be, for example, a non-heart beating donor (NHBD). Whether the donor is a living donor or a cadaveric donor (e.g., a BDD or NHBD), the donor can be from any animal, for example, a human or non-human animal. The donor can be in any stage of development, including, but not limited to fetal, perinatal, neonatal, pre-weaning, post-weaning, juvenile, young adult, or adult. A donor of cells used in the preparation of a tolerizing vaccine or preparatory regimen can be fully or partially MHC (major histocompatibility complex) matched to a transplant donor (e.g., a donor of cells, tissues, or organs used for transplantation). In some cases, the partially matched donor is haploidentical to the transplant donor. In some cases, the partially matched donor shares one or more MHC alleles with a transplant donor. For example, the partially matched donor can share one or more of a MHC class I A allele, a MHC class I B allele, a MHC class II DR allele, a MHC class II DQ allele, or a combination thereof with a transplant donor. The partially matched donor can share one DR allele with the transplant donors.
- A “recipient” can be a human or non-human animal that can receive, is receiving, or has received a transplant graft, a tolerizing vaccine, a preparatory regimen for transplantation, and/or other compositions provided in the present disclosure. A recipient can also be in need of a transplant graft, a tolerizing vaccine, a preparatory regimen for transplantation, and/or other compositions provided herein. In some cases, the recipient can be a human or non-human animal that can receive, is receiving, or has received a transplant graft. In some cases, the recipient can be a human or non-human animal that can receive, is receiving, or has received the presently described tolerizing vaccine or preparatory regimen for transplantation.
- The term “non-human animal” and its grammatical equivalents as used herein includes all animal species other than humans, including non-human mammals, which can be a native animal or a genetically modified non-human animal. A non-human mammal includes, an ungulate, such as an even-toed ungulate (e.g., pigs, peccaries, hippopotamuses, camels, llamas, chevrotains (mouse deer), deer, giraffes, pronghorn, antelopes, goat-antelopes (which include sheep, goats and others), or cattle) or an odd-toed ungulate (e.g., horse, tapirs, and rhinoceroses), a non-human primate (e.g., a monkey, or a chimpanzee), a Canidae (e.g., a dog) or a cat. A non-human animal can be a member of the Laurasiatheria superorder. The Laurasiatheria superorder can include a group of mammals as described in Waddell et al., Towards Resolving the Interordinal Relationships of Placental Mammals. Systematic Biology 48 (1): 1-5 (1999). Members of the Laurasiatheria superorder can include Eulipotyphla (hedgehogs, shrews, and moles), Perissodactyla (rhinoceroses, horses, and tapirs), Carnivora (carnivores), Cetartiodactyla (artiodactyls and cetaceans), Chiroptera (bats), and Pholidota (pangolins). A member of Laurasiatheria superorder can be an ungulate described herein, e.g., an odd-toed ungulate or even-toed ungulate. An ungulate can be a pig. A member can be a member of Carnivora, such as a cat, or a dog. In some cases, a member of the Laurasiatheria superorder can be a pig.
- The term “porcine”, “porcine animal”, “pig” and “swine” and its grammatical equivalents as used herein can refer to an animal in the genus Sus, within the Suidae family of even-toed ungulates. For example, a pig can be a wild pig, a domestic pig, mini pigs, a Sus scrofa pig, a Sus scrofa domesticus pig, or inbred pigs.
- The term “fetal animal” and its grammatical equivalents can refer to any unborn offspring of an animal. In some cases, pancreatic cell or tissue are isolated from 6 weeks old embryonic pig for transplantation. The term “perinatal animal” and its grammatical equivalents can refer to an animal immediately before or after birth. For example, a perinatal period can start from 20th to 28th week of gestation and ends 1 to 4 weeks after birth. The term “neonatal animal” and its grammatical equivalents can refer to any new born animals. For example, a neonatal animal can be an animal born within a month. The term “pre-weaning non-human animal” and its grammatical equivalents can refer to any animal before being withdrawn from the mother's milk. The term “juvenile animal” and its grammatical equivalents can refer to any animal before becoming a young adult animal. For example, a juvenile stage of pigs can refer to any pigs of 2 years of age or younger.
- The term “genetically modified”, “genetically engineered”, “transgenic”, “genetic modification” and its grammatical equivalents as used herein refer to having one or more alterations of a nucleic acid, e.g., the nucleic acid within an organism's genome. For example, genetic modification can refer to alterations, additions, and/or deletion of genes. A genetically modified cell can also refer to a cell with an added, deleted and/or altered gene. A genetically modified cell can be from a genetically modified non-human animal. A genetically engineered cell from a genetically engineered non-human animal can be a cell isolated from such genetically engineered non-human animal. A genetically modified cell from a genetically modified non-human animal can be a cell originated from such genetically modified non-human animal. A genetically engineered cell or a genetically engineered animal can comprise a transgene, or other foreign DNA, added or incorporated, or an endogenous gene modified, including, targeted, recombined, interrupted, deleted, disrupted, replaced, suppressed, enhanced, or otherwise altered, to mediate a genotypic or phenotypic effect in at least one cell of the animal, and typically into at least one germ line cell of the animal.
- The term “transgene” and its grammatical equivalents as used herein can refer to a gene or genetic material that can be transferred into an organism. For example, a transgene can be a stretch or segment of DNA containing a gene that is introduced into an organism. The gene or genetic material can be from a different species. The gene or genetic material can be synthetic. When a transgene is transferred into an organism, the organism can then be referred to as a transgenic organism. A transgene can retain its ability to produce RNA or polypeptides (e.g., proteins) in a transgenic organism. A transgene can comprise a polynucleotide encoding a protein or a fragment (e.g., a functional fragment) thereof. The polynucleotide of a transgene can be an exogenous polynucleotide. A fragment (e.g., a functional fragment) of a protein can comprise at least or at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% of the amino acid sequence of the protein. A fragment of a protein can be a functional fragment of the protein. A functional fragment of a protein can retain part or all of the function of the protein. An exogenous polypeptide can encode an exogenous protein or a functional fragment thereof.
- The term “exogenous nucleic acid sequence”, “exogenous polynucleotide” and its grammatical equivalents as used herein can refer to a gene or genetic material that was transferred into a cell or animal that originated outside of the cell or animal. An exogenous nucleic acid sequence can by synthetically produced. An exogenous nucleic acid sequence can be from a different species, or a different member of the same species. An exogenous nucleic acid sequence can be another copy of an endogenous nucleic acid sequence.
- The term “gene knock-out” or “knock-out” can refer to any genetic modification that reduces the expression of the gene being “knocked out.” Reduced expression can include no expression. The genetic modification can include a genomic disruption.
- The term “disrupting” and its grammatical equivalents as used herein can refer to a process of altering a gene, e.g., by deletion, insertion, mutation, rearrangement, or any combination thereof. For example, a gene can be disrupted by knockout. Disrupting a gene can be partially reducing or completely suppressing expression (e.g., mRNA and/or protein expression) of the gene. Disrupting can also include inhibitory technology, such as shRNA, siRNA, microRNA, dominant negative, or any other means to inhibit functionality or expression of a gene or protein.
- The term “gene editing” and its grammatical equivalents as used herein can refer to genetic engineering in which one or more nucleotides are inserted, replaced, or removed from a genome. For example, gene editing can be performed using a nuclease (e.g., a natural-existing nuclease or an artificially engineered nuclease).
- The term “condition” and its grammatical equivalents as used herein can refer to a disease, event, or change in health status.
- The term “diabetes” and its grammatical equivalents as used herein can refer to is a disease characterized by high blood sugar levels over a prolonged period. For example, the term “diabetes” and its grammatical equivalents as used herein can refer to all or any type of diabetes, including, but not limited to,
type 1,type 2, type 3c (pancreatogenic diabetes including cystic fibrosis-related, and surgical, and hemochromatosis-related), gestational diabetes, and monogenic diabetes (HNF1A-MODY, GCK-MODY 2, etc.), and other forms of mitochondrial diabetes. In some cases, diabetes can be a form of hereditary diabetes. - As used herein, the term “susceptible to” refers to an individual who is “susceptible to” a disease, disorder, and/or condition has not been diagnosed with the disease, disorder, and/or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and/or condition may not exhibit symptoms of the disease, disorder, and/or condition. In some embodiments, an individual who is susceptible to a disease, disorder, condition, or event (for example, Muscular Dystrophy) may be characterized by one or more of the following: (1) a genetic mutation associated with development of the disease, disorder, and/or condition; (2) a genetic polymorphism associated with development of the disease, disorder, and/or condition; (3) increased and/or decreased expression and/or activity of a protein associated with the disease, disorder, and/or condition; (4) habits and/or lifestyles associated with development of the disease, disorder, condition, and/or event (5) having undergone, planning to undergo, or requiring a transplant. In some embodiments, an individual who is susceptible to a disease, disorder, and/or condition will develop the disease, disorder, and/or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and/or condition will not develop the disease, disorder, and/or condition.
- The term “phenotype” and its grammatical equivalents as used herein can refer to a composite of an organism's observable characteristics or traits, such as its morphology, development, biochemical or physiological properties, phenology, behavior, and products of behavior. Depending on the context, the term “phenotype” can sometimes refer to a composite of a population's observable characteristics or traits.
- “Inflammatory disorder” means an immune-mediated inflammatory condition that affects humans and is generally characterized by dysregulated expression of one or more cytokines. Examples of inflammatory disorders include skin inflammatory disorders, inflammatory disorders of the joints, inflammatory disorders of the cardiovascular system, certain autoimmune diseases, lung and airway inflammatory disorders, intestinal inflammatory disorders. Examples of skin inflammatory disorders include dermatitis, for example atopic dermatitis and contact dermatitis, acne vulgaris, and psoriasis. Examples of inflammatory disorders of the joints include rheumatoid arthritis. Examples of inflammatory disorders of the cardiovascular system are cardiovascular disease and atherosclerosis. Examples of autoimmune diseases include
Type 1 diabetes, Graves disease, Guillain-Barre disease, Lupus, Psoriatic arthritis, and Ulcerative colitis. Examples of lung and airway inflammatory disorders include asthma, cystic fibrosis, COPD, emphysema, and acute respiratory distress syndrome. Examples of intestinal inflammatory disorders include colitis and inflammatory bowel disease. Other inflammatory disorders include cancer, hay fever, periodontitis, allergies, hypersensitivity, ischemia, depression, systemic diseases, post infection inflammation and bronchitis. The peptides and compositions of the invention may also be employed in the non-therapeutic treatment of inflammation. Examples of non-therapeutic treatment of inflammation include use to relieve normal, non-pathological, inflammation, for example inflammation in the muscles and joints following exercise - As used herein, the terms “protein”, “peptide” and “polypeptide” are used interchangeably to designate a series of amino acid residues connected to each other by peptide bonds between the alpha-amino and carboxy groups of adjacent residues. The terms “protein”, “peptide” and “polypeptide” refer to a polymer of amino acids, including modified amino acids (e.g., phosphorylated, glycated, glycosylated, etc.) and amino acid analogs, regardless of its size or function. “Protein” and “polypeptide” are often used in reference to relatively large polypeptides, whereas the term “peptide” is often used in reference to small polypeptides, but usage of these terms in the art overlaps. The terms “protein”, “peptide” and “polypeptide” are used interchangeably herein when referring to a gene product and fragments thereof.
- The terms “increased”/“increase”, “increasing” or “enhance” are all used herein to generally mean an increase by a statically significant amount; for the avoidance of doubt, the terms “increased”, “increase”, or “enhance”, mean an increase of at least 10% as compared to a reference level, for example an increase of at least about 10%, at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% increase or any increase between 10-100% as compared to a reference level, or at least about a 2-fold, or at least about a 3-fold, or at least about a 4-fold, or at least about a 5-fold or at least about a 10-fold increase, or any increase between 2-fold and 10-fold or greater as compared to a reference level. The increase can be, for example, at least 10%, at least 20%, at least 30%, at least 40% or more, and is preferably to a level accepted as within the range of normal for an individual without a given disease.
- The terms, “decrease”, “reduce”, “reduction”, “lower” or “lowering,” or “inhibit” are all used herein generally to mean a decrease by a statistically significant amount. For example, “decrease”, “reduce”, “reduction”, or “inhibit” means a decrease by at least 10% as compared to a reference level, for example a decrease by at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% decrease (e.g., absent level or non-detectable level as compared to a reference level), or any decrease between 10-100% as compared to a reference level. In the context of a marker or symptom, by these terms is meant a statistically significant decrease in such level. The decrease can be, for example, at least 10%, at least 20%, at least 30%, at least 40% or more, and is preferably down to a level accepted as within the range of normal for an individual without a given disease.
- As used herein, substantially pure means sufficiently homogeneous to appear free of readily detectable impurities as determined by standard methods of analysis, such as thin layer chromatography [TLC], gel electrophoresis and high performance liquid chromatography [HPLC], used by those of skill in the art to assess such purity, or sufficiently pure such that further purification would not detectably alter the physical and chemical properties, such as enzymatic and biological activities, of the substance. Methods for purification of the compounds to produce substantially chemically pure compounds are known to those of skill in the art. A substantially chemically pure compound may, however, be a mixture of stereoisomers. In such instances, further purification might increase the specific activity of the compound. In some embodiments, the compositions of the present disclosure are substantially pure.
- By “detectable agent” is meant a compound that is linked to a diagnostic agent to facilitate detection. Such a “detectable agent” may be covalently or non-covalently linked to a diagnostic agent, hi addition, the linkage may be direct or indirect Examples of “detectable agents” include, protein purification tags, cytotoxins, enzymes, paramagnetic labels, enzyme substrates, co-factors, enzymatic inhibitors, dyes, radionuclides, chemiluminescent labels, fluorescent markers, growth inhibitors, cytokines, antibodies, and biotin.
- As used herein, biological activity refers to the in vitro, ex vivo, or in vivo activities of a compound or physiological responses that result upon in vivo administration of a compound, composition or other mixture. Biological activity, thus, encompasses therapeutic effects and pharmaceutical activity of such compounds, compositions and mixtures.
- Some numerical values disclosed throughout are referred to as, for example, “X is at least or at least about 100; or 200 [or any numerical number].” This numerical value includes the number itself and all of the following:
-
- i) X is at least 100;
- ii) X is at least 200;
- iii) X is at least about 100; and
- iv) X is at least about 200.
- All these different combinations are contemplated by the numerical values disclosed throughout. All disclosed numerical values should be interpreted in this manner, whether it refers to an administration of a therapeutic agent or referring to days, months, years, weight, dosage amounts, etc., unless otherwise specifically indicated to the contrary.
- The ranges disclosed throughout are sometimes referred to as, for example, “X is administered on or on about
day 1 to 2; or 2 to 3 [or any numerical range].” This range includes the numbers themselves (e.g., the endpoints of the range) and all of the following: -
- i) X being administered on between
day 1 andday 2; - ii) X being administered on between
day 2 andday 3; - iii) X being administered on between about
day 1 andday 2; - iv) X being administered on between about
day 2 andday 3; - v) X being administered on between
day 1 and aboutday 2; - vi) X being administered on between
day 2 and aboutday 3; - vii) X being administered on between about
day 1 and aboutday 2; and - viii) X being administered on between about
day 2 and aboutday 3.
- i) X being administered on between
- All these different combinations are contemplated by the ranges disclosed throughout. All disclosed ranges should be interpreted in this manner, whether it refers to an administration of a therapeutic agent or referring to days, months, years, weight, dosage amounts, etc., unless otherwise specifically indicated to the contrary.
- Provided herein are peptides capable of binding CD40 and thereby inhibit activity of CD40, i.e., interaction of CD40 with CD154. The terms “CD40 binding peptide” are used interchangeably with “anti-CD40 peptide”. The present disclosure provides pharmaceutical compositions comprising a peptide which comprise an amino acid sequence selected from Table 1 or a portion thereof or a functional fragment thereof. As it relates to the composition and methods of the instant disclosure a peptide can be full length peptide selected from Table 1 or can be a portion thereof or a functional fragment thereof. In some embodiments, a peptide of the compositions and methods disclosed herein are less than 20 amino acids in length. In some embodiments, the peptide is 4, 6, 8, 10, 12, 14, or 15 amino acids in length. In some embodiments, the peptide comprises an amino acid sequence selected from group consisting of SEQ ID NOs: 1-6 and 8-17. The sequences of such peptides are shown in Table 1.
- In some embodiments, the peptide of the compositions and methods disclosed herein have an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% identical to that of SEQ ID NO:1. In some embodiments, the peptide of the compositions and methods disclosed herein have an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% identical to that of SEQ ID NO: 2. In some embodiments, the peptide of the compositions and methods disclosed herein have an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% identical to that of SEQ ID NO:3. In some embodiments, the peptide of the compositions and methods disclosed herein have an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% identical to that of SEQ ID NO:4. In some embodiments, the peptide of the compositions and methods disclosed herein have an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% identical to that of SEQ ID NO:5. In some embodiments, the peptide of the compositions and methods disclosed herein have an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% identical to that of SEQ ID NO: 6.
- In some embodiments, the peptide of the compositions and methods disclosed herein have an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% identical to that of SEQ ID NO:8.
- In some embodiments, the peptide of the compositions and methods disclosed herein have an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% identical to that of SEQ ID NO:9.
- In some embodiments, the peptide of the compositions and methods disclosed herein have an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% identical to that of SEQ ID NO:10.
- In some embodiments, the peptide of the compositions and methods disclosed herein have an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% identical to that of SEQ ID NO:11.
- In some embodiments, the peptide of the compositions and methods disclosed herein have an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% identical to that of SEQ ID NO:12.
- In some embodiments, the peptide of the compositions and methods disclosed herein have an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% identical to that of SEQ ID NO:13.
- In some embodiments, the peptide of the compositions and methods disclosed herein have an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% identical to that of SEQ ID NO:14.
- In some embodiments, the peptide of the compositions and methods disclosed herein have an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% identical to that of SEQ ID NO:15.
- In some embodiments, the peptide of the compositions and methods disclosed herein have an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% identical to that of SEQ ID NO:16.
- In some embodiments, the peptide of the compositions and methods disclosed herein have an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% identical to that of SEQ ID NO:17.
- In some embodiments, the peptide or functional fragment thereof of the present disclosure retain, the ability at a minimum, to bind a CD40 protein. In some embodiments, the peptide comprises a portion (e.g., a functional fragment of the peptide) of the wild type sequence selected from SEQ ID NOs 1-6 and 8-17.
- Percent (%) amino acid sequence identity for a given peptide sequence relative to a reference sequence (e.g., unmodified, full length peptide selected from SEQ ID NOs:1-6 and 8-17 is defined as the percentage of identical amino acid residues identified after aligning the two sequences and introducing gaps if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Percent (%) amino acid sequence homology for a given polypeptide sequence relative to a reference sequence is defined as the percentage of identical or strongly similar amino acid residues identified after aligning the two sequences and introducing gaps if necessary, to achieve the maximum percent homology. Non identities of amino acid sequences include conservative substitutions, deletions or additions that do not affect the biological activity of the peptide. Strongly similar amino acids can include, for example, conservative substitutions known in the art. Percent identity and/or homology can be calculated using alignment methods known in the art, for instance alignment of the sequences can be conducted using publicly available software such as BLAST, Align, ClustalW2. Those skilled in the art can determine the appropriate parameters for alignment, but the default parameters for BLAST are specifically contemplated.
- As used herein, the term “wild type peptide” refers to the native, un-modified peptide comprising amino acid sequence selected from SEQ ID NOs 1-6 and 8-17.
- As used herein, a “fragment” or “portion” is one that substantially retains at least one biological activity normally associated with that peptide. In particular embodiments, the “fragment” or “portion” substantially retains all of the activities possessed by the wild type peptide. By “substantially retains” biological activity, it is meant that the fragment or portion retains at least about 50%, 60%, 75%, 85%, 90%, 95%, 97%, 98%, 99%, or more, of the biological activity of the wild type peptide (and can even have a higher level of activity than the wild type peptide). In some embodiments, a fragment or portion of the protein or polypeptide described herein is at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 or more contiguous amino acids and/or less than about 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, or 4 contiguous amino acids, including any combination of the foregoing as long as the lower limit is less than the upper limit and retains at least one biological activity associated with the wild type peptide.
- By “retains” biological activity, it is meant that the peptide retains at least about 50%, 60%, 75%, 85%, 90%, 95%, 97%, 98%, 99%, or more, of the biological activity of the native peptide (and can even have a higher level of activity than the native polypeptide). A “non-functional” peptide is one that exhibits essentially no detectable biological activity normally associated with the peptide (e.g., at most, only an insignificant amount, e.g., less than about 10% or even 5%).
- As used herein, a “functional” peptide or fragment is one that retains at least one biological, activity normally associated with wild type peptide. Preferably, a “functional” peptide or fragment thereof retains all of the activities possessed by the native, unmodified or full-length wild type peptide. A “non-functional” peptide is one that exhibits essentially no detectable biological activity normally associated with the wild type peptide (e.g., at most, only an insignificant amount, e.g., less than about 10% or even 5%). The functional activity can be tested by one of ordinary skill in the art by the assays described below and in the Examples herein.
- The compositions and methods of the present disclosure contemplate a functional derivative, functional fragment, variant, analogue or chemical derivative of a peptide disclosed herein (e.g., a peptide selected from Table 1).
- In one embodiment, a peptide useful in the methods and compositions described herein consists of, consists essentially of, or comprises an amino acid sequence, or is a fragment thereof derived from sequence selected from SEQ ID NOs: 1-15, provided that the peptide or functional fragment retains at least one biological activity of corresponding full length wild type peptide, the biological activity being selected from at a minimum, to bind CD40, inhibiting interaction of CD40 and CD154 or modulate immune response mediated by CD40.
- The peptides or functional fragment thereof described herein can comprise conservative amino acid substitutions at one or more amino acid residues, e.g., at essential or non-essential amino acid residues but will retain a therapeutically or physiologically relevant activity of a peptide as that term is described herein. A “conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, in a conservative substitution variant, a nonessential amino acid residue in the polypeptide is preferably replaced with another amino acid residue from the same side chain family.
- In some embodiments, peptide can be a variant of wild type peptide. The term “variant” as used herein refers to a peptide or nucleic acid that is “substantially similar” to a wild-type peptide. A molecule is said to be “substantially similar” to another molecule if both molecules have substantially similar structures (i.e., they are at least 50% similar in amino acid sequence as determined by BLASTp alignment set at default parameters) and are substantially similar in at least one therapeutically or physiologically relevant biological activity. A variant differs from the naturally occurring polypeptide or nucleic acid by one or more amino acid or nucleic acid deletions, additions, substitutions or side-chain modifications, yet retains one or more therapeutically relevant, specific functions or desired biological activities of the wild type peptide (e.g., binding CD40).
- Amino acid substitutions include alterations in which an amino acid is replaced with a different naturally-occurring or a non-conventional amino acid residue. Some substitutions can be classified as “conservative,” in which case an amino acid residue contained in a polypeptide is replaced with another naturally occurring amino acid of similar character either in relation to polarity, side chain functionality or size. Substitutions encompassed by variants as described herein can also be “non-conservative,” in which an amino acid residue which is present in a peptide is substituted with an amino acid having different properties (e.g., substituting a charged or hydrophobic amino acid with an uncharged or hydrophilic amino acid), or alternatively, in which a naturally-occurring amino acid is substituted with a non-conventional amino acid.
- “Conservative amino acid substitutions” result from replacing one amino acid with another having similar structural and/or chemical properties, such as the replacement of a leucine with an isoleucine or valine, an aspartate with a glutamate, or a threonine with a serine. Thus, a “conservative substitution” of a particular amino acid sequence refers to substitution of those amino acids that are not critical for polypeptide activity or substitution of amino acids with other amino acids having similar properties (e.g., acidic, basic, positively or negatively charged, polar or non-polar, etc.) such that the substitution of even critical amino acids does not reduce the activity of the peptide, (i.e. the ability of the peptide to penetrate the blood brain barrier (BBB)). Conservative substitution tables providing functionally similar amino acids are well known in the art. For example, the following six groups each contain amino acids that are conservative substitutions for one another: 1) Alanine (A), Serine (S), Threonine (T); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W). (See also Creighton, Proteins, W. H. Freeman and Company (1984), incorporated by reference in its entirety.) In some embodiments, individual substitutions, deletions or additions that alter, add or delete a single amino acid or a small percentage of amino acids can also be considered “conservative substitutions” if the change does not reduce the activity of the peptide. Insertions or deletions are typically in the range of about 1 to 5 amino acids. The choice of conservative amino acids may be selected based on the location of the amino acid to be substituted in the peptide, for example if the amino acid is on the exterior of the peptide and expose to solvents, or on the interior and not exposed to solvents.
- The term “insertions” or “deletions” are typically in the range of about 1 to 5 amino acids. The variation allowed can be experimentally determined by producing the peptide synthetically while systematically making insertions, deletions, or substitutions of nucleotides in the sequence using recombinant DNA techniques.
- The term “substitution” when referring to a peptide, refers to a change in an amino acid for a different entity, for example another amino acid or amino-acid moiety. Substitutions can be conservative or non-conservative substitutions.
- In alternative embodiments, one can select the amino acid which will substitute an existing amino acid based on the location of the existing amino acid, i.e. its exposure to solvents (i.e. if the amino acid is exposed to solvents or is present on the outer surface of the peptide or polypeptide as compared to internally localized amino acids not exposed to solvents). Selection of such conservative amino acid substitutions are well known in the art, for example as disclosed in Dordo et al, J. Mol Biol, 1999, 217, 721-739 and Taylor et al, J. Theor. Biol. 119(1986); 205-218 and S. French and B. Robson, J. Mol. Evol. 19(1983)171. Accordingly, one can select conservative amino acid substitutions suitable for amino acids on the exterior of a protein or peptide (i.e. amino acids exposed to a solvent), for example, but not limited to, the following substitutions can be used: substitution of Y with F, T with S or K, P with A, E with D or Q, N with D or G, R with K, G with N or A, T with S or K, D with N or E, I with L or V, F with Y, S with T or A, R with K, G with N or A, K with R, A with S, K or P.
- In alternative embodiments, one can also select conservative amino acid substitutions encompassed suitable for amino acids on the interior of a protein or peptide, for example one can use suitable conservative substitutions for amino acids is on the interior of a protein or peptide (i.e. the amino acids are not exposed to a solvent), for example but not limited to, one can use the following conservative substitutions: where Y is substituted with F, T with A or S, I with L or V, W with Y, M with L, N with D, G with A, T with A or S, D with N, I with L or V, F with Y or L, S with A or T and A with S, G, T or V. In some embodiments, non-conservative amino acid substitutions are also encompassed within the term of variants. The term “derivative” as used herein refers to peptides which have been chemically modified, for example but not limited to by techniques such as ubiquitination, labeling, pegylation (derivatization with polyethylene glycol), lipidation, glycosylation, or addition of other molecules. A molecule also a “derivative” of another molecule when it contains additional chemical moieties not normally a part of the molecule. Such moieties can improve the molecule's solubility, absorption, biological half-life, etc. The moieties can alternatively decrease the toxicity of the molecule, eliminate or attenuate any undesirable side effect of the molecule, etc. Moieties capable of mediating such effects are disclosed in Remington's Pharmaceutical Sciences, 18th edition, A. R. Gennaro, Ed., MackPubl., Easton, Pa. (1990), incorporated herein, by reference, in its entirety.
- A “derivative” is defined as a molecule having the amino acid sequence of a wild-type peptide (e.g., peptide selected from Table 1) or analog thereof, but additionally having a chemical modification of one or more of its amino acid side groups, alpha-carbon atoms, terminal amino group, or terminal carboxylic acid group for example by ubiquitination, labeling, pegylation (derivatization with polyethylene glycol) or addition of other molecules. A chemical modification includes, but is not limited to, adding chemical moieties, creating new bonds, and removing chemical moieties. Furthermore, one or more side groups, or terminal groups, may be protected by protective groups known to the ordinarily-skilled synthetic chemist. The term “functional” when used in conjunction with “derivative” or “variant” refers to a peptide which possesses a biological activity that is substantially similar to a biological activity of the entity or molecule of which it is a derivative or variant. By “substantially similar” in this context is meant that at least 50% of the relevant or desired biological activity of a corresponding wild-type peptide is retained. In some embodiments, the derivatives retains at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or more, including 100% or even more (i.e., the derivative or variant has improved activity relative to wild-type) of the wild type peptide.
- In some embodiments a peptide useful in the composition and methods of the present disclosure is an analog of a wild type peptide (e.g., selected from Table 1). An “analog” of a molecule such as a peptide refers to a molecule similar in function to either the entire molecule or to a fragment thereof. The term “analog” is also intended to include allelic species and induced variants. Analogs typically differ from wild type peptides at one or a few positions, often by virtue of conservative substitutions. Analogs typically exhibit at least 80 or 90% sequence identity with wild type peptides. Some analogs also include unnatural amino acids or modifications of N or C terminal amino acids. Examples of unnatural amino acids are, for example but not limited to; disubstituted amino acids, N-alkyl amino acids, lactic acid, 4-hydroxyproline, γ-carboxyglutamate, ε-N,N,N-trimethyllysine, ε-N-acetyllysine, O-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, σ-N-methylarginine. Other examples of unnatural amino acids include, but are not limited to the D-amino acids, 2,4-diaminobutyric acid, a-amino isobutyric acid, 2-aminoisobutyric acid, 4-aminobutyric acid, Abu, 2-amino butyric acid, g-Abu, e-Ahx, 6-amino hexanoic acid, Aib, 2-amino isobutyric acid, 3-amino propionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, naphthalene, L-1-naphthalene, citrulline, homocitrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, b-alanine, fluoro-amino acids, designer amino acids such as b-methyl amino acids, Ca-methyl amino acids, Na-methyl amino acids, and amino acid analogs in general. By “covalently bonded” is meant joined either directly or indirectly (e.g., through a linker) by a covalent chemical bond.
- The compositions and methods of the instant disclosure also contemplate a peptidomimetic. As used herein, a “peptidomimetic” is a compound containing non-peptidic structural elements that is capable of mimicking the biological action of a parent peptide. A peptidomimetic may or may not comprise peptide bonds.
- A peptide useful for compositions and methods of the present disclosure should be of a size sufficient to interact with CD40 protein in such a manner as to inhibit interaction of CD40 with CD154. In some embodiments, a peptide of the present disclosure is less than 20 amino acids in length. A peptide can be, for example, 4, 6, 13 or 15 amino acids in length. In one embodiment, the peptide consists of an amino acid selected from the group consisting of SEQ ID NO:1-6 and 8-17. The sequences of such peptides are shown below in Table 1. In some embodiments, the peptide comprises at least a portion that is capable of binding CD40 and inhibiting interaction of CD40 with CD154. In some embodiments, the peptide comprises at least a portion of peptide comprising an amino acid sequence selected from SEQ ID NOs: 1-6 and 8-17. In some embodiments, the peptide of the present disclosure is as short as possible, yet at a minimum, retains the biological activity or functional activity of binding to CD40. In some embodiments, the peptide is a shortest region of contiguous sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, or SEQ ID NO: 17 which retain at a minimum the biological ability or functional activity of binding CD40. Useful peptides can also comprise additional regions of sequence from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15, SEQ ID NO: 16, or SEQ ID NO: 17 which surround the shortest possible contiguous sequence capable of binding CD40. In some embodiments, the peptide or portion thereof bind to CD40 at a site which overlaps the binding site of CD154. In some embodiments, the peptide or portion thereof bind CD40 at a site which is the binding site of CD154. In some embodiments, the peptide or portion thereof bind the CD40 at a site which is not a site of CD154 binding. The peptides of the present disclosure can comprise additional sequences and modifications that do not bind a CD40 protein, so long as the additional sequence do not lower the biological activity of the peptide than that of unmodified peptide, for example, biological activity of binding CD40. Non-limiting examples of additional sequences include sequences coding for B-galactosidase, luciferase, glutathione-s-transferase, thioredoxin, HIS-tags, biotin tags, and fluorescent proteins.
- In some embodiments, the composition comprises at least one peptide or portion thereof or functional fragment thereof is selected from Table 1. In some embodiments, the composition can comprise for example more than one peptides or portion thereof or functional fragment thereof disclosed herein (e.g., a peptide selected from Table 1). In some embodiments, the compositions disclosed herein can comprise at least 2 peptides, at least 3 peptides, at least 4 peptides, at least 5 peptides, at least 6 peptides, at least 7 peptides, at least 8 peptides, at least 9 peptides, at least 10 peptides, at least 11 peptides, at least 12 peptides, at least 13 peptides, at least 14 peptides, at least 15 peptides, or portion thereof (e.g., peptides selected from Table 1). In some embodiments, the composition can comprise all the peptides or portion thereof disclosed herein (e.g., peptides in Table 1).
- The term “biological activity,” as used herein, refers to one having structural, regulatory, or biochemical functions of an unmodified molecule or any function related to or associated with a metabolic or physiological process. The biological activity can include an improved desired activity, or a decreased undesirable activity. For example, an entity demonstrates biological activity when it participates in a molecular interaction with another molecule (e.g., binding CD40), when it has therapeutic value in alleviating a disease condition, when it has prophylactic value or therapeutic value in modulating an immune response, when it has diagnostic and/or prognostic value in determining the presence of a molecule (e.g., cell expressing CD40).
- The biological activity and/or biological effect of a peptide disclosed herein include at least in part binding to a CD40 protein. As such, the peptides or functional fragment thereof disclosed herein are CD40 binding peptides or functional fragment thereof. In such a binding, the peptide associates with CD40 to form a complex. An example of complex formation is association of an antigen with an antibody. The binding of the peptides disclosed herein to CD40 can be covalent binding interactions between proteins include, for example, disulfide bonds, ester bonds, amide bonds and the like or non-covalent bonding interactions between proteins include, for example, hydrophobic interactions, van der Waals interactions, ionic interactions, hydrogen bonding interactions and the like. The binding of peptides disclosed herein with CD40 can be reversible (e.g., non-covalent binding interactions) or non-reversible (e.g., covalent bonds). Moreover, a reversible interaction can be strong or weak, the strength of the interaction being determined by the forces (e.g., ionic charges, hydrogen binding, van der Walls interactions, etc.) exerted by each protein on the other protein in the complex. Factors affecting the strength of an interaction between two molecules are known to those skilled in the art. One useful measure of the strength of binding between two molecules, such as a peptide and a protein, is the dissociation constant (Kd). The term “dissociation constant,” “kinetic off-rate”, “off-rate”, or “koff” as used interchangeably herein, refers to the value indicating the dissociation rate of a ligand (e.g., peptides disclosed herein) from its target protein (e.g., CD40) or separation of the ligand and protein complex (e.g., complex of peptide disclosed herein with a CD40 protein) over time into free ligand and free protein. In some embodiments, peptides of the present disclosure are those that bind to a CD40 protein with a Kd of no more than about 1×10˜6 M, about 1×10˜7 M, or about 1×10˜8 M. In some embodiments of the compositions disclosed herein, a peptide has a Kd of less than about 1×10˜9 M. In one embodiment, a peptide of the present disclosure binds to a CD40 protein with a Kd of less than 100 nM, less than 50 nM, less than 25 nM, less than 10 nM, less than 5 nM, less than 3 nM, less than 2 nM, or less than 1 nM. Methods of measuring and analyzing binding interactions between a peptide and a CD40 protein are known by those of skill in the art e.g., US20180172683A1. Kd can be determined by measurement of the kinetics of complex formation and dissociation, e.g. by the SPR method (Biacore). In one aspect provided herein is a complex comprising a peptide disclosed herein or a portion thereof non-covalently bound to a CD40 protein.
- In some embodiments, peptide of the present disclosure selectively binds with a CD40 protein in solution, as determined using an assay such as an immunosorbent assay, or on the surface of a cell. As used herein, the terms selectively, selective, specific, and the like, indicate the peptide has a greater affinity for a CD40 protein than it does for proteins unrelated to the CD40 protein. More specifically, the terms selectively, selective, specific, and the like indicate that the affinity of the peptide for CD40 is statistically significantly higher than its affinity for a negative control (e.g., an unrelated protein such as albumin) as measured using a standard assay (e.g., ELISA). Suitable techniques for assaying the ability of a peptide to selectively interact with a CD40 protein are known to those skilled in the art. Such assays can be in vitro or in vivo assays. Methods of determining selective binding of peptides with CD40 are described in Examples herein. Other non-limiting assays to determine selective binding include, but are not limited to, an enzyme-linked immunoassay, a competitive enzyme-linked immunoassay, a radioimmunoassay, a fluorescence immunoassay, a chemiluminescent assay, a lateral flow assay, a flow-through assay, an agglutination assay, a particulate-based assay (e.g., using particulates such as, but not limited to, magnetic particles or plastic polymers, such as latex or polystyrene beads), an immunoprecipitation assay, an immunoblot assay (e.g., a western blot), a phosphorescence assay, a flow-through assay, a chromatography assay, a polyacrylamide gel electrophoresis (PAGE)-based assay, a surface plasmon resonance assay, a spectrophotometric assay, a particulate-based assay, an electronic sensory assay and a flow cytometric assay. Methods of performing such assays are well known to those skilled in the art. In one embodiment, an assay can be performed using isolated CD40 protein, of CD40 expressed on a surface of a cell in culture, or it can be performed in a whole animal. Assays can be designed to give qualitative, quantitative or semi-quantitative results, depending on how they are used and the type of result that is desired.
- Human CD40 antigen (CD40) is a peptide of 277 amino acids having a molecular weight of 30,600, and a 19 amino acid secretory signal peptide comprising predominantly hydrophobic amino acids. CD40 is a member of the tumor necrosis factor (TNF)/nerve growth factor (NGF) receptor family, which is defined by the presence of cysteine-rich motifs in the extracellular region.
- As used herein, “CD40” or “CD40 protein” refers to a cell surface glycoprotein that is a member of the tumor necrosis factor receptor family. Other names in the art for CD40 include: TNFRSF5, p50, CDW40, CD40 receptor and Bp50. As used herein, the term “CD40 protein”, generally refers to an CD40 polypeptide that is similar or identical in sequence to a wild-type CD40. In some embodiments, the term “CD40 protein” refers to a CD40 polypeptide having an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100%, identical to that of a wild-type CD40 and that retains the ability, at a minimum, to bind CD154. Accordingly in some embodiments, “CD40” can be full length CD40. In some embodiments, “CD40” can be a functional fragment of a full length CD40, a species homologue and/or functional fragments thereof, an ortholog of CD40 and/or functional fragments thereof. The CD40 protein can be a mammalian CD40 protein. The CD40 polypeptide can also be a functional isoform of the full length CD40 or functional fragment thereof.
- In some embodiments, “CD40” is a wild-type CD40 of human origin, having the following amino acid sequence (SEQ ID NO:18), or a functional fragment thereof.
-
(SEQ ID NO: 18) MVRLPLQCVLWGCLLTAVHPEPPTACREKQYLINSQCCSLCQPGQKLVS DCTEFTETECLPCGESEFLDTWNRETHCHQHKYCDPNLGLRVQQKGTSE TDTICTCEEGWHCTSEACESCVLHRSCSPGFGVKQIATGVSDTICEPCP VGFFSNVSSAFEKCHPWTSCETKDLVVQQAGTNKTDVVCGPQDRLRALV VIPIIFGILFAILLVLVFIKKVAKKPTNKAPHPKQEPQEINFPDDLPGS NTAAPVQETLHGCQPVTQEDGKESRISVQERQ - (See GenBank Accession No. AAH12419.1 (sequence includes signal peptide), which is encoded by nucleic acid sequence GENBANK® Accession no. BC012419.1. The sequences are incorporated herein by reference in their entirety).
- A “functional fragment” or a “portion thereof” as it relates to CD40 refers to fragment of the full length CD40 (e.g. corresponding to SEQ ID NO: 18 of at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, at least 160, at least 170, at least 180, at least 190, at least 200 or more consecutive amino acids of full length wild-type CD40, that has at least about 70%, 80%, 90%, 100% or more than 100% of the function of wild-type CD40 of binding CD154 The functional activity can be tested by one of ordinary skill in the art by the assays described in the examples.
- The polypeptide and coding nucleic acid sequences of CD40 protein of human origin and those of a number of animals are publicly available, e.g., from the NCBI website and are contemplated for use in the methods and compositions herein. Examples include, but are not limited to, Mouse (GenBank Accession No. AAB08705.1), Rat (GenBank Accession No. AAH97949.1), monkey (Genbank Accession No. AWU67715.1), Porcine (GenBank Accession No. AWU67715.1) Bovine (GenBank Accession No. AAI34766.1).
- In some embodiments, the CD40 can be a mammalian homolog of human CD40 or a functional fragment thereof. In some embodiments, the CD40 polypeptide has an amino acid sequence at least 85%, at least 90%, at least 95%, at least 97% or at least 99% identical to the amino acid sequence of SEQ ID NO:18 and is capable of binding its cognate ligand CD154. In some embodiments, the CD40 is a functional fragment or a portion of SEQ ID NO: 18 of at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 110, at least 120, at least 130, at least 140 consecutive amino acids of SEQ ID NO: 18, that has at least about 50%, 60%, 70%, 80%, 90%, 100% or more than 100% of the function of wild type CD40 of binding its ligand CD154. The functional activity can be tested by one of ordinary skill in the art by the assays described above.
- Percent (%) amino acid sequence identity for a given polypeptide sequence relative to a reference sequence is defined as the percentage of identical amino acid residues identified after aligning the two sequences and introducing gaps if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Percent (%) amino acid sequence homology for a given polypeptide sequence relative to a reference sequence is defined as the percentage of identical or strongly similar amino acid residues identified after aligning the two sequences and introducing gaps if necessary, to achieve the maximum percent homology. Non identities of amino acid sequences include conservative substitutions, deletions or additions that do not affect the biological activity of CD40. Strongly similar amino acids can include, for example, conservative substitutions known in the art. Percent identity and/or homology can be calculated using alignment methods known in the art, for instance alignment of the sequences can be conducted using publicly available software software such as BLAST, Align, ClustalW2. Those skilled in the art can determine the appropriate parameters for alignment, but the default parameters for BLAST are specifically contemplated.
- In one embodiment, “CD40 protein” useful in the methods and compositions described herein consists of, consists essentially of, or comprises an amino acid sequence, or is a portion thereof derived from SEQ ID NO: 18, provided that the polypeptide retains at least one biological activity of full length CD40 of SEQ ID NO: 18, the biological activity being selected from at a minimum, binding to CD154. The CD40 described herein can comprise conservative amino acid substitutions at one or more amino acid residues, e.g., at essential or non-essential amino acid residues but will retain a therapeutically or physiologically relevant activity of an inhibitory peptide as that term is described herein. A “conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, in a conservative substitution variant, a nonessential amino acid residue in the polypeptide is preferably replaced with another amino acid residue from the same side chain family.
- In some embodiments, CD40 can be a variant of wild type CD40. The term “variant” as used herein refers to a polypeptide or nucleic acid that is “substantially similar” to a wild-type CD40. A molecule is said to be “substantially similar” to another molecule if both molecules have substantially similar structures (i.e., they are at least 50% similar in amino acid sequence as determined by BLASTp alignment set at default parameters) and are substantially similar in at least one therapeutically or physiologically relevant biological activity. A variant differs from the naturally occurring polypeptide or nucleic acid by one or more amino acid or nucleic acid deletions, additions, substitutions or side-chain modifications, yet retains one or more therapeutically relevant, specific functions or desired biological activities of the naturally occurring molecule (e.g., maintains primitive HSCs in a quiescent state, enhances hematopoietic reconstitution in vivo).
- Amino acid substitutions include alterations in which an amino acid is replaced with a different naturally-occurring or a non-conventional amino acid residue. Some substitutions can be classified as “conservative,” in which case an amino acid residue contained in a polypeptide is replaced with another naturally occurring amino acid of similar character either in relation to polarity, side chain functionality or size. Substitutions encompassed by variants as described herein can also be “non-conservative,” in which an amino acid residue which is present in a peptide is substituted with an amino acid having different properties (e.g., substituting a charged or hydrophobic amino acid with an uncharged or hydrophilic amino acid), or alternatively, in which a naturally-occurring amino acid is substituted with a non-conventional amino acid. Also encompassed within the term “variant,” when used with reference to a polynucleotide or polypeptide, are variations in primary, secondary, or tertiary structure, as compared to a reference polynucleotide or polypeptide, respectively (e.g., as compared to a wild-type polynucleotide or polypeptide). Polynucleotide changes can result in amino acid substitutions, additions, deletions, fusions and truncations in the polypeptide encoded by the reference sequence. Variants can also include insertions, deletions or substitutions of amino acids in the peptide sequence. To be therapeutically useful, such variants will retain a therapeutically or physiologically relevant activity as that term is used herein.
- In some embodiments, CD40 can be an agonist of wild-type CD40, an analog or a derivative thereof. In some embodiments, the agonist of wild-type CD40, an analog or a derivative thereof, retains at least one biological activity of full length CD40 of SEQ ID NO: 18, the biological activity being selected from at a minimum, to bind CD154. In some embodiments, the agonist of wild-type CD40, an analog or a derivative thereof, retains at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100% or more than 100% of the biological activity of full length CD40 of SEQ ID NO: 18.
- The CD40 polypeptide can be recombinant, purified, isolated, naturally occurring or synthetically produced. The term “recombinant” when used in reference to a nucleic acid, protein, cell or a vector indicates that the nucleic acid, protein, vector or cell containing them have been modified by introduction of a heterologous nucleic acid or protein or the alteration of a native nucleic acid or a protein, or that the cell is derived from a cell so modified. The term “heterologous” (meaning ‘derived from a different organism’) refers to the fact that often the transferred protein was initially derived from a different cell type or a different species from the recipient. Typically the protein itself is not transferred, but instead the genetic material coding for the protein (often the complementary DNA or cDNA) is added to the recipient cell. Methods of generating and isolating recombinant polypeptides are known to those skilled in the art and can be performed using routine techniques in the field of recombinant genetics and protein expression. For standard recombinant methods, see Sambrook et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY (1989); Deutscher, Methods in Enzymology 182:83-9(1990); Scopes, Protein Purification: Principles and Practice, Springer-Verlag, NY (1982). The CD40 polypeptide can be immobilized onto a solid surface, e.g., a bead or array plate.
- In some embodiments, the CD40 protein or a portion thereof is expressed on the surface of a cell. Accordingly, the methods and compositions of the present disclosure contemplates a cell expressing CD40 on its surface. The disclosure is not limiting as it relates to the type of cell expressing CD40 on its surface. The cell can be an immune cell (e.g., B-cell, T-cell, neutrophil, monocyte, macrophage, dendritic cell, platelet), non-immune cell (e.g., an endothelial cell, epithelial cell, smooth muscle cell, fibroblast), or a cancer cell (e.g., B lymphoma cell, carcinoma cell, melanoma cell).
- In some embodiments, the CD40 expressing cell is in vitro under cell culture conditions. In other embodiments, the cell expressing CD40 protein is in vivo. CD40 may be detected on the surface of a cell by any one of several means known in the art. For example, an antibody specific for CD40 may be used in a fluorescence-activated cell sorting technique to determine whether cells express CD40 and isolate cells expressing CD40. Other methods of detecting cell surface molecules are also useful in detecting and isolating cell expressing CD40.
- As used herein, “CD154” refers to a cell surface glycoprotein that is a member of the tumor necrosis factor ligand family. CD40 ligand is the cognate ligand for CD40. Other names in the art for CD40 ligand include: CD40L, CD40LG, CD 40 ligand, TNFSF5, TRAP, gp39, HIGM1, IGM, IMD3, and T-BAM. An exemplary protein sequence for human CD40L is NP_000065.1, which is encoded by nucleic acid sequence NM_000074.2.
- A CD40 ligand “protein, polypeptide or peptide”, or CD154 as used herein, refers to a proteinaceous CD40 ligand component that has sufficient biological activity to be biologically effective. Accordingly, “CD154” include full-length CD154 proteins and polypeptides and also CD154 that have been subject to non-native processing or biological modification. Such modifications include truncations, extensions, active domains or fragments, fusion proteins, mutants with substantial or sufficient biological activity, peptidomimetics and the like. Any form of CD154 can be used methods disclosed herein, including those isolated and purified from natural sources. CD154 prepared by recombinant expression can also be used, i.e., those obtained by expressing a CD40 ligand nucleic acid in a recombinant host cell and collecting the expressed CD40 ligand protein. CD40 ligands prepared by automated peptide synthesis are also included.
- CD40 is well known in the art to interact with its cognate ligand CD154. One type of interaction is binding interaction and the said binding or interaction can result in activation of immune response at least, for example, by B-cell development, lymphocyte activation and proliferation; modulation of antigen presenting cells function; regulation of activity of dendritic cells, macrophages and B cells; induction of production of inflammatory cytokines in macrophages and dendritic cells; up-regulating antigen presentation; up-regulating T cell stimulation; and promoting immunoglobulin class switching in B cells. The binding can result, for example, in the inhibition of biological activity mediated by CD40. In one embodiment, peptides of the present disclosure bind to CD40 in such a manner as to inhibit the interaction of CD40 with CD154. Accordingly, in some embodiments, the biological activity of the peptides of the instant disclosure can be inhibiting the interaction of a CD40 protein with a CD154 protein. Therefore the peptides or functional fragment thereof disclosed herein are anti-CD40 peptides or functional fragments thereof. The peptide can interact with the CD40 protein in such a manner that the strength of the interaction between the CD40 protein and a CD154 protein is increased or decreased.
- In some embodiments, the peptides disclosed herein can inhibit the interaction between CD40 and CD154. As used herein, the term “interact” or “interaction”, and the like mean that two molecules come into sufficient physical proximity such that they cause a biological downstream effect e.g., activation of an immune cell. The interaction can be, for example, a binding interaction. The interaction can be covalent binding interactions between proteins include, for example, disulfide bonds, ester bonds, amide bonds and the like or non-covalent bonding interactions between proteins include, for example, hydrophobic interactions, van der Waals interactions, ionic interactions, hydrogen bonding interactions and the like. The interaction can be reversible (e.g., non-covalent binding interactions) or non-reversible (e.g., covalent bonds). Moreover, a reversible interaction can be strong or weak, the strength of the interaction being determined by the forces (e.g., ionic charges, hydrogen binding, van der Walls interactions, etc.) exerted by each protein on the other protein in the complex. Factors affecting the strength of an interaction between two molecules are known to those skilled in the art. One useful measure of the strength of binding between two molecules, such as a peptide and a protein, is the dissociation constant (Kd). KD can be determined by measurement of the kinetics of complex formation and dissociation, e.g. by the SPR method (Biacore). “Inhibit” or “inhibiting,” as used herein, e.g., as in “inhibiting an interaction” between two binding partners such as proteins, refers to a process of lowering or reducing the ability of a first protein (e.g., CD40 protein) and a second protein (e.g., CD154 protein) to bind or associate, or disrupting an interaction between a first protein and a second protein. “Inhibiting an interaction” between two proteins may involve disrupting one or more covalent or non-covalent interactions between the first protein and the second protein. Covalent bonding interactions between proteins include, for example, disulfide bonds, ester bonds, amide bonds and the like. Non-covalent bonding interactions between proteins include, for example, hydrophobic interactions, van der Waals interactions, ionic interactions, hydrogen bonding interactions and the like. As it relates to the present disclosure, inhibiting an interaction binding of one molecule (e.g., CD40) with another molecule (e.g., CD154) or the inhibition of stimulation of one cell (e.g., cell expressing a CD40 protein on its surface) by another cell (e.g., cell expressing CD154 on its surface) can be through steric hindrance, conformational alterations or other biochemical mechanism (e.g., through binding of peptides of instant disclosure to CD40 protein or cell expressing CD40 protein on its surface).
- In some embodiments, the peptides of the present disclosure can inhibit an interaction between a CD40 protein and a CD154 protein by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%), at least 90%>, or at least 95%. In some embodiments, the peptides disclosed herein can completely inhibit binding of CD40 to CD154. Complete inhibition of binding between CD40 and CD154 means that when a CD40 protein is contacted (treated or brought into proximity with) a peptide disclosed herein in the presence of a CD154 protein under conditions that would normally allow the interaction of CD40 and CD154, no such interaction occurs and activation signals are not stimulated in the CD40-expressing cell. Consequently CD40/CD154 mediated modulation of immune response does not occur. Methods to measure strength of binding between two proteins, for example, CD40 and CD154, has been discussed above and are known to those skilled in the art.
- The term “contacting” as used herein, refers to bringing a disclosed agent (e.g., peptides disclosed herein or a portion thereof) and a cell, a target receptor, or other biological entity (e.g., CD40 protein or cell expressing CD40 on its surface) together in such a manner that the compound can affect the activity of the target, either directly; i.e., by interacting with the target itself, or indirectly; i.e., by interacting with another molecule, co-factor, factor, or protein on which the activity of the target is dependent. “Contacting” as used herein, e.g., as in “contacting a CD40 protein” refers to contacting directly or indirectly in vitro, ex vivo, or in vivo (i.e. within a subject as defined herein). In some embodiments, the CD40 protein can be isolated, recombinant, synthesized, or immobilized on a solid surface. In some embodiments, the CD40 protein can be expressed on a surface of cell (e.g. an immune cell or a cancer cell). The cell expressing CD40 can be present in an in vitro cell culture condition or in vivo in a subject. Contacting a CD40 protein can include contacting a cell expressing said CD40 protein. Contacting a CD40 protein can include addition of a peptide or a portion thereof, disclosed herein to a sample comprising a cell expressing a CD40 protein, or administration to a subject. Contacting encompasses administration to a solution, cell, tissue, mammal, subject, patient, or human. Further, contacting a cell includes adding a peptide or portion thereof, disclosed herein to a cell culture.
- In one aspect, provided herein is a method of inhibiting an interaction between a CD40 protein and a CD154 protein comprising contacting the CD40 protein with a peptide disclosed herein.
- In some embodiments, peptides disclosed herein can interact with any site on the CD40 protein. In some embodiments, peptides disclosed herein bind with the CD40 protein at a site that overlaps with the CD154 binding site. In some embodiments, a peptide disclosed herein binds the CD40 protein at the CD154 binding site.
- CD40 is well known to play an important role in activation of immune response. B cells are known to constitutively express the CD40 molecule on the cells membrane (van Kooten C. et al, J. Leukoc. Biol. 2000). Stimulation of CD40 by CD154-expressing activated T cells, results in B cells activation, proliferation and isotype switching. Recent studies have indicated that CD40-CD154 interaction can upregulate costimulatory molecules, activate antigen presenting cells (APCs) and influence T-cell priming and T-cell-mediated effector of CD40 activities include modulating cell survival (e.g., functions as cell survival signal), antibody production, antibody isotype switching, production of cytokines (e.g., IL-2, IL-6, IL-8, IL-12, TNF-α, IL-4, IL-5 and IL-10), metalloproteases (e.g., MMP-I/collagenase and MMP-9/gelatinase B) and establishment of immune memory. CD40 activities further include modulating production of proteins involved in cell-cell contact or adhesion (e.g., E-selectin, VCAM-1 and ICAM-1). This interaction can activate macrophages, natural killer (NK) cells, and endothelial cells. CD40 plays a significant role in immune cell function and signaling, including B-cell and T-cell activation by antigen presenting cells, such as macrophages and dendritic cells. CD40 activation stimulates antibody production, isotype switching, and establishment of memory. CD40 activation stimulates production of cytokines, such as IL-2, IL-6, IL-8, IL-12, TNF-α, IL-4, IL-S and IL-10; and metalloproteases, such as MMP-I/collagenase and MMP-9/gelatinase B. CD40 activation stimulates production of proteins involved in cell-cell contact or adhesion, such as E-selectin, VCAM-1 and ICAM-1. CD40 recognition on target cells provides an activation pathway for NK cell cytotoxic activity. Thus, increasing or decreasing cell survival, antibody production, isotype switching, establishment of memory, production of cytokines, metalloproteases or proteins involved in cell-cell contact or adhesion, NK cell cytotoxic activity, B-cell activation, B-cell proliferation, T-cell activation, T-cell proliferation, macrophage activation, migration of immune cell can all be effected or inhibited by contacting an appropriate cell expressing CD40 with a peptide of the present disclosure (e.g., peptide selected from Table 1 or a pharmaceutical composition disclosed herein
- The term “immune response” encompasses but is not limited to one or both of the following responses: antibody production (e.g., humoral immunity), and induction of cell-mediated immunity (e.g., cellular immunity including helper T cell and/or cytotoxic T cell responses). “Immune response” generally refers to innate and acquired immune responses including, but not limited to, both humoral immune responses (mediated by B lymphocytes) and cellular immune responses (mediated by T lymphocytes). An immune response may be beneficial and lead to immunity against infectious pathogens, or an immune response may be pathogenic and lead to autoimmune or hypersensitivity disease. Immune responses against foreign viruses, bacteria, fungi, parasites typically represent beneficial adaptive immune responses. Immune responses against self-tissues, innocuous foreign objects (e.g., dust mite or pollen allergens, etc.), or tissue transplants represent examples of adverse maladaptive immune responses. The term “modulate,” “modulates” or “modulation” refers to enhancement (e.g., an increase) or inhibition (e.g., a reduction) in the specified activity or suppression of a specified activity. As it relates to the present disclosure, immunosuppression also refers to inhibition of an immune response mediated by CD40 activity or one that is mediated by interaction of CD40 with CD154
- As used herein, “modulation of immune response” can be change in the level of an immune cell (e.g., B-cell, T-cell, antigen presenting cell, activated B-cell, activated T-cell, activated macrophage), a change in level of immunomodulatory molecules (e.g., inflammatory cytokines, chemokines), or a combination thereof. In some embodiments, modulation of immune response can be suppression of immune response or immunosuppression. As used herein, the terms level, number, count and concentration can be used interchangeably. The term “immunomodulatory molecule” as used herein refers to any molecule which is capable of effecting the proliferation or activation of the cells of a subject's immune system. Such molecules include, without limitation, prostaglandin E2 (PGE2), transforming growth factor-0 (TGF-β),
indoleamine 2,3-dioxygenase (IDO), nitric oxide, hepatocyte growth factor (HGF), interleukin 6 (IL-6) and interleukin 10 (IL-10). - Modulation of immune response can mean increase or decrease of in the level of an immune cell. In some embodiments, the peptides of the present disclosure inhibit or decrease the immune response by inhibiting the level of immune cell (e.g., T-cell, B-cell, antigen presenting cell, activated T-cell, activated B-cell, or activated macrophage), inhibiting the level of immunomodulatory molecules, or a combination thereof).
- Accordingly, in some embodiments the biological activity of peptides disclosed herein can be inhibition of CD40 activity such as CD40 mediated immune response (e.g., B-cell activation, B-cell proliferation, T-cell activation, T-cell proliferation, macrophage activation, inhibition of inflammatory cytokine production, or a combination thereof).
- It will be appreciated by those skilled in the art that both a cell culture system and the immune system of a subject comprise basal levels of immune cells and immunomodulatory molecules. The phrases basal level and normal level can be used interchangeably. As used herein, the basal level of a type of immune cell, or a immunomodulatory molecule, refers to the average number of that cell type, or immunomodulatory molecule, present in a population of individuals considered healthy (i.e., free of metabolic, autoimmune, or infectious disease) or the basal level of a type of immune cell, or an immunomodulatory molecule, refers to the average level of that cell type, or immunomodulatory molecule, present in a population of cells that is not-activated. Those skilled in the art are capable of determining if an immune cell, or a population of such cells, is activated. For example, the expression of CD69, CD25 and/or CD154 proteins by a T-cell indicates that the cell has been activated. For example, the expression of MHC-class II, B220 and CD3 proteins by B-cell indicates that the B-cell has been activated. For example, the expression of IL-12, iNOS, Arg-1, or IL-1 proteins by macrophage indicates the macrophage has been activated.
- Methods to measure immune cells are well known in the art including methods based on identifying expression of specific surface marker proteins e.g., by flow cytometry. Level of immune cell can be measure, for example, by measuring proliferation by 3H-Thymidine Uptake, Bromodeoxyuridine Uptake (BrdU), ATP Luminescence, Fluorescent Dye Reduction (carboxyfluorescein succinimidyl ester (CFSE)-like dyes); cytokine measurement, for example, using Multi-Analyte ELISArray Kits, bead-based multiplex assay; measuring surface antigen expression, for example, by flow cytometry; measuring cell cytotoxicity, for example, by Two-Label Flow Cytometry, Calcein AM Dye Release, Luciferase Transduced Targets, or Annexin V. Methods to measure T-cell responses and B-cell responses are well known in the art, for example see Expert Rev. Vaccines 9(6), 595-600 (2010), mBio. 2015 Jul.-Aug.; 6(4).
- The reference level or basal level of a cell or molecule can be a specific amount (e.g., a specific concentration) or it can encompass a range of amounts. Basal levels, or ranges, of immune cells and immunoregulatory molecules are known to those in the art. For example, in a healthy individual, the normal level of CD4+ T-cells present in human blood is 500-1500 cells/ml. Basal levels of cells can also be reported as a percentage of a total cell population.
- Immune cell number and function, for example may be monitored by assays that detect immune cells by an activity such as cytokine production, proliferation, or cytotoxicity. For example, Lymphoproliferation Assay, which assays the ability of T cells to proliferate in response to an antigen can be used as an indicator of the presence of antigen-specific CD4+ helper T cells. Typically, the specimen of purified T cells or PBMCs is mixed with various dilutions of antigen or antigen in the presence of stimulator cells (irradiated autologous or HLA matched antigen-presenting cells). After 72-120 h, [3H] thymidine is added, and DNA synthesis (as a measure of proliferation) is quantified by using a gamma counter to measure the amount of radiolabeled thymidine incorporated into the DNA. A stimulation index can be calculated by dividing the number of cpm for the specimen by the number of counts per minute in a control. As it relates to a treatment of an inflammatory disease, the proliferation assay can be used to compare T-cell responses before and after treatment with peptides of the present disclosure or pharmaceutical compositions disclosed herein. Another example of assay that can be employed for detection of proliferation of immune cells (e.g., T-cell, B-cells) include use of intracellular fluorescent dye, 5,6-carboxyfluorescein diacetate succinimidyl ester (CFSE) in mixed lymphocyte reaction (CFSE-MLR) and determination of proliferating cells using flow cytometry. Another example of an assay that can be employed is detection of secreted cytokines by ELISA and ELISPOT Assays.
- Cytokine secretion by immune cells in a subject suffering from inflammatory disease or in response to a transplant) may be detected by measuring either bulk cytokine production (by an ELISA) or enumerating individual cytokine producing immune cells (by an ELISPOT assay). Generally, in an ELISA assay, PBMC specimens are incubated with antigen (with or without antigen-presenting cells), and after a defined period of time, the supernatant from the culture is harvested and added to microtiter plates coated with antibody for cytokines of interest such as IFN-γ, TNF-α, or IL-2. Antibodies ultimately linked to a detectable label or reporter molecule are added, and the plates are washed and read. In a modification of the assay, cytokine secretions can be measured in samples (e.g., serum or other body fluids) obtained from a subject using ELISA or ELISPOT before and after treatment with the peptides disclosed herein or pharmaceutical compositions disclosed herein. Other useful assays include, measurement of detection of intracellular cytokine assay by flow cytometry, measurement of cytokine mRNA levels by RT-PCR and direct cytotoxicity assays of T-cell (See: Clay et al., 2001). Macrophage activation can be determined, for example, by measuring levels of chemokines such as IL-8/CXCL8, IP-10/CXCL10, MIP-1 alpha/CCL3, MIP-1 beta/CCL4, and RANTES/CCL5, which are released as chemoattractants for neutrophils, immature dendritic cells, natural killer cells, and activated T cells. Levels of pro-inflammatory cytokines are released including IL-1 beta/IL-1F2, IL-6, and TNF-alpha can also be measured by assays well known in the art. Levels of proteolytic enzymes, MMP-1, −2, −7, −9, and −12, which degrade Collagen, Elastin, Fibronectin, and other ECM components can also be measured to determine macrophage activation. Leukocytes are attracted by the macrophage via its release of chemokines including MDC/CCL22, PARC/CCL18, and TARC/CCL17. Levels of activated B-cell can be determined, for example, by measuring antigen specific antibody secretion or detecting activated B-cell specific surface markers such as CD27, CD19, CD20, CD25, CD30, CD69, CD80, CD86, CD135, by assays such as flow cytometry.
- In some embodiments, the peptides disclosed herein can reduce the level of an immune cell to less than about 50%, less than about 45%, less than about 40%, less than about 35%, less than about 30%, less than about 27%, or equal to about 25% of the total immune cell population. Methods of measuring different types of T-cells in the T-cell population are known to those skilled in the art. In some embodiments, binding of a peptide disclosed herein with a CD40 protein decreases the number of immune cells by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%. In another embodiment, binding of a peptide disclosed herein with a CD40 protein decreases the number of immune cells by a factor of at least 10, at least 100, at least 1,000, at least 10,000. In some embodiments, the level of immune cells is reduced so that immune cells comprise no more than about 20%>, about 25%, about 30%>, about 35%, or about 40%> of the total immune cell population.
- The peptides can generally be prepared following techniques known in the art. The peptides can also be prepared using standard genetic engineering techniques known to those skilled in the art. For example, the peptide can be produced enzymatically by inserting nucleic acid encoding the peptide into an expression vector, expressing the DNA, and translating the DNA into the peptide in the presence of the required amino acids. The peptide is then purified using chromatographic or electrophoretic techniques, or by means of a carrier protein which can be fused to, and subsequently cleaved from, the peptide. This can be done by inserting into the expression vector in phase with the peptide encoding sequence, a nucleic acid sequence encoding the carrier protein. The fusion protein-peptide may be isolated using chromatographic, electrophoretic or immunological techniques (such as binding to a resin via an antibody to the carrier protein). The peptide can be cleaved using chemical methodology or enzymatically, as by, for example, using hydrolases.
- Peptides of the disclosure can also be prepared using solution methods, by either stepwise or fragment condensations. An appropriately alpha amino-protected amino acid is coupled to an appropriately alpha carboxyl protected amino acid (such protection may not be required depending on the coupling method chosen) using diimides, symmetrical or unsymmetrical anhydrides, BOP, or other coupling reagents or techniques known to those skilled in the art. These techniques may be either or enzymatic. The alpha amino and/or alpha carboxyl protecting groups are removed and the next suitably protected amino acid or block of amino acids are coupled to extend the growing peptide. Various combinations of protecting groups and of chemical and/or enzymatic techniques and assembly strategies can be used in each synthesis.
- A peptide of the instant disclosure can be synthesized by conventional techniques. For example, the peptides can be synthesized by chemical synthesis using solid phase peptide synthesis. These methods employ either solid or solution phase synthesis methods (see for example, J. M. Stewart, and J. D. Young, Solid Phase Peptide Synthesis, 2nd Ed., Pierce Chemical Co., Rockford 111. (1984) and G. Barany and R. B. Merrifield, The Peptides: Analysis Synthesis, Biology editors E. Gross and J. Meienhofer Vol. 2 Academic Press, New York, 1980, pp. 3-254 for solid phase synthesis techniques; and M Bodansky, Principles of Peptide Synthesis, Springer-Verlag, Berlin 1984, and E. Gross and J. Meienhofer, Eds., The Peptides: Analysis, Synthesis, Biology, suprs,
Vol 1, for classical solution synthesis.) - The peptides can be chemically synthesized by Merrifield-type solid phase peptide synthesis. This method may be routinely performed to yield peptides up to about 60-70 residues in length, and may, in some cases, be utilized to make peptides up to about 100 amino acids long. Larger peptides may also be generated synthetically via fragment condensation or native chemical ligation (Dawson et al., 2000, Ann. Rev. Biochem. 69:923-960). An advantage to the utilization of a synthetic peptide route is the ability to produce large amounts of peptides, even those that rarely occur naturally, with relatively high purities, i.e., purities sufficient for research, diagnostic or therapeutic purposes. Solid phase peptide synthesis is described by Stewart et al. in Solid Phase Peptide Synthesis, 2nd Edition, 1984, Pierce Chemical Company, Rockford, III; and Bodanszky and Bodanszky in The Practice of Peptide Synthesis, 1984, Springer-Verlag, New York. At the outset, a suitably protected amino acid residue is attached through its carboxyl group to a derivatized, insoluble polymeric support, such as cross-linked polystyrene or polyamide resin. “Suitably protected” refers to the presence of protecting groups on both the alpha-amino group of the amino acid, and on any side chain functional groups. Side chain protecting groups are generally stable to the solvents, reagents and reaction conditions used throughout the synthesis, and are removable under conditions which will not affect the final peptide product. Stepwise synthesis of the oligopeptide is carried out by the removal of the N-protecting group from the initial amino acid, and coupling thereto of the carboxyl end of the next amino acid in the sequence of the desired peptide. This amino acid is also suitably protected. The carboxyl of the incoming amino acid can be activated to react with the N-terminus of the support-bound amino acid by formation into a reactive group, such as formation into a carbodiimide, a symmetric acid anhydride, or an “active ester” group, such as hydroxybenzotriazole or pentafluorophenyl esters.
- Appropriate protective groups usable in such syntheses and their abbreviations will be found in the above text, as well as in J. F. W. McOmie, Protective Groups in Organic Chemistry, (Plenum Press, New York, 1973). The common protective groups used herein are t-butyloxycarbonyl (Boc), fluorenylmethoxycarboyl (FMOC), benzyl (Bzl), tosyl (Tos), o-bromo-phenylmethoxycarbonyl (BrCBZ), phenylmethoxycarbonyl (CBZ), 2-chloro-phenylmethoxycarbonyl (2-Cl—CBZ), 4-methoxy-2,3,6-trimethylbenzenesulfonyl (Mtr), trityl (Trt), formyl (CHD), and tertiary butyl (t-Bu). N-terminal acetylation on the deprotected N.sup.α-amino group of peptides synthesized using the Boc strategy can be accomplished with 10% Ac2 O and 5% DIEA in NMP, followed by washing of the peptide resin with NMP and/or CH2 Cl2.
- Examples of solid phase peptide synthesis methods include the BOC method which utilized tert-butyloxcarbonyl as the alpha-amino protecting group, and the FMOC method which utilizes 9-fluorenylmethyloxcarbonyl to protect the alpha-amino of the amino acid residues, both which methods are well-known by those of skill in the art. Incorporation of N- and/or C-blocking groups may also be achieved using protocols conventional to solid phase peptide synthesis methods. For incorporation of C-terminal blocking groups, for example, synthesis of the desired peptide is typically performed using, as solid phase, a supporting resin that has been chemically modified so that cleavage from the resin results in a peptide having the desired C-terminal blocking group. To provide peptides in which the C-terminus bears a primary amino blocking group, for instance, synthesis is performed using a p-methylbenzhydrylamine (MBHA) resin, so that, when peptide synthesis is completed, treatment with hydrofluoric acid releases the desired C-terminally amidated peptide. Similarly, incorporation of an N-methylamine blocking group at the C-terminus is achieved using N-methylaminoethyl-derivatized DVB, resin, which upon HF treatment releases a peptide bearing an N-methylamidated C-terminus. Blockage of the C-terminus by esterification can also be achieved using conventional procedures. This entails use of resin/blocking group combination that permits release of side-chain peptide from the resin, to allow for subsequent reaction with the desired alcohol, to form the ester function. FMOC protecting group, in combination with DVB resin derivatized with methoxyalkoxybenzyl alcohol or equivalent linker, can be used for this purpose, with cleavage from the support being effected by TFA in dicholoromethane. Esterification of the suitably activated carboxyl function, e.g. with DCC, can then proceed by addition of the desired alcohol, followed by de-protection and isolation of the esterified peptide product.
- In one embodiment, the peptides of the present disclosure are manufactured by solid phase peptide synthesis using Fmoc chemistry. In certain embodiments, after synthesis the Fmoc group is deprotected at the N-terminus, the side chain protection group is deprotected, and the peptide is cleaved from the resin. In one embodiment, the resin is a Cl-resin. In one embodiment, the condensation reaction reagent is DIC+HOBT. In one embodiment deprotection is done using Pip. In certain embodiments, the synthesized peptides are purified by RP-HPLC using a solvent of acetonitrile+deionized with TFA as the buffer. In one embodiment, the peptides are purified by gradient elution.
- The peptides of the disclosure may be prepared by standard chemical or biological means of peptide synthesis. Biological methods include, without limitation, expression of a nucleic acid encoding a peptide in a host cell or in an in vitro translation system.
- Included in the disclosure are nucleic acid sequences that encode the peptide of the instant disclosure. In one embodiment, the methods and compositions herein contemplate use of a nucleic acid fragment encoding a peptide disclosed herein (e.g., peptide selected from Table 1). Accordingly, subclones of a nucleic acid sequence encoding a peptide of the disclosure can be produced using conventional molecular genetic manipulation for subcloning gene fragments, such as described by Sambrook et al, Molecular Cloning: A Laboratory Manual, Cold Springs Laboratory, Cold Springs Harbor, N.Y. (2012), and Ausubel et al. (ed.), Current Protocols in Molecular Biology, John Wiley & Sons (New York, N.Y.) (1999 and preceding editions), each of which is hereby incorporated by reference in its entirety. The subclones then are expressed in vitro or in vivo in bacterial cells to yield a smaller protein or polypeptide that can be tested for a particular activity.
- Accordingly, in one aspect provided herein is an isolated nucleic acid fragment encoding a peptide comprising an amino acid sequence selected from group consisting of SEQ ID NOs: 1-6 and 8-17 or a portion thereof. Further, the disclosure encompasses an isolated nucleic acid encoding a peptide having substantial homology to a peptide selected from group consisting of SEQ ID NOs: 1-6 and 8-17. In some embodiments, the isolated nucleic acid fragment encodes a peptide having a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% identical to that of SEQ ID NO: 1. In some embodiments, the isolated nucleic acid fragment encodes a peptide having an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% identical to that of SEQ ID NO: 2. In some embodiments, the nucleic acid fragment encodes a peptide having an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% identical to that of SEQ ID NO:3. In some embodiments, the nucleic acid fragment encodes a peptide having an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% identical to that of SEQ ID NO: 4. In some embodiments, the nucleic acid fragment encodes a peptide having an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% identical to that of SEQ ID NO: 5. In some embodiments, the isolated nucleic acid fragment encodes a peptide having an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% identical to that of SEQ ID NO: 6. In some embodiments, the isolated nucleic acid fragment encodes a peptide having an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% identical to that of SEQ ID NO: 8. In some embodiments, the isolated nucleic acid fragment encodes a peptide having an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% identical to that of SEQ ID NO: 9. In some embodiments, the isolated nucleic acid fragment encodes a peptide having an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% identical to that of SEQ ID NO: 10. In some embodiments, the isolated nucleic acid fragment encodes a peptide having an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% identical to that of SEQ ID NO: 11. In some embodiments, the isolated nucleic acid fragment encodes a peptide having an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% identical to that of SEQ ID NO: 12. In some embodiments, the isolated nucleic acid fragment encodes a peptide having an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% identical to that of SEQ ID NO: 13. In some embodiments, the isolated nucleic acid fragment encodes a peptide having an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% identical to that of SEQ ID NO: 14. In some embodiments, the isolated nucleic acid fragment encodes a peptide having an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% identical to that of SEQ ID NO: 15. In some embodiments, the isolated nucleic acid fragment encodes a peptide having an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% identical to that of SEQ ID NO: 16. In some embodiments, the isolated nucleic acid fragment encodes a peptide having an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% identical to that of SEQ ID NO: 17.
- As used herein, an “isolated nucleic acid” refers to a nucleic acid segment or fragment which has been separated from sequences which flank it in a naturally occurring state, i.e., a DNA fragment which has been removed from the sequences which are normally adjacent to the fragment, i.e., the sequences adjacent to the fragment in a genome in which it naturally occurs. The term also applies to nucleic acids which have been substantially purified from other components which naturally accompany the nucleic acid, i.e., RNA or DNA or proteins, which naturally accompany it in the cell. The term therefore includes, for example, a recombinant DNA which is incorporated into a vector, into an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (i.e., as a cDNA or a genomic or cDNA fragment produced by PCR or restriction enzyme digestion) independent of other sequences. It also includes a recombinant DNA which is part of a hybrid gene encoding additional polypeptide sequence. In the context of the present invention, the following abbreviations for the commonly occurring nucleic acid bases are used, “A” refers to adenosine, “C” refers to cytosine, “G” refers to guanosine, “T” refers to thymidine, and “U” refers to uridine.
- The present disclosure also encompasses recombinant nucleic acid encoding the peptides described herein. A “recombinant” nucleic acid is one that has been created using genetic engineering techniques.
- Biological preparation of a peptide disclosed herein involves expression of a nucleic acid encoding a desired peptide. An expression cassette comprising such a coding sequence may be used to produce a desired peptide for use in the method of the disclosure. In the context of an expression vector, the vector can be readily introduced into a host cell, e.g., mammalian, bacterial, yeast or insect cell by any method in the art. Coding sequences for a desired peptide of the disclosure can be codon optimized based on the codon usage of the intended host cell in order to improve expression efficiency as demonstrated herein. Codon usage patterns can be found in the literature (Nakamura et al, 2000, Nuc Acids Res. 28:292). Representative examples of appropriate hosts include bacterial cells, such as streptococci, staphylococci, E. coli, Streptomyces and Bacillus subtilis cells; fungal cells, such as yeast cells and Aspergillus cells; insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animal cells such as CHO, COS, HeLa, C127, 3T3, BHK, HEK 293 and Bowes melanoma cells; and plant cells.
- Numerous vectors are known in the art including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term “vector” includes an autonomously replicating plasmid or a virus. The term should also be construed to include non-plasmid and non-viral compounds which facilitate transfer of nucleic acid into cells, such as, for example, polylysine compounds, liposomes, and the like. Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno-associated virus vectors, retroviral vectors, and the like. The expression vector can be transferred into a host cell by physical, biological or chemical means, discussed in detail elsewhere herein. To ensure that the peptide obtained from either chemical or biological synthetic techniques is the desired peptide, analysis of the peptide composition can be conducted. Such amino acid composition analysis may be conducted using high resolution mass spectrometry to determine the molecular weight of the peptide.
- Alternatively, or additionally, the amino acid content of the peptide can be confirmed by hydrolyzing the peptide in aqueous acid, and separating, identifying and quantifying the components of the mixture using HPLC, or an amino acid analyzer. Protein sequenators, which sequentially degrade the peptide and identify the amino acids in order, can also be used to determine definitely the sequence of the peptide.
- The isolated nucleic acid can comprise any type of nucleic acid, including, but not limited to DNA and RNA. For example, in one embodiment, the composition comprises an isolated DNA molecule, including for example, an isolated cDNA molecule, encoding a peptide of the disclosure, or functional fragment thereof. In one embodiment, the composition comprises an isolated RNA molecule encoding a peptide of the invention, or a functional fragment thereof. The isolated nucleic acids can be synthesized using any method known in the art.
- The nucleic acid molecules of the present disclosure can be modified to improve stability in serum or in growth medium for cell cultures. Modifications can be added to enhance stability, functionality, and/or specificity and to minimize immunostimulatory properties of the nucleic acid molecule of the disclosure. For example, in order to enhance the stability, the 3′-residues may be stabilized against degradation, e.g., they may be selected such that they consist of purine nucleotides, particularly adenosine or guanosine nucleotides. Alternatively, substitution of pyrimidine nucleotides by modified analogues, e.g., substitution of uridine by 2′-deoxythymidine is tolerated and does not affect function of the molecule.
- In one embodiment of the present disclosure the nucleic acid molecule can contain at least one modified nucleotide analogue. For example, the ends may be stabilized by incorporating modified nucleotide analogues. Non-limiting examples of nucleotide analogues include sugar- and/or backbone-modified ribonucleotides (i.e., include modifications to the phosphate-sugar backbone). For example, the phosphodiester linkages of natural RNA may be modified to include at least one of a nitrogen or sulfur heteroatom. In preferred backbone-modified ribonucleotides the phosphoester group connecting to adjacent ribonucleotides is replaced by a modified group, e.g., of phosphothioate group. In preferred sugar-modified ribonucleotides, the 2′ OH-group is replaced by a group selected from H, OR, R, halo, SH, SR, NH2, NHR, NR2 or ON, wherein R is Ci-C6 alkyl, alkenyl or alkynyl and halo is F, CI, Br or I.
- Other examples of modifications are nucleobase-modified ribonucleotides, i.e., ribonucleotides, containing at least one non-naturally occurring nucleobase instead of a naturally occurring nucleobase. Bases can be modified to block the activity of adenosine deaminase. Exemplary modified nucleobases include, but are not limited to, uridine and/or cytidine modified at the 5-position, e.g., 5-(2-amino)propyl uridine, 5-bromo uridine; adenosine and/or guanosines modified at the 8 position, e.g., 8-bromo guanosine; deaza nucleotides, e.g., 7-deaza-adenosine; O- and N-alkylated nucleotides, e.g., N6-methyl adenosine are suitable. It should be noted that the above modifications may be combined.
- In some instances, the nucleic acid molecule comprises at least one of the following chemical modifications: 2′-H, 2′-O-methyl, or 2′-OH modification of one or more nucleotides. In certain embodiments, a nucleic acid molecule of the invention can have enhanced resistance to nucleases. For increased nuclease resistance, a nucleic acid molecule, can include, for example, 2′-modified ribose units and/or phosphorothioate linkages. For example, the 2′ hydroxyl group (OH) can be modified or replaced with a number of different “oxy” or “deoxy” substituents. For increased nuclease resistance the nucleic acid molecules of the invention can include 2′-O-methyl, 2′-fluorine, 2′-O-methoxyethyl, 2′-O-aminopropyl, 2′-amino, and/or phosphorothioate linkages. Inclusion of locked nucleic acids (LNA), ethylene nucleic acids (ENA), e.g., 2′-4′-ethylene-bridged nucleic acids, and certain nucleobase modifications such as 2-amino-A, 2-thio (e.g., 2-thio-U), G-clamp modifications, can also increase binding affinity to a target.
- In one embodiment, the nucleic acid molecule includes a 2′-modified nucleotide, e.g., a 2′-deoxy, 2′-deoxy-2′-fluoro, 2′-0-methyl, 2′-0-methoxyethyl (2′-0-MOE), 2′-0-aminopropyl (2′-0-AP), 2′-0-dimethylaminoethyl (2′-0-DMAOE), 2′-0-dimethylaminopropyl (2′-O-DMAP), 2′-0-dimethylaminoethyloxyethyl (2′-0-DMAEOE), or 2′-0-N-methylacetamido (2′-0-NMA). In one embodiment, the nucleic acid molecule includes at least one 2′-0-methyl-modified nucleotide, and in some embodiments, all of the nucleotides of the nucleic acid molecule include a 2′-0-methyl modification.
- Nucleic acid fragments discussed herein include otherwise unmodified RNA and DNA as well as RNA and DNA that have been modified, e.g., to improve efficacy, and polymers of nucleoside surrogates. Unmodified RNA refers to a molecule in which the components of the nucleic acid, namely sugars, bases, and phosphate moieties, are the same or essentially the same as that which occur in nature, preferably as occur naturally in the human body. The art has referred to rare or unusual, but naturally occurring, RNAs as modified RNAs, see, e.g., Limbach et al. {Nucleic Acids Res., 1994, 22:2183-2196). Such rare or unusual RNAs, often termed modified RNAs, are typically the result of a post-transcriptional modification and are within the term unmodified RNA as used herein. Modified RNA, as used herein, refers to a molecule in which one or more of the components of the nucleic acid, namely sugars, bases, and phosphate moieties, are different from that which occur in nature, preferably different from that which occurs in the human body. While they are referred to as “modified RNAs” they will of course, because of the modification, include molecules that are not, strictly speaking, RNAs. Nucleoside surrogates are molecules in which the ribophosphate backbone is replaced with a non-ribophosphate construct that allows the bases to be presented in the correct spatial relationship such that hybridization is substantially similar to what is seen with a ribophosphate backbone, e.g., non-charged mimics of the ribophosphate backbone.
- Modifications of the nucleic acid of the disclosure can be present at one or more of, a phosphate group, a sugar group, backbone, N-terminus, C-terminus, or nucleobase.
- Also provided herein is a vector comprising the isolated nucleic acid fragment of the present disclosure inserted. As used herein, a “vector” or “delivery vector” can be a viral or non-viral vector that is used to deliver a nucleic acid to a cell, tissue or subject. Exemplary vectors include, but are not limited to, adeno-associated virus vectors, adenovirus vectors, lentivirus vectors, paramyxovirus vectors, alphavirus vectors and herpes virus vectors.
- A “recombinant” vector or delivery vector refers to a viral or non-viral vector that comprises one or more heterologous nucleotide sequences (i.e., transgenes), e.g., two, three, four, five or more heterologous nucleotide sequences. In an embodiment of the disclosure, the recombinant vectors and delivery vectors of the invention encode a fusion polypeptide of NAg and a cytokine such as IFN-β, but can also include one or more additional heterologous nucleotide sequences, for example, sequences encoding C- or N-terminal modifications and linker moieties. As used herein, the term “viral vector” or “viral delivery vector” can refer to a virus particle that functions as a nucleic acid delivery vehicle, and which comprises the vector genome packaged within a virion. Alternatively, these terms can be used to refer to the vector genome when used as a nucleic acid delivery vehicle in the absence of the virion. The art is replete with suitable vectors that are useful in the present disclosure.
- In brief summary, the expression of natural or synthetic nucleic acids encoding a peptide is typically achieved by operably linking a nucleic acid encoding the peptide or portions thereof to a promoter, and incorporating the construct into an expression vector. The vectors to be used are suitable for replication and, optionally, integration in eukaryotic cells. Typical vectors contain transcription and translation terminators, initiation sequences, and promoters useful for regulation of the expression of the desired nucleic acid sequence. The vectors of the present invention may also be used for nucleic acid immunization and gene therapy, using standard gene delivery protocols. Methods for gene delivery are known in the art. See, e.g., U.S. Pat. Nos. 5,399,346, 5,580,859, 5,589,466, incorporated by reference herein in their entireties. In another embodiment, the disclosure provides a gene therapy vector.
- The isolated nucleic acid of the disclosure can be cloned into a number of types of vectors. For example, the nucleic acid can be cloned into a vector including, but not limited to a plasmid, a phagemid, a phage derivative, an animal virus, and a cosmid. Vectors of particular interest include expression vectors, replication vectors, probe generation vectors, and sequencing vectors. Further, the vector may be provided to a cell in the form of a viral vector. Viral vector technology is well known in the art and is described, for example, in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York), and in other virology and molecular biology manuals. Viruses, which are useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses. In general, a suitable vector contains an origin of replication functional in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers, (e.g., WO 01/96584; WO 01/29058; and U.S. Pat. No. 6,326,193).
- A number of viral based systems have been developed for gene transfer into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. A selected gene can be inserted into a vector and packaged in retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to cells of the subject either in vivo or ex vivo. A number of retroviral systems are known in the art. In some embodiments, adenovirus vectors are used. A number of adenovirus vectors are known in the art. In one embodiment, lentivirus vectors are used.
- For example, vectors derived from retroviruses such as the lentivirus are suitable tools to achieve long-term gene transfer since they allow long-term, stable integration of a transgene and its propagation in daughter cells. Lentiviral vectors have the added advantage over vectors derived from onco-retroviruses such as murine leukemia viruses in that they can transduce non-proliferating cells, such as hepatocytes. They also have the added advantage of low immunogenicity. In one embodiment, the composition includes a vector derived from an adeno-associated virus (AAV). Adeno-associated viral (AAV) vectors have become powerful gene delivery tools for the treatment of various disorders. AAV vectors possess a number of features that render them ideally suited for gene therapy, including a lack of pathogenicity, minimal immunogenicity, and the ability to transduce postmitotic cells in a stable and efficient manner. Expression of a particular gene contained within an AAV vector can be specifically targeted to one or more types of cells by choosing the appropriate combination of AAV serotype, promoter, and delivery method
- In certain embodiments, the vector also includes conventional control elements which are operably linked to a regulatory control sequence in a manner which permits its transcription, translation and/or expression in a cell transfected with the plasmid vector or infected with the virus produced by the disclosure. As used herein, “operably linked” sequences include both expression control sequences that are contiguous with the gene of interest and expression control sequences that act in trans or at a distance to control the gene of interest. Expression control sequences include appropriate transcription initiation, termination, promoter and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation (poly A) signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., Kozak consensus sequence); sequences that enhance protein stability; and when desired, sequences that enhance secretion of the encoded product. A great number of expression control sequences, including promoters which are native, constitutive, inducible and/or tissue-specific, are known in the art and may be utilized. Additional promoter elements, e.g., enhancers, regulate the frequency of transcriptional initiation. Typically, these are located in the region 30-110 bp upstream of the start site, although a number of promoters have recently been shown to contain functional elements downstream of the start site as well. The spacing between promoter elements frequently is flexible, so that promoter function is preserved when elements are inverted or moved relative to one another. In the thymidine kinase (tk) promoter, the spacing between promoter elements can be increased to 50 bp apart before activity begins to decline. Depending on the promoter, it appears that individual elements can function either cooperatively or independently to activate transcription.
- One example of a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strong constitutive promoter sequence capable of driving high levels of expression of any polynucleotide sequence operative ly linked thereto. Another example of a suitable promoter is Elongation Growth Factor-1 (EF-1a). However, other constitutive promoter sequences may also be used, including, but not limited to the simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, an avian leukemia virus promoter, an Epstein-Barr virus immediate early promoter, a Rous sarcoma virus promoter, as well as human gene promoters such as, but not limited to, the actin promoter, the myosin promoter, the hemoglobin promoter, and the creatine kinase promoter. Further, the invention should not be limited to the use of constitutive promoters. Inducible promoters are also contemplated as part of the invention. The use of an inducible promoter provides a molecular switch capable of turning on expression of the polynucleotide sequence which it is operatively linked when such expression is desired, or turning off the expression when expression is not desired. Examples of inducible promoters include, but are not limited to a metallothionine promoter, a glucocorticoid promoter, a progesterone promoter, and a tetracycline promoter.
- Enhancer sequences found on a vector also regulates expression of the gene contained therein. Typically, enhancers are bound with protein factors to enhance the transcription of a gene. Enhancers may be located upstream or downstream of the gene it regulates. Enhancers may also be tissue-specific to enhance transcription in a specific cell or tissue type. In one embodiment, the vector of the present invention comprises one or more enhancers to boost transcription of the gene present within the vector.
- In order to assess the expression of the peptide, the expression vector to be introduced into a cell can also contain either a selectable marker gene or a reporter gene or both to facilitate identification and selection of expressing cells from the population of cells sought to be transfected or infected through viral vectors. In other aspects, the selectable marker may be carried on a separate piece of DNA and used in a co-transfection procedure. Both selectable markers and reporter genes may be flanked with appropriate regulatory sequences to enable expression in the host cells. Useful selectable markers include, for example, antibiotic-resistance genes, such as neo and the like.
- Reporter genes are used for identifying potentially transfected cells and for evaluating the functionality of regulatory sequences. In general, a reporter gene is a gene that is not present in or expressed by the recipient organism or tissue and that encodes a polypeptide whose expression is manifested by some easily detectable property, e.g., enzymatic activity. Expression of the reporter gene is assayed at a suitable time after the DNA has been introduced into the recipient cells. Suitable reporter genes may include genes encoding luciferase, beta-galactosidase, chloramphenicol acetyl transferase, secreted alkaline phosphatase, or the green fluorescent protein gene (e.g., Ui-Tei et al, 2000 FEBS Letters 479: 79-82). Suitable expression systems are well known and may be prepared using known techniques or obtained commercially. In general, the construct with the minimal 5′ flanking region showing the highest level of expression of reporter gene is identified as the promoter. Such promoter regions may be linked to a reporter gene and used to evaluate agents for the ability to modulate promoter-driven transcription.
- Methods of introducing and expressing genes into a cell are known in the art. In the context of an expression vector, the vector can be readily introduced into a host cell, e.g., mammalian, bacterial, yeast, or insect cell by any method in the art. For example, the expression vector can be transferred into a host cell by physical, chemical, or biological means. Physical methods for introducing a polynucleotide into a host cell include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods for producing cells comprising vectors and/or exogenous nucleic acids are well-known in the art. See, for example, Sambrook et al. (2012, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York). A preferred method for the introduction of a polynucleotide into a host cell is calcium phosphate transfection. Biological methods for introducing a polynucleotide of interest into a host cell include the use of DNA and RNA vectors. Viral vectors, and especially retroviral vectors, have become the most widely used method for inserting genes into mammalian, e.g., human cells. Other viral vectors can be derived from lentivirus, poxviruses, herpes simplex virus I, adenoviruses and adeno-associated viruses, and the like. See, for example, U.S. Pat. Nos. 5,350,674 and 5,585,362.
- Chemical means for introducing a polynucleotide into a host cell include colloidal dispersion systems, such as macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (e.g., an artificial membrane vesicle). In the case where a non-viral delivery system is utilized, an exemplary delivery vehicle is a liposome. The use of lipid formulations is contemplated for the introduction of the nucleic acids into a host cell (in vitro, ex vivo or in vivo). In another aspect, the nucleic acid may be associated with a lipid. The nucleic acid associated with a lipid may be encapsulated in the aqueous interior of a liposome, interspersed within the lipid bilayer of a liposome, attached to a liposome via a linking molecule that is associated with both the liposome and the oligonucleotide, entrapped in a liposome, complexed with a liposome, dispersed in a solution containing a lipid, mixed with a lipid, combined with a lipid, contained as a suspension in a lipid, contained or complexed with a micelle, or otherwise associated with a lipid. Lipid, lipid/DNA or lipid/expression vector associated compositions are not limited to any particular structure in solution. For example, they may be present in a bilayer structure, as micelles, or with a “collapsed” structure. They may also simply be interspersed in a solution, possibly forming aggregates that are not uniform in size or shape. Lipids are fatty substances which may be naturally occurring or synthetic lipids. For example, lipids include the fatty droplets that naturally occur in the cytoplasm as well as the class of compounds which contain long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.
- Lipids suitable for use can be obtained from commercial sources. For example, dimyristyl phosphatidylcholine (“DMPC”) can be obtained from Sigma, St. Louis, Mo.; dicetyl phosphate (“DCP”) can be obtained from K & K Laboratories (Plainview, N.Y.); cholesterol (“Choi”) can be obtained from Calbiochem-Behring; dimyristyl phosphatidylglycerol (“DMPG”) and other lipids may be obtained from Avanti Polar Lipids, Inc. (Birmingham, Ala.). Stock solutions of lipids in chloroform or chloroform/methanol can be stored at about −20° C. Chloroform is used as the only solvent since it is more readily evaporated than methanol. “Liposome” is a generic term encompassing a variety of single and multilamellar lipid vehicles formed by the generation of enclosed lipid bilayers or aggregates. Liposomes can be characterized as having vesicular structures with a phospholipid bilayer membrane and an inner aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-rearrangement before the formation of closed structures and entrap water and dissolved solutes between the lipid bilayers (Ghosh et al, 1991 Glycobiology 5: 505-10). However, compositions that have different structures in solution than the normal vesicular structure are also encompassed. For example, the lipids may assume a micellar structure or merely exist as nonuniform aggregates of lipid molecules. Also contemplated are lipofectamine-nucleic acid complexes.
- Regardless of the method used to introduce exogenous nucleic acids into a host cell, in order to confirm the presence of the recombinant DNA sequence in the host cell, a variety of assays may be performed. Such assays include, for example, “molecular biological” assays well known to those of skill in the art, such as Southern and Northern blotting, RT-PCR and PCR; “biochemical” assays, such as detecting the presence or absence of a particular peptide, e.g., by immunological means (ELISAs and Western blots) or by assays described herein to identify agents falling within the scope of the disclosure.
- The present disclosure includes a composition comprising a host cell which comprises a peptide of the disclosure, a nucleic acid encoding a peptide of the disclosure, or a combination thereof. As used herein, the term “host cell” comprises prokaryotic cells and eukaryotic cells. Exemplary prokaryotic host cells include E. coli, Bacillus subtilis, etc. Exemplary eukaryotic cells include yeast cells, insect cells, mammal cells, etc. In one embodiment, the cell is genetically modified to comprise a peptide and/or nucleic acid of the disclosure. In certain embodiments, genetically modified cell is autologous to a subject being treated with the composition of the invention. Alternatively, the cells can be allogeneic, syngeneic, or xenogeneic with respect to the subject. In certain embodiment, the cell is able to secrete or release the expressed peptide of the invention into extracellular space in order to deliver the peptide to one or more other cells.
- The genetically modified cell may be modified in vivo or ex vivo, using techniques standard in the art. Genetic modification of the cell may be carried out using an expression vector or using a naked isolated nucleic acid construct. In one embodiment, the cell is obtained and modified ex vivo, using an isolated nucleic acid encoding a peptide. In one embodiment, the cell is obtained from a subject, genetically modified to express the peptide and/or nucleic acid, and is re-administered to the subject. In certain embodiments, the cell is expanded ex vivo or in vitro to produce a population of cells, wherein at least a portion of the population is administered to a subject in need. In one embodiment, the cell is genetically modified to stably express the peptide. In another embodiment, the cell is genetically modified to transiently express the peptide.
- The present disclosure provides a scaffold or substrate composition comprising a peptide of the disclosure, an isolated nucleic acid of the disclosure, a host cell comprising the peptide or isolated nucleic acid fragment of the disclosure, or a combination thereof. For example, in one embodiment, a peptide of the disclosure, an isolated nucleic acid of the disclosure, a host cell producing the peptide of the disclosure, or a combination thereof is incorporated within a scaffold. In another embodiment, a peptide of the disclosure, an isolated nucleic acid of the disclosure, a host cell producing the peptide of the disclosure, or a combination thereof is applied to the surface of a scaffold. The scaffold of the disclosure can be of any type known in the art. Non-limiting examples of such a scaffold includes a, hydrogel, electrospun scaffold, foam, mesh, sheet, patch, and sponge.
- The compositions and methods of the present disclosure contemplate use of modified peptides, so long as the modified peptide, retains, the biological activity of the wild type unmodified peptide e.g., binding CD40. In some aspects of the disclosure, the peptide is altered. The term “altered polypeptide” refers to a peptide that includes alterations, such as deletions, additions, and substitutions (generally conservative in nature as would be known to a person in the art, such as alanines), to the wild type sequence, as long as the peptide retains the desired biological activity, i.e., binding CD40, or inhibiting interaction of CD40 and CD154, and/or modulating immune responses directed by interaction of CD40 and CD154. These modifications can be deliberate, as through site-directed mutagenesis, or can be accidental, such as through mutations of artificial hosts, such as genetically engineered bacteria, yeast or mammalian cells, that produce the proteins, or errors due to PCR amplification or other recombinant DNA methods.
- In some aspects, a peptide of the instant disclosure is a “modified peptide” comprising non-naturally occurring amino acids. In some aspects, the peptides comprise a combination of naturally occurring and non-naturally occurring amino acids, and in some embodiments, the peptides comprise only non-naturally occurring amino acids.
- “Modified peptide” may include the incorporation of non-natural amino acids into the peptides of the disclosure, including synthetic non-native amino acids, substituted amino acids, or one or more D-amino acids into the peptides (or other components of the composition, with exception for protease recognition sequences) is desirable in certain situations. D-amino acid-containing peptides exhibit increased stability in vitro or in vivo compared to L-amino acid-containing forms. Thus, the construction of peptides incorporating D-amino acids can be particularly useful when greater in vivo or intracellular stability is desired or required. More specifically, D-peptides are resistant to endogenous peptidases and proteases, thereby providing better oral trans-epithelial and transdermal delivery of linked drugs and conjugates, improved bioavailability of membrane-permanent complexes (see below for further discussion), and prolonged intravascular and interstitial lifetimes when such properties are desirable. The use of D-isomer peptides can also enhance transdermal and oral trans-epithelial delivery of linked drugs and other cargo molecules. Additionally, D-peptides cannot be processed efficiently for major histocompatibility complex class Il-restricted presentation to T helper cells, and are therefore less likely to induce humoral immune responses in the whole organism. Peptide conjugates can therefore be constructed using, for example, D-isomer forms of cell penetrating peptide sequences, L-isomer forms of cleavage sites, and D-isomer forms of therapeutic peptides.
- Therefore, in some embodiments the peptides as disclosed comprise L and D amino acids, wherein no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 D-amino acids are included. In certain aspects, the peptides comprise more than 10 D-amino acids, and in certain aspects all the amino acids of the peptides are D-amino acids.
- Also included are peptides which have been modified using ordinary molecular biological techniques so as to improve their resistance to proteolytic degradation or to optimize solubility properties or to render them more suitable as a therapeutic agent. Such variants include those containing residues other than naturally-occurring L-amino acids, e.g., D-amino acids or non-naturally-occurring synthetic amino acids. The peptides disclosed herein may include unnatural amino acids formed by post-translational modification or by introducing unnatural amino acids during translation. A variety of approaches are available for introducing unnatural amino acids during protein translation. By way of example, special tRNAs, such as tRNAs which have suppressor properties, suppressor tRNAs, have been used in the process of site-directed non-native amino acid replacement (SNAAR). In SNAAR, a unique codon is required on the mRNA and the suppressor tRNA, acting to target a non-native amino acid to a unique site during the protein synthesis (described in WO90/05785). However, the suppressor tRNA must not be recognizable by the aminoacyl tRNA synthetases present in the protein translation system. In certain cases, a non-native amino acid can be formed after the tRNA molecule is aminoacylated using chemical reactions which specifically modify the native amino acid and do not significantly alter the functional activity of the aminoacylated tRNA. These reactions are referred to as post-aminoacylation modifications. For example, the epsilon-amino group of the lysine linked to its cognate tRNA (tRNALYs), could be modified with an amine specific photoaffinity label.
- In some embodiments, a peptide of the composition and methods disclosed herein is in a cyclic form. In some embodiments, the cyclic peptide comprises a linkage between amino acids. Cyclic derivatives of the peptides disclosed herein are also part of the present disclosure. Cyclization may allow the peptide to assume a more favorable conformation for association with other molecules. Cyclization may be achieved using techniques known in the art. For example, disulfide bonds may be formed between two appropriately spaced components having free sulfhydryl groups, or an amide bond may be formed between an amino group of one component and a carboxyl group of another component. Cyclization may also be achieved using an azobenzene-containing amino acid as described by Ulysse, L., et al, J. Am. Chem. Soc. 1995, 117, 8466-8467. The components that form the bonds may be side chains of amino acids, non-amino acid components or a combination of the two. In an embodiment of the aspects of the disclosure, cyclic peptides may comprise a beta-turn in the right position. Beta-turns may be introduced into the peptides of the invention by adding the amino acids Pro-Gly at the right position.
- It may be desirable to produce a cyclic peptide which is more flexible than the cyclic peptides containing peptide bond linkages as described above. A more flexible peptide may be prepared by introducing cysteines at the right and left position of the peptide and forming a disulphide bridge between the two cysteines. The two cysteines are arranged so as not to deform the beta-sheet and turn. The peptide is more flexible as a result of the length of the disulfide linkage and the smaller number of hydrogen bonds in the beta-sheet portion. The relative flexibility of a cyclic peptide can be determined by molecular dynamics simulations.
- The peptides of the invention may be converted into pharmaceutical salts by reacting with inorganic acids such as hydrochloric acid, sulfuric acid, hydrobromic acid, phosphoric acid, etc., or organic acids such as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, succinic acid, malic acid, tartaric acid, citric acid, benzoic acid, salicylic acid, benezenesulfonic acid, and toluenesulfonic acids.
- In yet a further aspect, the peptides or fragments or derivatives thereof can be “retro-inverso peptides.” A “retro-inverso peptide” refers to a peptide with a reversal of the direction of the peptide bond on at least one position, i.e., a reversal of the amino- and carboxy-termini with respect to the side chain of the amino acid. Thus, a retro-inverso analogue has reversed termini and reversed direction of peptide bonds while approximately maintaining the topology of the side chains as in the native peptide sequence. The retro-inverso peptide can contain L-amino acids or D-amino acids, or a mixture of L-amino acids and D-amino acids, up to all of the amino acids being the D-isomer. Partial retro-inverso peptide analogues are polypeptides in which only part of the sequence is reversed and replaced with enantiomeric amino acid residues. Since the retro-inverted portion of such an analogue has reversed amino and carboxyl termini, the amino acid residues flanking the retro-inverted portion are replaced by side-chain-analogous a-substituted geminal-diaminomethanes and malonates, respectively. Retro-inverso forms of cell penetrating peptides have been found to work as efficiently in translocating across a membrane as the natural forms. Synthesis of retro-inverso peptide analogues are described in Bonelli, F. et al., Int J Pept Protein Res. 24(6):553-6 (1984); Verdini, A and Viscomi, G. C, J. Chem. Soc. Perkin Trans. 1:697-701 (1985); and U.S. Pat. No. 6,261,569, which are incorporated herein in their entirety by reference. Processes for the solid-phase synthesis of partial retro-inverso peptide analogues have been described (EP 97994-B) which is also incorporated herein in its entirety by reference.
- By Conjugation with Polymers-PEGylation
- In certain embodiments, the peptides of the disclosure comprise an N-terminal and/or C-terminal modifications. Such modification can that in certain instances improve activity. For example, in one embodiment, the peptide of the disclosure comprises a butyric acid at the N-terminus of the peptide. In one embodiment, the peptide is PEGylated at the C-terminus of the peptide. In one embodiment, the carboxy terminus of the peptide of the present disclosure is amidated. The present disclosure encompasses variants of the peptide analogs, including those with terminal modifications, without terminal modifications, or having different terminal modifications.
- For example, in one embodiment, a peptide of the present disclosure can have, for example, a butyric acid at the N-terminus and is PEGylated and/or amidated at the C-terminus. However, the present disclosure also encompasses analogs and derivatives of peptides disclosed herein, including peptides having different terminal modifications or no terminal modifications. In some embodiments, a peptide or compositions disclosed herein can be PEGylated. PEGylation is considered as a gold standard for chemical modifications of peptides. It involves one or more polyethylene glycol (PEG) chains linked to a protein, peptide or non-peptide molecule. PEGylation is the term given to conjugation with monomethoxy poly (ethyleneglycol) or mPEG. PEG polymer, a polyether compound, or a polymer of ethylene oxide is highly soluble in water.
- The peptides of the disclosure may further be conjugated to non-amino acid moieties that are useful in their therapeutic application. In particular, moieties that improve the stability, biological half-life, water solubility, and/or immunologic characteristics of the peptide are useful. A non-limiting example of such a moiety is polyethylene glycol (PEG).
- For maximum benefits, a stable bond is formed between PEG and polypeptide. To prepare an active PEG derivative with a functional group—such as active ester, active carbonate, tresylate or aldehyde suitable for coupling to a given peptide. Preferred positions for PEGylation are the N-terminal amino group of the polypeptide backbone and the ε-amino group in the side chain of the amino acid residue lysine. Being highly labile, mild chemical reaction conditions are used by proteins and peptides for the conjugation at the hydrophobic sites.
- It is a synthetic polymer, made up of styrene and maleic anhydride polymer, with molecular weight of 1.5 kDa, which on conjugation with proteins and peptide increases systemic circulation time and elimination half life of the conjugated peptide to natural long-circulating blood plasma components such as serum albumin or lipoproteins.
- It involves the conjugation of peptides and proteins to the naturally occurring, biodegradable α-(2→8) linked polysialic acid3. Polysialic acid is highly hydrophilic in nature which increases the systemic circulation. It has the advantage of being biodegradable and its catabolic products are not toxic (e.g. NeuNAc). It markedly reduces proteolysis, prolongs the half life in systemic circulation (up to 40 h) and retains the activity of protein and peptide in-vivo. It also reduces immunogenicity and antigenicity. Polysialic acids are linear polymers of N-acetylneuraminic acid (sialic acid) abundantly present on the surface of cells and many proteins. Polysialylation has been tested and are used in therapeutics for the treatment of various diseases. It includes preservation of stability and function, optimal pharmacokinetics and pharmacodynamics, and
- Covalent attachment of biologically active compounds to water-soluble polymers is one method for alteration and control of biodistribution, pharmacokinetics, and often, toxicity for these compounds (Duncan et al, 1984, Adv. Polym. Sci. 57:53-101). Many water-soluble polymers have been used to achieve these effects, such as poly(sialic acid), dextran, poly(N-(2-hydroxypropyl)methacrylamide) (PHPMA), poly(N-vinylpyrrolidone) (PVP), poly(vinyl alcohol) (PVA), poly(ethylene glycol-co-propylene glycol), poly(N-acryloyl morpholine (PAcM), and poly(ethylene glycol) (PEG) (Powell, 1980, Polyethylene glycol. In R. L. Davidson (Ed.) Handbook of Water Soluble Gums and Resins. McGraw-Hill, New York, chapter 18). PEG possess an ideal set of properties: very low toxicity (Pang, 1993, J. Am. Coll. Toxicol. 12: 429-456) excellent solubility in aqueous solution (Powell, supra), low immunogenicity and antigenicity (Dreborg et al, 1990, Crit. Rev. Ther. Drug Carrier Syst. 6: 315-365). PEG-conjugated or “PEGylated” protein therapeutics, containing single or multiple chains of polyethylene glycol on the protein, have been described in the scientific literature (Clark et al., 1996, J. Biol. Chem. 271: 21969-21977; Hershfield, 1997, Biochemistry and immunology of poly(ethylene glycol)-modified adenosine deaminase (PEG-ADA). In J. M. Harris and S. Zalipsky (Eds) Poly(ethylene glycol): Chemistry and Biological Applications. American Chemical Society, Washington, D.C., p 145-154; Olson et al, 1997, Preparation and characterization of poly(ethylene glycosylated human growth hormone antagonist. In J. M. Harris and S. Zalipsky (Eds) Poly(ethylene glycol): Chemistry and Biological Applications. American Chemical Society, Washington, D.C., p 170-181).
- In some aspects of all the embodiments of the disclosure, the peptides or modified peptides further comprise co-translational and post-translational (C-terminal peptide cleavage) modifications, such as, for example, disulfide-bond formation, glycosylation, acetylation, phosphorylation, proteolytic cleavage (e.g., cleavage by furins or metalloproteases), and the like. The main post-translational modifications associated with peptides are: Acetylation, acylation, ADP-ribosylation, Amidation, γ-Carboxylation and β-hydroxylation, Disulfide bond formation, Glycosylation, Phosphorylation, Proteolytic processing, Sulfation, Methylation, Acyl Lipidation. A peptide modification includes phosphorylation (e.g., on a Tyr, Ser or Thr residue), N-terminal acetylation, C-terminal amidation, C-terminal hydrazide, C-terminal methyl ester, fatty acid attachment, sulfonation (tyrosine), N-terminal dansylation, N-terminal succinylation, tripalmitoyl-S-Glyceryl Cysteine (
PAMb 3 Cys-OH) as well as famesylation of a Cys residue. Systematic chemical modification of a peptide can, for example, be performed in the process of peptide optimalization. For example, post-translational modifications that fall within the scope of the present disclosure include signal peptide cleavage, glycosylation, acetylation, isoprenylation, proteolysis, myristoylation, protein folding and proteolytic processing, etc. Some modifications or processing events require introduction of additional biological machinery. For example, processing events, such as signal peptide cleavage and core glycosylation, are examined by adding canine microsomal membranes or Xenopus egg extracts (U.S. Pat. No. 6,103,489) to a standard translation reaction. Modifications (which do not normally alter primary sequence) include in vivo, or in vitro chemical derivatization of polypeptides, e.g., acetylation, or carboxylation. Also included are modifications of glycosylation, e.g., those made by modifying the glycosylation patterns of a polypeptide during its synthesis and processing or in further processing steps; e.g., by exposing the polypeptide to enzymes which affect glycosylation, e.g., mammalian glycosylating or deglycosylating enzymes. Also embraced are sequences which have phosphorylated amino acid residues, e.g., phosphotyrosine, phosphoserine, or phosphothreonine. - The peptides of the disclosure can be conjugated with other molecules, such as peptide tags or proteins, to prepare fusion proteins. This may be accomplished, for example, by the synthesis of N-terminal or C-terminal fusion proteins provided that the resulting fusion protein retains the functionality of the peptide of the invention. The term “fusion protein” as used herein refers to a recombinant protein of two or more proteins. Fusion proteins can be produced, for example, by a nucleic acid sequence encoding one protein is joined to the nucleic acid encoding another protein such that they constitute a single open-reading frame that can be translated in the cells into a single polypeptide harboring all the intended proteins. The order of arrangement of the proteins can vary. Fusion proteins can include an epitope tag or a half-life extender. Epitope tags include biotin, FLAG tag, c-myc, hemaglutinin, His6, digoxigenin, FITC, Cy3, Cy5, green fluorescent protein, V5 epitope tags, GST, β-galactosidase, AU1, AUS, and avidin. Half-life extenders include Fc domain and serum albumin. Accordingly, in some embodiments, the peptides disclosed herein can be conjugated to a peptide tag. Other modifications can be made, for example, Fusion of a peptide to a solubilising protein fusion partner such a maltose-binding protein (MBP), glutathione S-transferase (GST), green fluorescent protein (GFP). In some embodiments, a peptide of the present disclosure further comprises a peptide tag. Addition of short solubility enhancement peptide tags containing 5-10 positively charged amino acids to N- or C-terminus of proteins and peptides. Such peptide tags are small relative to large proteins but are in significant fraction for smaller proteins. Tolbert et al used a betaine moiety, a small molecule, containing a positively charged quaternary ammonium group (MW-100) is introduced as a small solubility enhancement tag onto the N-terminus of polypeptides by chemical ligation or expressed protein ligation. The modified forms of polypeptides are more water soluble. Modification with small ubiquitin related modifier (SUMO) has the ability to enhance protein expression and solubility.
- In some embodiments, a peptide of the present disclosure can be conjugated to a detectable agent. The term “detectable agent” as used herein that is conjugated directly or indirectly to a probe (e.g., peptide) to generate a “labeled” probe. The label may be detectable by itself (e.g. radioisotope labels, fluorescent agent or chemiluminescent agent) or, in the case of an enzymatic label, may catalyze chemical alteration of a substrate compound or composition that is detectable (e.g., avidin-biotin). In some instances, primers can be labeled to detect a PCR product.
- In some embodiments, the peptides of the compositions and methods disclosed herein is modified by linkage to a carrier peptide. The term “conjugated” are used to refer to any method known in the art for functionally connecting moieties (such as detectable agent, carrier peptide), including, without limitation, recombinant fusion, covalent bonding, disulfide bonding, ionic bonding, hydrogen bonding, and electrostatic bonding. The term “carrier peptide” as used herein indicates peptides that transport a specific substance or group of substances (e.g., peptides of the present disclosure) through intracellular compartments or in extracellular fluids (e.g. in the blood) or else across the cell membrane. Exemplary carrier proteins comprise subunit B of cholera toxin, Avidin, BTG protein, Bovine G globulin, Bovine Immunoglobulin G, Bovine Thyroglobulin, Bovine Serum Albumin (BSA), Conalbumin, Edestein, Exoprotein A from Pseudomonas aeruginosa, HC (Hemocyanin from crab Paralithodes camtschatica), Helix Promatia Haemocyanin (HPH), Human Serum Albumin (HSA), KTI (Kunits trypsin inhibitor from soybeans), Keyhole Limpet Heamocyanin (KLH), LPH (Haemocyanin from Limulus polyphemus) Ovalbumin, Pam3Cys-Th, Polylysine, porcine Thyroglobulin (PTG), Purified Protein Derivative (PPD), Rabbit Serum Albumin (RSA), Soybeab Trypsin Inhibitor (STI) (Sunflower Globulin (SFG) and additional molecules identifiable by a skilled person. Additional carriers comprise molecule having immunogenic activities including cytokines such as IL-10, IL12, IL-4 IL-16 and Transforming Growth Factor Beta (TGFP).
- Accordingly, in some embodiments a peptide of the compositions and methods disclosed herein is fused to a carrier peptide. In some embodiments, attachment of the carrier is performed at the C-terminus or N-terminus of the fragment. In an embodiment the fusion protein (i.e., peptides of the present disclosure and a carrier peptide) can be provided as a single polypeptide through recombinant DNA technology and related processes, such as cloning, chimeric constructs, Polymerase Chain Reaction and additional procedures identifiable by a skilled person. In some embodiments, attachment can be performed through chemical linkage of the fragment to the carrier using methods also identifiable by a skilled person.
- Non-limiting examples of such transportable peptides, or vectors, suitable for coupling to the pharmaceutical agent include transferrin, insulin-like growth factors I and II, basic albumin and prolactin. The conjugation can be carried out using bifunctional reagents which are capable of reacting with each of the polypeptides and forming a bridge between the two. The preferred method of conjugation involves polypeptide thiolation, wherein the two polypeptides are treated with a reagent such as N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP) to form a disulfide bridge between the two polypeptides. Other known conjugation agents can be used, so long as they provide linkage of the two polypeptides (i.e. therapeutic polypeptide drug and the transportable peptide) together without denaturing them. Preferably, the linkage can be easily broken once the chimeric polypeptide has entered the region of interest. In some embodiments, the peptides of the present disclosure can be linked to short cell penetrating peptides, which have the ability to cross cell membrane bilayers. Non limiting examples of such peptides include TAT (HIV-1 transactivating transcriptor) SynB3, Tat, transportan.
- Variants of suitable peptides of the invention can also be expressed. Variants may be made by, for example, the deletion, addition, or alteration of amino acids that have either (i) minimal influence on certain properties, secondary structure, and hydropathic nature of the polypeptide or (ii) substantial effect on one or more properties of the peptide mimetics of the invention. Variants may also include, for example, a peptide conjugated to a linker or other sequence for ease of synthesis, purification, identification, or therapeutic use (i.e., delivery) of the peptide. The variants of the peptides according to the present invention may be (i) one in which one or more of the amino acid residues are substituted with a conserved or non-conserved amino acid residue (preferably a conserved amino acid residue) and such substituted amino acid residue may or may not be one encoded by the genetic code, (ii) one in which there are one or more modified amino acid residues, e.g., residues that are modified by the attachment of substituent groups, (iii) one in which the peptide is an alternative splice variant of the peptide of the present invention, (iv) fragments of the peptides and/or (v) one in which the peptide is fused with another peptide, such as a leader or secretory sequence or a sequence which is employed for purification (for example, His-tag) or for detection (for example, Sv5 epitope tag). The fragments include peptides generated via proteolytic cleavage (including multi-site proteolysis) of an original sequence. Variants may be post-translationally, or chemically modified. Such variants are deemed to be within the scope of those skilled in the art from the teaching herein.
- Conjugation with Fatty Acids (Lipidization)
- Delivery to and uptake of peptides is favored by low molecular weight, lack of ionization at physiological pH and lipophilicity. Therefore one possible strategy to improve targeting of the compositions of the present disclosure is to modify the peptides to increase its lipophilicity. Accordingly, in some embodiments, the peptides disclosed herein can comprise a lipophilic molecular group. As used herein the term “lipophilic molecular group” refers to a lipid moiety, such as a fatty acid, glyceride or phospholipid which when coupled to a therapeutic molecule, increases its lipophilicity. The lipophilic molecular group can be attached to the therapeutic molecule through an ester bond. Examples of such modifications include, among others, esterification, or amidation of the hydroxy-, amino-, or carboxylic acid-groups of the polypeptide. Lipophilic molecular groups can comprise lipid moieties such as fatty acid, glyceride or phospholipids.
- Lipidization can involve acylation of the ε-amino group of lysine residues or acylation of N-terminal α-amino groups. The presence of a lipid group in peptides changes their secondary structures, hydrophobicity and self-assembling propensities but retains its ability to bind to target receptors.
- Reversible Aqueous lipidization technology (REAL) This is a new method for developing fatty acids-peptide conjugates which is carried out in aqueous solution and can regenerate the original active peptides in tissues or the blood and thus called REAL Technology. Lipidization can be carried out at cysteine residues following cleavage of intra-disulfide bonds with a reducing agent. This type of lipidization is typically reversible. Methods of REAL are described in, for example, Shen et al. In some embodiments, a modification combining site specific modification and lipidization technique is carried out, e.g., Chang et. al. L Pharm Sci 2002.
- In some embodiments, modification of a peptide disclosed herein is a modification by adding an opposite charged surfactant that binds to peptide to obtain neutral hydrophobicity entity. Solubility of ionic compounds is high due to ease of solvation of the counter ions with ionic detergents. Positively charged peptides and negatively charged surfactants should be employed, since cationic surfactants might have toxic side effects. Replacement of counter ions decreases the aqueous solubility and increases the lipophilicity. Thus, it changes the partition coefficient by orders of magnitude. HIP method is inexpensive and reversible. The process involves replacement of counter ions (e.g. chloride, acetate, nitrate) with an ionic detergent of similar charge. For many proteins, dissolution in organic solvents occurs with retention of native-like structure and maintenance of enzymatic activity without any chemical modification. Thus, ion-pairing is a useful method for increasing the bioavailability of drugs and enhancing permeation of certain drugs. Dai et al., (Int. J. Pharm. 2007; 336: 58-66), Sun et al. (Int. J. Nanomed, 2011) prepared insulin hydrophobic ion paired complex using different anionic surfactants like sodium lauryl sulphate, surfoplex, sodium deoxycholate and sodium oleate respectively in 1:6 ratio.
- In some embodiments, peptides disclosed herein can be modified by attaching one or more amphiphilic oligomers, for example, Nobex Technology. Creation of amphiphilic oligomers is accomplished by binding a lipophilic alkyl unit to a hydrophilic polyethylene glycol (PEG) unit. The resulting oligomer is then covalently bound to a specific site or sites on the target molecule to facilitate the desired changes in properties of the peptide. The two types of bonds are involved in this technology are hydrolyzable bond at the site of attachment, and non-hydrolyzable bond to create a ‘micro-pegylated’ peptide. Methods for this modification are described in, for example, Still J G. et al. Diabetes Metab. Res. Rev. 2002. In some embodiments, the peptide disclosed herein can be modified by contacting with small hydrophobic organic compounds non-covalently. In Emisphere'seligen™ technology. See Singh B et al. Oral Delivery of Therapeutic Macromolecules: A Perspective Using the Eligen™ Technology; 2014.
- Disclosed herein are compositions comprising peptides or portion thereof capable of biding CD40 and methods of their use. In some embodiments, the compositions disclosed herein are formulated for administration via intracerebroventricular, intravenous, intradermal, intraperitoneal, oral, intramuscular, subcutaneous, intranasal, intracranial, intracelial, intracerebellar, intrathecal, transdermal, pulmonary, or topical administration route.
- The compositions provided herein can be prepared in a variety of ways depending on the intended use of the compositions. For example, a composition useful in practicing the technology herein may be a liquid comprising a peptide disclosed herein in solution, in suspension, or both (solution/suspension). The term “solution/suspension” refers to a liquid composition where a first portion of the active agent is present in solution and a second portion of the active agent is present in particulate form, in suspension in a liquid matrix. A liquid composition also includes a gel. The liquid composition may be aqueous or in the form of an ointment, salve, cream, or the like. An aqueous suspension or solution/suspension useful for practicing the methods disclosed herein may contain one or more polymers as suspending agents. Useful polymers include water-soluble polymers such as cellulosic polymers and water-insoluble polymers such as cross-linked carboxyl-containing polymers. An aqueous suspension or solution/suspension of the present disclosure can be viscous or muco-adhesive, or both viscous and muco-adhesive.
- In some embodiments, the pharmaceutical compositions herein comprise a pharmaceutically acceptable carrier. The term “pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly, in humans. The phrase “pharmaceutically acceptable carrier” as used herein means a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent, media, encapsulating material, manufacturing aid (e.g., lubricant, talc magnesium, calcium or zinc stearate, or steric acid), or solvent encapsulating material, involved in maintaining the stability, solubility, or activity of, active agents in the compositions. Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials which can serve as pharmaceutically-acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, methylcellulose, ethyl cellulose, microcrystalline cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) excipients, such as cocoa butter and suppository waxes; (8) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (9) glycols, such as propylene glycol; (10) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol (PEG); (11) esters, such as ethyl oleate and ethyl laurate; (12) agar; (13) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (14) alginic acid; (15) pyrogen-free water; (16) isotonic saline; (17) Ringer's solution; (19) pH buffered solutions; (20) polyesters, polycarbonates and/or polyanhydrides; (21) bulking agents, such as polypeptides and amino acids (22) serum components, such as serum albumin, HDL and LDL; (23) C2-C12 alcohols, such as ethanol; and (24) other non-toxic compatible substances employed in pharmaceutical formulations. Release agents, coating agents, preservatives, and antioxidants can also be present in the formulation. The terms such as “excipient”, “carrier”, “pharmaceutically acceptable carrier” or the like are used interchangeably herein. Examples of suitable pharmaceutical carriers are described in “Remington's Pharmaceutical Sciences” by E. W. Martin, and still others are familiar to skilled artisans.
- The compositions of the present disclosure can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like, including those adapted for the following: (1) parenteral administration, for example, by subcutaneous, intramuscular, intravenous or epidural injection as, for example, a sterile solution or suspension, or sustained-release formulation; (2) topical application, for example, as a cream, ointment, or a controlled-release patch or spray applied to the skin; (3) intravaginally or intrarectally, for example, as a pessary, cream or foam; (4) ocularly; (5) transdermally; (6) transmucosally; or (7) nasally. The pharmaceutical compositions of the invention can be formulated as neutral or salt forms. Pharmaceutically acceptable salts include those formed with free amino groups such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with free carboxyl groups such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc.
- The compositions disclosed herein can comprise a preservative. A “preservative” is a compound which can be added to the diluent to essentially reduce bacterial action in the reconstituted formulation, thus facilitating the production of a multi-use reconstituted formulation, for example. Examples of potential preservatives include octadecyldimethylbenzyl ammonium chloride, hexamethonium chloride, benzalkoniurn chloride (a mixture of alkylbenzyldimethylammonium chlorides in which the alkyl groups are long-chain compounds), and benzethonium chloride. Other types of preservatives include aromatic alcohols such as phenol, butyl and benzyl alcohol, alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol. The most preferred preservative herein is benzyl alcohol. One important purpose of the present formulation is to stabilize these peptide or analogue against spontaneous dimerization. This is preferably accomplished by formulating the peptides at low pH that will prevent disulfide bond formation between two Cys residues. Such acid buffers are preferably biocompatible. Examples include citrate, acetate, 2-(N-morpholino)ethanesulfonic acid (MES), or any similar buffer with a pK of about 5, wherein in the presence of the buffer, the pH of the solution is <7.5 but preferably not below 3.0. A preferred acidic formulation comprises citrate, preferably at about 25 mM. The buffer is preferably supplemented with glycine (GIy) as an excipient and bulking agent. A preferred concentration is about 50 mg/ml GIy. Another advantage of GIy is that it is an accepted excipient for intravenous infusion or injection in humans. Other amino acids or compounds can be used in place of GIy. Examples of desirable acids or combinations are citrate+acetate, acetate and Tris, and the like.
- A pharmaceutical composition of the disclosure is formulated to be compatible with its intended route of administration. Examples of routes of administration include, but are not limited to, parenteral, e.g., intravenous, intradermal, subcutaneous, oral, intranasal (e.g., inhalation), transdermal (e.g., topical), transmucosal, and rectal administration. In a specific embodiment, the composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous, subcutaneous, intramuscular, oral, intranasal, or topical administration to human beings. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. Where necessary, the composition may also include a solubilizing agent and a local anesthetic such as lignocaine to ease pain at the site of the injection.
- If the compositions of the disclosure are to be administered topically, the compositions can be formulated in the form of an ointment, cream, transdermal patch, lotion, gel, shampoo, spray, aerosol, solution, emulsion, or other form well-known to one of skill in the art. See, e.g., Remington's Pharmaceutical Sciences and Introduction to Pharmaceutical Dosage Forms, 19th ed., Mack Pub. Co., Easton, Pa. (1995). For non-sprayable topical dosage forms, viscous to semi-solid or solid forms comprising a carrier or one or more excipients compatible with topical application and having a dynamic viscosity preferably greater than water are typically employed. Suitable formulations include, without limitation, solutions, suspensions, emulsions, creams, ointments, powders, liniments, salves, and the like, which are, if desired, sterilized or mixed with auxiliary agents (e.g., preservatives, stabilizers, wetting agents, buffers, or salts) for influencing various properties, such as, for example, osmotic pressure. Other suitable topical dosage forms include sprayable aerosol preparations wherein the active ingredient, preferably in combination with a solid or liquid inert carrier, is packaged in a mixture with a pressurized volatile (e.g., a gaseous propellant, such as freon) or in a squeeze bottle. Moisturizers or humectants can also be added to pharmaceutical compositions and dosage forms if desired. Examples of such additional ingredients are well known in the art.
- If the compositions disclosed herein are to be formulated for intranasal administration of a composition, the composition can be formulated in an aerosol form, spray, mist or in the form of drops. In particular, prophylactic or therapeutic agents for use according to the present invention can be conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable propellant (e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas). In the case of a pressurized aerosol the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges (composed of, e.g., gelatin) for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.
- If the compositions disclosed herein are to be formulated for oral administration, compositions can be formulated orally in the form of tablets, capsules, cachets, gel caps, solutions, suspensions, and the like. Tablets or capsules can be prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g., pregelatinized maize starch, polyvinylpyrrolidone, or hydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystalline cellulose, or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc, or silica); disintegrants (e.g., potato starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulphate). The tablets may be coated by methods well-known in the art. Liquid preparations for oral administration may take the form of, but not limited to, solutions, syrups or suspensions, or they may be presented as a dry product for constitution with water or other suitable vehicle before use. Such liquid preparations may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives, or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters, ethyl alcohol, or fractionated vegetable oils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid). The preparations may also contain buffer salts, flavoring, coloring, and sweetening agents as appropriate. Preparations for oral administration may be suitably formulated for slow release, controlled release, or sustained release of a prophylactic or therapeutic agent(s).
- The method of the disclosure may comprise pulmonary administration, e.g., by use of an inhaler or nebulizer, of a composition formulated with an aerosolizing agent. See, e.g., U.S. Pat. Nos. 6,019,968, 5,985,320, 5,985,309, 5,934,272, 5,874,064, 5,855,913, 5,290,540, and 4,880,078; and PCT Publication Nos. WO 92/19244, WO 97/32572, WO 97/44013, WO 98/31346, and WO 99/66903, each of which is incorporated herein by reference their entireties. In a specific embodiment, an antibody of the invention, combination therapy, and/or composition of the invention is administered using Alkermes AIR pulmonary drug delivery technology (Alkermes, Inc., Cambridge, Mass.).
- The method of the disclosure may comprise administration of a composition formulated for parenteral administration by injection (e.g., by bolus injection or continuous infusion). Formulations for injection may be presented in unit dosage form (e.g., in ampoules or in multi-dose containers) with an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle (e.g., sterile pyrogen-free water) before use.
- The methods of the disclosure may additionally comprise of administration of compositions formulated as depot preparations. Such long acting formulations may be administered by implantation (e.g., subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the compositions may be formulated with suitable polymeric or hydrophobic materials (e.g., as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives (e.g., as a sparingly soluble salt).
- The methods of the disclosure encompass administration of compositions formulated as neutral or salt forms. Pharmaceutically acceptable salts include those formed with anions such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with cations such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc.
- Generally, the ingredients of compositions are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent. Where the mode of administration is infusion, composition can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the mode of administration is by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.
- In particular, the disclosure also provides that one or more of the peptides or pharmaceutical compositions of the disclosure is packaged in a hermetically sealed container such as an ampoule or sachette indicating the quantity of the agent. In one embodiment, one or more of the peptides, or pharmaceutical compositions of the disclosure is supplied as a dry sterilized lyophilized powder or water free concentrate in a hermetically sealed container and can be reconstituted (e.g., with water or saline) to the appropriate concentration for administration to a subject. Preferably, one or more of the peptides or pharmaceutical compositions of the disclosure is supplied as a dry sterile lyophilized powder in a hermetically sealed container at a unit dosage of at least 5 mg, more preferably at least 10 mg, at least 15 mg, at least 25 mg, at least 35 mg, at least 45 mg, at least 50 mg, at least 75 mg, or at least 100 mg. The lyophilized peptides or pharmaceutical compositions of the invention should be stored at between 2° C. and 8° C. in its original container and the peptides, or pharmaceutical compositions of the disclosure should be administered within 1 week, preferably within 5 days, within 72 hours, within 48 hours, within 24 hours, within 12 hours, within 6 hours, within 5 hours, within 3 hours, or within 1 hour after being reconstituted. In an alternative embodiment, one or more of the peptides or pharmaceutical compositions of the disclosure is supplied in liquid form in a hermetically sealed container indicating the quantity and concentration of the agent. Preferably, the liquid form of the administered composition is supplied in a hermetically sealed container at least 0.25 mg/ml, more preferably at least 0.5 mg/ml, at least 1 mg/ml, at least 2.5 mg/ml, at least 5 mg/ml, at least 8 mg/ml, at least 10 mg/ml, at least 15 mg/ml, at least 25 mg/ml, at least 50 mg/ml, at least 75 mg/ml or at least 100 mg/ml. The liquid form should be stored at between 2° C. and 8° C. in its original container.
- The pharmaceutical compositions can be administered in various ways, depending on the preference for local or systemic treatment, and on the area to be treated. Administration may be done topically (including ophthalmically, vaginally, rectally, intranasally), orally, by inhalation, or parenterally, for example by intravenous drip or intraperitoneal, subcutaneous, subdural, intramuscular or intravenous injection, or via an implantable delivery device. Formulations for topical administration may include, but are not limited to, lotions, ointments, gels, creams, suppositories, drops, liquids, sprays and powders Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable. Compositions for oral administration include powders or granules, suspensions or solutions in water or nonaqueous media, sachets, capsules or tablets. Thickeners, diluents, flavorings, dispersing aids, emulsifiers or binders may be desirable. Formulations for parenteral administration may include, but are not limited to, sterile solutions, which may also contain buffers, diluents and other suitable additives. Formulations for implantable delivery devices may similarly include, but are not limited to, sterile solutions, which may also contain buffers, diluents and other suitable additives.
- In some embodiments, a composition as described above is in a pharmaceutically acceptable medium suitable for administration to a recipient subject. Pharmaceutically acceptable mediums suitable for administration to a subject are known in the art. In some embodiments, compositions disclosed herein can be conveniently provided as sterile liquid preparations, e.g., isotonic aqueous solutions, suspensions, emulsions, dispersions, or viscous compositions, which may be buffered to a selected pH. Liquid preparations are normally easier to prepare than gels, other viscous compositions, and solid compositions. Additionally, liquid compositions are somewhat more convenient to administer, especially by injection (e.g., intravenous injection). Viscous compositions, on the other hand, can be formulated within the appropriate viscosity range to provide longer contact periods with specific tissues. Liquid or viscous compositions can comprise carriers, which can be a solvent or dispersing medium containing, for example, water, saline, phosphate buffered saline, polyol (for example, glycerol, propylene, glycol, liquid polyethylene glycol, and the like) and suitable mixtures thereof.
- Sterile injectable solutions can be prepared by incorporating the compositions disclosed herein in the required amount of the appropriate solvent with various amounts of the other ingredients, as desired. Such compositions may be in admixture with a suitable carrier, diluent, or excipient such as sterile water, physiological saline, glucose, dextrose, or the like. The compositions can also be lyophilized. The compositions can contain auxiliary substances such as wetting, dispersing, or emulsifying agents (e.g., methylcellulose), pH buffering agents, gelling or viscosity enhancing additives, preservatives, flavoring agents, colors, and the like, depending upon the route of administration and the preparation desired. Standard texts, such as “Remington's Pharmaceutical Science”, 17th edition, 1985, incorporated herein by reference, may be consulted to prepare suitable preparations, without undue experimentation.
- Various additives which enhance the stability and sterility of the compositions, including antimicrobial preservatives, antioxidants, chelating agents, and buffers, may be added. Prevention of the action of microorganisms can be ensured by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, and the like. The compositions can be isotonic, i.e., they can have the same osmotic pressure as blood and lacrimal fluid. The desired isotonicity of the compositions herein may be accomplished using sodium chloride, or other pharmaceutically acceptable agents such as dextrose, boric acid, sodium tartrate, propylene glycol or other inorganic or organic solutes. Sodium chloride is preferred particularly for buffers containing sodium ions.
- Parenteral dosage forms of the compositions can also be administered to a subject by various routes, including, but not limited to subcutaneous, intravenous (including bolus injection), intramuscular, and intraarterial. Since administration of parenteral dosage forms typically bypasses the subject's natural defenses against contaminants, parenteral dosage forms are preferably sterile or capable of being sterilized prior to administration to a subject. Examples of parenteral dosage forms include, but are not limited to solutions ready for injection, dry products ready to be dissolved or suspended in a pharmaceutically acceptable vehicle for injection, suspensions ready for injection, controlled-release parenteral dosage forms, and emulsions. Suitable vehicles that can be used to provide parenteral dosage forms of the disclosure are well known to those skilled in the art. Examples include, without limitation: sterile water; water for injection USP; saline solution; glucose solution; aqueous vehicles such as but not limited to sodium chloride injection, Ringer's injection, dextrose Injection, dextrose and sodium chloride injection, and lactated Ringer's injection; water-miscible vehicles such as, but not limited to ethyl alcohol, polyethylene glycol, and propylene glycol; and non-aqueous vehicles such as, but not limited to corn oil, cottonseed oil, peanut oil, sesame oil, ethyl oleate, isopropyl myristate, and benzyl benzoate.
- Compositions provided herein can be packaged in a pressurized aerosol container together with suitable propellants, for example, hydrocarbon propellants like propane, butane, or isobutane with conventional adjuvants. Compositions can also be administered in a non-pressurized form such as in a nebulizer or atomizer. Compositions can also be administered directly to the airways in the form of a dry powder, for example, by use of an inhaler. Suitable powder compositions include, by way of illustration, powdered preparations of a composition comprising a peptide disclosed herein thoroughly intermixed with lactose, or other inert powders acceptable for intrabronchial administration. The powder compositions can be administered via an aerosol dispenser or encased in a breakable capsule which can be inserted by the subject into a device that punctures the capsule and blows the powder out in a steady stream suitable for inhalation. The compositions can include propellants, surfactants, and co-solvents and can be filled into conventional aerosol containers that are closed by a suitable metering valve.
- Aerosols for the delivery to the respiratory tract are known in the art. See for example, Adjei, A. and Garren, J. Pharm. Res., 1: 565-569 (1990); Zanen, P. and Lamm, J.-W. J. Int. J. Pharm., 114: 111-115 (1995); Gonda, I. “Aerosols for delivery of therapeutic and diagnostic agents to the respiratory tract,” in Critical Reviews in Therapeutic Drug Carrier Systems, 6:273-313 (1990); Anderson et al, Am. Rev. Respir. Dis., 140: 1317-1324 (1989)) and have potential for the systemic delivery of peptides and proteins as well (Patton and Platz, Advanced Drug Delivery Reviews, 8: 179-196 (1992)); Timsina et. al, Int. J. Pharm., 101: 1-13 (1995); and Tansey, I. P., Spray Technol. Market, 4:26-29 (1994); French, D. L., Edwards, D. A. and Niven, R. W., Aerosol Sci., 27: 769-783 (1996); Visser, J., Powder Technology 58: 1-10 (1989)); Rudt, S, and R. H. Muller, J. Controlled Release, 22: 263-272 (1992); Tabata, Y, and Y. Ikada, Biomed. Mater. Res., 22: 837-858 (1988); Wall, D. A., Drug Delivery, 2: 10 1-20 1995); Patton, J. and Platz, R, Adv. Drug Del. Rev., 8: 179-196 (1992); Bryon, P., Adv. Drug. Del. Rev., 5: 107-132 (1990); Patton, J. S., et al, Controlled Release, 28: 15 79-85 (1994); Damms, B. and Bains, W., Nature Biotechnology (1996); Niven, R. W., et al., Pharm. Res., 12(9); 1343-1349 (1995); and Kobayashi, S., et al., Pharm. Res., 13(1): 80-83 (1996), contents of all of which are herein incorporated by reference in their entirety.
- The formulations of the compositions disclosed herein further encompass anhydrous pharmaceutical compositions and dosage forms comprising the disclosed compounds as active ingredients, since water can facilitate the degradation of some compounds. For example, the addition of water (e.g., 5%) is widely accepted in the pharmaceutical arts as a means of simulating long-term storage in order to determine characteristics such as shelf life or the stability of formulations over time. See, e.g., Jens T. Carstensen, Drug Stability: Principles & Practice, 379-80 (2nd ed., Marcel Dekker, NY, N.Y.: 1995). Anhydrous pharmaceutical compositions and dosage forms of the disclosure can be prepared using anhydrous or low moisture containing ingredients and low moisture or low humidity conditions. Pharmaceutical compositions and dosage forms that comprise lactose and at least one active ingredient that comprises a primary or secondary amine are preferably anhydrous if substantial contact with moisture and/or humidity during manufacturing, packaging, and/or storage is expected. Anhydrous compositions are preferably packaged using materials known to prevent exposure to water such that they can be included in suitable formulary kits. Examples of suitable packaging include, but are not limited to hermetically sealed foils, plastics, unit dose containers (e.g., vials) with or without desiccants, blister packs, and strip packs.
- In some embodiments of the aspects described herein, the compositions can be administered to a subject by controlled- or delayed-release means. Ideally, the use of an optimally designed controlled-release preparation in medical treatment is characterized by a minimum of drug substance being employed to cure or control the condition in a minimum amount of time. Advantages of controlled-release formulations include: 1) extended activity of the active agent; 2) reduced dosage frequency; 3) increased patient compliance; 4) usage of less total drug; 5) reduction in local or systemic side effects; 6) minimization of drug accumulation; 7) reduction in blood level fluctuations; 8) improvement in efficacy of treatment; 9) reduction of potentiation or loss of drug activity; and 10) improvement in speed of control of diseases or conditions. (Kim, Chemg-ju, Controlled Release Dosage Form Design, 2 (Technomic Publishing, Lancaster, Pa.: 2000)). Controlled-release formulations can be used to control a compound of formula (I)'s onset of action, duration of action, plasma levels within the therapeutic window, and peak blood levels. In particular, controlled- or extended-release dosage forms or formulations can be used to ensure that the maximum effectiveness of a compound of formula (I) is achieved while minimizing potential adverse effects and safety concerns, which can occur both from under-dosing a drug (i.e., going below the minimum therapeutic levels) as well as exceeding the toxicity level for the drug.
- A variety of known controlled- or extended-release dosage forms, formulations, and devices can be adapted for use with the compositions described herein. Examples include, but are not limited to those described in U.S. Pat. Nos. 3,845,770; 3,916,899; 3,536,809; 3,598, 123; 4,008,719; 5674,533; 5,059,595; 5,591,767; 5, 120,548; 5,073,543; 5,639,476; 5,354,556; 5,733,566; and 6,365,185
B 1, each of which is incorporated herein by reference in their entireties. These dosage forms can be used to provide slow or controlled-release of one or more active ingredients using, for example, hydroxypropylmethyl cellulose, other polymer matrices, gels, permeable membranes, osmotic systems (such as OROS). (Alza Corporation, Mountain View, Calif. USA)), multilayer coatings, microparticles, liposomes, or microspheres or a combination thereof to provide the desired release profile in varying proportions. Additionally, ion exchange materials can be used to prepare immobilized, adsorbed salt forms of the disclosed compounds and thus effect controlled delivery of the drug. Examples of specific anion exchangers include, but are not limited to Duolite. A568 and Duolite. AP143 (Rohm&Haas, Spring House, Pa. USA). In some embodiments, compositions described herein can be administered to a subject by sustained release or in pulses. Pulse therapy is not a form of discontinuous administration of the same amount of a composition over time, but comprises administration of the same dose of the composition at a reduced frequency or administration of reduced doses. Sustained release or pulse administrations are particularly preferred when the disorder occurs continuously in the subject, for example where the subject has continuous or chronic symptoms of an inflammatory disease. Each pulse dose can be reduced and the total amount of a peptide disclosed herein can be administered over the course of treatment to the subject is minimized. - The interval between pulses, when necessary, can be determined by one of ordinary skill in the art. Often, the interval between pulses can be calculated by administering another dose of the composition when the composition or the active component of the composition is no longer detectable in the subject prior to delivery of the next pulse. Intervals can also be calculated from the in vivo half-life of the composition. Intervals can be calculated as greater than the in vivo half-life, or 2, 3, 4, 5 and even 10 times greater the composition half-life. Various methods and apparatus for pulsing compositions by infusion or other forms of delivery to the patient are disclosed in U.S. Pat. Nos. 4,747,825; 4,723,958; 4,948,592; 4,965,251 and 5,403,590.
- Formulations suitable for parenteral administration, such as, for example, by intraarticular (in the joints), intravenous, intramuscular, intradermal, intraperitoneal, and subcutaneous routes, and carriers include aqueous isotonic sterile injection solutions, which can contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, preservatives, liposomes, microspheres and emulsions. The compositions described herein include, but are not limited to therapeutic compositions useful for practicing the therapeutic methods described herein. Therapeutic compositions contain a physiologically tolerable carrier together with an active agent as described herein, dissolved or dispersed therein as an active ingredient. In one embodiment, the therapeutic composition is not immunogenic (e.g., allergenic) when administered to a mammal or human patient for therapeutic purposes. As used herein, the terms “pharmaceutically acceptable”, “physiologically tolerable” and grammatical variations thereof, as they refer to compositions, carriers, diluents and reagents, are used interchangeably and represent that the materials are capable of administration to or upon a mammal without the production of undesirable physiological effects such as nausea, dizziness, gastric upset and the like. A pharmaceutically acceptable carrier will not promote the raising of an immune response to an agent with which it is admixed, unless so desired. The preparation of a pharmacological composition that contains active ingredients dissolved or dispersed therein is well understood in the art and need not be limited based on formulation. The active ingredient can be mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredient and in amounts suitable for use in the therapeutic methods described herein. Suitable excipients include, for example, water, saline, dextrose, glycerol, ethanol or the like and combinations thereof. In addition, if desired, the composition can contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents and the like which enhance the effectiveness of the active ingredient. The compositions described herein can include pharmaceutically acceptable salts of the components therein.
- Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the polypeptide) that are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, tartaric, mandelic and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine and the like. Physiologically tolerable carriers are well known in the art. Exemplary liquid carriers are sterile aqueous solutions that contain no materials in addition to the active ingredients and water, or contain a buffer such as sodium phosphate at physiological pH value, physiological saline or both, such as phosphate-buffered saline. Still further, aqueous carriers can contain more than one buffer salt, as well as salts such as sodium and potassium chlorides, dextrose, polyethylene glycol and other solutes. Liquid compositions can also contain liquid phases in addition to and to the exclusion of water. Exemplary of such additional liquid phases are glycerin, vegetable oils such as cottonseed oil, and water-oil emulsions. The amount of an active agent used in the methods described herein that will be effective in the treatment of a particular disorder or condition will depend on the nature of the disorder or condition, and can be determined by standard clinical techniques.
- While any suitable carrier known to those of ordinary skill in the art can be employed in the pharmaceutical compositions provided herein, the type of carrier will vary depending on the mode of administration. Compositions can be formulated for any appropriate manner of administration, including for example, topical, oral, nasal, intravenous, intracranial, intraperitoneal, subcutaneous or intramuscular administration. For parenteral administration, such as subcutaneous injection, the carrier preferably comprises water, saline, alcohol, a fat, a wax or a buffer. For oral administration, any of the above carriers or a solid carrier, such as mannitol, lactose, starch, magnesium stearate, sodium saccharine, talcum, cellulose, glucose, sucrose, and magnesium carbonate, may be employed. Biodegradable microspheres (e.g., polylactate polyglycolate) can also be employed as carriers for the pharmaceutical compositions of this invention. Suitable biodegradable microspheres are disclosed, for example, in U.S. Pat. Nos. 4,897,268 and 5,075,109. Such compositions can also comprise buffers (e.g., neutral buffered saline or phosphate buffered saline), carbohydrates (e.g., glucose, mannose, sucrose or dextrans), mannitol, proteins, polypeptides or amino acids such as glycine, antioxidants, chelating agents such as EDTA or glutathione, adjuvants (e.g., aluminum hydroxide) and/or preservatives. Alternatively, compositions as described herein can be formulated as a lyophilizate. Compounds can also be encapsulated within liposomes using well known technology. The compositions described herein can be administered as part of a sustained release formulation (i.e., a formulation such as a capsule or sponge that effects a slow release of compound following administration). Such formulations can generally be prepared using well known technology and administered by, for example, oral, rectal or subcutaneous implantation, or by implantation at the desired target site. Sustained-release formulations can contain a polypeptide, polynucleotide dispersed in a carrier matrix and/or contained within a reservoir surrounded by a rate controlling membrane. Carriers for use within such formulations are biocompatible, and can also be biodegradable; preferably the formulation provides a relatively constant level of active component release. The amount of active compound contained within a sustained release formulation depends upon the site of implantation, the rate and expected duration of release and the nature of the condition to be treated or prevented.
- In some embodiments, the pharmaceutical composition is contained in an implantable pump. The pump can be designed to deliver to the intended site of action, at the required rate of administration, and in the proper therapeutic dose. One example of such pump includes but is not limited to the commercially available, the Alzet osmotic mini pump to delivering drugs at a controlled rate and dose over extended periods.
- In some embodiments, the peptides of the compositions disclosed herein can be contained in a continuous flow pump. The delivery mechanism of one such pump is based on the expansion of Freon gas at 37° C. that pushes a diaphragm “plunger/pusher” plate. Usually, the pump reservoir is implanted subcutaneously and is connected to a catheter to deliver the therapeutic molecules. In some embodiments, the peptides can be contained in a programmable pump. Programmable pumps include electromechanical pumps of the peristaltic type, powered by batteries. Their built-in electronics can be remotely controlled from an external programming unit. An example is the SynchroMed system (Medtronic Inc.). The infusion can be programmed in various modes: continuous hourly infusions, repeated bolus infusions with a specified delay, multiple doses over a programmed interval, or a single bolus infusion. In some embodiments, the pharmaceutical composition comprising peptides disclosed herein is contained in a syringe, including a blunt tip syringe for injection to the target site. Microspheres can be implanted stereotactically in the target site.
- In certain embodiments, the compositions disclosed herein can be formulated in liposomes, for example to improve stability and promote delivery. As used herein, the term “liposome” refers to a vesicular structure having lipid-containing membranes enclosing an aqueous interior. In cell biology, a vesicular structure is a hollow, lamellar, spherical structure, and provides a small and enclosed compartment, separated from the cytosol by at least one lipid bilayer. Liposomes can have one or more lipid membranes. Oligolamellar large vesicles and multilamellar vesicles have multiple, usually concentric, membrane layers and are typically larger than 100 nm. Liposomes with several nonconcentric membranes, i.e., several smaller vesicles contained within a larger vesicle, are termed multivesicular vesicles. Liposomes can further comprise one or more additional lipids and/or other components such as sterols, e.g., cholesterol. Additional lipids can be included in the liposome compositions for a variety of purposes, such as to prevent lipid oxidation, to stabilize the bilayer, to reduce aggregation during formation or to attach ligands onto the liposome surface. Any of a number of additional lipids and/or other components can be present, including amphipathic, neutral, cationic, anionic lipids, and programmable fusion lipids. Such lipids and/or components can be used alone or in combination. One or more components of the liposome can comprise a ligand, e.g., a targeting ligand.
- Liposome compositions can be prepared by a variety of methods that are known in the art. See e.g., U.S. Pat. No. 6,339,069B1. Niosomes are non-phospholipid based synthetic vesicles that have properties and function like liposomes. Liposomes are vesicular structures with an aqueous core surrounded by a hydrophobic lipid membrane created by extrusion of phospholipids and known in the art to be used for drug delivery purposes. In some embodiments of the compositions and methods described herein, the peptide is encapsulated in a liposome. Liposomes can vary in size from 15 nm to 100μπι and are contemplated to have either a single layer (uni-lamellar), or multiple phospholipid bilayer membranes (mutilamellar structure). In one aspect, the peptide can be encapsulated in a niosome, a non-phospholipid-based synthetic vesicle
- In some embodiments, the compositions disclosed herein are formulated using micelles formed from lipid-associated peptides disclosed herein, e.g., peptides of the present disclosure conjugated to at least one amphiphilic carrier, in which the micelles have an average diameter of less than about 100 nm, preferably. More preferred embodiments provide micelles having an average diameter less than about 50 nm, and even more preferred embodiments provide micelles having an average diameter less than about 100 nm, or even less than about 20 nm. As used herein, “micelles” are a particular type of molecular assembly in which amphipathic molecules are arranged in a spherical structure such that all hydrophobic portions on the molecules are directed inward, leaving the hydrophilic portions in contact with the surrounding aqueous phase. The converse arrangement exists if the environment is hydrophobic.
- In one aspect, the peptides of the present disclosure are encapsulated in a micelle. Micelles are spherical aggregates of amphiphilic molecules dispersing in water with their hydrophilic head groups on the surface of the sphere, and their hydrophobic tails collected inside. An important property of micelles is their ability to increase the solubility and bioavailability of poorly soluble pharmaceuticals. The amphiphilic molecules in micelles are in constant exchange with those in the bulk solution. On the other hand, polymeric micelles, also known as polymersomes, are self-assembled polymer shells composed of block copolymer amphiphiles such as polyethylene glycol-polylactic acid (PEG-PLA) and PEG-polycaprolactone (PEG-PCL). Polymeric micelles differ from nanoparticles that are either more solid or monolithic (nanospheres) or contain an oily or aqueous core and are surrounded by a polymer shell (nanocapsules). However, in practice, polymeric micelles also be referred to as nanoparticle or nanocarriers because of their particle size.
- Accordingly, in some embodiments the peptides of the compositions disclosed herein is encapsulated in a nanoparticle. In some embodiment, the peptide disclosed herein can be encapsulated in a microcapsule or a microsphere, which are free flowing powders consisting of spherical particles of 2 millimeters or less in diameter, usually 500 microns or less in diameter. As used herein, the term “nanoparticle” refers to a particle having a size between 1 and 1000 nm which can be manufactured from artificial or natural macromolecular substances. To such nanoparticles can be bound drugs or other biologically active materials by covalent, ionic or adsorptive linkage, or the latter can be incorporated into the material of the nanoparticles. Nanoparticles may or may not exhibit size-related properties that differ significantly from those observed in fine particles or bulk materials. Nanoparticles provide improved bioavailability by enhancing aqueous solubility, increasing resistance time in the body (increasing half-life for clearance/increasing specificity for its cognate receptors and targeting drug to specific location in the body (its site of action). This results in concomitant reduction in quantity of the drug required and dosage toxicity, enabling the safe delivery of toxic therapeutic drugs and protection of non-target tissues and cells from severe side effects. Non-limiting examples of nanoparticles include solid lipid nanoparticles (comprise lipids that are in solid phase at room temperature and surfactants for emulsification, the mean diameters of which range from 50 nm to 1000 nm for colloid drug delivery applications), liposomes, nanoemulsions (oil-in-water emulsions done on a nano-scale), albumin nanoparticles, and polymeric nanoparticles. Nanoparticles can be surface coated to modulate their stability, solubility, and targeting. A coating that is multivalent or polymeric confers high stability. A non-limiting example includes coating with hydrophilic polymer such as polyethylene glycol or ploysorbate-80.
- Methods of making nanoparticles encompassing therapeutic molecules are known in the art. See for example, U.S. Pat. No. 6,207,195B1, US20080311214A1, US20080311214A1, Tan et al, 2010. Nanoparticles are solid matrix colloidal particles with diameters ranging from 1-100 nm formed using various polymers like degradable starch, dextran, chitosan, microcrystalline cellulose (MCC), hydroxypropyl cellulose (HPC), hydroxypropyl ethylcellulose (HPMC), carbomer, and wax-like starch, gelatin polymers. In these carrier systems, the drug can be loaded via either incorporation with the system or its adsorption on the particulate system. The encapsulating nanoparticle can be, for example, solid-lipid nanoparticles (SLNs), polymeric nanoparticles, or oil-in-water nanoemulsions. Solid-lipid nanoparticles are surfactant-stabilized aqueous colloidal dispersions of lipid nanoparticles that solidify upon cooling. They contain a lipid phase dispersed in an aqueous environment. Polymeric nanoparticles are solid colloidal particles created from polymeric systems. These nanoparticles are made from biocompatible polymers that encapsulate or adsorb drugs for prolonged release. Nanoemulsions are oil-in-water (OAV) or water-in-oil (W/O) formulations made with edible or otherwise pharmaceutically acceptable oils, surface-active agents (surfactants), and water, where the diameter of the inner phase is reduced to nanometer length scale. The versatility of nanoemulsions is based on the different types of oils and surface modifiers that can be used. For instance, oils that are rich in omega-3 polyunsaturated fatty acids (PUFA) can play a very important role in overcoming biological barriers, including the blood brain barrier.
- For effective targeting, the liposomes and nanoparticles encapsulating the peptides disclosed herein can be further linked with and/or coated with other agents. One example of such an agent can be an antibody binding fragment such as Fab, F(ab′)2, Fab′ or a single antibody chain polypeptide which binds to a receptor molecule present on a target cell of interest, (e.g., immune cell expressing CD40 or a neoplastic cell expressing CD40 on its surface). In some embodiments, the nanoparticles encapsulating the peptides of the compositions and methods disclosed herein can be coated with poly(ethylene glycol) or polysorbate 80 or albumin or its functional groups. PEG-containing surfactants, poly(oxy-ethylene)-poly(oxy-propylene) can also be used for coating nanoparticles. Polysorbate 80-coated poly(n-butylcyanoacrilate) nanoparticles have been formulated by emulsion polymerization method to target selectively rivastigmine or tacrine. Nanoparticles and liposomes can also be linked to carrier peptides for examples TAT, to improve their lipophilicity.
- In some embodiments, the peptides of the compositions and methods disclosed herein can be fused to a carrier peptide, for example for transdermal administration. See for example, US20080305989A1, EP2857033A2
- In some embodiments, the compositions of the present disclosure are formulated for intranasal administration. Therefore, in order to enhance the absorption of a therapeutic drug or agent into the olfactory neurons, the drug or agent should be capable of at least partially dissolving in the fluids that are secreted by the mucous membrane that surround the cilia of the olfactory receptor cells of the olfactory epithelium. Therefore the therapeutic peptide can be linked to a carrier that increases its dissolution within nasal secretions. Non-limiting examples of such carriers include GM-1 ganglioside, phosphotidylserine (PS), and emulsifiers such as polysorbate 80. Linkage with lipophilic carriers such as gangliosides or phosphotidylserine can improve the adsorption of the therapeutic drug into the olfactory neurons and through the olfactory epithelium. In some embodiments, the compositions of the present disclosure comprises a ganglioside or a phosphatidylserine (e.g., composition comprising a peptide disclosed herein formulated for intranasal administration).
- In some embodiments, the peptides of the present disclosure can be encapsulated in nanoparticles, liposomes, micelles, microspheres, niosomes, cyclodextrin-inclusion complexes, or nanoemulsions. The nanoparticles can be coated with polymers such as polyethylene glycol-polylactic acid (PEG-PLA) or Chitosan (CS). Methods for preparation of (PEG-PLA) nanoparticles are well known in the art. The chitosan nanoparticles can be complexed with cyclodextrins. In some embodiments, the compositions of the present disclosure further comprise cyclodextrin. Use of cyclodextrin for delivery of peptide compositions is known in the art (US20120302505A1) The term “cyclodextrins” refers to cyclic oligosaccharides, like α-, β- and γ-cyclodextrin and their derivatives, preferably β-cyclodextrin and its derivatives, preferably methylated β-cyclodextrin, with a degree of CH3-substitution between 0.5 and 3.0, more preferably between 1.7 and 2.1. The term “saccharides” refers to disaccharides, like lactose, maltose, saccharose and also refers to polysaccharides, like dextrans, with an average molecular weight between 10,000 and 100,000, preferably 40,000 and 70,000. The term “sugar alcohols” refers to mannitol and sorbitol. In some embodiments, the compositions of the present disclosure further comprises saccharides selected from the group consisting of cyclodextrins, disaccharides, polysaccharides and combinations thereof. Peptides can be encapsulated in carriers, like cyclodextrins inclusion complexes containing a hydrophobic core and a hydrophilic shell which can help improve upon the solubility problems.
- The compositions can be dispensed intranasally as a powdered or liquid nasal spray, nose drops, a gel or ointment, injection or infusion contained in a tube or catheter, by syringe, by pledge, or by submucosal infusion. Also the composition can made viscous using vehicles such as natural gums, methylcellulose and derivatives, acrylic polymers (carbopol) and vinyl polymers (polyvinylpyrrolidone). Many other excipients, known in the pharmaceutical literature, can be added, such as preservatives, surfactants, co-solvents, adhesives, antioxidants, buffers, viscosity enhancing agents, and agents to adjust the pH or the osmolarity.
- Nasal powder compositions can be made by mixing the active agent and the excipient, both possessing the desired particle size. Other methods to make a suitable powder formulation can be selected. Firstly, a solution of the active agent and the cyclodextrin and/or the other saccharide and/or sugar alcohol is made, followed by precipitation, filtration and pulverization. It is also possible to remove the solvent by freeze drying, followed by pulverization of the powder in the desired particle size by using conventional techniques, known from the pharmaceutical literature. The final step is size classification for instance by sieving, to get particles that are less than 100 microns in diameter, preferably between 50 and 100 microns in diameter. Powders can be administered using a nasal insufflator. Powders may also be administered in such a manner that they are placed in a capsule. The capsule is set in an inhalation or insufflation device. A needle is penetrated through the capsule to make pores at the top and the bottom of the capsule and air is sent to blow out the powder particles. Powder formulation can also be administered in a jet-spray of an inert gas or suspended in liquid organic fluids. In some embodiments, the composition for intranasal administration can be adapted for aerosolization and inhalation. The composition can be administered nasally via pressurized aerosol, aqueous pump spray or other standard methods known to those skilled in the art.
- The compositions of present disclosure can be administered in the form of spray in a non-pressurized aerosol device, for example a Pfeiffer pump. In some embodiments, the compositions can be contained in a syringe, catheter, an inhaler, a nebulizer, a nasal spray pump, a nasal irrigation pump, or a nasal lavage pump. Non-limiting example for intranasal administration of liquid formulation can include (a) delivering drops with a drop pipette, (b) rhinyle catheter and squirt tube which involves inserting the tip of a fine catheter or micropipette to the desired area under visual control and squirt the liquid into the desired location, (c) squeeze bottles which involve squeezing a partly air-filled plastic bottle, to deliver the atomized drug from a jet outlet, (d) metered-dose spray pumps which deliver a unit dose of drug per use, (e) single- and duo-dose spray devices which is used for a single administration of a unit dose of drug, (f) nasal pressurized metered-dose inhalers delivers a nasal aerosol preparation, and (g) powered nebulizers and atomizers to administer drug in the form of a mist. Non-limiting examples for intranasal administration of liquid formulations can include nasal powder inhalers (e.g., Rhinocort Turbuhaler®; BiDose™/Prohaler™) from Pfeiffer/Aptar), nasal powder sprayers (e.g., Fit-lizer™ device, Unidose-DP™), and nasal powder insufflators (e.g., Bi-Directional™ nasal delivery, Optinose).
- The compositions of the present disclosure can be provided in a lyophilized form. The composition in a lyophilized form can further comprise a lyoprotectant. A “lyoprotectant” is a molecule which, when combined with a protein or peptide of interest, significantly prevents or reduces chemical and/or physical instability of the protein or peptide upon lyophilization and subsequent storage. Exemplary lyoprotectants include sugars such as sucrose or trehalose; an amino acid such as monosodium glutamate or histidine; a methylamine such as betaine; a lyotropic salt such as magnesium sulfate; a polyol such as trihydric or higher sugar alcohols, e.g., glycerin, erythritol, glycerol, arabitol, xylitol, sorbitol, and mannitol; propylene glycol; polyethylene glycol; pluronics; and combinations thereof. The preferred lyoprotectant is a non-reducing sugar, such as trehalose or sucrose. The lyoprotectant is added to the pre-lyophilized formulation in a “lyoprotecting amount” which means that, following lyophilization of the peptide in the presence of the lyoprotecting amount of the lyoprotectant, the peptide essentially retains its physical and chemical stability and integrity upon lyophilization and storage. The composition in a lyophilized form can comprise a bulking agent. A “bulking agent” is a compound which adds mass to the lyophilized mixture and contributes to the physical structure of the lyophilized cake (e.g., facilitates the production of an essentially uniform lyophilized cake which maintains an open pore structure). Exemplary bulking agents include mannitol, glycine, polyethylene glycol and xorbitol.
- At the desired stage, typically when it is time to administer the peptide to a subject, the lyophilized formulation may be reconstituted with a diluent such that the peptide concentration in the reconstituted formulation is at least 10 mg/mL, for example from about 10 mg/mL to about 1000 mg/mL, more preferably from about 50 mg/mL to about 500 mg/mL, and most preferably from about 100 mg/mL to about 500 mg/mL. Such high peptide concentrations in the reconstituted formulation are considered to be particularly useful where subcutaneous delivery of the reconstituted formulation is intended. However, for other routes of administration, such as intravenous (i.v.) administration, lower concentrations of the peptide in the reconstituted formulation may be desired (for example from about 1-100 mg/mL, or from about 5-50 mg/mL peptide in the reconstituted formulation). In certain embodiments, the peptide concentration in the reconstituted formulation is significantly higher than that in the pre-lyophilized formulation. For example, the peptide concentration in the reconstituted formulation may be about 2-40 times, preferably 3-10 times and most preferably 3-6 times (e.g., at least three fold or at least four fold) that of the pre-lyophilized formulation.
- Reconstitution generally takes place at a temperature of about 25° C. to ensure complete hydration, although other temperatures may be employed as desired. The time required for reconstitution will depend, for example, on the type of diluent, amount of excipient(s) and peptide. The “diluent” of interest is one which is pharmaceutically acceptable (safe and non-toxic for administration to a human) and is useful for diluting a peptide in solution or the preparation of a reconstituted formulation. Exemplary diluents include sterile water, bacteriostatic water for injection (BWFI), a pH buffered solution (e.g. phosphate-buffered saline, PBS), sterile saline solution, Ringer's solution or dextrose solution. The diluent optionally contains a preservative. Exemplary preservatives have been described above, with aromatic alcohols such as benzyl or phenol alcohol being the preferred preservatives. The amount of preservative employed is determined by assessing different preservative concentrations for compatibility with the peptide and preservative efficacy testing. For example, if the preservative is an aromatic alcohol (such as benzyl alcohol), it can be present in an amount from about 0.1-2.0% and preferably from about 0.5-1.5%, but most preferably about 1.0-1.2%. Preferably, the reconstituted formulation has less than 6000 particles per vial which are >10 μm in size.
- In general, the composition, comprising the peptides, may be packaged in sterile form in a sterile container such as a vial, syringe (for injection) etc. In some embodiments, the composition has a shelf life in packaged form of at least 2, 3 or 4 days, up to 1, 2 or 3 weeks or one month, or in some embodiments a shelf life in packaged form of at least one or two months up to 6 months or one to two years, or more, at a temperature of 37° C., or at room temperature. “Shelf life” has its conventional meaning in the art and refers to the time period during which the active agent retains sufficient activity for accurate and effective administration thereof, and the formulation retains sufficient stability (e.g., a single phase solution remains as a single phase solution) for accurate and effective administration thereof at the recited temperature conditions.
- The peptides of the present disclosure selectively bind CD40 protein. In some embodiments, the CD40 protein is expressed on a surface of a cell (e.g., an immune cell, a non-immune cell, a cancer cell). It will be well appreciated by those skilled in the art that the peptides or portion thereof disclosed herein can bind CD40 protein in vitro, (for example when used in a cell culture system), ex vivo, and in vivo. In some embodiments, peptides of the present disclosure can be used to detect CD40 expressing cells. In some embodiments, the peptide is conjugated to a detectable agent, for example, fluorescent agent, chemiluminescent agent, radioistope, or enzyme-substrate agent such as alkaline phosphatase. An assay for detecting a cell expressing CD40 in a sample comprises, for example, a) contacting the sample with a peptide of the present disclosure under condition suitable to allow binding of the peptide with CD40 on the cell surface; b) washing the unbound peptide; and c) detecting the peptide bound to cells. The peptide can be detected, for example, by screening for the detectable agent conjugated to the peptide. The presence of detectable agent indicates presence of CD40 expressing cell. In some embodiments, a secondary detection agent (e.g., an antibody binding the peptide disclosed herein) can be used to detect the peptide bound to CD40 expressing cell. In some embodiments, the peptide useful for such assays can be immobilized onto a solid surface. The solid surface can be in the form of tubes, beads, discs, silicon chips, microplates, polyvinylidene difluoride (PVDF) membrane, nitrocellulose membrane, nylon membrane, other porous membrane, non-porous membrane, e.g., plastic, polymer, perspex, silicon, amongst others, a plurality of polymeric pins, or a plurality of microtitre wells, or any other surface suitable for immobilizing polypeptide or polynucleotides.
- Activated immune cells expressing CD40 are associated with risk of developing inflammatory diseases, autoimmune disease or cancer. Accordingly, in some embodiments, the peptides disclosed herein can be used to identify patients at risk of developing inflammatory diseases, autoimmune disease or a cancer. A sample obtained from candidate patients can be assayed for presence of CD40 expressing cells using, for example, an assay described above. In some embodiments, the sample can be screened for a specific type of cell expressing CD40 alone or in combination with other surface markers specific for that cell type. For example, a sample can be assayed for a population of activated T-cell by using the peptides disclosed herein to detect expression of CD40 alone or using the peptides disclosed herein in combination with molecules detecting activated T-cell specific surface proteins such as CD25, CD38, CD69 and the like. In another embodiment, a sample can be assayed to identify a CD40 expressing cancer cell using the peptides disclosed herein alone or by using the peptides disclosed herein to detect the CD40 in combination with molecules detecting cancer markers on cell surface. An increase in number of a specific type of cell (e.g., activated T-cell) relative to the total cell population in a sample can be indicative of a disease condition (e.g. inflammatory diseases, autoimmune disease or a cancer).
- In some embodiments, the peptides disclosed herein can be used to screen for molecules (e.g., small molecule, proteins, peptides) that bind CD40 and modulate CD40 activity. The assay can be designed, for example, as a competitive binding assay. The assay can be designed, to test the ability of a test molecule to compete with the peptides disclosed herein for binding to a CD40 protein. The peptides disclosed herein useful for such an assay can be one that is fused to a detectable agent. In one embodiment, the method comprises: a) contacting a cell or biological sample with a peptide disclosed herein in the presence or absence of a test agent under conditions which allow for their binding to CD40; b) washing the sample to remove any unbound peptides; and c) screening the sample for presence of detectable agent. In one embodiment, absence of a detectable agent, indicates the test molecule is able to compete with the peptide for binding to a CD40 protein. The assay can be used, for example, to screen for molecules that partially inhibit binding of peptides to the CD40 protein. Methods of competitive binding assay are well known in the art. The test agents can be obtained using any of the numerous approaches in combinatorial-library methods known in the art, including: biological libraries; spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; the “one-bead one-compound” library method; and synthetic library methods using affinity chromatography selection. The biological library approach is limited to peptide libraries, while the other four approaches are applicable to peptide, non-peptide oligomer or small molecule libraries of compounds (Lam et al., 1997, Anticancer Drug Des.12:45). Examples of methods for the synthesis of molecular libraries can be found in the art, for example, in: DeWitt et al, 1993, Proc. Natl. Acad. USA 90:6909; Erb et al, 1994, Proc. Natl. Acad. Sci. USA 91: 11422; Zuckermann et al, 1994, J. Med. Chem. 37:2678; Cho et al, 1993, Science 261: 1303; Carrell et al, 1994, Angew. Chem. Int. Ed. Engl. 33:2059; Carell et al, 1994, Angew. Chem. Int. Ed. Engl. 33:2061; and Gallop et al, 1994, J. Med. Chem. 37: 1233. Libraries of compounds may be presented in solution (e.g., Houghten, 1992, Biotechniques 13:412-421), or on beads (Lam, 1991, Nature 354:82-84), chips (Fodor, 1993, Nature 364:555-556), bacteria (Ladner U.S. Pat. No. 5,223,409), spores (Ladner U.S. Pat. No. '409), plasmids (Cull et al, 1992, Proc. Natl. Acad. Sci. USA 89: 1865-1869) or on phage (Scott and Smith, 1990, Science 249:386-390; Devlin, 1990, Science 249:404-406; Cwirla et al, 1990, Proc. Natl. Acad. Sci. USA 87:6378-6382; Felici, 1991, J. Mol. Biol. 222:301-310; and Ladner supra).
- In situations where “high-throughput” modalities are preferred, it is typical that new chemical entities with useful properties are generated by identifying a chemical compound (called a “lead compound”) with some desirable property or activity, creating variants of the lead compound, and evaluating the property and activity of those variant compounds. The current trend is to shorten the time scale for all aspects of drug discovery. In one embodiment, high throughput screening methods involve providing a library containing a large number of compounds (candidate compounds) potentially having the desired activity. Such “combinatorial chemical libraries” are then screened in one or more assays, as described herein, to identify those library members (particular chemical species or subclasses) that display a desired characteristic activity. The compounds thus identified can serve as conventional “lead compounds” or can themselves be used as potential or actual therapeutics. Assays disclosed herein are meant as examples of useful assays and it is understood that other assays or modifications of assays described above using the peptides disclosed herein can be employed. Suitable assay techniques are known to those skilled in the art and are disclosed for example in Molecular Cloning: A Laboratory Manual, Sambrook, J., Fritsch, E. F., and Maniatis, T, Cold Spring Harbor Laboratory Press; 2nd Edition (December 1989). All referenced cited herein are incorporated herein in their entirety.
- The term “immunosuppression” as used herein relates to an inhibition in the activation and/or maintenance of an immune response (for example, immune response directed towards a transplant). As used herein, the term “immune response” refers to the alteration in the reactivity of an organism's immune system upon exposure to an antigen. The term “immune response” encompasses but is not limited to one or both of the following responses: antibody production (e.g., humoral immunity), and induction of cell-mediated immunity (e.g., cellular immunity including helper T cell and/or cytotoxic T cell responses). “Immune response” generally refers to innate and acquired immune responses including, but not limited to, both humoral immune responses (mediated by B lymphocytes) and cellular immune responses (mediated by T lymphocytes). An immune response may be beneficial and lead to immunity against infectious pathogens, or an immune response may be pathogenic and lead to autoimmune or hypersensitivity disease. Immune responses against foreign viruses, bacteria, fungi, parasites typically represent beneficial adaptive immune responses. Immune responses against self tissues, innocuous foreign objects (e.g., dust mite or pollen allergens, etc.), or tissue transplants represent examples of adverse maladaptive immune responses. The term “modulate,” “modulates” or “modulation” refers to enhancement (e.g., an increase) or inhibition (e.g., a reduction) in the specified activity or suppression thereof. As it relates to the present disclosure, immunosuppression also refers to inhibition of an immune response mediated by CD40 activity or one that is mediated by interaction of CD40 with CD154.
- CD40 is well known to play an important role in activation of immune response. B cells are known to constitutively express the CD40 molecule on the cells membrane (van Kooten C. et al, J. Leukoc. Biol. 2000). Stimulation of CD40 by CD154-expressing activated T cells, results in B cells activation, proliferation and isotype switching. Recent studies have indicated that CD40-CD154 interaction can upregulate costimulatory molecules, activate APCs, and influence T-cell priming and T-cell-mediated effector CD40 activities include modulating cell survival (e.g., functions as cell survival signal), antibody production, antibody isotype switching, production of cytokines (e.g., IL-2, IL-6, IL-8, IL-12, TNF-α, IL-4, IL-5 and IL-10), metalloproteases (e.g., MMP-I/collagenase and MMP-9/gelatinase B) and establishment of immune memory. CD40 activities further include modulating production of proteins involved in cell-cell contact or adhesion (e.g., E-selectin, VCAM-1 and ICAM-1). This interaction can activate macrophages, natural killer (NK) cells, and endothelial cells. CD40 plays a significant role in immune cell function and signaling, including B-cell and T-cell activation by antigen presenting cells, such as macrophages and dendritic cells. CD40 activity stimulates antibody production, isotype switching, and establishment of memory. CD40 activity stimulates production of cytokines, such as IL-2, IL-6, IL-8, IL-12, TNF-α, IL-4, IL-S and IL-10; and metalloproteases, such as MMP-I/collagenase and MMP-9/gelatinase B. CD40 activity stimulates production of proteins involved in cell-cell contact or adhesion, such as E-selectin, VCAM-1 and ICAM-1. CD40 recognition on target cells provides an activation pathway for NK cell cytotoxic activity. Thus, increasing or decreasing cell survival, antibody production, isotype switching, establishment of memory, production of cytokines, metalloproteases or proteins involved in cell-cell contact or adhesion, NK cell cytotoxic activity, B-cell activation, B-cell proliferation, T-cell activation, T-cell proliferation, macrophage activation, migration of immune cell can all be effected or inhibited by contacting an appropriate cell expressing CD40 with the peptide of the present disclosure or a pharmaceutical composition disclosed herein
- Thus, the disclosure further provides methods of modulating cell survival, antibody production, isotype switching, establishment of memory, production of cytokines, metalloproteases or proteins involved in cell-cell contact or adhesion, NK cell cytotoxic activity, B-cell activation, B-cell proliferation, T-cell activation, T-cell proliferation, macrophage activation, or migration of an immune cell, comprising contacting a cell expressing CD40 with an effective amount of a peptide disclosed herein. By “a cell expressing CD40” herein is intended any normal or malignant cells that express detectable levels of the CD40 antigen. The cell can be an immune cell, a nonimmune cell, or a cancer cell, for example, lymphoma cell. Methods for detecting CD40 expression in cells are well known in the art and include, but are not limited to, PCR techniques, immunohistochemistry, flow cytometry, Western blot, ELISA, and the like. These methods allow for the detection of CD40 mRNA, CD40 antigen and cell-surface CD40 antigen. Preferably, the CD40-expressing cells are cells that express detectable levels of cell-surface CD40 antigen. The cell to be contacted can be informed by the immune response to be modulated. For example, in embodiments related to B-cell mediated immune response, such as B-cell proliferation, B-cell activation, the cell contacted with the peptides or compositions disclosed herein can be a B-cell. For example, in embodiments related to inhibiting activation of macrophage activation or production of cytokines, the cell contacted with the peptides or compositions disclosed herein can be a macrophage. The cell to be contacted can be easily determined by one of skill in the art.
- Assays for monitoring immune responses are well known in the art and are described above. Methods of immunosuppression can be practiced on a subject in order to achieve the effect in the subject. Accordingly, the disclosure provides for a method for inducing immunosuppression in a subject in need thereof, the method comprising administering to the subject an effective amount of a peptide of the present disclosure or a pharmaceutical composition disclosed herein.
- Compositions, methods, kits, and systems provided herein can be utilized to prevent and/or treat an autoimmune disorder. The term “autoimmune disorder”, “autoimmune disease”, “autoimmune condition”, and their grammatical equivalents as used herein can be used interchangeably. In some cases, the tolerance vaccine provided herein can be crosslinked to autoantigenic peptides, autoantigens, or other cellular carriers and used as a tolerance therapy for an autoimmune disorder. In some cases, the cellular carrier is an apoptotic cellular carrier. In some cases, the cellular carrier is a syngeneic apoptotic cellular carrier.
- The tolerizing vaccine as described herein can be used with a cellular carrier (e.g., autoantigens, autoantigenic peptides, apoptotic cellular carriers) to induce antigen-specific T cell tolerance for treatment of an autoimmune condition. Without being bound by theory, the tolerizing vaccine with or without the carrier can be taken up, processed, and presented in a tolerogenic manner by host splenic antigen presenting cells, thereby inducing regulatory T cells, and the secretion of immune suppressive cytokines (e.g., IL-4, IL-10, IL-13, TGF-β).
- Methods and composition of the present disclosure can be employed to treat a CD40 associated disorder. The disclosure therefore also provides methods of treating a CD40 associated disorder. As used herein, the term “CD40 associated disorder” means any undesirable physiological condition or pathological disorder in which modulating a CD40 activity (e.g., binding CD154 or activation of immune response mediated by CD40 may improve or reduce one or more undesirable symptoms of the condition or disorder.
- Thus, where CD40 is associated with an undesirable immune response or process in vivo, such as autoimmunity, hypersensitivity, inflammation or transplant rejection, a peptide disclosed herein or a pharmaceutical composition disclosed herein can be administered to a subject having, or at risk of having autoimmunity, hypersensitivity, inflammation or transplant rejection in order to induce immunosuppression and inhibit or prevent autoimmunity, hypersensitivity, inflammation or transplant rejection in the subject.
- Accordingly, in one embodiment, the method of inducing immunosuppression disclosed herein can be useful to a subject who is suffering from or is at a risk of developing an inflammatory disease. “Inflammatory disease” means an immune-mediated inflammatory condition, generally characterized by dysregulated expression of one or more cytokines. Examples of inflammatory disease include skin inflammatory disorders, inflammatory disorders of the joints, and inflammatory disorders of the cardiovascular system, autoimmune diseases, lung and airway inflammatory disorders, intestinal inflammatory disorders. Examples of skin inflammatory disorders include dermatitis, for example atopic dermatitis and contact dermatitis, acne vulgaris, and psoriasis. Examples of inflammatory disorders of the joints include rheumatoid arthritis. Examples of inflammatory disorders of the cardiovascular system are cardiovascular disease and atherosclerosis. Examples of autoimmune diseases include
Type 1 diabetes, Graves' disease, Guillain-Barre disease, Lupus, Psoriatic arthritis, and Ulcerative colitis. Examples of lung and airway inflammatory disorders include asthma, cystic fibrosis, COPD, emphysema, and acute respiratory distress syndrome. Examples of intestinal inflammatory disorders include colitis and inflammatory bowel disease. Other inflammatory disorders include cancer, hay fever, periodontitis, allergies, hypersensitivity, ischemia, depression, systemic diseases, post infection inflammation, amyotrophic lateral sclerosis and bronchitis. - As used herein, the term “autoimmune disease” refers generally to those diseases characterized by the failure of one or more B- and/or T-cell populations, or gene products thereof, to distinguish between self and non-self antigenic determinants. Autoimmune diseases are often characterized by the infiltration of the target cells with inflammatory lymphoid cells, for example, mononuclear phagocytes, lymphocytes and plasma cells as well as secondary lymphoid follicles. Exemplary autoimmune diseases include, but are not limited to, organ specific disorders such as Hashimoto's thyroiditis, primary myxoedema thyrotoxicosis, pernicious anemia, Addison's disease, and insulin-dependent diabetes mellitus as well as non-organ specific disorders such as systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), multiple sclerosis, dermatomyositis, scleroderma and psoriasis. Non-limiting examples of autoimmune disorders treatable with a peptide or pharmaceutical composition of the disclosure include rheumatoid arthritis, lupus (e.g., SLE, lupus nephritis), production of auto-antibodies which, in the case of antibodies against myelin basic protein contribute to multiple sclerosis and in the case of antibodies against insulin contribute to diabetes, and Crohn's disease. Non-limiting examples of hypersensitivity treatable with a peptide disclosed herein or pharmaceutical compositions of the disclosure include allergic reactions to antigens, antibiotics, etc.
- Non-limiting examples of inflammation treatable with a peptide or pharmaceutical composition of the present disclosure include vascular inflammatory disease (e.g., artherosclerotic lesions, plaque disruption and thrombus formation), production of inflammatory cytokines (e.g., LIF, GM-CSF, and IL-6), lung fibrosis and inflammation associated with multiple sclerosis or a tissue or organ transplant. CD40 activity can be associated with inflammation caused by viral myocarditis and, as such, a peptide or pharmaceutical composition disclosed herein can be used to inhibit inflammation associated with viral infection.
- Additional situations exist in which it may be desired to inhibit an immune response. For example, production of neutralizing antibodies against therapeutic agents, such as anti-insulin antibodies in diabetics administered insulin repeatedly, or in subjects that produce anti-virus antibodies (e.g., adenovirus or adeno-associated virus) being treated with a gene therapy virus vector, may be inhibited using a peptide or pharmaceutical composition herein. Accordingly, provided herein is a method of treating a subject suffering from an inflammatory disease, the method comprising, administering to the subject an effective amount of a pharmaceutical composition disclosed herein.
- CD40 is also present in various other tissues and cells. For example, CD40 is present in epithelial cells, vascular endothelium and smooth muscle cells, and CD40 ligand (CD 154) was expressed by thrombin-activated platelets. These findings indicate a role for CD40 activity in vascular thrombotic-atheromatic pathophysiology. Therefore, a peptide disclosed herein or a pharmaceutical composition of the present disclosure that decreases a CD40 activity can be used to inhibit thrombus formation or artherosclerosis in a subject.
- CD40 is also expressed in human renal tubules, thymic epithelia and neural cells. Interestingly, CD40 activity appears to induce apoptosis in neural cells, in contrast to its cell survival role in the immune system. Thus, a peptide or pharmaceutical composition herein can be used to inhibit apoptosis in neural cells. Accordingly, CD40 can be used to treat neural disorders characterized by cell degeneration or undesirable or excessive cell death, such as Parkinson's disease, Alzheimer's, Huntington disease, spinocerebellar ataxias/atrophies, etc.
- CD40 is also expressed in carcinomas, such as melanoma, Kaposi's sarcoma, osteosarcoma and Ewing' sarcoma. CD40 in malignant melanoma appears to be predictive of a negative prognosis. CD40 in human bladder carcinoma cells inhibits fas-mediated apoptosis. CD40 has been detected in tumor vasculature in a renal carcinoma mass. Stimulation of CD40 in B-cell lymphomas stimulates growth. CD40 can therefore function as a cell survival or growth factor in some tumors, and may promote angiogenesis. Other data indicate that CD40 may induce cell death in transformed cells.
- Thus, a peptide or compositions disclosed herein that decreases a CD40 activity associated with tumor survival will be useful in treating cell proliferative disorders (e.g., tumors) in which CD40 functions as a cell survival signal or growth promoter, either directly (e.g., in the tumor cell) or indirectly (i.e., through stimulation of angiogenesis within a tumor mass). Other biological pathways and physiological conditions that CD40 participates in are described in Biancone, et al. (Int. J. Mol. Med. 3:343 (1999)), Laman et al. (Dev. Immunol. 6:215 (1998)), Kooten and Bachereau (J. Leukoc. Biol. 67:2 (2000)), Noelle et al. (Ann. NY Acad. Sci. 815:384 (1997)), Noelle (Immunity 4:415 (1996)), Grewal et al. (Curr. Opin. Immunol. 9:491 (1997)); Grewal et al. (Ann. Rev. Immunol. 16:111 (1998); Grewal et al. (Immunol. Rev. 153:85 (1996)); Gruss et al. (Leuk. Lymphoma 24(5-6):393 (1997)); van Kooten et al. (Curr. Opin. Immunol. 9:330 (1997)). Such pathways as well as others known in the art are amenable to modulation using the peptides disclosed herein or compositions comprising said peptides disclosed herein, as are the physiological conditions associated with CD40 activity described therein or otherwise known in the art.
- The peptides or compositions disclosed can find use in treatment of a neoplastic disease. In one aspect, provided herein is a method of treating a subject suffering from or is at risk of developing a neoplastic disease characterized by a neoplastic cell that expresses CD40, the method comprising administering to the subject an effective amount of peptides disclosed herein or a composition comprising the peptides disclosed herein. CD40 has been shown to be expressed not only on immune cells, but also on tumor cells, including B-cell malignancies and solid tumors.
- As used herein, the term “neoplastic cells that expresses CD40,” refers to a cell (e.g., an immune cell such as B-cell, T-cell, or an non-immune cell, such as epithelial cell, endothelial cell, smooth muscle cell) that expresses a CD40 protein on its cell surface and exhibits abnormal cell growth, cell division and proliferation not undergoing apoptosis, or both, under inappropriate conditions. For example, a “neoplastic cell” may undergo cell division when a corresponding non-neoplastic cell does not undergo cell division, or, alternatively, a “neoplastic cell” may not respond to normal cellcycle checkpoint controls. Accordingly a neoplastic cell is one that exhibits abnormal proliferation of cells. The growth of neoplastic cells exceeds that of normal tissue around it and it is not coordinated with that of the normal tissue around it resulting in neoplasms. Neoplasms may be benign (e.g., benign tumor and atypical hyperplasia), pre-malignant (e.g., carcinoma in situ and pre-cancer) or malignant (e.g., cancer). The term “cancer” refers to any of the various malignant neoplasms characterized by the proliferation of cells that have the capability to invade surrounding tissue and/or metastasize to new colonization sites, including but not limited to leukemias, lymphomas, carcinomas, melanomas, sarcomas, germ cell tumors and blastomas. Exemplary cancers include cancers of the brain, bladder, breast, cervix, colon, head and neck, kidney, lung, non-small cell lung, mesothelioma, ovary, prostate, stomach and uterus, leukemia and medulloblastoma.
- Neoplastic tissues can originate from any cell type or tissue found in a mammal, including, but not limited to hepatic, skin, breast, prostate, neural, optic, intestinal, cardiac, vasculature, lymph, spleen, renal, bladder, lung, muscle, connective, tissue, pancreatic, pituitary, endocrine, reproductive organs, bone, and blood. The neoplastic tissue for analysis may include any type of solid tumor or hematological cancer. In some embodiments, the neoplastic tissue is a breast cancer tissue. In other embodiments, the neoplastic tissue is a breast tissue with atypical hyperplasia.
- The term “leukemia” refers to broadly progressive, malignant diseases of the blood-forming organs and is generally characterized by a distorted proliferation and development of leukocytes and their precursors in the blood and bone marrow. Leukemia diseases include, for example, acute nonlymphocytic leukemia, chronic lymphocytic leukemia, acute granulocytic leukemia, chronic granulocytic leukemia, acute promyelocytic leukemia, adult T-cell leukemia, aleukemic leukemia, a leukocythemic leukemia, basophylic leukemia, blast cell leukemia, bovine leukemia, chronic myelocytic leukemia, leukemia cutis, embryonal leukemia, eosinophilic leukemia, Gross' leukemia, hairy-cell leukemia, hemoblastic leukemia, hemocytoblastic leukemia, histiocytic leukemia, stem cell leukemia, acute monocytic leukemia, leukopenic leukemia, lymphatic leukemia, lymphoblastic leukemia, lymphocytic leukemia, lymphogenous leukemia, lymphoid leukemia, lymphosarcoma cell leukemia, mast cell leukemia, megakaryocytic leukemia, micromyeloblastic leukemia, monocytic leukemia, myeloblastic leukemia, myelocytic leukemia, myeloid granulocytic leukemia, myelomonocytic leukemia, Naegeli leukemia, plasma cell leukemia, plasmacytic leukemia, promyelocytic leukemia, Rieder cell leukemia, Schilling's leukemia, stem cell leukemia, subleukemic leukemia, and undifferentiated cell leukemia.
- The term “carcinoma” refers to a malignant new growth made up of epithelial cells tending to infiltrate the surrounding tissues and give rise to metastases. Exemplary carcinomas include, for example, acinar carcinoma, acinous carcinoma, adenocystic carcinoma, adenoid cystic carcinoma, carcinoma adenomatosum, carcinoma of adrenal cortex, alveolar carcinoma, alveolar cell carcinoma, basal cell carcinoma, carcinoma basocellulare, basaloid carcinoma, basosquamous cell carcinoma, bronchioalveolar carcinoma, bronchiolar carcinoma, bronchogenic carcinoma, cerebriform carcinoma, cholangiocellular carcinoma, chorionic carcinoma, colloid carcinoma, comedo carcinoma, corpus carcinoma, cribriform carcinoma, carcinoma en cuirasse, carcinoma cutaneum, cylindrical carcinoma, cylindrical cell carcinoma, duct carcinoma, carcinoma durum, embryonal carcinoma, encephaloid carcinoma, epiennoid carcinoma, carcinoma epitheliale adenoides, exophytic carcinoma, carcinoma ex ulcere, carcinoma fibrosum, gelatiniform carcinoma, gelatinous carcinoma, giant cell carcinoma, carcinoma gigantocellulare, glandular carcinoma, granulosa cell carcinoma, hair-matrix carcinoma, hematoid carcinoma, hepatocellular carcinoma, Hurthle cell carcinoma, hyaline carcinoma, hypemephroid carcinoma, infantile embryonal carcinoma, carcinoma in situ, intraepidermal carcinoma, intraepithelial carcinoma, Krompecher's carcinoma, Kulchitzky-cell carcinoma, large-cell carcinoma, lenticular carcinoma, carcinoma lenticulare, lipomatous carcinoma, lymphoepithelial carcinoma, carcinoma medullare, medullary carcinoma, melanotic carcinoma, carcinoma molle, mucinous carcinoma, carcinoma muciparum, carcinoma mucocellulare, mucoepidermoid carcinoma, carcinoma mucosum, mucous carcinoma, carcinoma myxomatodes, naspharyngeal carcinoma, oat cell carcinoma, carcinoma ossificans, osteoid carcinoma, papillary carcinoma, periportal carcinoma, preinvasive carcinoma, prickle cell carcinoma, pultaceous carcinoma, renal cell carcinoma of kidney, reserve cell carcinoma, carcinoma sarcomatodes, schneiderian carcinoma, scirrhous carcinoma, carcinoma scroti, signet-ring cell carcinoma, carcinoma simplex, small-cell carcinoma, solanoid carcinoma, spheroidal cell carcinoma, spindle cell carcinoma, carcinoma spongiosum, squamous carcinoma, squamous cell carcinoma, string carcinoma, carcinoma telangiectaticum, carcinoma telangiectodes, transitional cell carcinoma, carcinoma tuberosum, tuberous carcinoma, verrucous carcinoma, and carcinoma villosum.
- The term “sarcoma” generally refers to a tumor which arises from transformed cells of mesenchymal origin. Sarcomas are malignant tumors of the connective tissue and are generally composed of closely packed cells embedded in a fibrillar or homogeneous substance. Sarcomas include, for example, chondrosarcoma, fibrosarcoma, lymphosarcoma, melanosarcoma, myxosarcoma, osteosarcoma, Abemethy's sarcoma, adipose sarcoma, liposarcoma, alveolar soft part sarcoma, ameloblastic sarcoma, botryoid sarcoma, chloroma sarcoma, chorio carcinoma, embryonal sarcoma, Wilms' tumor sarcoma, endometrial sarcoma, stromal sarcoma, Ewing's sarcoma, fascial sarcoma, fibroblastic sarcoma, giant cell sarcoma, granulocytic sarcoma, Hodgkin's sarcoma, idiopathic multiple pigmented hemorrhagic sarcoma, immunoblastic sarcoma of B cells, lymphomas (e.g., Non-Hodgkin Lymphoma), immunoblastic sarcoma of T-cells, Jensen's sarcoma, Kaposi's sarcoma, Kupffer cell sarcoma, angiosarcoma, leukosarcoma, malignant mesenchymoma sarcoma, parosteal sarcoma, reticulocytic sarcoma, Rous sarcoma, serocystic sarcoma, synovial sarcoma, and telangiectaltic sarcoma.
- The term “melanoma” is taken to mean a tumor arising from the melanocytic system of the skin and other organs. Melanomas include, for example, acral-lentiginous melanoma, amelanotic melanoma, benign juvenile melanoma, Cloudman's melanoma, S91 melanoma, Harding-Passey melanoma, juvenile melanoma, lentigo maligna melanoma, malignant melanoma, nodular melanoma subungal melanoma, and superficial spreading melanoma.
- The Examples herein show that the CD40 binding peptides disclosed herein can bind CD40 expressed on B-cell and inhibit, for example, B-cell proliferation and B-cell activation. The peptides of the present disclosure can find use, for example, in the treatment of B-cell lymphomas, prevention of EBV-induced B-cell lymphoma that can occur after transplantation or in other instances of immunosuppression, such as AIDS, and which present a significant risk in such patient populations. The methods disclosed herein can be useful in prevention of immunoblastic B-cell lymphomas that frequently arise in immunocompromised individuals. In such preventative methods, a mammal at risk of developing an immunoblastic B-cell lymphoma is administered an effective amount of peptides disclosed herein. The peptides can be administered for as long as the state of immunocompromise that places the individual at risk exists.
- The peptides of the present disclosure or compositions comprising the peptides can be useful for treatment of cancer characterized by “neoplastic B-cell growth” is intended any disease or condition (including pre-malignant conditions) involving uncontrolled growth of cells of B-cell lineage. Such diseases and conditions include, but are not limited to, acute lymphoblastic leukemia (ALL), acute myelogenous leukemia (AML), chronic myelogenous leukemia (CIVIL), chronic lymphocytic leukemia (CLL), prolymphocytic leukemia (PLL), small lymphocytic leukemia (SLL), diffuse small lymphocytic leukemia (DSLL), diffuse large B-cell lymphoma (DLBCL), hairy cell leukemia, non-Hodgkin's lymphomas, Hodgkin's disease, Epstein-Barr Virus (EBV) induced lymphomas, myelomas such as multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, mucosal associated lymphoid tissue lymphoma, monocytoid B cell lymphoma, splenic lymphoma, lymphomatoid granulomatosis, intravascular lymphomatosis, immunoblastic lymphomas, AIDS-related lymphomas, and the like.
- In another aspect, the compositions and methods used to prevent occurrence (or reoccurrence) or treatment of neoplastic disease characterized by other types of malignant cells that express CD40 in individuals at risk for such disease. Individuals that are considered at risk in these instances include those with family history or other genetic characteristics indicating predisposition to cancers in which the neoplastic cells express CD40, and individuals that develop drug-resistant neoplastic disease as a result of chemotherapy, in which the drug-resistant neoplastic cells express CD40.
- Individuals afflicted with disease characterized by neoplastic cells that express CD40 may also be treated according to the methods disclosed herein. The term treatment, as it is generally understood in the art, refers to initiation of therapy after clinical symptoms or signs of disease have been observed. The methods may be used in conjunction with other therapies appropriate for afflicted individuals, including chemotherapy, radiation therapy, and immunotherapy.
- The “effective amount” to be administered will be governed by such considerations, and refers to the minimum amount necessary to ameliorate, treat, or stabilize, the cancer; to increase the time until progression (duration of progression free survival) or to treat or prevent the occurrence or recurrence of a tumor, a dormant tumor, or a micrometastases. The peptides, disclosed herein, can be optionally formulated with one or more additional therapeutic agents currently used to prevent or treat cancer or a risk of developing a cancer. The effective amount of such other agents depends on the amount of peptides present in the formulation, the type of disorder or treatment, and other factors such age, weight etc. These are generally used in the same dosages and with administration routes as used herein before or about from 1 to 99% of the heretofore employed dosages.
- The efficacy of the treatment methods for cancer, comprising administering the peptides disclosed herein or pharmaceutical compositions of the present disclosure can be measured by various endpoints commonly used in evaluating cancer treatments, including but not limited to, tumor regression, tumor weight or size shrinkage, time to progression, duration of survival, progression free survival, overall response rate, duration of response, and quality of life. The peptides disclosed herein can require unique measures and definitions of clinical responses to drugs. In the case of cancers, the therapeutically effective amount of the peptides disclosed herein or compositions comprising the same can reduce the number of cancer cells; reduce the tumor size; inhibit (i.e., slow to some extent and preferably stop) cancer cell infiltration into peripheral organs; inhibit (i.e., slow to some extent and preferably stop) tumor metastasis; inhibit, to some extent, tumor growth; and/or relieve to some extent one or more of the symptoms associated with the disorder. To the extent the peptides disclosed herein, act to prevent growth and/or kill existing cancer cells; it can be cytostatic and/or cytotoxic. For cancer therapy, efficacy in vivo can, for example, be measured by assessing the duration of survival, duration of progression free survival (PFS), the response rates (RR), duration of response, and/or quality of life.
- In other embodiments, described herein are methods for increasing progression free survival of a human subject susceptible to or diagnosed with a cancer, for example, skin cutaneous melanoma. Time to disease progression is defined as the time from administration of the drug until disease progression or death. In a preferred embodiment, the combination treatment of the disclosure using a peptide or compositions disclosed herein, and one or more chemotherapeutic agents may significantly increase progression free survival by at least about 1 month, 1.2 months, 2 months, 2.4 months, 2.9 months, 3.5 months, such as by about 1 to about 5 months, when compared to a treatment with chemotherapy alone. In another embodiment, the methods described herein may significantly increase response rates in a group of human subjects susceptible to or diagnosed with a cancer that are treated with various therapeutics. Response rate is defined as the percentage of treated subjects who responded to the treatment. In one embodiment, the combination treatment described herein using a peptide or compositions disclosed herein, and one or more chemotherapeutic agents significantly increases response rate in the treated subject group compared to the group treated with chemotherapy alone.
- For example, in some embodiments, the methods described herein comprise administering an effective amount of the peptides of the instant disclosure or compositions comprising said peptides, to a subject in order to alleviate a symptom of a cancer. As used herein, “alleviating a symptom of a cancer” is ameliorating or reducing any condition or symptom associated with the cancer. As compared with an equivalent untreated control, such reduction or degree of prevention is at least 5%, 10%, 20%, 40%, 50%, 60%, 80%, 90%, 95%, or 100% as measured by any standard technique. Ideally, the cancer is completely cleared as detected by any standard method known in the art, in which case the cancer is considered to have been treated. A patient who is being treated for a cancer is one who a medical practitioner has diagnosed as having such a condition. Diagnosis can be by any suitable means. Diagnosis and monitoring can involve, for example, detecting the level of cancer cells in a biological sample (for example, a tissue or lymph node biopsy, blood test, or urine test), detecting the level of a surrogate marker of the cancer in a biological sample, detecting symptoms associated with the specific cancer, or detecting immune cells involved in the immune response typical of such a cancer.
- The treatment and/or prevention of cancer includes, but is not limited to, alleviating symptoms associated with cancer, the inhibition of the progression of cancer, the promotion of the regression of cancer, the promotion of the immune response, inhibition of tumor growth, inhibition of tumor size, inhibition of metastasis, inhibition of cancer cell growth, inhibition of cancer cell proliferation, or cause cancer cell death.
- Described herein are compositions, systems, and methods for inducing graft tolerance. In particular, the present disclosure relates to administering a tolerizing vaccine or a preparatory regimen before, during, and/or after administration of donor transplant cells, tissue(s), or organ(s). The tolerizing vaccine or preparatory regimen can induce tolerance to the allograft or xenograft in the graft recipient.
- An organ, tissue, or cell can be differentiated from stem cells, grown de novo, or isolated from an animal (e.g., a human or non-human animal) and can be transplanted into a recipient in need of a transplant from the same species (an allotransplant) or a different species (a xenotransplant). The donor of the organ, tissue, or cell can be referred to herein as a transplant donor. The transplanted organ, tissue, or cell can be referred to herein as a transplant or a graft.
- The donor of transplant or graft can be any animal, including human and non-human animals. In some cases the donor is a human. In some cases, the transplant donor is a non-human animal. The transplant donor (e.g., a non-human animal donor) can be genetically modified, for example, to reduce or eliminate the likelihood that the transplant or graft can be recognized by the recipient's immune system, or to reduce the immune response by the recipient's immune system upon recognizing the transplant or graft.
- The donor of a transplant or graft can be a living donor or a cadaveric donor. In some cases, the transplant donor is a living donor. In some cases, the transplant donor is a cadaveric donor. The cadaveric donor may be a brain dead, heart beating donor (BDD). Alternatively, the cadaveric donor may be a non-heart beating donor (NHBD). Whether the transplant donor is a living donor or a cadaveric donor (e.g., a BDD or NHBD), the donor can be from any animal, for example, a human or non-human animal.
- The donor of a transplant or graft can be at any age or stage of development. For example, the transplant donor can be a fetal, perinatal, neonatal, pre-weaning, post-weaning, juvenile, young adult, or adult human or animal.
- Transplants or grafts can be used to treat diseases or disorders in recipients in need thereof. Suitable diseases that can be treated are any in which an organ, tissue, or cell of a recipient is defective or injured, and the recipient can be treated by transplantation of an organ, tissue, or cell (e.g., a kidney, heart, lung, liver, vein, skin, endocrine pancreas, pancreatic islet cell, or a combination thereof). In some cases, the transplant comprises a kidney, liver, heart, lung, pancreas, endocrine pancreas, islet cell, small bowel, bone marrow, hematopoietic stem cell, embryonic or induced pluripotent stem cell-derived cells such as islet beta cells or hepatocytes, embryonic or induced pluripotent stem cell-derived islet, embryonic or induced pluripotent stem cell-derived hepatocyte, or a combination thereof.
- In some embodiments, methods for inducing immunosuppression can be useful to prevent graft rejection in a subject who has undergone, is undergoing or will be undergoing a cell, organ or tissue transplant.
- The peptides and compositions disclosed herein can find use in preventing transplant rejection by inhibiting a CD40 mediated immune response in a recipient, towards an antigen on a transplanted material (e.g., cells, tissues, an/or organs). In one aspect provided herein is a method of inhibiting an immune response to a donor cell or tissue transplantation in a recipient, the method comprising administering to the recipient a peptide or compositions disclosed herein. Examples of transplant rejection (acute or chronic) treatable with a peptide disclosed herein include blood vessels, kidney, liver, heart, lung, pancreas and skin. Transplantation includes grafting of tissues or organ from the body of an individual to a different place within the same individual, or a different individual. Transplantation also involves grafting of tissues or organs from one area of the body to another. Transplantation of tissues or organs between genetically dissimilar animals of the same species is termed as allogeneic transplantation. Transplantation of animal organs into humans is termed xenotransplants.
- Transplant/graft rejection can involve recognition of donor-specific antigens, for example, recognition of donor-specific antigens presented to T cells by host antigen presenting cells (indirect) or donor antigen presenting cells (direct). T cell activation in response to donor-specific antigens can lead to a pro-inflammatory response and transplant rejection. Transplant rejection (e.g., T cell-mediated transplant rejection) can be prevented by chronic immunosuppression with one or more immunomodulatory molecules. However, immunosuppression is costly and associated with the risk of serious side effects.
- In some embodiments, a method described herein to prevent transplantation rejection or prolong the time to transplantation rejection without or with minimal immunosuppressive drug use (e.g., one or more immunomodulatory molecules) involves using a genetically modified animal as a cell, organ, or tissue donor. The cells, organs, and/or tissues of the altered donor animal, e.g., a donor non-human animal, can be harvested and used in allografts or xenografts. Alternatively, a cell, organ, or tissue can be extracted from an animal, and used to generate a genetically-altered cell, organ or tissue. In some cases, primary cells can be extracted from an animal, and used to make genetically altered cells. In some cases, a cell, organ, or tissue derived from an animal (e.g., a cell line) can be used to create a genetically altered cell, organ, or tissue.
- Transplant rejection can also be reduced or eliminated by inducing tolerance to a transplant or graft using a tolerizing vaccine or preparatory regimen. A tolerizing vaccine or preparatory regimen of the disclosure can be used to prevent transplant rejection or delay rejection, for example, by reducing a pro-inflammatory immune response to the transplant, and/or enhancing a tolerance-promoting immune response. In some cases, a tolerizing vaccine or preparatory regimen of the disclosure can circumvent the need for long-term immunosuppression of the recipient.
- An effective amount, which is determined by these considerations, is the minimum amount necessary to inhibit an immune response in a recipient that would result in rejection of the transplant, but as much as necessary to achieve a longer transplant survival time. Such amount is preferably below the amount that is toxic to the recipient or renders the recipient significantly more susceptible to infections. The amount of peptides or compositions disclosed herein used for inhibiting immune response directed to a transplant can be lower than the amount of an immunosuppressive agent normally required for transplanted grafts and depends on the individual circumstances surrounding the transplant. A treatment with the peptides or compositions disclosed herein can also lessen or prevent the side effects frequently observed in transplant recipients who undergo immune suppressive therapy,
- The present disclosure therefore provides a method for inhibiting transplant rejection and a method to increase transplant survival. In one aspect, the peptides or compositions disclosed herein can administered as a part of a transplantation protocol. Accordingly, provided herein is a method for transplantation of a cell, organ, or tissue from a mammalian donor in a recipient, the method comprising administering an effective amount of the peptides or pharmaceutical compositions disclosed herein. In some embodiments, the method of transplantation further comprises transplanting the donor cell tissue or organ in a recipient.
- An organ, tissue, or cell isolated from an animal (e.g., a human or non-human animal) can be transplanted into a recipient in need of a transplant from the same species (an allotransplant) or a different species (a xenotransplant). Transplants or grafts can be used to treat diseases or disorders in a recipient in need thereof. Suitable diseases that can be treated are any in which an organ, tissue, or cell of a recipient is defective or injured, (e.g., a kidney, heart, lung, liver, vein, skin, pancreatic islet cell, or a combination thereof) and the recipient can be treated by transplantation of an organ, tissue, or cell. In some cases, the transplant comprises a kidney, liver, heart, lung, pancreas, islet cell, small bowel, bone marrow, hematopoietic stem cell, embryonic or induced pluripotent stem cell-derived cells, embryonic or induced pluripotent stem cell-derived islet, embryonic or induced pluripotent stem cell-derived hepatocyte, or a combination thereof.
- The methods herein can comprise administering one or more dose of a peptide or compositions disclosed herein to a recipient before, after, and/or during transplant of donor cells, organs, and/or tissues to inhibit donor-specific immune response in a recipient. In some cases, a first dose of a peptide or compositions disclosed herein can be given on or on about day −100, day −90, day −80, day −70, day −60, day −50, day −40, day −30, day −20, day −15, day −14, day −13, day −12, day −11, day −10, day −9, day −8, day −7, day −6, day −5, day −4, day −3, day −2 or day −1, relative to transplant of donor cells, organs, and/or tissues on
day 0. In some cases, a first dose of a peptide or compositions disclosed herein can be given on or on about day −100 to −50; −50 to −40; −40 to −30; −30 to −20; −20 to −10; −10 to −5; −7 to −1, relative to transplant of donor cells, organs, and/or tissues onday 0. For example, a first dose of a peptide or compositions disclosed herein can be given 8 days (e.g., day −8) before transplant of donor cells, organs, and/or tissues. In some embodiments, a peptide or compositions of the present disclosure can be administered on the same day (e.g., day 0) as transplant of donor cells, organs, and/or tissues. In some cases, a peptide or a composition disclosed herein can be administered on or on about day 300, day 200, day 100, day 90, day 80, day 70, day 60, day 50, day 40, day 30,day 20,day 15, day 14, day 13, day 12, day 11,day 10, day 9, day 8,day 7,day 6,day 5,day 4,day 3,day 2 orday 1, relative to transplant of donor cells, organs, and/or tissues onday 0. For example, a peptide or compositions disclosed herein can be administered on or on about day 400 to 350; 350 to 300; 300 to 250; 250 to 200; 200 to 150; 150 to 100; 100 to 50; 50 to 40; 40 to 30; 30 to 20; 20 to 10; 10 to 5; 7 to 1, relative to transplant of donor cells, organs, and/or tissues onday 0. For example, a peptide or compositions disclosed herein can be administered on 1 day after (e.g., day 1) transplant of donor cells, organs, and/or tissues. In some embodiments, a peptide or compositions disclosed herein can be administered of day 14 after transplant of donor cells, organs, and/or tissues. In some embodiments, a peptide or compositions disclosed herein can be administered for a lifetime of the recipient. - A recipient (e.g., a human or a non-human animal) can require administration with a peptide or composition disclosed herein for at least or at least about 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 900, or 1,000 days after transplantation, e.g., for at least or at least about 1 to 5; 5 to 10; 10 to 20; 20 to 30; 30 to 60; 60 to 100; 100 to 200; 200 to 300; 300 to 400; 400 to 500; 500 to 600; 600 to 700; 700 to 800; 800 to 900; 900 to 1,000 days. A recipient (e.g., a human or a non-human animal) can require administration of a peptide or composition disclosed herein for at least or at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 months after transplantation, e.g., for at least or at least about 1 to 2; 2 to 3; 3 to 6; 6 to 9; 9 to 12; 12 to 18; 18 to 24; 24 to 30; 30 to 36 months after transplantation. A recipient (e.g., a human or a non-human animal) can require administration of a peptide or a composition disclosed herein for at least or at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or 30 years after transplantation, e.g. for at least or at least about 1 to 2; 2 to 3; 3 to 4; 4 to 5; 1 to 5; 5 to 10; 10 to 15; 15 to 20; 20 to 25; 25 to 30 years after transplantation. In some cases, a recipient (e.g., a human or a non-human animal) can require administration of a peptide or a composition disclosed herein for up to the lifetime of the recipient.
- A recipient (e.g., a human or a non-human animal) can require administration for at least or at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 months after transplantation e.g., for at least or at least about 1 to 2; 2 to 3; 3 to 6; 6 to 9; 9 to 12; 12 to 18; 18 to 24; 24 to 30; 30 to 36 months after transplantation. A recipient (e.g., a human or a non-human animal) can require administration of a peptide or a composition disclosed herein for at least or at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or 30 years after transplantation, e.g. for at least or at least about 1 to 2; 2 to 3; 3 to 4; 4 to 5; 1 to 5; 5 to 10; 10 to 15; 15 to 20; 20 to 25; 25 to 30 years after transplantation.
- The transplantation method of the present disclosure can comprise multiple doses of a peptide or a composition disclosed herein before, and/or during and/or after transplantation of a graft cell, tissue, or organ. The multiple doses can be referred to as comprising an initial dose and one or more booster doses. Typically, the initial dose occurs prior to or concurrently with the transplant of the graft cell tissue or organ. The booster dose(s), when administered, occur after the initial dose. In some embodiments, a booster dose is administered to achieve and maintain a particular serum/blood trough level of the peptides disclosed herein.
- Depending upon when the initial dose of the peptide or composition disclosed herein is administered, one or more booster doses can be administered before, and/or concurrently with, and/or after transplant of the graft cell, tissue, or organ.
- Subsequent (e.g., booster) dose(s) of the peptide or compositions disclosed herein can be administered in any interval of time following a preceding dose (e.g., an initial dose). For example, the subsequent dose can be administered 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 31 days, 32 days, 33 days, 34 days, 35 days, 36 days, 37 days, 38 days, 39 days, 40 days, 41 days, 42 days, 43 days, 44 days, 45 days, 46 days, 47 days, 48 days, 49 days, 50 days, 51 days, 52 days, 53 days, 54 days, 55 days, 56 days, 57 days, 58 days, 59 days, 60 days, 90 days, 120 days, 150 days, or 180 days after the preceding dose. Depending upon when the initial dose is administered subsequent (booster) dose(s) can be administered before, concurrently with, or after transplantation of the graft cell, tissue, or organ. In some cases, the methods of the present disclosure comprise at least one dose of the peptide or compositions disclosed herein prior to transplantation. In some cases, the methods of the present disclosure comprises at least two doses of a peptide or compositions disclosed herein prior to transplantation (e.g., an initial dose and a booster dose). In some cases, the methods herein comprises at least three doses of the peptide or compositions disclosed herein prior to transplantation (e.g., an initial dose and two booster doses). In some cases, the preparatory regimen comprises an initial dose of the peptide or compositions disclosed herein prior to transplantation and at least one dose of booster concurrently with or after transplantation of the graft cell, tissue, or organ.
- In some cases, two doses of a peptide or compositions thereof can be administered. For example, the first dose can be administered on day −8 relative to transplant of donor cells, organs, and/or tissues on
day 0. For example, the second dose can be administered on day −1 relative to transplant of donor cells, organs, and/or tissues onday 0. For example, the first dose can be administered on day −8, −9, −10, −11, −12, −13, or −14 relative to transplant of donor cells, organs, and/or tissues onday 0. For example, the second dose can be administered on day −1, −2, −3, −4, −5, or −6 relative to transplant of donor cells, organs, and/or tissues onday 0. In some cases, a second dose (e.g., a booster dose) can be administered on day 100, day 90, day 80, day 70, day 60, day 50, day 40, day 30, day 29, day 28, day 27, day 26,day 25, day 24, day 23, day 22, day 21,day 20, day 19, day 18, day 17, day 16,day 15, day 14, day 13, day 12, day 11,day 10, day 9, day 8,day 7,day 6,day 5,day 4,day 3,day 2 orday 1, relative to transplant of donor cells, organs, and/or tissues onday 0. For example, the second dose (e.g., a booster dose) can be administered on 1 day after (e.g., day 1) transplant of donor cells, organs, and/or tissues. - In some cases, a third dose of a peptide or compositions disclosed herein (e.g., a booster dose) can be administered on day 300, day 200, day 100, day 90, day 80, day 70, day 60, day 50, day 40, day 30, day 29, day 28, day 27, day 26,
day 25, day 24, day 23, day 22, day 21,day 20, day 19, day 18, day 17, day 16,day 15, day 14, day 13, day 12, day 11,day 10, day 9, day 8,day 7,day 6,day 5,day 4,day 3,day 2 orday 1, relative to transplant of donor cells, organs, and/or tissues onday 0. For example, the peptide or compositions disclosed herein can be administered on or on about day 300 to 200; 200 to 100; 100 to 50; 50 to 40; 40 to 30; 30 to 20; 20 to 10; 10 to 5; 7 to 1, relative to transplant of donor cells, organs, and/or tissues onday 0. - In some cases, a fourth dose of a peptide or compositions disclosed herein (e.g., a booster vaccine) can be administered on day 600,
day 500, day 400, day 300, day 200, 100, day 90, day 80, day 70, day 60, day 50, day 40, day 30, day 29, day 28, day 27, day 26,day 25, day 24, day 23, day 22, day 21,day 20, day 19, day 18, day 17, day 16,day 15, day 14, day 13, day 12, day 11,day 10, day 9, day 8,day 7,day 6,day 5,day 4,day 3,day 2 orday 1, relative to transplant of donor cells, organs, and/or tissues onday 0. For example, the peptide or compositions disclosed herein can be administered on or on about day 600 to 500; 500 to 400; 400 to 300; 300 to 200; 200 to 100; 100 to 50; 50 to 40; 40 to 30; 30 to 20; 20 to 10; 10 to 5; 7 to 1, relative to transplant of donor cells, organs, and/or tissues onday 0. - In some cases, a fifth dose of a peptide or compositions disclosed herein (e.g., a booster dose) can be administered on day 1,000, day 900, day 800, day 700, day 600,
day 500, day 400, day 300, day 200, 100, day 90, day 80, day 70, day 60, day 50, day 40, day 30, day 29, day 28, day 27, day 26,day 25, day 24, day 23, day 22, day 21,day 20, day 19, day 18, day 17, day 16,day 15, day 14, day 13, day 12, day 11,day 10, day 9, day 8,day 7,day 6,day 5,day 4,day 3,day 2 orday 1, relative to transplant of donor cells, organs, and/or tissues onday 0. For example, the peptide or compositions disclosed herein can be administered on or on about day 1,000 to 900; 900 to 800; 800 to 700; 700 to 600; 600 to 500; 500 to 400; 400 to 300; 300 to 200; 200 to 100; 100 to 50; 50 to 40; 40 to 30; 30 to 20; 20 to 10; 10 to 5; 7 to 1, relative to transplant of donor cells, organs, and/or tissues onday 0. In some cases, a second dose of a booster is not required. In some cases, a second dose of a booster is given concomitantly onday 0 with transplant donor cells, organs, and/or tissues. - Booster doses of a peptide or compositions disclosed herein can be lower dose than the initial or preceding dose of the peptide or compositions disclosed herein. For example, a booster or subsequent dose can be about: 1%, 2%, 3%, 4%, 5%, 7.5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, or 75% lower than the initial or preceding dose.
- In some embodiments, the transplantation method comprises administering a tolerizing agent in the subject. The term “tolerizing agent” or a “toleragen” as used herein means any donor antigen (such as a protein, nucleic acid, carbohydrate, lipid, or combination of any thereof) that mediates host unresponsiveness. By way of example, a tolerizing agent works by inducing the tolerized host not to produce antibodies or cell-mediated immune responses specific for the toleragen. Additional discussion of tolerizing may be found, for instance, in PCT publication WO 2006/052668, which is incorporated herein in its entirety. In some embodiments, the tolerizing agent can be a tolerizing vaccine comprising inactivated or apoptotic donor cells that upon intravenous injection can result in tolerance to transplanted donor cells or tissues or compositions comprising said cells with similar marks. In some embodiments, the tolerizing agent can be an anti-CD40 agent (e.g., agent comprising one or more amino acid sequences listed in any one of SEQ ID 1-6 and 8-17). In some embodiments, the tolerizing agent can be mammalian leukocytes fixed with a crosslinking agent. In some cases, the tolerizing agent can be mammalian leukocytes fixed with a crosslinking agent, administered together with an anti-CD40 binding agent (e.g., agent comprising one or more amino acid sequences listed in any one of SEQ ID 1-6 and 8-17). In some cases, the tolerizing agent can be mammalian leukocytes fixed with a crosslinking agent, surface-conjugated with an anti-CD40 binding agent (e.g., agent comprising one or more amino acid sequences listed in any one of SEQ ID 1-6 and 8-17).
- In some embodiments, the preparatory regimen can be inactivated or apoptotic donor cells that upon intravenous injection can result in tolerance to transplanted donor cells or tissues or compositions comprising said cells with similar marks. In some embodiments, the preparatory regimen can be an anti-CD40 agent (e.g., agent comprising one or more amino acid sequences listed in any one of SEQ ID 1-6 and 8-17). In some embodiments, the preparatory regimen can be mammalian leukocytes fixed with a crosslinking agent. In some cases, the preparatory regimen can be mammalian leukocytes fixed with a crosslinking agent, administered together with an anti-CD40 binding agent (e.g., agent comprising one or more amino acid sequences listed in any one of SEQ ID 1-6 and 8-17). In some cases, the preparatory regimen can be fixed mammalian leukocytes surface-conjugated with an anti-CD40 binding agent (e.g., agent comprising one or more amino acid sequences listed in any one of SEQ ID 1-6 and 8-17).
- Robust tolerance to allografts can be achieved without requiring same donor bone marrow or hematopoietic stem cell transplantation with the associated intense conditioning therapy by employing a negative vaccine strategy. In an exemplary embodiment, a tolerizing vaccine or preparatory regimen of the disclosure can comprise administering an anti-CD40 agent or an anti-CD40 ligand agent, e.g., any one or more of the peptides comprising an amino acid sequence with at least 80%, at least 85%, at least 90% or at least 95% sequence identity to the sequences set forth in any one of SEQ ID NOs. 1-6 and 8-17. In addition, the tolerizing vaccine or preparatory regimen can further comprise immunosuppression agents such as (i) an mTOR inhibitor, (ii) an anti-tumor necrosis factor agent or an anti-tumor necrosis factor receptor agent, or (iii) an
anti-interleukin 6 agent or an anti-interleukin 6 receptor agent. - In another embodiment, a tolerizing vaccine or preparatory regimen of the disclosure can comprise administering apoptotic cells (e.g., apoptotic donor leukocytes) to a recipient conjugated to an anti-CD40 agent or an anti-CD40 ligand agent, e.g., any one or more of the peptides comprising an amino acid sequence with at least 80%, at least 85%, at least 90% or at least 95% sequence identity to the sequences set forth in any one of SEQ ID NOs. 1-6 and 8-17. The short-term administration of these agents along with apoptotic donor leukocytes as disclosed herein can promote long-term tolerance to a transplanted cell, organ, or tissue despite not administering long term maintenance immunosuppression to the recipient.
- Cells for the preparation of a tolerizing vaccine or preparatory regimen can be obtained from any source, including animals, cells lines, and/or lab-generated cells. For example, the cells can be obtained from a human or non-human animal. In another example, the cells can be from a cell line (e.g., a human or non-human cell line). In some cases, the cells are human cells. In other cases, the cells are non-human cells. In some cases, the cells are of a non-human primate. In some cases, the cells are of a member of the Laurasiatheria superorder. In some cases, the cells are of an ungulate, for instance a camelid or a pig. In some cases, the cells are of a pig. In some cases, the cells are from the same species as the transplant donor. In some cases, the cells are from the same species as the transplant recipient. In some cases, the cells are from the same species as the transplant donor and the transplant recipient. In some cases, the cells are from a different species than the transplant donor. In some cases, the cells are from a different species than the transplant recipient. In some cases, the cells are from a different species than the transplant donor and the transplant recipient.
- Cells for the preparation of a tolerizing vaccine or preparatory regimen can be obtained from living donors or cadaveric donors. In some cases, the donor is a living donor. In some cases, the donor is a cadaveric donor. The cadaveric donor may be, for example, a brain dead, heart beating donor (BDD). The cadaveric donor may be, for example, a non-heart beating donor (NHBD). Whether the donor is a living donor or a cadaveric donor (e.g., a BDD or NHBD), the donor can be from any animal, for example, a human or non-human animal. In some cases, cells for the preparation of a tolerizing vaccine can be from the same donor as a graft or transplant. In some cases, cells for the preparation of a tolerizing vaccine can be from a different donor than the graft or transplant.
- Cells for the preparation of a tolerizing vaccine or preparatory regimen can be obtained from a donor animal of any age or stage of development. For example, the donor animal can be a fetal, perinatal, neonatal, pre-weaning, post-weaning, juvenile, young adult, or adult animal. In some cases, non-human animals can be past weaning age. For example, donor animals can be at least or at least about six months old. In some cases, donor animals can be at least or at least about 18 months old. In some cases, cells for the preparation of a tolerizing vaccine or preparatory regimen can be obtained (for example, differentiated) from stem cells (e.g., embryonic stem cells, induced pluripotent stem cells, and/or mesenchymal stem cells).
- The cells used to make a tolerizing vaccine or preparatory regimen can include one or more cells from tissues, organs, or bodily fluids. For example, the cells can be from tissues, organs, or bodily fluids including, but not limited to: brain, lung, liver, heart, spleen, pancreas, small intestine, large intestine, skeletal muscle, smooth muscle, skin, bones, adipose tissues, hairs, thyroid, trachea, gall bladder, kidney, ureter, bladder, aorta, vein, esophagus, diaphragm, stomach, rectum, adrenal glands, bronchi, ears, eyes, retina, genitals, hypothalamus, larynx, nose, tongue, spinal cord, or ureters, uterus, ovary, testis, blood, spinal fluid, lymph fluid, or a combination thereof.
- The cells used to make a tolerizing vaccine or preparatory regimen can include one or more types of cells. For example, the cells can include, but are not limited to: trichocytes, keratinocytes, gonadotropes, corticotropes, thyrotropes, somatotropes, lactotrophs, chromaffin cells, parafollicular cells, glomus cells melanocytes, nevus cells, Merkel cells, odontoblasts, cementoblasts corneal keratocytes, retina Muller cells, retinal pigment epithelium cells, neurons, glias (e.g., oligodendrocyte astrocytes), ependymocytes, pinealocytes, pneumocytes (e.g., type I pneumocytes, and type II pneumocytes), clara cells, goblet cells, G cells, D cells, ECL cells, gastric chief cells, parietal cells, foveolar cells, K cells, D cells, I cells, goblet cells, paneth cells, enterocytes, microfold cells, hepatocytes, hepatic stellate cells (e.g., Kupffer cells from mesoderm), cholecystocytes, centroacinar cells, pancreatic stellate cells, pancreatic α cells, pancreatic β cells, pancreatic δ cells, pancreatic F cells (e.g., PP cells), pancreatic c cells, thyroid (e.g., follicular cells), parathyroid (e.g., parathyroid chief cells), oxyphil cells, urothelial cells, osteoblasts, osteocytes, chondroblasts, chondrocytes, fibroblasts, fibrocytes, myoblasts, myocytes, myosatellite cells, tendon cells, cardiac muscle cells, lipoblasts, adipocytes, interstitial cells of cajal, angioblasts, endothelial cells, mesangial cells (e.g., intraglomerular mesangial cells and extraglomerular mesangial cells), juxtaglomerular cells, macula densa cells, stromal cells, interstitial cells, telocytes simple epithelial cells, podocytes, kidney proximal tubule brush border cells, sertoli cells, leydig cells, granulosa cells, peg cells, germ cells, spermatozoon ovums, lymphocytes, myeloid cells, endothelial progenitor cells, endothelial stem cells, angioblasts, mesoangioblasts, pericyte mural cells, mesenchymal stromal cells, or splenocytes (e.g., T lymphocytes, B lymphocytes, dendritic cells, microphages, leukocytes). In some cases, the cells used to make a tolerizing vaccine or preparatory regimen comprise a cell type that expresses MHC class II. In some cases, the cells used to make a tolerizing vaccine or preparatory regimen comprise a cell type that does not expresses MHC class II.
- A tolerizing vaccine or preparatory regimen can comprise leukocytes. Leukocytes can include, for example, neutrophils, eosinophils, basophils, lymphocytes, monocytes, or a combination thereof. Lymphocytes can include, for example, B lymphocytes (B cells), T lymphocytes (T cells), natural killer (NK) cells, or a combination thereof.
- Leukocytes in a tolerizing vaccine or preparatory regimen can be obtained from any source, including, for example, a donor, a cell line, or a differentiated stem cell. Leukocytes obtained from a donor can include leukocytes obtained from a spleen (e.g., splenocytes, splenic B cells); a liver; peripheral blood (including peripheral blood B cells); a lymph node; a thymus; bone marrow; or any other organ, tissue, or bodily fluid; or any combination thereof. In some cases, the tolerizing vaccine or preparatory regimen comprises splenic B cells, peripheral blood B cells, or a combination thereof. In some cases, the tolerizing vaccine or preparatory regimen comprises cells mobilized from the bone marrow to peripheral blood with a mobilization agent, e.g., cells mobilized with granulocyte colony-stimulating factor (G-CSF), granulocyte macrophage colony-stimulating factor (GM-CSF), mozobil, or a combination thereof. The leukocytes in the tolerizing vaccine or preparatory regimen that are obtained from a donor can comprise primary cells, cells expanded ex vivo, or a combination thereof.
- A donor of cells used in the preparation of a tolerizing vaccine or preparatory regimen can be genetically modified. Alternatively, or additionally, cells obtained from a donor can be genetically modified ex vivo. In some cases, cell lines are genetically modified to produce cells for use in a tolerizing vaccine or preparatory regimen. The genetically modified donors and/or cells can be produced using any method known in the art, including those described herein. Regardless of whether the genetically modified cells are isolated from a genetically modified animal, produced in culture, or a combination thereof, the genetically modified cells can be of any animal species, including human and non-human animals.
- Genetically modified cells used in a tolerizing vaccine or preparatory regimen can comprise one or more genetic modifications that reduce or eliminate expression or a gene or gene product (e.g., a protein). The genetic modification(s) can be modifications to the gene whose expression is reduced or eliminated. Such genes can be referred to as disrupted genes. The genetic modification(s) can also be to areas of the genome separate from the gene whose expression is reduced or eliminated (for example, modification to a promoter, enhancer, silencer, transcription factor, etc.). The genetically modified cells used in the tolerizing vaccine or preparatory regimen can comprise, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, or more genes whose expression is reduced or eliminated by genetic modification.
- Non-limiting examples of genes whose expression can be reduced or eliminated by genetic modification in the cells used in a tolerizing vaccine or preparatory regimen include, but are not limited to:
alpha CXC chemokine receptor 3 ligand (CXCL3); a CXC motif chemokine ligand10 (CXCL10) gene; MHC II transactivator (MHCIITA); a MHC class I polypeptide-related sequence A (MICA) gene; a MEW class I polypeptide-related sequence B (MICB) gene; a natural killer (NK) group 2D ligand (NKG2DL); a tumor necrosis factor receptor (TNF-R); a pig endogenous retrovirus (PERV); B2M, PD-1, PD-L1 or any combination thereof. - In some cases, the genetically modified cells used in a tolerizing vaccine can comprise disruptions in one or more genes comprising GGTA1, CMAH, B4GALNT2, B2M, NLRC5 or any combination thereof. For example, the genetically modified cells used to make a tolerizing vaccine or preparatory regimen can have disrupted GGTA1 only, or disrupted CMAH only, or disrupted B4GALNT2, B2M or NLRC5 only. The genetically modified cells used to make a tolerizing vaccine or preparatory regimen can also have disrupted GGTA1 and CMAH, disrupted GGTA1 and B4GALNT2, or disrupted CMAH and B4GALNT2, or disrupted NLRC5 and B2M. The genetically modified cells used to make a tolerizing vaccine or preparatory regimen can have disrupted GGTA1, CMAH, and B4GALNT2. Without wishing to be bound by any particular theory, such disruptions can minimize or eliminate cell-mediated immunity, antibody-mediated immunity, antibody-dependent cell-mediated immunity, and/or cell-dependent antibody-mediated immunity to organ, tissue, cell, and cell line grafts (e.g., xenografts or allografts comprising the same genetic modification(s) as the cells used in the tolerizing vaccine).
- Genetically modified cells used in a tolerizing vaccine or preparatory regimen can comprise, or further comprise, one or more genetic modifications that increase expression of one or more genes or gene products. The increased expression can be from zero expression, e.g., the increased expression can be of a gene or gene product that is not normally expressed in the cell without genetic modification. The increased expression can be compared to a threshold level, e.g., a level normally expressed in the cell without genetic modification. The genetic modification(s) can comprise one or more exogenous polynucleotides encoding a polypeptide (e.g., an endogenous or exogenous polypeptide).
- Non-limiting examples of exogenous polynucleotides include, but are not limited to, polynucleotides encoding one or more of an MHC I formation suppressor (e.g., an infected cell protein 47 (ICP47)); a regulator of complement activation (e.g., CD46, CD55, or CD59); an inhibitory ligand for NK cells; a B7 family member (e.g., a programmed death ligand such as PD-L1 or PD-L2); a serine protease inhibitor (e.g., Spi9); a galectin; an interleukin (e.g., IL-37); a CD40:CD40L blocking agent (e.g., a CD40 antagonist polypeptide, an anti-CD40 ligand polypeptide); a Fas ligand (FasL); any functional fragment thereof; or any combination thereof. In some embodiments, an inhibitory ligand for NK cells is a human leukocyte antigen (HLA), such as human leukocyte antigen E (HLA-E), human leukocyte antigen G (HLA-G), β-2-microglobulin (B2M) or any combination thereof. In some embodiments, the HLA-G is HLA-G1, HLA-G2, HLA-G3, HLA-G4, HLA-G5, HLA-G6, HLA-G7, or any combination thereof. In some cases, galectins is galectin-1, galectin-2, galectin-3, galectin-4, galectin-5, galectin-6, galectin-7, galectin-8, galectin-9, galectin-10, galectin-11, galectin-12, galectin-13, galectin-14, or galectin-15. For example, a galectin can be galectin-9.
- Donor leukocytes can be retrieved from a living donor's spleen. In one embodiment, B lymphocytes can be taken from the donor in one or more blood draws and/or apheresis procedures and optionally expanded ex vivo, or a separate cell donor can be identified that is a suitable match or partial match to the transplant donor. In some cases, separate donors can be used for the tolerogenic leukocytes and the allograft. For example, splenocytes from a cadaveric donor who is fully matched or partially matched (e.g., haploidentical) with the prospective living transplant donor are a clinically viable source of tolerogenic leukocytes. Upon availability of a matched, partially matched, or haploidentical spleen, a tolerization protocol can be initiated with the infusion of splenocytes on e.g., day −7 followed by the living donor transplant (e.g., a kidney transplant) on e.g.,
day 0 and the infusion of ex vivo expanded splenic B cells on e.g., onday + 1. - Cells (e.g., leukocytes) used in preparing a tolerizing vaccine/regimen or preparatory regimen can be expanded ex vivo. In some cases, leukocyte cells can be expanded in vitro in the presence of one or more reagents for a predetermined amount of time prior to use as a tolerizing vaccine. For instance, the cells can be contacted with at least one cytokine for about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 160, 165, 170, 175, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 390, or 300 hours. In some cases, the cells can be contacted with at least one cytokine for about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more days. In some cases, the cells can be contacted with at least one cytokine for about 1 to 2, 2 to 3, 3 to 4, 1 to 4, 1 to 3, or 2 to 4 weeks. In some cases, the cytokine is one or more interleukin. In some cases, the interleukin (IL) is at least one of IL-2, IL-4, IL-21, BAFF, multimer CD40L, IL-10, IL-12, and IL-15. In some cases, the cells are contacted with IL-2, IL-4, IL-21, BAFF, and multimer CD40L.
- Leukocytes used in a tolerizing vaccine/regimen or preparatory regimen can comprise at least or at least about 10%, e.g., 25%, CD19 positive, CD20 positive, or CD21 positive MHC Class II positive B cells. For example, donor splenocytes can comprise at least or at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or 80% CD19, CD20, and/or CD21 positive MHC Class II positive B cells, e.g., about, at least, or at least about 10 to 20; 20 to 30; 30 to 40; 40 to 50; 50 to 60%, or 60 to 70%. In some cases, splenic B cells or leukocytes used in a tolerizing vaccine/regimen or preparatory regimen can comprise at least or at least about 60%, e.g., 90%, CD19, CD20, or CD21 positive MHC Class II positive B cells. For example, splenic B cells or leukocytes used in a tolerizing vaccine/regimen or preparatory regimen can comprise can comprise about, at least, or at least about 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% CD19, CD20, or CD21 positive MEW Class II positive B cells e.g., at least or at least about 60 to 70; 70 to 80; 80 to 90; or 90 to 95%. In some cases, donor splenocytes or leukocytes used in a tolerizing vaccine/regimen or preparatory regimen can comprise from or from about 50% to 100%, e.g., from or from about 60% to 100% or 80% to 100%, CD19, CD20, or CD21 positive MEW Class II positive B cells. In some embodiments the MEW class II is swine leukocyte antigen (SLA) Class II. In some embodiments the MEW class II is human leukocyte antigen (HLA) Class II.
- A tolerizing agent of the methods herein can comprise apoptotic cells, non-apoptotic cells, or a combination thereof. Cells for a tolerizing vaccine or preparatory regimen can be made apoptotic a number of different ways. For example, the cells can be contacted with a chemical (e.g., a fixative or cross-linking agent, a cellular damaging agent, or a combination thereof), to make some or all of the cells apoptotic. In another example, the cells can be irradiated (e.g., with ultraviolet radiation, gamma radiation, etc.) to make some or all of the cells apoptotic.
- Cells for a tolerizing vaccine or preparatory regimen can be contacted with a chemical, such as a fixative or cross-linking agent, a carbodiimide, or a carbodiimide derivative, a diimidoester, a sulfhydryl-to-sulfhydryl crosslinker, an amine-to-sulfhydryl crosslinker, a sulfhydryl-to-carbohydrate crosslinker, a photoreactive crosslinker, an in vivo crosslinker, a cellular damaging agent or an apoptosis inducer. The contacting can make some or all of the cells apoptotic. Suitable fixatives or cross-linking agents include, but are not limited to: an amine-to-amine crosslinker, a sulfhydryl-to-sulfhydryl crosslinker, an amine-to-sulfhydryl crosslinker, an in vivo crosslinker, a sulfhydryl-to-carbohydrate crosslinker, a photoreactive crosslinker, a chemoselective ligation crosslinking agent, a carboxyl-to-amine crosslinker, a carbodiimide, genipin, acrylic aldehyde, diformyl, osmium tetroxide, a diimidoester, mercuric chloride, zinc sulphate, zinc chloride, trinitrophenol (picric acid), potassium dichromate, ethanol, methanol, acetone, acetic acid, or a combination thereof.
- The carbodiimide can comprise ethylcarbodiimide; ethylene carbodiimide; N,N′-diisopropylcarbodiimide (DIC); N,N′-dicyclohexylcarbodiimide (DCC); 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDCI, EDC, ECDI, or EDAC); or a combination thereof. In some cases, the carbodiimide comprises ethylcarbodiimide. In some cases, the carbodiimide comprises ethylene carbodiimide. In some cases, the carbodiimide comprises N,N′-diisopropylcarbodiimide (DIC). In some cases, the carbodiimide comprises N,N-dicyclohexylcarbodiimide (DCC). In some cases, the carbodiimide comprises 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDCI, EDC, ECDI, or EDAC). In some cases, the tolerizing vaccine comprises cells treated with EDCI derivatives and/or functionalized EDCI. Methods of preparation, administration and use of tolerizing agent e.g., a tolerizing vaccine is well known in the art, for example, see U.S. Pat. No. 8,734,786B2, U.S. Pat. No. 9,888,673B2.
- Cells for a tolerizing vaccine or preparatory regimen can be contacted with a diimidoester. The contacting can be for a pre-determined time. The contacting can make some or all of the cells apoptotic. The diimidoester can comprise cyanuric chloride; diisocyanate; diethylpyrocarbonate (DEPC) or diethyl dicarbonate; a maleimide; benzoquinone; or a combination thereof.
- Cells for a tolerizing vaccine or preparatory regimen can be contacted with an amine-to-amine crosslinker. The contacting can be for a pre-determined time. The contacting can make some or all of the cells apoptotic. In some cases, the amine-to-amine-crosslinker comprises disuccinimidyl glutarate (DSG); disuccinimidyl suberate (DSS); bis(sulfosuccinimidyl)suberate (BS3); tris-(succinimidyl) aminotriacetate (TSAT); BS(PEG)5; BS(PEG)9; dithiobis(succinimidyl propionate) (DSP); 3,3′-dithiobis(sulfosuccinimidyl propionate) (DTSSP); disuccinimidyl tartrate (DST); bis(2-(succinimidooxycarbonyloxy)ethyl)sulfone (BSOCOES); ethylene glycol bis(succinimidyl succinate) (EGS); sulfo-EGS; or any combination thereof. In some cases, the amine-to-amine crosslinker comprises an imidoester, such as dimethyl adipimidate (DMA); dimethyl pimelimidate (DMP); dimethyl suberimidate (DMS);
dimethyl - Cells for a tolerizing vaccine or preparatory regimen can be contacted with a sulfhydryl-to-sulfhydryl crosslinker. The contacting can be for a pre-determined time. The contacting can make some or all of the cells apoptotic. In some cases, the sulfhydryl-to-sulfhydryl crosslinker comprises a maleimide, such as bismaleimidoethane (BMOE); 1,4-bismaleimidobutane (BMB); bismaleimidohexane (BMH); tris(2-maleimidoethyl)amine (TMEA); BM(PEG)2 (such as 1,8-bismaleimido-diethyleneglycol); BM(PEG)3 (such as 1,11-bismaleimido-triethyleneglycol), dithiobismaleimidoethane (DTME); or any combination thereof.
- Cells for a tolerizing vaccine or preparatory regimen can be contacted with an amine-to-sulfhydryl crosslinker. The contacting can be for a pre-determined time. The contacting can make some or all of the cells apoptotic. In some cases, the amine-to-sulfhydryl crosslinker comprises a NHS-haloacetyl crosslinker, a NHS-maleimide, a NHS-pyridyldithiol crosslinker, a sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) crosslinker, or any combination thereof. The NHS-haloacetyl crosslinkers can comprise succinimidyl iodoacetate (SIA); succinimidyl 3-(bromoacetamido)propionate (SBAP); succinimidyl (4-iodoacetyl)aminobenzoate (SIAB); sulfo-SIAB; or a combination thereof. The NHS-maleimide can comprise N-α-maleimidoacet-oxysuccinimide ester (AMAS); N-β-maleimidopropyl-oxysuccinimide ester (BMPS); N-γ-maleimidobutyryl-oxysuccinimide ester (GMBS); sulfo-GMBS; m-maleimidobenzoyl-N-hydrosuccinimide ester (MBS); sulfo-MBS; SMCC; sulfo-SMCC; N-ε-malemidocaproyl-oxysuccinimide ester (EMCS); sulfo-EMCS; succinimidyl 4-(p-maleimidophenyl)butyrate (SMPB); sulfo-SMPB; succinimidyl 6-((beta-maleimidopropionamido)hexanoate) (SMPH); sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxy-(6-amidocaproate) (LC-SMCC); N-κ-maleimidoundecanoyl-oxysulfosuccinimide ester (sulfo-KMUS); or a combination thereof. The NHS-pyridyldithiol crosslinker can comprise succinimidyl 3-(2-pyridyldithio)propionate (SPDP), succinimidyl 6-(3(2-pyridyldithio)propionamido)hexanoate (LC-SPDP), sulfo-LC-SPDP, or 4-succinimidyloxycarbonyl-alpha-methyl-α(2-pyridyldithio)tolune (SMPT).
- Cells for a tolerizing vaccine or preparatory regimen can be contacted with a sulfhydryl-to-carbohydrate crosslinker. The contacting can be for a pre-determined time. The contacting can make some or all of the cells apoptotic. In some cases, the sulfhydryl-to-carbohydrate crosslinker comprises (N-β-maleimidopropionic acid hydrazide (BMPH), N-ε-maleimidocaproic acid hydrazide (EMCH), 4-(4-N-maleimidophenyl)butyric acid hydrazide (MPBH), N-κ-maleimidoundecanoic acid hydrazide (KMUH), 3-(2-pyridyldithio)propionyl hydrazide (PDPH), or any combination thereof.
- In some cases, the carboxyl-to-amine crosslinker is dicyclohexylcarbodiimide (DCC), 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDCI, EDC, or EDAC), N-hydroxysuccinimide (NHS), sulfo-NHS, or any combination thereof.
- Cells for a tolerizing vaccine or preparatory regimen can be contacted with a photoreactive crosslinker. The contacting can be for a pre-determined time. The contacting can make some or all of the cells apoptotic. In some cases, the photoreactive crosslinker comprises a NHS ester/aryl azide, a NHS ester/diazirine, or a combination thereof. The NHS ester/aryl azide can comprise N-5-azido-2-nitrobenzoyloxysuccinimide (ANB-NOS), sulfo-SANPAH, or a combination thereof. The NHS ester/diazirine can comprise SDA (NHS-diazirine/
succinimidyl - Cells for a tolerizing vaccine or preparatory regimen can be contacted with an in vivo crosslinker. The contacting can be for a pre-determined time. The contacting can make some or all of the cells apoptotic. The in vivo crosslinker can comprise BS3, DTSSP, sulfo-EGS, DSG, DSP, DSS, EGS, sulfo-SDA, sulfo-LC-SDA, sulfo-SDAD, SDA, LC-SDA, SDAD, NHS-ester diazirine, or any combination thereof.
- In some cases, the cells for use in a tolerizing vaccine or preparatory regimen are treated with a cellular damaging agent or an apoptosis inducer. In some cases, the cellular damaging agent induces apoptosis in some or all of the contacted cells. Non-limiting exemplary cellular damaging agents include doxorubicin, staurosporine, etoposide, comptothecin, paclitaxel, vinblastine, or any combination thereof. Non-limiting exemplary apoptosis inducers include marinopyrrole A, maritoclax, (E)-3,4,5,4′-tetramethoxystilbene, 17-(Allylamino)-17-demethoxygeldanamycin, 2,4,3′,5′-tetramethoxystilbene, 2OHOA, 6,8-bis(benzylthio)-octanoic acid, AT101, apoptolidin, FU 40A, ara-G hydrate, arylquin 1, BAD, BAM7, BAX activator molecule 7, BH3I-1, BID, BMS-906024, BV02, bendamustine, borrelidin, borrelidine, cyclopentanecarboxylic acid, NSC 216128, treponemycin, brassinin, brassinine, brefeldin A, ascotoxin, BFA, cyanein, decumbin, bufalin, CCF642, CCT007093, CD437, CHM-1 hydrate, 2-(2-fluorophenyl)-6,7-methylenedioxy-2-4-quinolone hydrate, NSC 656158, CIL-102, CP-31398, dihydrochloride hydrate, camalexin, 3-(Thiazol-2-yl)-1H-indole, camalexine, carboxyatractyloside, cepharanthine, cepharanthine, cinnabarinic acid, cirsiliol, combretastatin A4, costunolide, DBeQ, DIM-C-pPhtBu, DMXAA, DPBQ, enniatin A1, enniatin A, enniatin B1, enniatin B, erastin, eupatorin, FADD, fluticasone propionate, fosbretabulin disodium, GO-201 trifluoroacetate, gambogic acid, HA 14-1, HMBA, hexaminolevulinate (HAL), IMB5046, IMS2186, ikarugamycin, imiquimod, iniparib, kurarinone, LLP-3, lipocalin-2, lometrexol, MI-4F, ML 210, ML291, mollugin, muristerone A, NA-17, NID-1, NPC26, NSC59984, Nap-FF, neocarzinostatin, nifetepimine, nitidine chloride, nutlin-3, nutlin-3a, PKF118-310, PRIMA-1, PRT4165, pemetrexed, penta-O-galloyl-β-D-glucose hydrate, phenoxodiol, prodigiosin (PG), psoralidin, pterostilbene, raltitrexed, raptinal, ridaifen-B, rifabutin, roslin 2, s-p-bromobenzylglutathione cyclopentyl diester, SJ-17255, SMBA1, STF-62247, suprafenacine, syrosingopine, talniflumate, taurolidine, temoporfin, temozolomide, tetrazanbigen, thaxtomin A, thiocolchicine, tirapazamine, UCD38B, UMI-77, undecylprodigiosin, VK3-OCH3, vacquinol-1, violacein, vosaroxin, zerumbone, gAcrp30, gAcrp30/adipolean, or any combination thereof. Cells contacted with a cellular damaging agent or an apoptosis inducer may subsequently be contacted with a fixative or cross-linking agent.
- Cells for a tolerizing vaccine or preparatory regimen can be made apoptotic by contacting the cells with a chemical (e.g., a fixative or cross-linking agent, a cellular damaging agent, or a combination thereof) for a predetermined amount of time. In some embodiments, the cells in the tolerizing vaccine or the preparatory regimen are made apoptotic by fixing for a predetermined amount time with the crosslinking agent (e.g., ECDI). In some cases, the predetermined amount of time is about 30 minutes, about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 12 hours, about 18 hours, about 24 hours, about 36 hours, about 48 hours, about 60 hours, or about 72 hours. In some cases, the predetermined amount of time is less than an hour. In some cases, the predetermined time is at least about 1 minute, 5 minutes, 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 60 minutes, 75, minutes, 90 minutes, 120 minutes, 150 minutes, 180 minutes, 210 minutes or 240 minutes. In some cases, the predetermined time is at most about 30 minutes, 40 minutes, 50 minutes, 60 minutes, 75, minutes, 90 minutes, 120 minutes, 150 minutes, 180 minutes, 210 minutes or 240 minutes. In some cases, the predetermined amount of time is about 1 minute to about 240 minutes, 1 minute to about 10 minutes, 10 minutes to about 240 minutes, about 10 minutes to about 180 minutes, about 10 minutes to about 120 minutes, about 10 minutes to about 90 minutes, about 10 minutes to about 60 minutes, about 10 minutes to about 30 minutes, about 30 minutes to about 240 minutes, about 30 minutes to about 180 minutes, about 30 minutes to about 120 minutes, about 30 minutes to about 90 minutes, about 30 minutes to about 60 minutes, about 50 minutes to about 240 minutes, about 50 minutes to about 180 minutes, about 50 minutes to about 120 minutes, about 50 minutes to about 90 minutes, about 50 minutes to about 60 minutes, about 10 minutes to about 20 minutes, about 20 minutes to about 30 minutes, about 30 minutes to about 40 minutes, about 40 minutes to about 50 minutes, about 50 minutes to about 60 minutes, about 60 minutes to about 70 minutes, about 70 minutes to about 80 minutes, about 80 minutes to about 90 minutes, about 90 minutes to about 100 minutes, about 100 minutes to about 110 minutes, about 110 minutes to about 120 minutes, about 10 minutes to about 30 minutes, about 30 minutes to about 50 minutes, about 50 minutes to about 70 minutes, about 70 minutes to about 90 minutes, about 90 minutes to about 110 minutes, about 110 minutes to about 130 minutes, about 130 minutes to about 150 minutes, about 150 minutes to about 170 minutes, about 170 minutes to about 190 minutes, about 190 minutes to about 210 minutes, about 210 minutes to about 240 minutes, up to about 30 minutes, about 30 minutes to about 60 minutes, about 60 minutes to about 90 minutes, about 90 minutes to about 120 minutes, or about 120 minutes to about 150 minutes.
- The contacting can be at any temperature. In some cases the contacting is performed on ice (e.g., at 4° C.). In other cases, the contacting is performed at room temperature. In some cases, the contacting is performed at a temperature of at least about 0° C., 2° C., 4° C., 8° C., 15° C., 20° C., 25, 30° C., 35° C., or 37° C. In some cases, the contacting is performed at a temperature of at most about 4° C., 8° C., 15° C., 20° C., 25, 30° C., 35° C., 37° C., or 40° C. In some cases, the contacting is performed at a temperature of about 0° C. to about 37° C., about 0° C. to about 25° C., about 0° C. to about 15° C., about 0° C. to about 10° C., about 0° C. to about 8° C., about 0° C. to about 6° C., about 0° C. to about 4° C., about 0° C. to about 2° C., about 2° C. to about 10° C., about 2° C. to about 8° C., about 2° C. to about 6° C., about 4° C. to about 25° C., about 4° C. to about 10° C., about 15° C. to about 37° C., about 15° C. to about 25° C., about 20° C. to about 40° C., about 20° C. to about 37° C., or about 20° C. to about 30° C.
- Cells in a tolerizing vaccine or preparatory regimen can aggregate as a result of the method of making some or all of the cells apoptotic. For example, cells can aggregate after contacting with a chemical, such as a fixative or crosslinking agent. The predetermined amount of time that the cells are contacted with the chemical can be selected to minimize the amount of aggregation in the tolerizing vaccine or preparatory regimen. In some cases, aggregates can be removed, for example, by washing and/or filtration.
- In some cases, a tolerizing vaccine or preparatory regimen can comprise from or from about 0.01 to 10 aggregates, per μl. For example, the tolerizing vaccine or preparatory regimen can comprise from or from about 0.01 to 1, 0.1 to 1, 0.25 to 1, 0.5 to 1, 1 to 5; or 1 to 10 aggregate per μl. The tolerizing vaccine or preparatory regimen can comprise less than about 0.1, 0.5, 0.75, 1, 5, or 10 aggregates per μL.
- In some cases, the tolerizing vaccine or preparatory regimen can comprise less than 5 aggregates per μL. For example, the tolerizing vaccine or preparatory regimen can comprise less than about: 5, 4, 3, 2, 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.05, or 0.01 aggregates per μL.
- In some case, the tolerizing vaccine or preparatory regimen comprises 1 or fewer aggregates per μL. For example, the tolerizing vaccine or preparatory regimen can comprise about 0.01, about 0.05, about 0.1, about 0.2, about 0.3, about 0.4, about 0.5 about 0.6, about 0.7, about 0.8, about 0.9, or about 1.0 aggregates per μL.
- The tolerizing vaccine or preparatory regimen can include from or from about 0.01% to 10%, e.g., from or from about 0.01% to 2%, necrotic cells. For example, cells of a tolerizing vaccine or preparatory regimen can comprise from or from about 0.01% to 10%; 0.01% to 7.5%, 0.01% to 5%; 0.01% to 2.5%; or 0.01% to 1% necrotic cells. In some embodiments, the cells of a tolerizing vaccine or preparatory regimen of the disclosure can comprise at most about 0.01%, 0.1%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% necrotic cells.
- As used herein, the term “conjugate” or “conjugated to,” and the like refer to molecular entities (.e.g, peptides of the present disclosure and an apoptotic leucocyte) being linked together through covalent or non-covalent bonds. Conjugation may be accomplished by directly coupling the two molecular entities, e.g., creating an ester or amide from an hydroxyl group, amino group, and a carboxylic acid. Conjugation may be accomplished by indirectly coupling the two molecular entities, e.g., instituting a linking group such as a polyethylene glycol. Conjugation may be accomplished by modifying the molecular entities with chemical groups that react with one another, e.g., alkyne-functionalized entity with an azide-functionalized entity or the reduction of thiol groups on individual entities to form a disulfide bond. Conjugates such as ethylene carbodiimide (ECDI), hexamethylene diisocyanate, propyleneglycol di-glycidylether which contain 2 epoxy residues, and epichlorohydrin can be used for fixation of peptides or proteins to the apoptotic leucocyte surface. Reactive carboxyl groups on the surface of an apoptotic leukocyte can be joined to free amines (e.g., from Lys residues) on the peptide or protein, by reacting them with, for example, 1-ethyl-3-[3,9-dimethyl aminopropyl] carbodiimide hydrochloride (EDC) or N-hydroxysuccinimide ester (NHS). Similarly, the same chemistry may be used to conjugate free amines on the surface of an apoptotic leukocyte with free carboxyls (e.g., from the C-terminus, or from Asp or GIu residues) on the peptide or protein. Alternatively, free amine groups on the surface of an apoptotic leuckocyte may be covalently bound to peptides and proteins, or peptide or protein fusion proteins, using sulfo-SIAB chemistry, essentially as described by Arano et al. (1991) Chem. 2:71-6. A great variety of means, well known in the art, may be used to conjugate the peptides to surface of apoptotic leuckocytes. These methods include any standard chemistry which do not destroy or severely limit the biological activity of the peptides and that of the apoptotic leuckocytes, and which allow for a sufficient number of peptides to be conjugated to the surface in an orientation which allows for inducing tolerance. In some embodiments the C-terminal regions of a peptide are conjugated. In other embodiments, the N-terminus of a peptide can be conjugated onto the surface of the apoptotic leucocyte.
- In one aspect, the present disclosure provides preparatory regimen and/or tolerizing vaccines and regimen comprising apoptotic cells such as leucocytes or mesenchymal stromal cells comprising one or more peptides conjugated to the surface of the apoptotic cell. In some cases, the one or more peptides can comprise an amino acid sequence set forth in any one of SEQ ID NOs. 1-6 and 8-17. In some cases, the peptide conjugated to the surface of an apoptotic cell such as a leukocyte can vary in length. In some cases, the one or more peptides can comprise a sequence set forth in any one of SEQ ID NOs. 1-6 and 8-17 and can be at least about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 275, 200, or 250 amino acids in length. In some cases, the one or more peptides can comprise a sequence from 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, 99.9%, 99.95% or 100% identical to the sequences set forth in any of SEQ ID NOs. 1-6 and 8-17. For example, in some cases, the one or more peptides can comprise a sequence set forth in any one of SEQ ID NOs. 1-6 and 8-17 and can be about 9, about 10, about 1, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 75, about 80, about 90, about 100, about 120, about 140, about 150, about 160, about 170, about 180, about 200, about 220, about 250, about 270, about 300, about 330, about 350, about 400, about 440, about 500, about 600, about 700, about 800, about 900 or about 1000 amino acids in length. In some cases, the one or more peptides can comprise a sequence set forth in any one of SEQ ID NOs. 1-6 and 8-17 and be at most about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 275, 200, or 250 amino acids in length. In some cases, the peptides be about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 275, 200, or 250 amino acids in length. In some embodiments, the one or more peptides can comprise a sequence set forth in any one of SEQ ID NOs. 1-6 and 8-17 and can be about 5-250, 10-250, 20-250, 30-250, 50-250, 100-250, 5-100, 10-100, 20-100, 30-100, 50-100, 5-50, 10-50, 20-50, 30-50, 5-30, 10-30, 15-30, or 20-30 amino acids in length.
- In some embodiments, the peptides conjugated on the surface of apoptotic leukocytes are derived from a MHC molecule. The term “MHC molecule” refers to a molecule comprising Major Histocompatibility Complex (MHC) glycoprotein protein sequences. The term “MHC” as used herein will be understood to refer to the Major Histocompability Complex, which is defined as a set of gene loci specifying major histocompatibility complex glycoprotein antigens including the human leukocyte antigen (HLA). The term “HLA” as used herein will be understood to refer to Human Leukocyte Antigens, which is defined as the major histocompatibility antigens found in humans. As used herein, “HLA” is the human form of “MHC” and therefore can be used interchangeably. Examples of HLA proteins that can be utilized in accordance with the presently disclosed and claimed inventive concept(s) include, but are not limited to, an HLA class I α chain, an HLA class II α chain and an HLA class II β chain. Specific examples of HLA class II α and/or β proteins that may be utilized in accordance with the presently disclosed and claimed inventive concept(s) include, but are not limited to, those encoded at the following gene loci: HLA-DRA; HLA-DRB1; HLA-DRB3,4,5; HLA-DQA; HLA-DQB; HLA-DPA; and HLA-DPB.
- In some embodiments, the apoptotic cells further comprise one or more peptides derived from a MHC class I molecule. As such, the sequences of amino acid residues in the peptide can be substantially similar or functionally comparable to a polypeptide sequence in the MHC molecule. Thus, “a peptide derived from a MHC class II molecule” refers to a peptide that has a sequence “from a region in an MHC class II molecule” (e.g., the hypervariable region), and is a peptide that has a sequence 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, 99.9%, 99.95% or 100% identical to the naturally occurring MHC amino acid sequence of the region. In some embodiments, the peptide derived from a MHC class II molecule can comprise a sequence from 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, 99.9%, 99.95% or 100% identical to the hypervariable region of the WIC class II molecule. Thus, “a peptide derived from a MHC class I molecule” refers to a peptide that has a sequence “from a region in an WIC class I molecule” (e.g., the hypervariable region), and is a peptide that has a sequence 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, 99.9%, 99.95% or 100% identical to the naturally occurring WIC amino acid sequence of the region. In some embodiments, the peptide derived from a MHC class I molecule will comprise a sequence from the hypervariable region of the MHC class I molecule.
- The peptides derived from a recipient's MHC class II molecule may comprise an entire WIC class II molecule. The peptides derived from a recipient's WIC class II molecule may comprise an entire α chain of DR, DQ or DP. The peptides derived from a recipient's MHC class II molecule may comprise entire β chain of DR, DQ, or DP. The peptides derived from a recipient's WIC class II molecule may comprise entire α1 and/or α2 domains of DR, DQ or DP. The peptides derived from a recipient's WIC class II molecule may comprise entire β1 and/or β2 domains of DR, DQ, or DP. The peptides derived from a recipient's WIC class II molecule may comprise MHC-DR1, MHC-DR2, MHC-DR3, MHC-DR4, and/or MHC-DRS.
- The peptides derived from a recipient's MHC class II molecule may comprise a fragment of an α1 and/or α2 domain of DR, DQ or DP. The peptides derived from a recipient's WIC class II molecule may comprise a fragment of a β1 and/or β2 domain of DR, DQ, or DP. The peptides derived from a recipient's WIC class II molecule may comprise a fragment of MHC-DR1, MHC-DR2, MHC-DR3, MHC-DR4, and/or MHC-DRS. The peptides derived from a recipient's MHC class II molecule may comprise a sequence from a hypervariable region. The peptides derived from a recipient's WIC class II molecule can comprise an in silico-identified high, medium, or low affinity peptides from the hypervariable region of the DRB molecule (e.g., a 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid peptide).
- The peptides derived from a recipient's MHC class II molecule may be synthesized or recombinant. In some cases, the peptides derived from a recipient's MHC class II molecule may between about 10 and 30 amino acids in length. The peptides derived from a recipient's MHC class II molecule may be at least 10 to 30 amino acids in length. In some embodiments, the peptides derived from a recipient's MHC class II molecule can be at least about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 275, 200, or 250 amino acids in length. In some embodiments, the peptides derived from a recipient's MHC class II molecule can be at most about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 275, 200, or 250 amino acids in length. In some embodiments, the peptides derived from a recipient's MHC class II molecule can be about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 275, 200, or 250 amino acids in length. In some embodiments, the peptides derived from a recipient's MHC class II molecule can be about 5-250, 10-250, 20-250, 30-250, 50-250, 100-250, 5-100, 10-100, 20-100, 30-100, 50-100, 5-50, 10-50, 20-50, 30-50, 5-30, 10-30, 15-30, or 20-30 amino acids in length.
- Cells (e.g., leukocytes) for a tolerizing vaccine or preparatory regimen can be treated with a fixative or crosslinking agent (e.g., a carbodiimide such as ECDI) in the presence of one or more antigens and/or epitopes. In some cases, the epitopes can comprise a peptide containing an amino acid sequence set forth in any one of SEQ ID NOs. 1-6 and 8-17. In some cases, the antigens and/or epitopes can comprise antigens and/or epitopes from a transplant donor, a transplant recipient, a third party, or a combination thereof. In some cases, the cells in the tolerizing vaccine or preparatory regimen are coupled to recipient antigens and/or epitopes. In some cases, the cells in the tolerizing vaccine or preparatory regimen are coupled to third party antigens and/or epitopes. In some cases, the cells in the tolerizing vaccine or preparatory regimen are coupled to transplant donor antigens and/or epitopes.
- In some embodiments, a “cocktail” of peptides can be conjugated on the surface of the apoptotic leucocytes. In some embodiments, the apoptotic cells such as leucocytes can comprise one or more peptides derived from MHC class II molecule. In some embodiments, the apoptotic cells such as leucocytes can comprise, for example, at least 2, 3, 5, 7, 10, 15, 20, 30, 40, 50, 100 or more peptides derived from a MHC class II molecule. In some embodiments, the apoptotic leucocytes can comprise one or more peptides derived from a MHC class I molecule. In some embodiments, the apoptotic cells such as leucocytes can comprise, for example, at least 2, 3, 5, 7, 10, 15, 20, 30, 40, 50, 100 or more peptides derived from a MHC class I molecule. A mixture of more than one peptide derived from a MHC class II molecule, has the advantage of inducing increased immune tolerance response in the recipient. The increased tolerance can be, for example, through a mechanism called linked supersession. The mechanism of linked suppression will be known to an artisan skilled in the art. For instance, peptides comprising sequences from hypervariable regions of α and β chains may be used in combination.
- In certain embodiments, the size of a protein or a peptide that is conjugated to the surface of the apoptotic leukocyte may comprise, but is not limited to 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, 800, 825, 850, 875, 900, 925, 950, 975, 1000, 1100, 1200, 1300, 1400, 1500, 1750, 2000, 2250, 2500 amino molecules or greater, including any range or value derivable therein, or derivative thereof. It is contemplated that peptides might be altered by fusing or conjugating a heterologous peptide sequence with a particular function (e.g., for presentation as a protein complex, for enhanced immunogenicity, etc.).
- Cells (e.g., leukocytes) for a tolerizing vaccine or preparatory regimen can be treated with a fixative or crosslinking agent (e.g., a carbodiimide such as ECDI) in the presence of one or more immunomodulatory molecules. In some cases, the one or more immunomodulatory molecules can comprise one or more peptides comprising an amino acid sequence set forth in any one of SEQ ID NOs. 1-6 and 8-17. In some cases, the one or more immunomodulatory molecules can comprise all or a portion of: IFN-γ, an NF-kB inhibitor, vitamin D3, siCD40, anti-CD40 antibody, cobalt protoporphyrin, insulin B9-23, α1-antitrypsin, a cluster of differentiation protein, a gp39 antagonist, α1-antitrypsin, CD47, PD-L1, PD-L2, CTLA-4, rapamycin, compstatin, abatacept, ipilimumab, or a combination thereof. The NF-kB inhibitor can comprise dehydroxymethylepoxyquinomicin (DHMEQ), curcumin, triptolide, Bay-117085, or a combination thereof. The cluster of differentiation protein can comprise CD4, CD46, CD47, CD55, CD59, or a portion thereof, or a combination thereof.
- In some embodiments, the one or more immunomodulatory molecules can comprise all or a portion of a calcineurin inhibitor (e.g., cyclosporine or tacrolimus), a costimulatory signal blockade, an IL-2 signaling inhibitor (e.g., daclizumab or basiliximab), a cell cycle blocker (e.g., mycophenolate mofetil (MMF) or azathioprine), a T cell recirculation inhibitor (e.g., FTY720 or another sphingosine 1-phosphate (S1P) receptor agonist), a nitrogen mustard alkylating agent (e.g., cyclophosphamide), a complement C3 or C5 inhibitor, or any combination thereof.
- In some embodiments, the immunomodulatory molecules can target T cell receptor (TCR), CD3e, FK506-binding protein 12 (FKBP12), cytotoxic T lymphocyte associated protein 4 (CTLA-4), programmed cell death protein 1 (PD-1, e.g., pembrolizumab), programmed death ligand 1 (PD-L1, e.g., MPDL3280A), CD40L (CD154), CD40, inducible costimulatory (ICOS), IL-2, TNF-α (e.g., infliximab), IL-6 or IL-6R (e.g., tocilizumab, actemra, clazakizumab, ALD518, siltuximab, elsilimomab, sirukumab, sarilumab, olokizumab), IL-7, CD2, CD20, CD52, α-4 integrin, mTOR (e.g., rapamycin or everolimus), DNA synthesis, molecules in pro-inflammatory pathways (e.g., cytokines, α1-antitrypsin, NFkB), or any combination thereof.
- Cells (e.g., leukocytes) for tolerizing vaccine or preparatory regimen can be treated with a fixative or crosslinking agent (e.g., a carbodiimide such as ECDI) in the presence of an agent that increases expression of anti-inflammatory cytokines in a recipient. In some cases, the agent can comprise one or more peptides comprising an amino acid sequence set forth in any one of SEQ ID NOs. 1-6 and 8-17. In some cases, the anti-inflammatory cytokines may include, for example, TGF-β, IL-10, IL-13, or a combination thereof. In some cases, the agent that increases expression of anti-inflammatory cytokines in the recipient comprises α1-antitrypsin.
- In some cases where ADLs are administered to a transplant recipient multiple times, ADLs from all the doses can all be conjugated with the same immunomodulatory molecules, agents that increases expression of anti-inflammatory cytokines, and/or antigens or epitopes. In some cases where ADLs are administered to a transplant recipient multiple times, ADLs from one or more doses can be conjugated with a first set of immunomodulatory molecules, agents that increases expression of anti-inflammatory cytokines, and/or antigens or epitopes, and ADLs from other dose(s) can be conjugated with a different set of immunomodulatory molecules, agents that increases expression of anti-inflammatory cytokines, and/or antigens or epitopes.
- Short Term Immunosuppression
- In some cases, a preparatory regimen or tolerizing vaccine of the disclosure can comprise administering one or more immunosuppression agents and/or anti-inflammatory agents to a transplant recipient. For example, in some cases, the regimen may include administering one or more immunosuppression agents and/or anti-inflammatory agents in addition to apoptotic cells (such as apoptotic donor leukocytes or mesenchymal stromal cells). In other cases, the regimen may include administering one or more immunosuppression agents and/or anti-inflammatory agents in addition to an anti-CD40 agent, for e.g., a peptide comprising an amino acid sequence with at least 80%, at least 85%, at least 90% or at least 95% sequence identity to the sequences set forth in any one of SEQ ID NOs. 1-6 and 8-17.
- In some embodiments, the short term immunosuppression comprises administering an antagonistic anti-CD40 agent. An anti-CD40 agent or an anti-CD40 ligand agent can be, for example, a peptide comprising an amino acid sequence with at least 80%, at least 85%, at least 90% or at least 95% sequence identity to the sequences set forth in any one of SEQ ID NOs. 1-6 and 8-17. In some cases, the anti-CD40 agent or anti-CD40 ligand agent can be, for example, a peptide comprising an amino with at least 80%, 85%, 88%, 90%, 93%95% or 99% sequence identity to the sequences set forth in any one of SEQ ID NOs. 1-6 and 8-17.
- In some cases, an anti-CD40 agent or an anti-CD40 ligand agent can be, for example, an antagonistic anti-CD40 antibody, and antagonistic anti-CD40 ligand antibody, or an antigen binding fragmented thereof. An anti-CD40 agent or an anti-CD40 ligand agent can be an antagonistic anti-CD40 antibody or antigen-binding fragment thereof. Non-limiting examples of antagonistic anti-CD40 antibodies include 2C10, 2C10R4, ASKP1240, 4D11, bleselumab, BI-655064, HCD122, CFZ533, ch5D12, CDP7657, and FFP104. In some cases, the anti-CD40 agent is ASKP1240. An anti-CD40 agent or an anti-CD40 ligand agent can be an antagonistic anti-CD40 ligand antibody or antigen-binding fragment thereof. Non-limiting examples of antagonistic anti-CD40 ligand antibodies include BG9588, ruplizumab, toralizumab, IDEC-131, dapirolizumab, letolizumab, BMS-986004, VIB4920, and MEDI4920.
- In some cases, an immunosuppression agent used for short term immunosuppression can be any one or more of MMF (mycophenolate mofetil (Cellcept)), ATG (anti-thymocyte globulin), anti-CD154 (CD40L), alemtuzumab (Campath), CTLA4-Ig (Abatacept/Orencia), belatacept (LEA29Y), daclizumab (Ze-napax), basiliximab (Simulect), infliximab (Remicade), cyclosporin, deoxyspergualin,
soluble complement receptor 1, cobra venom factor, compstatin, anti C5 antibody (eculizumab/Soliris), methylprednisolone, FTY720, everolimus, anti-CD154-Ab, leflunomide, anti-IL-2R-Ab, anti-CXCR3 antibody, anti-ICOS antibody, anti-OX40 antibody, and anti-CD122 antibody, human anti-CD154 monoclonal antibody, CD40 antagonist, and CD40L (CD154) antagonist. - In some cases, an immunosuppression agent used for short term immunosuppression can target T cell receptor (TCR), CD3e, FK506-binding protein 12 (FKBP12), cytotoxic T lymphocyte associated protein 4 (CTLA-4), programmed cell death protein 1 (PD-1), programmed death ligand 1 (PD-L1), CD40L (CD154), CD40, inducible costimulatory (ICOS), IL-2, TNF-α, IL-6, IL-7, CD2, CD20, CD52, α-4 integrin, mTOR (mechanistic target of rapamycin, everolimus, serolimus), DNA synthesis, or any combination thereof. In some cases, an immunosuppression agent used for short term immunosuppression can be a MHC/TCR interaction blockade, a nonselective depleting agent, calcineurin inhibitor, costimulatory signal blockade, cytokine blockade, lymphocyte depleting agent, cell adhesion inhibitor, IL-2 signaling inhibitor, cell cycle blocker, or any combination thereof. For example, the MHC/TCR interaction blockade can be anti-abTCR mAb T10B9. For example, the nonselective depleting agent can be anti-CD3 mAb (OKT3) or antithymocyte globulin (ATG). For example, the calcineurin inhibitor can be cyclosporine or tacrolimus. For example, the costimulatory signal blockade can be anti-CTLA-4 mAb, abatacept, ipilimumab, anti-PD-1 (such as pembrolizumab), anti-PD-L1 (such as MPDL3280A), anti-CD154 mAb, anti-CD40 mAb, or anti-ICOS mAb. For example, the cytokine blockade can be anti-CD25 mAb (such as daclizumab or basiliximab), anti-TNF (etanercept, infliximab, adalimumab, certolizumab pegol, and golimumab), anti-IL-6/IL-6R mAb (such as clazakizumab, ALD518, siltuximab, elsilimomab, sirukumab, olokizumab, sarilumab, tocilizumab, actemra), or anti-IL-7 mAb. For example, the lymphocyte depleting agent can be anti-CD2 mAb, fusion protein with IgG1 (such as alefacept), anti-CD20 mAb (such as rituximab), or anti-CD52 mAb (such as alemtuzumab). For example, the cell adhesion inhibitor can be anti-very large antigen 4 (VLA4) (such as natalizumab). For example, the IL-2 signaling inhibitor can be sirolimus (rapamycin) or everolimus. For example, the cell cycle blocker can be mycophenolate mofetil (MMF) or azathioprine.
- Short term immunosuppression can comprise administering one or more immunosuppression agents and/or anti-inflammatory agents of the disclosure for at most about 100 days after a transplant. Short term immunosuppression can comprise administering one or more immunosuppression agents and/or anti-inflammatory agents of the disclosure, for example, for at most about 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 25, 28, 29, 30, 31, 32, 33, 34, 35, 40, 42, 49, 50, 55, 60, 70, 80, 90, or 100 days after a transplant. In some cases, short term immunosuppression can conclude within 28 days after a transplant. In some cases, short term immunosuppression can conclude about 21 days after a transplant.
- In some embodiments, short term immunosuppression can begin prior to transplantation. For example, short term immunosuppression can commence about or at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 25, 28, 29, 30, 35, or 50 days prior to a transplant. In some cases, short term immunosuppression can commence at most about 10 days prior to a transplant. In some cases, short term immunosuppression can commence about 7 days prior to a transplant.
- The duration of short term immunosuppression (e.g., the length of time between administering a first dose and a final dose of an immunosuppression agents and/or anti-inflammatory agents can be about or at most about 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 25, 28, 29, 30, 31, 32, 33, 34, 35, 40, 42, 49, 50, 55, 60, 70, 80, 90, or 100 days. In some cases, the duration of short term immunosuppression can be about or at most about 30 days. In some cases, the duration of short term immunosuppression can be about 28 days.
- Administration of Compositions in Combination with a Tolerizing Agent
- In one aspect, a peptide or compositions disclosed herein can be administered as a tolerizing agent. In another embodiment, a peptide or compositions disclosed herein can be administered before, after, and/or during the administration of a tolerizing agent. In some cases, the peptide of compositions disclosed herein can be administered between day −100 and
day 0, e.g., on day −90, day −80, day −70, day −60, day −50, day −40, day −30, day −20, day −15, day −14, day −13, day −12, day −11, day −10, day −9, day −8, day −7, day −6, day −5, day −4, day −3, day −2, day −1, orday 0 relative to the administration of a tolerizing agent. In some cases, the peptide or compositions disclosed herein can be administered concomitantly with the tolerizing agent. In some cases, the peptide or compositions disclosed herein can be administered on or on about day −100 to −50; −50 to −40; −40 to −30; −30 to −20; −20 to −10; −10 to −5; −7 to −1, relative to the administration of a tolerizing agent. - In some cases, the peptide or composition disclosed herein can be administered between
day 0 and day 100, e.g., on day 100, day 90, day 80, day 70, day 60, day 50, day 40, day 30,day 20,day 15, day 14, day 13, day 12, day 11,day 10, day 9, day 8,day 7,day 6,day 5,day 4,day 3,day 2 orday 1 relative to the administration of a tolerizing agent. For example, the peptide or compositions disclosed herein can be administered on or on about day 100 to 50; 50 to 40; 40 to 30; 30 to 20; 20 to 10; 10 to 5; 7 to 1, relative to the administration of a tolerizing agent. - In some cases, the peptide or compositions disclosed herein can be administered between
day 0 and day 300, e.g., on day 300, day 200, day 100, day 90, day 80, day 70, day 60, day 50, day 40, day 30,day 20,day 15, day 14, day 13, day 12, day 11,day 10, day 9, day 8,day 7,day 6,day 5,day 4,day 3,day 2 orday 1 relative to the administration of a tolerizing agent. For example, the peptide or compositions disclosed herein can be administered on or on about day 300 to 200; 200 to 100; 100 to 50; 50 to 40; 40 to 30; 30 to 20; 20 to 10; 10 to 5; 7 to 1, relative to the administration of a tolerizing agent. - In some cases, a peptide or compositions disclosed herein can be administered between
day 0 and day 600, e.g., on day 600,day 500, day 400, day 300, day 200, day 100, day 90, day 80, day 70, day 60, day 50, day 40, day 30,day 20,day 15, day 14, day 13, day 12, day 11,day 10, day 9, day 8,day 7,day 6,day 5,day 4,day 3,day 2 orday 1 relative to the administration of a tolerizing agent. For example, a peptide or composition disclosed herein can be administered on or on about day 600 to 500;day 500 to 400; day 400 to 300; 300 to 200; 200 to 100; 100 to 50; 50 to 40; 40 to 30; 30 to 20; 20 to 10; 10 to 5; 7 to 1, relative to the administration of a tolerizing agent. - In some cases, a peptide or compositions disclosed herein can be administered between
day 0 and day 1,000, e.g., on day 1,000, day 900, day 800, day 700, day 600,day 500, day 400, day 300, day 200, day 100, day 90, day 80, day 70, day 60, day 50, day 40, day 30,day 20,day 15, day 14, day 13, day 12, day 11,day 10, day 9, day 8,day 7,day 6,day 5,day 4,day 3,day 2 orday 1 relative to the administration of a tolerizing agent. For example, the a peptide or compositions disclosed herein can be administered on or on about day 1,000 to 900; 900 to 800; 800 to 700; 700 to 600; 600 to 500;day 500 to 400; day 400 to 300; 300 to 200; 200 to 100; 100 to 50; 50 to 40; 40 to 30; 30 to 20; 20 to 10; 10 to 5; 7 to 1, relative to the administration of a tolerizing agent. - In some cases, a peptide or compositions disclosed herein can be administered on the day when a tolerizing agent is administered. In other cases, a peptide or can be administered before and after the administration of the tolerizing vaccine or preparatory regimen.
- A peptide or compositions disclosed herein can be administered before, after, and/or during the day of the transplantation of the graft cell, tissue, or organ. In some cases, the peptide of compositions disclosed herein can be administered between day −100 and
day 0, e.g., on day −90, day −80, day −70, day −60, day −50, day −40, day −30, day −20, day −15, day −14, day −13, day −12, day −11, day −10, day −9, day −8, day −7, day −6, day −5, day −4, day −3, day −2, day −1, orday 0 relative to the day of the transplantation of the graft cell, tissue, or organ. In some cases, the peptide or compositions disclosed herein can be administered concomitantly with the day of the transplantation of the graft cell, tissue, or organ. In some cases, the peptide or compositions disclosed herein can be administered on or on about day −100 to −50; −50 to −40; −40 to −30; −30 to −20; −20 to −10; −10 to −5; −7 to −1, relative to the day of the transplantation of the graft cell, tissue, or organ. - In some cases, the peptide or composition disclosed herein can be administered between
day 0 and day 100, e.g., on day 100, day 90, day 80, day 70, day 60, day 50, day 40, day 30,day 20,day 15, day 14, day 13, day 12, day 11,day 10, day 9, day 8,day 7,day 6,day 5,day 4,day 3,day 2 orday 1 relative to the day of the transplantation of the graft cell, tissue, or organ. For example, the peptide or compositions disclosed herein can be administered on or on about day 100 to 50; 50 to 40; 40 to 30; 30 to 20; 20 to 10; 10 to 5; 7 to 1, relative to the day of the transplantation of the graft cell, tissue, or organ. - In some cases, the peptide or compositions disclosed herein can be administered between
day 0 and day 300, e.g., on day 300, day 200, day 100, day 90, day 80, day 70, day 60, day 50, day 40, day 30,day 20,day 15, day 14, day 13, day 12, day 11,day 10, day 9, day 8,day 7,day 6,day 5,day 4,day 3,day 2 orday 1 relative to the day of the transplantation of the graft cell, tissue, or organ. For example, the peptide or compositions disclosed herein can be administered on or on about day 300 to 200; 200 to 100; 100 to 50; 50 to 40; 40 to 30; 30 to 20; 20 to 10; 10 to 5; 7 to 1, relative to the day of the transplantation of the graft cell, tissue, or organ. - In some cases, a peptide or compositions disclosed herein can be administered between
day 0 and day 600, e.g., on day 600,day 500, day 400, day 300, day 200, day 100, day 90, day 80, day 70, day 60, day 50, day 40, day 30,day 20,day 15, day 14, day 13, day 12, day 11,day 10, day 9, day 8,day 7,day 6,day 5,day 4,day 3,day 2 orday 1 relative to the day of the transplantation of the graft cell, tissue, or organ. For example, a peptide or composition disclosed herein can be administered on or on about day 600 to 500;day 500 to 400; day 400 to 300; 300 to 200; 200 to 100; 100 to 50; 50 to 40; 40 to 30; 30 to 20; 20 to 10; 10 to 5; 7 to 1, relative to the day of the transplantation of the graft cell, tissue, or organ. - In some cases, a peptide or compositions disclosed herein can be administered between
day 0 and day 1,000, e.g., on day 1,000, day 900, day 800, day 700, day 600,day 500, day 400, day 300, day 200, day 100, day 90, day 80, day 70, day 60, day 50, day 40, day 30,day 20,day 15, day 14, day 13, day 12, day 11,day 10, day 9, day 8,day 7,day 6,day 5,day 4,day 3,day 2 orday 1 relative to the day of the transplantation of the graft cell, tissue, or organ. For example, the a peptide or compositions disclosed herein can be administered on or on about day 1,000 to 900; 900 to 800; 800 to 700; 700 to 600; 600 to 500;day 500 to 400; day 400 to 300; 300 to 200; 200 to 100; 100 to 50; 50 to 40; 40 to 30; 30 to 20; 20 to 10; 10 to 5; 7 to 1, relative to the day of the transplantation of the graft cell, tissue, or organ. - In some cases, a peptide or compositions disclosed herein can be administered on the day of the transplantation of the graft cell, tissue, or organ. In other cases, a peptide or can be administered before and after the day of the transplantation of the graft cell, tissue, or organ.
- A preparatory regimen can comprise multiple doses of a peptide or composition described herein, before, and/or during and/or after transplantation of a graft cell, tissue, or organ. In some cases, a preparatory regimen can comprise multiple doses of a peptide comprising a sequence with at least 80%, 85% 90% or 95% sequence identity set forth in any one of SEQ ID NOs. 1-6 and 8-17, before, and/or during and/or after transplantation of a graft cell, tissue, or organ. A tolerizing regimen can comprise multiple doses of a peptide or composition described herein, before, and/or during and/or after transplantation of a graft cell, tissue, or organ. In some cases, a tolerizing regimen can comprise multiple doses of a peptide comprising a sequence with at least 80%, 85% 90% or 95% sequence identity set forth in any one of SEQ ID NOs. 1-6 and 8-17, before, and/or during and/or after transplantation of a graft cell, tissue, or organ. The multiple doses can be referred to as comprising an initial dose and one or more booster doses. The initial dose can occur prior to or concurrently with the transplant of the graft cell tissue or organ. The booster dose(s), when administered, occur after the initial dose. Depending upon when the initial dose of the tolerizing vaccine is administered, one or more booster doses can be administered before, and/or concurrently with, and/or after transplant of the graft cell, tissue, or organ.
- Subsequent (e.g., booster) dose(s) of a preparatory regimen or tolerizing vaccine can be administered in any interval of time following a preceding dose (e.g., an initial dose). For example, the subsequent dose can be administered about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 31 days, 32 days, 33 days, 34 days, 35 days, 36 days, 37 days, 38 days, 39 days, 40 days, 41 days, 42 days, 43 days, 44 days, 45 days, 46 days, 47 days, 48 days, 49 days, 50 days, 51 days, 52 days, 53 days, 54 days, 55 days, 56 days, 57 days, 58 days, 59 days, 60 days, 90 days, 120 days, 150 days, or 180 days after the preceding dose.
- Depending upon when the initial dose is administered subsequent (booster) dose(s) can be administered before, concurrently with, or after transplantation of the graft cell, tissue, or organ. In some cases, the preparatory regimen comprises at least one dose, at least two doses, at least three doses of a preparatory regimen or tolerizing vaccine prior to transplantation.
- In some cases, two doses of the tolerizing vaccine or preparatory regimen can be administered. The first dose can be administered, for example, on day −14, −10, −9, −8, −7, −6, −5, −4, −3, −2, −1, 0, 1, 2, 3, 4, 5, 6, or 7 relative to transplant of donor cells, organs, and/or tissues on
day 0. The second dose can be administered, for example, on day −3, −2, −1, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 relative to transplant of donor cells, organs, and/or tissues onday 0. In some embodiments, the first dose is administered on day −8 and the second dose is administered on day −1. In some embodiments, the first dose is administered on day −8 and the second dose is administered onday 0. In some embodiments, the first dose is administered on day −8 and the second dose is administered onday 1. In some embodiments, the first dose is administered on day −7 and the second dose is administered on day −1. In some embodiments, the first dose is administered on day −7 and the second dose is administered onday 0. In some embodiments, the first dose is administered on day −7 and the second dose is administered onday 1. In some embodiments, the first dose is administered on day −12 and the second dose is administered on day −4. In some embodiments, the first dose is administered on day −11, −12, −13, or −14 and the second dose is administered on day −3, −4, −5, or −6. - In some cases, a second dose of the tolerizing vaccine/preparatory regimen (e.g., a booster vaccine) can be administered on day 100, day 90, day 80, day 70, day 60, day 50, day 40, day 30, day 29, day 28, day 27, day 26,
day 25, day 24, day 23, day 22, day 21,day 20, day 19, day 18, day 17, day 16,day 15, day 14, day 13, day 12, day 11,day 10, day 9, day 8,day 7,day 6,day 5,day 4,day 3,day 2 orday 1, relative to transplant of donor cells, organs, and/or tissues onday 0. For example, the second dose of the tolerizing vaccine (e.g., a booster vaccine) can be administered 1 day after transplant of donor cells, organs, and/or tissues. In some cases, a second dose of a tolerizing vaccine is given concomitantly onday 0 with transplant donor cells, organs, and/or tissues. In some cases, a second dose of a tolerizing vaccine is not required. - In some cases, three doses of the tolerizing vaccine/preparatory regimen or preparatory regimen can be administered. The first dose can be administered, for example, on day −14, −10, −9, −8, −7, −6, −5, −4, −3, −2, −1, 0, 1, 2, 3, 4, 5, 6, or 7 relative to transplant of donor cells, organs, and/or tissues on
day 0. The second dose can be administered, for example, on day −3, −2, −1, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 relative to transplant of donor cells, organs, and/or tissues onday 0. The third dose can be administered, for example, onday day 0. In some embodiments, the first dose is administered on day −8, the second dose is administered onday 1, and the third dose is administered onday 7. In some embodiments, the first dose is administered on day −8, the second dose is administered onday 1, and the third dose is administered on day 14. In some embodiments, the first dose is administered on day −8, the second dose is administered onday 1, and the third dose is administered on day 21. In some embodiments, the first dose is administered on day −7, the second dose is administered onday 1, and the third dose is administered onday 7. In some embodiments, the first dose is administered on day −7, the second dose is administered onday 1, and the third dose is administered on day 14. In some embodiments, the first dose is administered on day −7, the second dose is administered onday 1, and the third dose is administered on day 21. - In some cases, a third dose of the tolerizing vaccine/preparatory regimen (e.g., a booster vaccine) can be administered on day 300, day 200, day 100, day 90, day 80, day 70, day 60, day 50, day 40, day 30, day 29, day 28, day 27, day 26,
day 25, day 24, day 23, day 22, day 21,day 20, day 19, day 18, day 17, day 16,day 15, day 14, day 13, day 12, day 11,day 10, day 9, day 8,day 7,day 6,day 5,day 4,day 3,day 2 orday 1, relative to transplant of donor cells, organs, and/or tissues onday 0. For example, the tolerizing vaccine can be administered on or on about day 300 to 200; 200 to 100; 100 to 50; 50 to 40; 40 to 30; 30 to 20; 20 to 10; 10 to 5; or 7 to 1, relative to transplant of donor cells, organs, and/or tissues onday 0. - In some cases, a fourth dose of the tolerizing vaccine/preparatory regimen (e.g., a booster vaccine) can be administered on day 600,
day 500, day 400, day 300, day 200, 100, day 90, day 80, day 70, day 60, day 50, day 40, day 30, day 29, day 28, day 27, day 26,day 25, day 24, day 23, day 22, day 21,day 20, day 19, day 18, day 17, day 16,day 15, day 14, day 13, day 12, day 11,day 10, day 9, day 8,day 7,day 6,day 5,day 4,day 3,day 2 orday 1, relative to transplant of donor cells, organs, and/or tissues onday 0. For example, the tolerizing vaccine can be administered on or on about day 600 to 500; 500 to 400; 400 to 300; 300 to 200; 200 to 100; 100 to 50; 50 to 40; 40 to 30; 30 to 20; 20 to 10; 10 to 5; 7 to 1, relative to transplant of donor cells, organs, and/or tissues onday 0. - In some cases, a fifth dose of the tolerizing vaccine or preparatory regimen (e.g., a booster vaccine) can be administered on day 1,000, day 900, day 800, day 700, day 600,
day 500, day 400, day 300, day 200, 100, day 90, day 80, day 70, day 60, day 50, day 40, day 30, day 29, day 28, day 27, day 26,day 25, day 24, day 23, day 22, day 21,day 20, day 19, day 18, day 17, day 16,day 15, day 14, day 13, day 12, day 11,day 10, day 9, day 8,day 7,day 6,day 5,day 4,day 3,day 2 orday 1, relative to transplant of donor cells, organs, and/or tissues onday 0. For example, the tolerizing vaccine can be administered on or on about day 1,000 to 900; 900 to 800; 800 to 700; 700 to 600; 600 to 500; 500 to 400; 400 to 300; 300 to 200; 200 to 100; 100 to 50; 50 to 40; 40 to 30; 30 to 20; 20 to 10; 10 to 5; 7 to 1, relative to transplant of donor cells, organs, and/or tissues onday 0. - Administration of the tolerizing vaccine or preparatory regimen can result in long term tolerance to the cell, tissue, or organ transplant in the transplant recipient. In some cases, the long term tolerance is for a period of at least one month, at least two months, at least three months, at least four months, at least five months, at least six months, at least seven months, at least eight months, at least nine months, at least ten months, at least eleven months, at least twelve months, at least thirteen months, at least fourteen months, at least fifteen months, at least sixteen months, at least seventeen months, at least eighteen months, at least nineteen months, at least twenty months, at least twenty-one months, at least twenty-two months, at least twenty-three months, or at least twenty-four months. In some cases, the long term tolerance is for a period of at least 1 year, at least 2 years, at least 3 years, at least 4 years, at least 5 years, at least 6 years, at least 7 years, at least 8 years, at least 9 years, or at least 10 years. In some cases, the long term tolerance is achieved in the absence of a booster vaccine or booster regimen. In some cases, the long term tolerance is achieved with an administration of a booster vaccine or booster regimen in one or multiple doses. In some cases, one or more booster vaccine doses are administered on the day of, or at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 31 days, 32 days, 33 days, 34 days, 35 days, 36 days, 37 days, 38 days, 39 days, 40 days, 41 days, 42 days, 43 days, 44 days, 45 days, 46 days, 47 days, 48 days, 49 days, 50 days, 51 days, 52 days, 53 days, 54 days, 55 days, 56 days, 57 days, 58 days, 59 days, 60 days, 65 days, 70 days, 75 days, 80 days, 85 days, 90 days, 95 days, 100 days, 105 days, 110 days, 115 days, 120 days, 125 days, 130 days, 135 days, 140 days, 145 days, 150 days, 155 days, 160 days, 165 days, 170 days 175 days, 180 days, 185 days, 190 days, 195 days, 200 days, 205 days, 210 days, 215 days, 220 days, 230 days or 240 days after the transplantation. In certain specific cases, one or more (for instance three) doses of a preparatory regimen is administered prior to transplantation, and one or more booster vaccine doses are provided 1, 7, 14, 21, 90, or up to 180 days after transplantation.
- A tolerizing vaccine or preparatory regimen can be administered with or without an adjuvant (e.g., one or more immunomodulatory molecules). In some cases, the adjuvant enhances the tolerogenic properties of the tolerizing vaccine by inhibiting activation and maturation of antigen presenting cells.
- A dose of a tolerizing vaccine or preparatory regimen can vary based upon the weight of a recipient of a tolerizing vaccine. For example, the dose of the tolerizing vaccine or preparatory regimen can comprise about: 1×101 cells/kg, 1×102 cells/kg, 1×103 cells/kg, 1×104 cells/kg, 1×105 cells/kg, 1×106 cells/kg, 1×107 cells/kg, 1×108 cells/kg, 1×109 cells/kg, 1×1010 cells/kg, 1×1011 cells/kg, 1×1012 cells/kg, or more. In some cases, a dose of the tolerizing vaccine or preparatory regimen can comprises about: 1×101 to 1×102 cells/kg; 1×102 to 1×103 cells/kg; 1×103 to 1×104 cells/kg; 1×104 to 1×105 cells/kg; 1×105 to 1×106 cells/kg; 1×106 to 1×107 cells/kg; 1×107 to 1×108 cells/kg; 1×108 to 1×109 cells/kg; 1×109 to 1×1010 cells/kg, 1×1010 to 1×1011 cells/kg, 1×1011 to 1×1012 cells/kg. For example, a dose of the tolerizing vaccine or preparatory regimen for administration can be about 0.01×109 cells/kg, 0.02×109 cells/kg, 0.03×109 cells/kg, 0.04×109 cells/kg, 0.05×109 cells/kg, 0.06×109 cells/kg, 0.07×109 cells/kg, 0.08×109 cells/kg, 0.09×109 cells/kg, 0.1×109 cells/kg, 0.2×109 cells/kg, 0.21×109 cells/kg, 0.22×109 cells/kg, 0.23×109 cells/kg, 0.24×109 cells/kg, 0.25×109 cells/kg, 0.26×109 cells/kg, 0.27×109 cells/kg, 0.28×109 cells/kg, 0.29×109 cells/kg, 0.3×109 cells/kg, 0.4×109 cells/kg, 0.5×109 cells/kg, 0.6×109 cells/kg, 0.7×109 cells/kg, 0.8×109 cells/kg, 0.9×109 cells/kg, 1.0×109 cells/kg, 1.5×109 cells/kg, 2.0×109 cells/kg, 2.5×109 cells/kg, 3.0×109 cells/kg, 3.5×109 cells/kg, 4.0×109 cells/kg, 4.5×109 cells/kg, 5.0×109 cells/kg, 5.5×109 cells/kg, 6.0×109 cells/kg, 6.5×109 cells/kg, 7.0×109 cells/kg, 7.5×109 cells/kg, 8.0×109 cells/kg, 8.5×109 cells/kg, 9.0×109 cells/kg, 9.5×109 cells/kg, 10.0×109 cells/kg, or 25.0×109 cells/kg.
- In some cases, a dose of the tolerizing vaccine or preparatory regimen can comprise at least about: 1×104 cells/kg, 5×104 cells/kg, 1×105 cells/kg, 5×105 cells/kg, 1×106 cells/kg, 5×106 cells/kg, 1×107 cells/kg, 5×107 cells/kg, 1×108 cells/kg, 2×108 cells/kg, 3×108 cells/kg, 4×108 cells/kg, 5×108 cells/kg, 6×108 cells/kg, 7×108 cells/kg, 8×108 cells/kg, 9×108 cells/kg, 1×109 cells/kg, 1×1010 cells/kg, or more.
- The methods herein can comprise administering at least or at least about 0.25×109 cells (e.g., apoptotic donor leukocytes (ADLs), such as ECDI-treated cells, e.g., ECDI-treated leukocytes, or apoptotic mesenchymal stromal cells) per kg recipient body weight. For example, at least or at least about 1×107, 1×108, 0.25×109, 0.50×109, 0.75×109, 1.00×109, 1.25×109, 1.50×109, 1.75×109 or 2×109 cells (e.g., ECDI-treated cells, e.g., ECDI-treated leukocytes) per kg recipient body weight ECDI-treated cells can be administered.
- The cells can comprise leukocytes, e.g., splenocytes, peripheral blood mononuclear cells (PBMCs), stem-cell derived leukocytes, or a combination thereof. The splenocytes, PBMCs, stem-cell derived leukocytes, or the combination thereof can comprise B cells or B lymphocytes. The cells can comprise primary cells, cells expanded ex vivo, cells of a cell line, or a combination thereof. The cells can comprise mesenchymal stromal cells.
- Cells of tolerizing vaccine/regimen or preparatory regimen for each dose of administration can be suspended in a volume suitable for transfusion. For example, the cells can be suspended in a volume of about: 0.1 ml, 0.2 ml, 0.3 ml, 0.4 ml, 0.5 ml, 0.6 ml, 0.7 ml, 0.8 ml, 0.9 ml, 1 ml, 2 ml, 3 ml, 4 ml, 5 ml, 6 ml, 7 ml, 8 ml, 9 ml, 10 ml, 11 ml, 12 ml, 13 ml, 14 ml, 15 ml, 16 ml, 17 ml, 18 ml, 19 ml, 20 ml, 21 ml, 22 ml, 23 ml, 24 ml, 25 ml, 26 ml, 27 ml, 28 ml, 29 ml, 30 ml, 31 ml, 32 ml, 33 ml, 34 ml, 35 ml, 36 ml, 37 ml, 38 ml, 39 ml, 40 ml, 41 ml, 42 ml, 43 ml, 44 ml, 45 ml, 46 ml, 47 ml, 48 ml, 49 ml, 50 ml, 60 ml, 70 ml, 80 ml, 90 ml, 100 ml, 200 ml, 300 ml, 400 ml, or 500 ml. For example, the cells of tolerizing vaccine or preparatory regimen for each dose of administration can be suspended in a volume of about: 0.1 ml to 1 ml; 1 ml to 10 ml; 10 ml to 50 ml; 50 ml to 100 ml; 100 ml to 200 ml; 200 ml to 300 ml; 300 ml to 400 ml; or 400 ml to 500 ml. For example, 75×106 cells of tolerizing vaccine or preparatory regimen can be suspended in a volume of 0.5 ml.
- Tolerizing vaccines/regimen or preparatory regimens can be administered (e.g., by intravenous infusion) in a volume that varies depending upon the weight of the recipient. For example, the tolerizing vaccine or preparatory regimen can be given intravenously in a volume of at least or at least about 0.01 ml, 0.1 ml, 0.5 ml, 1 ml, 2 ml, 3 ml, 4 ml, 5 ml, 10 ml, 20 ml, 30 ml, 40 ml or 50 ml per kg recipient body weight, e.g., at least or at least about 0.01 to 0.1, 0.1 to 1, 1 to 2; 2 to 3; 3 to 4; 4 to 5; 1 to 5; 5 to 10; 10 to 20; 20 to 30; 30 to 40; or 40 to 50 ml per kg recipient body weight. In some cases, the tolerizing vaccine (e.g., comprising ECDI-treated cells) is given intravenously in a volume of about 7 ml per kg recipient body weight.
- Booster doses of a tolerizing vaccine or preparatory regimen can comprise fewer cells than an initial dose of the tolerizing vaccine or preparatory regimen. For example, a booster or subsequent dose of the tolerizing vaccine or preparatory regimen can comprise about: 1%, 2%, 3%, 4%, 5%, 7.5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, or 75% fewer cells, or less than the initial or preceding dose of the tolerizing vaccine or preparatory regimen.
- A cell of a tolerizing vaccine or a preparatory regimen can have a circulation half-life after it is administered to a subject. In some cases, a tolerizing vaccine or preparatory regimen described herein can have a circulation half-life of at least or at least about 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 18, 24, 36, 48, 60, or 72 hours. For example, the circulation half-life of the tolerizing vaccine or preparatory regimen cells can be from or from about 0.1 to 0.5; 0.5 to 1.0; 1.0 to 2.0; 1.0 to 3.0; 1.0 to 4.0; 1.0 to 5.0; 5 to 10; 10 to 15; 15 to 24; 24 to 36; 36 to 48; 48 to 60; or 60 to 72 hours. In some cases, a tolerizing vaccine or preparatory regimen described herein can have a circulation half-life of at least or at least about 3 hours.
- The cells of the tolerizing vaccine or preparatory regimen can be treated to enhance their circulation half-life. Such treatment can include coating the cell with a protein, e.g., CD47. The cell treated to enhance its circulation half-life can be a non-apoptotic cell. The cell treated to enhance its circulation half-life can be an apoptotic cell. Alternatively, the cell in a tolerizing vaccine or preparatory regimen can be genetically modified (e.g., insertion of a transgene such as CD47 in its genome) to enhance its circulation half-life. The cell genetically modified to enhance its circulation half-life can be a non-apoptotic cell. The cell genetically modified to enhance its circulation half-life can be an apoptotic cell.
- The tolerizing vaccine or the preparatory regimen can be advantageous in transplantation, for example, in xenotransplantation or in allotransplantation, by tolerizing a graft recipient and preventing or delaying graft rejection. The tolerization or the preparatory regimen can be conferred to a graft recipient without the use of immunosuppressive therapies (e.g., one or more immunomodulatory molecules). However, in some cases, other immunosuppressive therapies can be used in combination with tolerizing vaccines to prevent, decrease, or delay transplantation rejection.
- The dose of a peptide or composition thereof required in presence of a tolerizing agent can be lower than the dose of a peptide or composition disclosed herein required in its absence by at least or at least about 5%. For example, dose of a peptide or composition disclosed herein can be lower by at least or at least about 5%, 10%, 20%, 30%, 40%, 50%, or more. The term “lower” and its grammatical equivalents as used herein can refer to using less peptide or compositions thereof compared to a required dose when one or more wild-type cells, organs, and/or tissues is transplanted into a recipient (e.g., a human or a non-human animal).
- In some embodiments, the methods disclosed herein can increase the duration of survival of a transplant (e.g., a xenograft or an allograft transplant) in a recipient for a period of at least one month, at least two months, at least three months, at least four months, at least five months, at least six months, at least 1 year, at least 2 years, at least 3 years, at least 4 years, at least 5 years, at least 6 years, at least 7 years, at least 8 years, at least 9 years, or at least 10 years.
- In some cases, the transplant survives for a period of at least one month, at least two months, at least three months, at least four months, at least five months, at least six months, at least seven months, at least eight months, at least nine months, at least ten months, at least eleven months, at least twelve months, at least thirteen months, at least fourteen months, at least fifteen months, at least sixteen months, at least seventeen months, at least eighteen months, at least nineteen months, at least twenty months, at least twenty-one months, at least twenty-two months, at least twenty-three months, or at least twenty-four months. In some cases, the transplant survives for a period of at least 1 year, at least 2 years, at least 3 years, at least 4 years, at least 5 years, at least 6 years, at least 7 years, at least 8 years, at least 9 years, or at least 10 years. In some cases, the survival is achieved in the absence of a booster vaccine or booster regimen. In some cases, the transplant survival is achieved with an administration of a booster dose in one or multiple doses.
- Different doses of a peptide or compositions disclosed herein can be given to a recipient before, after, and/or during transplant of donor cells, organs, and/or tissues to induce donor-specific tolerance in a recipient. In some cases, a first dose of a peptide or compositions disclosed herein can comprise at least or at least about 1 mg, 5 mg, 10 mg, 20 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, 100 mg, or 200 mg of the a peptide or compositions disclosed herein per kg recipient body weight. In certain cases, a first dose of a peptide or compositions disclosed herein can comprise at least or at least about 30 mg, 40 mg, 50 mg, 60 mg, 70 mg of a peptide or compositions disclosed herein per kg recipient body weight. In some cases, a first dose of a peptide or compositions disclosed herein can comprise from or from about 1 mg to 200 mg, e.g., from or from about 20 mg to 100 mg; 30 mg to 80 mg; 30 mg to 70 mg; 40 mg to 70 mg; 40 mg to 60 mg; 50 mg to 70 mg; or 60 mg to 80 mg of the a peptide or compositions disclosed herein per kg recipient body weight.
- Methods of the present disclosure further comprises administering an immunomodulatory agent to the recipient. The immunomodulatory agent can be for example, an immunosuppressive agent. The term “immunosuppressive agent” as used herein refers to substances that act to suppress or mask the immune system of the host into which the graft is being transplanted. This would include substances that suppress cytokine production, down-regulate or suppress self-antigen expression, or mask the MHC antigens.
- Thus, peptides or compositions of the disclosure can be used alone or in some embodiments in combination with immunosuppressive agent that inhibits rejection of a transplant. The immunosuppressive agent can be administered simultaneously or at a separate time than the peptide or compositions herein. Non-limiting examples of immunosuppressive agents are azathioprine, corticosteroids, cyclosporine, 2-amino-6-aryl-5-substituted pyrimidines; azathioprine or cyclophosphamide; bromocryptine; glutaraldehyde; antiidiotypic antibodies for MHC antigens; cyclosporin A; one or more steroids, preferably corticosteroids and glucocorticosteroids such as prednisone, methyl prednisolone, and dexamethasone; anti-interferon-gamma antibodies; anti-tumor necrosis factor-alpha antibodies; anti-tumor necrosis factor-beta antibodies; anti-interleukin-2 antibodies; anticytokine receptor antibodies such as anti-IL-2 receptor antibodies; heterologous anti-lymphocyte globulin; pan-T antibodies, preferably OKT-3 monoclonal antibodies; antibodies to CD4; streptokinase; streptodomase; or RNA or DNA from the host.
- In some cases, the immunosuppressive agent inhibits T cell activation. The immunosuppressive agent that inhibits T cell activation can be an anti-CD40 agent or anti-CD40L (CD154) agent. In some embodiment, the anti-CD40 agent can be an anti-CD40 antibody. The anti-CD40 antibody can be an antagonistic antibody. The anti-CD40 antibody can be a Fab′ anti-CD40L monoclonal antibody fragment CDP7657. The anti-CD40 antibody can be a FcR-engineered, Fc silent anti-CD40L monoclonal domain antibody.
- In some cases, the peptide or compositions disclosed herein is administered with one or more additional immunosuppression agent described herein, such as further comprising providing to the recipient one or more of an anti-CD40 agent or anti-CD40L (CD154) agent (e.g., an anti-CD40 antibody), a B-cell targeting agent (e.g., B cell depleting biologics, for example, a biologic targeting CD20, CD19, or CD22, and/or B cell modulating biologics, for example, a biologic targeting BAFF, BAFF/APRIL, CD40, IgG4, ICOS, IL-21, B7RP1), an mTOR inhibitor, a TNF-alpha inhibitor, a IL-6 inhibitor, a nitrogen mustard alkylating agent (e.g., cyclophosphamide), a complement C3 or C5 inhibitor, IFNγ, an NFκB inhibitor, α1-antitrypsin, vitamin D3, siCD40, cobalt protoporphyrin, insulin B9-23, a cluster of differentiation protein (e.g., CD46, CD55, or CD59), any combination thereof, or any fragment thereof. In some cases, the NFκB inhibitor is dehydroxymethylepoxyquinomicin (DHMEQ), curcumin, triptolide, Bay-117085, or a combination thereof. In some cases, B-cell targeting biologic can be Rituximab, anti-CD20 antibody.
- In some cases, immunosuppressive agent can be MMF (mycophenolate mofetil (Cellcept)), ATG (anti-thymocyte globulin), anti-CD154 (CD40L), alemtuzumab (Campath), B-cell targeting agent (e.g., B cell depleting biologics, for example, a biologic targeting CD20, CD19, or CD22, and/or B cell modulating biologics, for example, a biologic targeting BAFF, BAFF/APRIL, CD40, IgG4, ICOS, IL-21, B7RP1), anti-IL-6R antibody (tocilizumab, Actemra), anti-IL-6 antibody (sarilumab, olokizumab), CTLA4-Ig (Abatacept/Orencia), belatacept (LEA29Y), mTOR inhibitor (sirolimus (e.g., Rapamune), rapamycin, everolimus), tacrolimus (Prograf), daclizumab (Zenapax), basiliximab (Simulect), infliximab (Remicade), cyclosporin, deoxyspergualin,
soluble complement receptor 1, cobra venom factor, compstatin, anti C5 antibody (eculizumab/Soliris), methylprednisolone, FTY720, everolimus, anti-CD154-Ab, leflunomide, anti-IL-2R-Ab, anti-CXCR3 antibody, anti-ICOS antibody, anti-OX40 antibody, and anti-CD122 antibody, human anti-CD154 monoclonal antibody, CD40 antagonist, and CD40L (CD154) antagonist. Non-limiting examples of B-cell targeting biologics include antagonistic anti-CD40 mAb antibody, Fc-engineered anti-CD40L antibodies, Rituximab, anti-CD20 antibody. One or more than one immunosuppressive agents/drugs can be used together or sequentially. One or more than one immunosuppressive agents/drugs can be used for induction therapy or for maintenance therapy. The same or different drugs can be used during induction and maintenance stages. For example, daclizumab (Zenapax) is used for induction therapy and tacrolimus (Prograf) and sirolimus (Rapamune), or everolimus, or any other mTOR inhibitor is used for maintenance therapy. In another example, daclizumab (Zenapax) is used for induction therapy and low dose tacrolimus (Prograf) and low dose sirolimus (Rapamune) is used for maintenance therapy. Immunosuppression can also be achieved using non-drug regimens including, but not limited to, whole body irradiation, thymic irradiation, and full and/or partial splenectomy. These techniques can also be used in combination with one or more immunosuppressive drug. - In some embodiments, one or more immunomodulatory molecules can target T cell receptor (TCR), CD3e, FK506-binding protein 12 (FKBP12), cytotoxic T lymphocyte associated protein 4 (CTLA-4), programmed cell death protein 1 (PD-1), programmed death ligand 1 (PD-L1), CD40L (CD154), CD40, inducible costimulatory (ICOS), IL-2, TNF-α, IL-6, IL-7, CD2, CD20, CD52, α-4 integrin, mTOR (mechanistic target of rapamycin, everolimus, serolimus), DNA synthesis, or any combination thereof. In some embodiments, the one or more immunomodulatory molecule can target B cell, (e.g., B cell depleting biologics, for example, a biologic targeting CD20, CD19, or CD22, and/or B cell modulating biologics, for example, a biologic targeting BAFF, BAFF/APRIL, CD40, IgG4, ICOS, IL-21, B7RP1). In some cases, the B cell targeting agent can be anti-CD20 mAb (such as rituximab) or other B-cell depleting antibody. In some embodiments, the immunosuppressive drugs can be a MHC/TCR interaction blockade, a nonselective depleting agent, calcineurin inhibitor, costimulatory signal blockade, cytokine blockade, lymphocyte depleting agent, cell adhesion inhibitor, IL-2 signaling inhibitor, cell cycle blocker, or any combination thereof. For example, the MHC/TCR interaction blockade can be anti-abTCR mAb T10B9. For example, the nonselective depleting agent can be anti-CD3 mAb (OKT3) or antithymocyte globulin (ATG). For example, the calcineurin inhibitor can be cyclosporine or tacrolimus. For example, the costimulatory signal blockade can be anti-CELA-4 mAb, abatacept, ipilimumab, anti-PD-1 (such as pembrolizumab), anti-PD-L1 (such as MPDL3280A), anti-CD154 mAb, anti-CD40 mAb, or anti-ICOS mAb. For example, the cytokine blockade can be anti-CD25 mAb (such as daclizumab or basiliximab), anti-TNF (infliximab), anti-IL-6 mAb (such as ALD518), or anti-IL-7 mAb. For example, the lymphocyte depleting agent can be anti-CD2 mAb, fusion protein with IgG1 (such as alefacept), anti-CD20 mAb (such as rituximab), or anti-CD52 mAb (such as alemtuzumab). For example, the cell adhesion inhibitor can be anti-very large antigen 4 (VLA4) (such as natalizumab). For example, the IL-2 signaling inhibitor can be sirolimus (rapamycin) or everolimus. For example, the cell cycle blocker can be mycophenolate mofetil (MMF) or azathioprine.
- The doses, time and mode of administration of a peptide or compositions disclosed herein or an immunomodulatory agent can be easily determined by one of skill in the art. The key factor in selecting an appropriate dose and scheduling is the result obtained, i.e., graft survival long-term. For example, a relatively high dose may be needed either initially for the treatment of hyperacute graft rejection, which can be attributed to antibody-mediated graft destruction, or at a later stage characterized by a sudden decline in graft function.
- When an immunosuppressive agent is used, it may be administered by any suitable means, including parenteral, and, if desired for local immunosuppressive treatment, intralesionally. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, and subcutaneous administration. In addition, when an immunosuppressive agent is used, it is suitably administered by pulse infusion, particularly with declining doses, or by continuous infusion.
- In one aspect, provided herein is a compositions disclosed herein can further comprise an immunomodulatory agent, for example, an immunosuppressive agent. An “effective amount” of an immunosuppressive agent and peptides disclosed herein is an amount which achieves a degree of immunosuppression sufficient to delay, inhibit, suppress or moderate tissue transplant rejection and/or delay, inhibit, suppress or moderate one or more symptoms of an inflammatory disease e.g., autoimmune disease described herein. Preferably, the peptides of the present disclosure in combination with an immunomodulatory agent are used at dosages suitable for inducing transplant survival or, alternatively, at dosages at which the immunosuppressive activities of the two agents synergize. Accordingly, sub-therapeutic dosages can be used in the disclosed methods, i.e., dosages which are lower than the amounts that would be effective when the peptide or immunomodulatory agent is used alone. Suitable sub-therapeutic dosages of one or both agents of the disclosed combination therapy are those which are sufficient, when the two agents are used in combination, to delay, inhibit, suppress or moderate an undesired immune response to an antigen, or inhibit transplant rejection as a result as described above. The skilled artisan will be able to determine such dosages using ordinary experimentation, such as by using animal models. One advantage of the disclosed combined therapy, therefore, is that the subject being treated can in some instances be spared the side-effects of higher levels of immunosuppression resulting from, for example, corticosteroids and inhibitors of calcineurin.
- Additional Therapeutic Agents
- Of course, the peptides or compositions of the present disclosure can be used in combination with other therapies for treating autoimmune disorders, inflammation, cancer or for inhibiting transplant rejection. Accordingly, the compositions and methods of the present disclosure can comprise an additional therapeutic agent. The therapeutic agent can be, for example, anti-inflammatory agent, immunosuppressive agent, anti-tumor agent as relevant for the condition to be treated. Immunosuppressive agents have been described above.
- Non-limiting example of anti-tumor agents include antibiotics and analogs (e.g., aclacinomycins, actinomycin f1, anthramycin, azaserine, bleomycins, cactinomycin, carubicin, carzinophilin, chromomycins, dactinomycin, daunorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, idarubicin, menogaril, mitomycins, mycophenolic acid, nogalamycin, olivomycines, peplomycin, pirarubicin, plicamycin, porfiromycin, puromycin, streptonigrin, streptozocin, tubercidin, zinostatin, zorubicin), antimetabolites (e.g. folic acid analogs (e.g., denopterin, edatrexate, methotrexate, piritrexim, pteropterin, Tomudex®, trimetrexate), purine analogs (e.g., cladribine, fludarabine, 6-mercaptopurine, thiamiprine, thioguanine), pyrimidine analogs (e.g., ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, doxifluridine, emitefur, enocitabine, floxuridine, fluorouracil, gemcitabine, tagafur).
- Anti-inflammatory agent can be Steroidal Anti-inflammatory Agents, such as 21-acetoxypregnenolone, alclometasone, algestone, amcinonide, beclomethasone, betamethasone, budesonide, chloroprednisone, clobetasol, clobetasone, clocortolone, cloprednol, corticosterone, cortisone, cortivazol, deflazacort, desonide, desoximetasone, dexamethasone, diflorasone, diflucortolone, difluprednate, enoxolone, fluazacort, flucloronide, flumethasone, flunisolide, fluocinolone acetonide, fluocinonide, fluocortin butyl, fluocortolone, fluorometholone, fluperolone acetate, fluprednidene acetate, fluprednisolone, flurandrenolide, fluticasone propionate, formocortal, halcinonide, halobetasol propionate, halometasone, halopredone acetate, hydrocortamate, hydrocortisone, loteprednol etabonate, mazipredone, medrysone, meprednisone, methylprednisolone, mometasone furoate, paramethasone, prednicarbate, prednisolone, prednisolone 25-diethylamino-acetate, prednisolone sodium phosphate, prednisone, prednival, prednylidene, rimexolone, tixocortol, triamcinolone, triamcinolone acetonide, triamcinolone benetonide, and triamcinolone hexacetonide and the like. Anti-inflamatory agent can be Non-Steroidal antiinflammatory agents such as Aminoarylcarboxylic acid derivatives (e.g., enfenamic acid, etofenamate, flufenamic acid, isonixin, meclofenamic acid, mefenamic acid, niflumic acid, talniflumate, terofenamate, tolfenamic acid), arylacetic acid derivatives (e.g., aceclofenac, acemetacin, alclofenac, amfenac, amtolmetin guacil, bromfenac, bufexamac, cinmetacin, clopirac, diclofenac sodium, etodolac, felbinac, fenclozic acid, fentiazac, glucametacin, ibufenac, indomethacin, isofezolac, isoxepac, lonazolac, metiazinic acid, mofezolac, oxametacine, pirazolac, proglumetacin, sulindac, tiaramide, tolmetin, tropesin, zomepirac), arylbutyric acid derivatives (e.g., bumadizon, butibufen, fenbufen, xenbucin), arylcarboxylic acids (e.g., clidanac, ketorolac, tinoridine), arylpropionic acid derivatives (e.g., alminoprofen, benoxaprofen, bermoprofen, bucloxic acid, carprofen, fenoprofen, flunoxaprofen, flurbiprofen, ibuprofen, ibuproxam, indoprofen, ketoprofen, loxoprofen, naproxen, oxaprozin, piketoprolen, pirprofen, pranoprofen, protizinic acid, suprofen, tiaprofenic acid, ximoprofen, zaltoprofen), pyrazoles (e.g., difenamizole, epirizole), pyrazolones (e.g., apazone, benzpiperylon, feprazone, mofebutazone, morazone, oxyphenbutazone, phenylbutazone, pipebuzone, propyphenazone, ramifenazone, suxibuzone, thiazolinobutazone), salicylic acid derivatives (erg., acetaminosalol, aspirin, benorylate, bromosaligenin, calcium acetylsalicylate, diflunisal, etersalate, fendosal, gentisic acid, glycol salicylate, imidazole salicylate, lysine acetylsalicylate, mesalamine, morpholine salicylate, 1-naphthyl salicylate, olsalazine, parsalmide, phenyl acetylsalicylate, phenyl salicylate, salacetamide, salicylamide o-acetic acid, salicylsulfuric acid, salsalate, sulfasalazine), thiazinecarboxamides (e.g., ampiroxicam, droxicam, isoxicam, lornoxicam, piroxicam, tenoxicam), ε-acetamidocaproic acid, s-adenosylmethionine, 3-amino-4-hydroxybutyric acid, amixetrine, bendazac, benzydamine, α-bisabolol, bucolome, difenpiramide, ditazol, emorfazone, fepradinol, guaiazulene, nabumetone, nimesulide, oxaceprol, paranyline, perisoxal, proquazone, superoxide dismutase, tenidap, and zileuton and the like.
- In some embodiments, the therapeutic agent can be a biologic. As used herein a biologic refers to any biological material suitable for a therapeutic or in vivo diagnostic purpose. Biologic therapeutics include peptides, proteins, vaccines, antibodies, aptamers, nucleic acids, DNA, RNA, antisense oligonucleotides, viruses and bacteria.
- As used herein, the terms “treat,” “treatment,” “treating,” or “amelioration” refer to therapeutic treatments, wherein the object is to reverse, alleviate, ameliorate, inhibit, slow down or stop the progression or severity of a condition associated with, a disease or disorder. The term “treating” includes reducing or alleviating at least one adverse effect or symptom of a condition, disease or disorder associated with a CD40 activity, such as, but not limited to, a inflammation, autoimmune disease or a cancer. Treatment is generally “effective” if one or more symptoms or clinical markers are reduced. Alternatively, treatment is “effective” if the progression of a disease is reduced or halted. That is, “treatment” includes not just the improvement of symptoms or markers, but also a cessation of at least slowing of progress or worsening of symptoms that would be expected in absence of treatment. Beneficial or desired clinical results include, but are not limited to, alleviation of one or more symptom(s), diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. The term “treatment” of a disease also includes providing relief from the symptoms or side-effects of the disease (including palliative treatment).
- The methods of the disclosure, including treating a CD40 associated disorder of a subject, likely results in an improvement in the subjects' condition, a reduction of symptoms or decreasing the subject's risk for developing symptoms associated with a CD40 associated disorder. Improvements therefore include one or more decreased symptoms associated with autoimmunity, allergy, cancer, etc. An improvement may also be reducing the frequency or amount of a drug used for treating a subject having or at risk of having a CD40 associated disorder. For example, autoimmune patients treated with steroids may require less steroid when treated in combination with a peptide or compositions disclosed herein. An improvement therefore would include reducing the dosage frequency or amount of steroid that the subject was administered in comparison to the dosage frequency or amount administered prior to treatment with a peptide or compositions disclosed herein. An improvement may be, for example, longer transplant survival, or reduction of side effects associated with conventional immunosuppressive regiments for transplantation.
- An improvement may be relatively short in duration, e.g., several hours, days or weeks, or extend over a longer period of time, e.g., months or years. The improvement need not be a complete ablation of any or all symptoms of the disorder. For example, reducing severe rheumatoid arthritis to a less severe form is an improvement. Thus, a satisfactory clinical endpoint is achieved when there is an incremental improvement or a partial reduction in the subject's condition or associated symptoms, over a short or long duration.
- The term “effective amount” as used herein refers to the amount needed to alleviate at least one or more symptom of the disease or disorder, and relates to a sufficient amount of pharmacological composition to provide the desired effect (e.g., binding to CD40 and/or inhibition of the interaction of CD40 and CD154). The term “therapeutically effective amount” therefore refers to an amount that is sufficient to effect a particular effect when administered to a typical subject. An effective amount as used herein would also include an amount sufficient to delay the development of a symptom of the disease, alter the course of a symptom disease (for example but not limited to, slow the progression of a symptom of the disease), or reverse a symptom of the disease. Thus, it is not possible to specify the exact “effective amount”. For any given case, however, an appropriate “effective amount” can be determined by one of ordinary skill in the art using only routine experimentation.
- Effective amounts, toxicity, and therapeutic efficacy can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dosage can vary depending upon the dosage form employed and the route of administration utilized. The dose ratio between toxic and therapeutic effects is the therapeutic index and can be expressed as the ratio LD50/ED50. Compositions and methods that exhibit large therapeutic indices are preferred. A therapeutically effective dose can be estimated initially from cell culture assays. Also, a dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the antibody or antigen binding fragment thereof), which achieves a half-maximal inhibition of symptoms as determined in cell culture, or in an appropriate animal model. Levels in plasma can be measured, for example, by high performance liquid chromatography. The effects of any particular dosage can be monitored by a suitable bioassay. The dosage can be determined by a physician and adjusted, as necessary, to suit observed effects of the treatment.
- The compositions of the disclosure are administered in effective amounts. An “effective amount” is that amount of a composition that alone, or together with further doses, produces the desired response. In the case of transplantation, a desired response is inhibition of transplant rejection or increasing transplant survival. In the case of treating a particular disease, such as arthritis, the desired response is inhibiting the progression of the disease. This may involve only slowing the progression of the disease temporarily, although more preferably, it involves halting the progression of the disease permanently. This can be monitored by routine methods.
- Such amounts will depend, of course, on the particular condition being treated, the severity of the disorder, the activity of the specific compound, the route of administration, the rate of clearance of the composition, the duration of treatment, the drugs used in combination or coincident with the compositions, the age, body weight, sex, diet, and general health of the subject, and like factors well known in the medical arts and sciences. Various general considerations taken into account in determining the “therapeutically effective amount” are known to those of skill in the art and are described, e.g., in Gilman et al, eds., Goodman And Gilman's: The Pharmacological Bases of Therapeutics, 8th ed., Pergamon Press, 1990; and Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Co., Easton, Pa., 1990. These factors are well known to those of ordinary skill in the art and can be addressed with no more than routine experimentation. It is generally preferred that a maximum dose of the individual components or combinations thereof be used, that is, the highest safe dose according to sound medical judgment. The compositions used in the foregoing methods preferably are sterile and contain an effective amount of the active agents for producing the desired response in a unit of weight or volume suitable for administration to a patient.
- In another embodiment of the disclosure, an article of manufacture which contains the pharmaceutical composition in a solution form or in a lyophilized form or a kit comprising an article of manufacture is provided. The kit can comprise instructions for diluting the composition or for its reconstitution and/or use. The article of manufacture comprises a container. Suitable containers include, for example, bottles, vials (e.g. dual chamber vials), syringes (such as dual chamber syringes) and test tubes. The container may be formed from a variety of materials such as glass or plastic. The container holds the lyophilized formulation and a label on, or associated with, the container may indicate directions for reconstitution and/or use. For example, the label may indicate that the lyophilized formulation is reconstituted to peptide concentrations as described above. The label may further indicate that the formulation is useful or intended for subcutaneous administration. The container holding the formulation may be a multi-use vial, which allows for repeat administrations (e.g., from 2-6 administrations) of the reconstituted formulation. The article of manufacture may further comprise a second container comprising a suitable diluent (e.g., BWFI). Upon mixing of the diluent and the lyophilized formulation, the final protein concentration in the reconstituted formulation will generally be at least 50 mg/mL. The article of manufacture may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use.
- Therapeutic kits may have a single container which contains the formulation of the peptide pharmaceutical compositions with or without other components (e.g., other compounds or pharmaceutical compositions of these other compounds) or may have distinct container for each component. In some embodiments, therapeutic kits of the disclosure include a formulation of peptide disclosed herein or compositions comprising said peptides or an acid addition salt thereof as disclosed herein packaged for use in combination with the co-administration of a second compound (such as an anti-inflammatory agent, immunomodulating agent, anti-tumor agent, a natural product, a hormone or antagonist, a anti-angiogenesis agent or inhibitor, a apoptosis-inducing agent or a chelator) or a pharmaceutical composition thereof. The components of the kit may be pre-complexed or each component may be in a separate distinct container prior to administration to a patient. The components of the kit may be provided in one or more liquid solutions, preferably, an aqueous solution, more preferably, a sterile aqueous solution. The components of the kit may also be provided as solids, which may be converted into liquids by addition of suitable solvents, which are preferably provided in another distinct container.
- The container of a therapeutic kit may be a vial, test tube, flask, bottle, syringe, or any other means of enclosing a solid or liquid. Usually, when there is more than one component, the kit will contain a second vial or other container, which allows for separate dosing. The kit may also contain another container for a pharmaceutically acceptable liquid. Preferably, a therapeutic kit will contain apparatus (e.g., one or more needles, syringes, eye droppers, pipette, etc.), which enables administration of the peptides of the disclosure which are components of the present kit.
- In another embodiment of the disclosure, an article of manufacture which contains compositions comprising apoptotic leukocytes and an anti-CD40 agent comprising one or more peptides with an amino sequence with at least 85%, at least 90% or at least 95% sequence identity to the sequences set forth in any one of SEQ ID NOs. 1-6 and 8-17, in a solution form or in a lyophilized form or a kit comprising an article of manufacture is provided. The kits of the instant disclosure can be for use in transplantation of a transplant in a recipient or reducing or inhibiting occurrence of GVHD in a recipient. In some embodiments, the kit is useful as a preparatory regimen prior to the transplantation. In some embodiments, the kit is useful as a tolerizing regimen post-transplantation. The kit can comprise instructions for diluting the composition or for its reconstitution and/or use. The article of manufacture comprises one or more containers. Suitable containers include, for example, bottles, vials (e.g. dual chamber vials), syringes (such as dual chamber syringes) and test tubes. The container may be formed from a variety of materials such as glass or plastic. The container holds the compositions e.g., in a lyophilized or solution form and a label on, or associated with, the container may indicate directions for reconstitution and/or use. For example, the label may indicate that the lyophilized composition is reconstituted to an effective amount as described above. The label may further indicate that the composition is useful or intended for subcutaneous administration or intravenous administration. The container holding the composition may be a multi-use vial, which allows for repeat administrations (e.g., from 2-6 administrations) of the composition. The article of manufacture may further comprise a container comprising a suitable diluent (e.g., BWFI). Upon mixing of the diluent and the composition, the final concentration in the composition can be, for example, an effective amount suitable for administration. The article of manufacture may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use.
- Kits may have a single container which contains, for example apoptotic leukocytes fixed in a crosslinking agent with or without other components (e.g., and an anti-CD40 agent or an anti-CD40 ligand agent). In some cases, the anti-CD40 agent conjugated to the apoptotic leukocytes may comprise a peptide comprising an amino acid sequence with at least 85%, at least 90% or at least 95% sequence identity to the sequences set forth in any one of SEQ ID NOs. 1-6 and 8-17. The components of the kit may be pre-complexed or each component may be in a separate distinct container prior to administration to a patient. The components of the kit may be provided in one or more liquid solutions, preferably, an aqueous solution, more preferably, a sterile aqueous solution. The components of the kit may also be provided as solids, which may be converted into liquids by addition of suitable solvents, which are preferably provided in another distinct container. In some embodiments, kits of the disclosure include the components disclosed herein packaged for use in combination with the co-administration of an additional component (such as an anti-inflammatory agent, immunomodulating agent, anti-tumor agent, a natural product, a hormone or antagonist, an anti-angiogenesis agent or inhibitor, a apoptosis-inducing agent, or a chelator). The container of a kit may be a vial, test tube, flask, bottle, syringe, or any other means of enclosing a solid or liquid. In some embodiments, the kit will contain a second vial or other container, which allows for separate dosing. The kit may also contain another container for a pharmaceutically acceptable liquid. Preferably, a kit will contain apparatus (e.g., one or more needles, syringes, eye droppers, pipette, etc.), which enables administration of the compositions of the disclosure which are components of the present kit.
- In some embodiments, the kit disclosed herein further comprises the transplant. In some embodiments, the transplant is a kidney, liver, heart, lung, pancreas, islet cell, small bowel, bone marrow, hematopoietic stem cell, embryonic or induced pluripotent stem cell-derived islet beta cell, embryonic or induced pluripotent stem cell-derived islet, embryonic or induced pluripotent stem cell-derived hepatocyte or a combination thereof. In some embodiments, the transplant can be autologous, allograft, or a xenograft.
- These examples are provided for illustrative purposes only and not to limit the scope of the claims provided herein.
- Table 1 lists the amino acid sequences of CD40 binding peptides. A control CD40-binding peptide,
Peptide 1 was included in the studies described below for comparison.Peptide 1 has the amino acid sequence of VLQWAEKGYYTMSNN (SEQ ID NO: 7). In silico prediction using the Amino Acid Complement Wave method was used to identify peptides 2-7 that exhibited high affinity to the human CD40 epitope. Peptides that can bind CD40 and block the CD40-CD40L signaling cascade were identified. Sequences of the peptides are presented in the table below. The identified peptides were synthesized with and without fluorescent labeling to study the binding and functional properties of the peptides to interfere in the CD40-CD40L signaling. Peptides 8-17 are exemplary fragments of peptide 2-7 expected to exhibit high affinity to the human CD40 epitope. -
TABLE 1 lists the amino acid sequences of the peptides identified to bind CD40. SEQ ID NO: 1 VLQWAERGFYTTSNN Peptide 2 SEQ ID NO: 2 LLQWAERGYFMTSNN Peptide 6 SEQ ID NO: 3 VVQWAEKGFFTMSNN Peptide 3 SEQ ID NO: 4 VLNWADKAYFTMSQN Peptide 4 SEQ ID NO: 5 LVNWADKAFYTTSNN Peptide 5 SEQ ID NO: 6 VVQWAEKGYYTMSQN Peptide 7 SEQ ID NO: 8 VLQWAERGFY Peptide 8 SEQ ID NO: 9 LLQWAERGYF Peptide 9 SEQ ID NO: 10 VVQWAEKGFF Peptide 10 SEQ ID NO: 11 VLNWADKAYF Peptide 11 SEQ ID NO: 12 LVNWADKAFY Peptide 12 SEQ ID NO: 13 RGFYTTSNN Peptide 13 SEQ ID NO: 14 RGYFMTSNN Peptide 14 SEQ ID NO: 15 KGFFTMSNN Peptide 15 SEQ ID NO: 16 KAYFTMSQN Peptide 16 SEQ ID NO: 17 KAFYTTSNN Peptide 17 - In order to screen the ability of the cyclic peptides to bind to CD40, peripheral blood mononuclear cells (PBMC) purified from 2 Rhesus macaques (RM) were incubated with 504 of fluorochrome tagged CD40 interacting peptides, Peptide 1-7 listed in Table 1 for 30 minutes at 4° C. The cells were washed 3× in PBS and analyzed by flow cytometry. Stained cells were acquired on a 3-laser BD Canto II instrument (BD Bioscience). A minimum of 200,000 events were acquired with FACSDIVA 6.1.3. Mean fluorescence intensity of the peptide binding to the cells were determined using FlowJo 10.1. software (Tree Star; Ashland, Oreg., USA). FACS analysis demonstrated that the synthetic peptides listed in SEQ ID NOs. 1-7 bind to the PBMC but at various intensities, confirming the ability of the synthetic peptides to bind to CD40 on mononuclear cells.
Peptide 1 andPeptide 3 showed high affinity for binding to mononuclear cells whereasPeptide 7 has the least binding affinity to the mononuclear cells. - In order to confirm the specificity of the peptides to interfere with CD40:CD154 interaction, the specificity of peptides 1-7 as listed in table 1 to bind to human CD154 by ELISA was tested. Briefly, 96 well ELISA (Nunc Immunolon) plates were coated with the peptides in carbonate buffer. The wells are washed and probed with FLAG-tagged rhsCD40 (1 μg/ml; Alexis) and developed with HRP conjugated rabbit anti-FLAG antibody. The plates were washed and the enzymatic activity was determined using 3,3′,5,5′-tetramethyl-benzidine substrate for 15 minutes and the reaction was stopped by the addition of 2 M H2SO4, and optical density was measured at 450 nm, with the correction wavelength set at 620 nm. Similar to the mononuclear cell binding studies, the
Peptide # 1 showed high binding affinity to recombinant human CD40, whereasPeptide 4 had the lowest binding affinity. These binding studies indicatePeptide 2,Peptide 3, andPeptide 7 showed specific binding to human CD40, and can therefore block CD40-CD154 interaction. - In order to determine the functional effect of co-stimulation blockade with peptides targeting CD40, one-way mixed lymphocyte reactions (MLR) was performed. CF SE-labeled PBMC from 5 different RM were stimulated with irradiated PBMC for 6 days. Stimulation with PHA was used as positive control. Initial screening with various concentration of peptides 1-7 listed in table 1 (15, 30 and 45 μM). These studies demonstrated that peptides added at 30 μM showed the maximum potential for costimulation blockade and this concentration used in further assays. The cells were incubated in the presence of peptides at 30 μM. Following stimulation, PBMC were stained with antibodies specific for CD4, CD8 and CD20 and proliferating cells were quantitated by determining the frequency of CF SE-low CD4, CD8 and CD20 cells. Stained cells were acquired on a 3-laser BD Canto II instrument (BD Bioscience). A minimum of 200,000 events were acquired with FACSDIVA 6.1.3. FACS analysis was performed with
FlowJo Version 10 software. Analysis of the results demonstrated that all the peptides 1-7 showed significant suppression of CD4, CD8 and CD20 cell proliferation in 4/5 donor recipient combinations. More significantly,Peptide 2 andPeptide 3 suppressed the proliferation of CD4, CD8, and CD20 cells at a higher efficacy compared to the other peptides. - The ability of CD40-binding peptides 1-7 as listed in Table 1 to block B cell activation was assessed by analyzing the expression of MHC class II molecules on the B cell when subsequently incubated in the presence of recombinant hCD40L. Briefly, B cells were purified from 3 RM PBMC by magnetic sorting. B cells were incubated with peptides at 30 μM for 15 minutes at 37° C., then activated for 30 minutes with recombinant hCD40L, and then analyzed for expression of MHC class II by staining the cells with specific antibody. Stained cells were acquired on a 3-laser BD Canto II instrument (BD Bioscience). A minimum of 200,000 events were acquired with FACSDIVA 6.1.3. FACS analysis was performed with
FlowJo Version 10 software. FACS analysis of the B cells demonstrated that pre-incubation with the peptides resulted in a 50% reduction in MHC class II expression on B cells following activation with CD40L. - This study identified new peptides that can bind to CD40 and inhibit CD40 activity (i.e., CD40 interaction with CD40L). The identified peptides bind specifically to CD40.
Peptide 2,Peptide 3, andPeptide 5 listed in Table 1 exhibited a higher affinity to bind to CD40 when compared to thecontrol Peptide 1. When compared withPeptide 1;Peptide 2,Peptide 3 andPeptide 5 show an enhanced ability to bind CD40 and inhibit CD40-CD40L interaction and down-stream signaling as evidenced by complete abrogation of T and B cell proliferation as well as activation of B cells. - This example examines effect of anti-CD40 peptides and the tolerogenic effect of ECDI-treated donor cells for islet transplantation in vivo. The effect of an exemplary CD40 binding peptide,
peptide 3 is illustrated. ECDI-treated splenocytes from a xenogeneic or allogeneic source are administered to a human or a non-human primate transplanted with islets, thereby minimizing the possibility of graft rejection in the human or non-human primate. The preparatory regimen of this example can readily be adapted to allotransplantation or xenotransplantation in human recipients (for example, transplant of islets, kidneys, or other cells, tissues, or organs). - In an illustrative example, a streptozotocin-induced model of diabetes is utilized with non-human primate subjects. Diabetes is induced by intravenous treatment with streptozotocin. Recipient subjects are transplanted with islets from a MHC-I disparate, one MHC-II DRB-allele matched donor. Recipients are treated with a short term immunosuppressive regimen comprising: (i) an anti-CD40 peptide (Peptide 3), given intravenously at a dose of 50 mg/kg on day −8, −1, 7, and 14 relative to transplant; (ii) rapamycin, given orally from day −7 to day 21 relative to transplant with a target trough level of 5-12 ng/mL; (iii) soluble TNF receptor, given intravenously at a dose of 1 mg/kg on days −7 and 0 relative to transplant and subcutaneously at a dose of 0.5 mg/kg on
days - Transplant recipients in experimental groups receive an intravenous infusion of ECDI-treated apoptotic donor leukocytes (ADLs) together with anti-CD40 peptide, peptide 3 (or any one or more peptide selected from peptides 1-17) before and optionally after transplantation. In some cases, ADLs together with an anti-CD40 peptide, peptide 3 (or any one or more peptide selected from peptides 1-17) are administered about 8 days before transplantation. In some cases, ADLs are administered about 7 days before transplantation. In some cases, ADLs together with an anti-CD40 peptide, peptide 3 (or any one or more peptide selected from peptides 1-17) are administered about 1 day after transplantation. In some cases, ADLs together with an anti-CD40 peptide are administered about 7 days after transplantation. In some cases, ADLs together with an anti-CD40 peptide are administered about 14 days after transplantation. In some cases, ADLs together with an anti-CD40 peptide are administered about 7 or 8 days before transplantation, and about 1, 7, and/or 14 days after transplantation. ADLs can be from the same donor as the islets or a different donor as disclosed herein.
- Transplant recipients in a control group do not receive ADLs. Small doses of exogenous insulin can be administered through day 21 after transplantation. Transplant recipients that receive ADLs together with an anti-CD40 peptide are expected to exhibit improved transplant survival compared to the group that do not receive ADLs. Transplant recipients that receive ADLs together with an anti-CD40 peptide are expected to exhibit improved rejection-free transplant survival compared to the group that do not receive ADLs. Transplant recipients that receive ADLs together with an anti-CD40 peptide are expected to exhibit long-term functional survival of islet allografts. For example, transplant recipients that receive ADLs together with an anti-CD40 peptide are expected to exhibit improved blood glucose control after transplant (e.g., become normoglycemic), including after they stop receiving exogenous insulin (e.g., past day 100 or day 365 post-transplant). Blood glucose control can be evaluated, for example, by intravenous glucose tolerance test (IVGTT), a mixed meal tolerance test (MMTT), or any other metabolic test established for monitoring pancreatic islet beta cell function. In IVGTT, exogenous glucose is injected intravenously, and the blood glucose level is measured over time after the injection. Transplant recipients that receive ADLs together with an anti-CD40 peptide are expected to exhibit rapid decreases in blood glucose levels and reduced area under the glucose concentration curve after IVGTT (e.g., comparable levels to those prior to streptozotocin treatment). Islet transplant recipients that receive ADLs together with an anti-CD40 peptide are expected to exhibit decreased hemoglobin A1C levels after transplant. Transplant recipients that receive ADLs together with an anti-CD40 peptide are expected to exhibit increased C-peptide levels after transplant that are maintained over time, indicating the transplanted islets are functional (e.g., fasted C-peptide levels, glucose-stimulated C-peptide levels, and/or increase in C-peptide levels upon glucose stimulation).
- This example demonstrates that conjugating anti-CD40 peptides (e.g., Peptides 1-17 listed in table 1) to apoptotic donor leukocytes (ADLs) can enhance the tolerance-inducing efficacy of a preparatory regimen. In addition, for donor-recipient pairs that are fully WIC class I and class II mismatched, additionally conjugating recipient-type WIC class II molecules to ADLs can enhance ADL efficacy in inducing tolerance to a transplanted cell, tissue, or organ.
- Coupling one or more peptides that can abrogate CD40-CD154 binding (e.g., Peptides 1-17 listed in table 1) to the surface of ADLs can enhance tolerance to a transplanted cell, tissue or organ, after uptake of ADLs (for example, by recipient spleen marginal zone antigen presenting cells or liven sinusoidal endothelial cells). Additionally, coupling recipient-type WIC class II peptides for presentation after uptake of ADLs, derived from one (or more) of the transplant recipient's WIC class II molecules can promote tolerance in the recipient, for example, via regulatory T cell subsets.
- To determine the ability of apoptotic donor leukocytes coupled via ECDI with anti-CD40 peptides (e.g. peptides listed in Table 1), and recipient-type DRB peptide to promote immune regulation and transplantation tolerance, the following fully mismatched monkey recipient/donor pair and experimental conditions are selected to test a mixed lymphocyte reaction (MLR) in vitro with WIC-defined stimulator (donor) and responder (recipient) peripheral blood mononuclear cells (PBMCs).
- Recipient PBMCs are Mauritian Cynomolgus Monkey (Mafa M4A, M4A, M4B, M4B, M4DR, M1DR). Apoptotic donor leukocytes are Mauritian Cynomolgus Monkey (M3A, M3A, M3B, M3B, M3DR, M3DR), with 14 μg of synthetic peptides (e.g., Peptides 1-17 listed in table 1) conjugated to the cell surface via ECDI.
- In the MLR assay, 3×106 apoptotic donor leukocytes with and without conjugated anti-CD40peptide are used to stimulate 3×106 recipient PBMCs at 37° C. in a CO2 incubator. Serial samples collected post stimulation on
day 1 andday 3 are analyzed for the induction of DC-10 (CD141+CD163+ of CD14+CD16+), Treg cells (CD25hi CD127− FoxP3+ of CD4+) and Tr1 cells (CD49b+LAG-3+ of CD4+) by flow cytometry. Stimulated cells show that in comparison to PBLs stimulated with control apoptotic donor leukocytes alone, there is an increase in the frequency of Tregs onday 1 and further onday 3 following stimulation with anti-CD40 peptide-conjugated apoptotic donor leukocytes. Similarly, a gradual increase in the frequency of Tr1 cells (onday 1 and day 3), is observed following stimulation with anti-CD40 peptide-conjugated versus control apoptotic donor leukocytes. These results demonstrate that conjugating anti-CD40 peptides (e.g., Peptides 1-17 listed in table 1) to apoptotic donor leukocytes can promote expansion of tolerance-promoting immune regulatory cell subsets. - The preparatory regimen of this example can readily be adapted to allotransplantation or xenotransplantation in mammalian recipients (for example, transplant of islets, kidneys, or other cells, tissues, or organs, such as embryonic stem cell, induced pluripotent stem cell (iPS)-derived, or mesenchymal stem cell-derived cells, tissues and organs).
- In an illustrative example, in addition to anti-CD40 peptides (e.g., Peptides 1-17 listed in table 1), recipient type MHC peptides are conjugated to ADLs, and the ADLs are used to induce tolerance to a human islet transplant recipient.
- Splenocytes are obtained from a fully MHC-I and MHC-II mismatched xenogeneic or allogeneic source and are conjugated during ECDI treatment to anti-CD40 peptides (e.g., Peptides 1-17 listed in table 1). Following that, MHC class II chains, domains, and/or peptides are conjugated during ECDI treatment to generate ADLs conjugated to anti-CD40 peptides and peptides derived from MHC-II. The ADLs conjugated to anti-CD40 peptides and peptides derived from MHC-II are administered to human subjects that receive islet transplants, thereby reducing the possibility of graft rejection. Transplant recipients receive an intravenous infusion of ADLs conjugated to anti-CD40 peptides and peptides derived from MHC-II before and optionally after transplantation, for example, on day −7 and day +1 relative to transplantation. ADLs can be from the same donor as the islets or a different donor as disclosed herein.
- Recipient subjects are transplanted with islets from the fully MHC class I and MHC class II mismatched donor. Recipients are treated with a short term immunosuppressive regimen comprising: (i) an antagonistic anti-CD40 peptide (e.g., Peptides 1-17 listed in table 1), given intravenously at a dose of 50 mg/kg on day −8, −1, 7, and 14 relative to transplant; (ii) rapamycin, given orally from day −7 to day 21 relative to transplant with a target trough level of 5-12 ng/mL; (iii) soluble TNF receptor, given intravenously at a dose of 1 mg/kg on days −7 and 0 relative to transplant and subcutaneously at a dose of 0.5 mg/kg on
days - Small doses of exogenous insulin can be administered through day 21 after transplantation. Transplant recipients in control groups do not receive ADLs, or receive ADLs without anti-CD40 peptide and MHC class II peptides conjugated. Transplant recipients that receive anti-CD40 peptide and MHC-II peptides conjugated ADLs are expected to exhibit improved survival compared to recipients that do not receive anti-CD40 peptide and MHC-II-conjugated ADLs. Transplant recipients that receive ADLs conjugated with anti-CD40 peptide and peptides derived from MHC-II are expected to exhibit improved rejection-free survival compared to recipients that do not receive anti-CD40 peptide and MHC-II-conjugated ADLs. Transplant recipients that receive ADLs conjugated to anti-CD40 peptides and peptides derived from MHC-II are expected to exhibit long-term functional survival of islet allografts compared to recipients that do not receive ADLs conjugated to anti-CD40 peptides and peptides derived from MHC-II. For example, transplant recipients that receive ADLs conjugated to anti-CD40 peptides and peptides derived from MHC-II are expected to exhibit improved blood glucose control after transplant (e.g., become normoglycemic), including after they stop receiving exogenous insulin (e.g., past day 100 or day 365 post-transplant). Blood glucose control can be evaluated, for example, by intravenous glucose tolerance test (IVGTT), a mixed meal tolerance test (MMTT), or any other metabolic test established for monitoring pancreatic islet beta cell function. In IVGTT, exogenous glucose is injected intravenously, and the blood glucose level is measured over time after the injection. Transplant recipients that receive ADLs conjugated to anti-CD40 peptides and peptides derived from MHC-II are expected to exhibit rapid decreases in blood glucose levels and reduced area under the glucose concentration curve after IVGTT (e.g., comparable levels to a healthy subject). Transplant recipients that receive ADLs conjugated to anti-CD40 peptides and peptides derived from MHC-II are expected to exhibit decreased hemoglobin A1C levels after transplant. Transplant recipients that receive anti-CD40L and MHC-II conjugated ADLs are expected to exhibit increased C-peptide levels after transplant that are maintained over time, indicating the transplanted islets are functional (e.g., fasted C-peptide levels, glucose-stimulated C-peptide levels, and/or increase in C-peptide levels upon glucose stimulation).
- Stem cells from one donor can be differentiated into a first population of cells for use as apoptotic donor leukocytes (ADLs), and separately differentiated into a second population of cells for transplant. This technique can be used to induce tolerance to any universal cell-derived cell, tissue, or organ transplant.
- The stem cells can be embryonic stems cells, induced pluripotent stem cells (iPSCs), and/or mesenchymal stem cells. The stem cells are differentiated into a first population of cells that express both MHC class I and MHC class II antigens. For example, iPSCs from a transplant donor are differentiated into B lymphocytes that express both MHC class I and II antigens. Methods of differentiating iPSCs into B cells are described, for example, in French A et al. (2015), Stem Cells and Development 24(9):1082-95. Preferably, for increased tolerogenic efficacy, the donor stem cell-derived B cells share one MHC class II antigen with the recipient (e.g., at least one MHC class II DR allele, MHC class II DQ allele, or MHC class II DP allele). Separately, the iPSCs are differentiated into a second population of cells to be transplanted. The stem-cell derived B cells are treated with ECDI in the presence of an anti-CD40 peptide (e.g., Peptides 1-17 listed in table 1) to generate anti-CD40 conjugated apoptotic donor leukocytes (anti-CD40 peptide conjugated ADLs).
- The stem cell-derived, anti-CD40 peptide conjugated ADLs are administered to a subject that receives a transplant of cells, tissues, or organs derived from the same stem cell donor, thereby reducing the possibility of transplant rejection. The transplant recipient receives an intravenous infusion of stem cell-derived anti-CD40 peptide conjugated ADLs before and optionally after transplantation, for example, on day −7 and day +1 relative to transplantation.
- The recipients can optionally be treated with a short term immunosuppressive regimen comprising: (i) an antagonistic anti-CD40 peptide (e.g., Peptides 1-17 listed in table 1), given intravenously at a dose of 50 mg/kg on day −8, −1, 7, and 14 relative to transplant; (ii) rapamycin, given orally from day −7 to day 21 relative to transplant with a target trough level of 5-12 ng/mL; (iii) soluble TNF receptor, given intravenously at a dose of 1 mg/kg on days −7 and 0 relative to transplant and subcutaneously at a dose of 0.5 mg/kg on
days - The recipient is expected to exhibit improved rejection-free survival compared to the recipients that do not receive the stem-cell derived, anti-CD40 peptide conjugated ADLs. This technique can be used to induce tolerance to any universal cell-derived cell, tissue, or organ transplant.
- ECDI-fixed B cell products conjugated with CD40 binding peptide (e.g., Peptides 1-17 listed in table 1) generated under different experimental conditions are compared for their ability to induce maturation-arrest in dendritic cells (DC). Human monocyte-derived DC are generated with IL-4 and GCSF. These DC are incubated in the presence or absence of various ECDI-fixed B cell products coupled to inhibitors of DC maturation (e.g., rapamycin, curcumin, vitamin D3, Bay-117085, siCD40, cobalt protoporphyrin, and α1-antitrypsin). Readouts of DC maturation arrest include i) expression of DC phenotypic markers (CD83, CD80, CD86, MHC class II, CD40), ii) STAT-6 phosphorylation, iii) RELP nuclear translocation, iv) IL-12p70 production, v) allostimulatory capacity, and vi) priming of T cells with regulatory phenotypes (CD4+CD25hiCD127lowFoxp3+ Tregs and CD4+CD49b+Lag-3+CD45RA− Tr1 cells). ECDI-treated B cell products are identified that induce maturation-arrest in DC.
- The effects of ECDI-fixed donor B lymphocytes on immune profiles in responder peripheral blood lymphocytes (PBL) is evaluated. ECDI-fixed donor B cells are generated, including B cells with various anti-CD40 peptides conjugated to their surface by ECDI (e.g., Peptides 1-17 listed in table 1). One-way mixed lymphocyte reactions (MLRs) are performed using PBL from fully mismatched, one DRB-matched, or one-DQ matched donor-recipient pairs, with or without the addition of increasing doses of ECDI-fixed anti-CD40 peptide conjugated donor B cells.
- Cells are phenotyped and proliferation evaluated at various time points by multi-parametric flow cytometry (e.g., including CFSE dilution and staining for markers that differentiate cell subsets of interest). Blocking antibodies are added and distinct cell subsets are depleted to dissect underlying mechanisms and to determine how the enhanced ECDI-fixed B cell products influence T cell immunity (e.g., B cell products with or without coupled anti-CD40 peptides). Readouts include i) fold-proliferation of CD4+ and CD8+ T cells with effector phenotypes as determined by surface markers, intracellular cytokines, and/or transcription factors; ii) fold-proliferation of CD4+ and CD8+ T cells with regulatory phenotypes as determined by surface markers, intracellular cytokines, and/or transcription factors; and iii) CD8+ T cell-mediated cytotoxicity against target cells in the MLR.
- The efficacy of peri-transplantation infusions of ADL products coupled to anti-CD40 peptides (e.g., Peptides 1-17 listed in table 1), for inducing stable renal allograft tolerance is evaluated in one DRB-matched, SI non-human primates. An experimental group receives ADL products comprising ex vivo expanded donor B cells coupled to anti-CD40 peptides (e.g., Peptides 1-17 listed in table 1), while a control group receives saline infusions. Group sizes of n=5 are studied; up to 2 additional non-human primates are added per group to replace non-informative recipients (e.g., recipients that contract unrelated diseases). SI mammalian recipients are identical in both groups and long-term maintenance drugs are not given to any recipient. Renal allograft failure is defined by serum creatinine>2.5 mg/dL and confirmed by graft histology.
- Purpose-bred, qualified non-human primate donors and recipients (exam, labs, microbial screen, vaccination, etc.) are selected from qualified vendors. The donors and recipients have a defined MHC disparity (MHC class I-disparate and one MHC class II DRB allele-matched donor-recipient pairs, based on high-resolution MHC class I and II genotyping using Fluidigm Access Arrays to generate amplicons for deep sequencing).
- Recipients with evidence of existing allo-reactive memory can be excluded from the study. Eligibility criteria for recipients can include ABO compatibility, low memory alloreactivity as defined by negative panel reactive antibodies (PRA; OneLambda Bead assay), negative donor-specific antibodies (DSA) by flow, and IFN-γ ELISPOT≤12 SFC/106 PBMC (B cell ELISPOT) against donor non-human primate. Male or female non-human primate recipients are trained for cooperation and instrumented with indwelling central and intraportal vascular access.
- Kidney transplantation in non-human primates follows established procedures. Briefly, following systemic heparinization of both donor and recipient, the donor organ is excised and the vessels are anastomosed to the recipient's infrarenal aorta and vena cava. Typically, this is performed in a left-to-right fashion owing to the longer length of the left renal vessels. The donor ureter is tunneled through the retroperitoneum and a primary ureteroneocystostomy is formed typically on the posterior wall of the bladder using a modified Leadbetter-Politano approach. Particular attention is paid to avoid urine leakage and ureteral stenosis. Bilateral native nephrectomy is completed prior to closure.
- Ex vivo expanded and ECDI-fixed donor B cells coupled to anti-CD40 peptides (e.g., Peptides 1-17 listed in table 1) are infused IV into experimental recipients on days −7 and +1 at a dose of 0.25×109/kg recipient body weight. Approximately 60 ml of blood (corresponding to 1% of body weight) is drawn from donors on day −21 or −22 (±2 days) relative to planned renal transplant, and B cells are purified by magnetic sorting using non-human primate CD20 beads. Alternatively or additionally, B cells can be enriched from leukapheresis products. A B cell expansion protocol is adapted from the culture system reported by Su et al (J Immunol 2016, 197:4163-76). Purified B cells (approximately 24×106 B cells from 60 ml of blood) are expanded ex vivo in a GREX100M flask (Wilson Wolf) until day −7 in RPMI 1640 medium with added 5% rhesus macaque serum, 55 μM 2-ME, 2 mM L-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, 10 mM HEPES, 1 mM sodium pyruvate, and 1% MEM nonessential amino acids. The culture medium is supplemented with anti-CD40 peptides (e.g., Peptides 1-17 listed in table 1), IL-2 (50 ng/ml), IL-4 (10 ng/ml), IL-21 (10 ng/ml), and BAFF (10 ng/ml). Input cell numbers, medium volume, and the concentration of CD40L-multimeric are optimized in feasibility studies. Cells are counted after 7 and 14 days, split after 14 days (day −7), and the cells not infused on day −7 are expanded for another 8 days for infusion on
day + 1. On the day of infusion, cells are agitated on ice for 1 hour with ECDI (30 mg/mL per 3.2×108 cells) in DPBS in the presence of the CD40 binding peptides (e.g., Peptides 1-17 listed in table 1), washed, cleaned to remove necrotic cells and microaggregates, and assessed for viability/necrosis by AO/PI fluorescent microscopy. ECDI-fixed B cells coupled to CD40 binding peptides, meeting all release criteria, are loaded into cold syringes for IV infusion with a maximum concentration of 20×106 cells/mL; the cells remain on ice until recipient administration. Induction of apoptosis is monitored in vitro by incubating an aliquot of ECDI-fixed cells coupled to CD40 binding peptides at 37° C. for 4-6 hours, labelling with Annexin V/PI, and analyzing via fluorescent microscopy or flow cytometry. - Identical short-term immunosuppression and anti-inflammatory therapies are administered to control and experimental subjects. The first dose of each drug is given on day −8 or −7 relative to transplantation on
day 0. The antagonistic anti-CD40 peptides are given intravenously at 50 mg/kg on days −8, −1, 7, and 14. Rapamycin (Rapamune®) is given orally (PO) from day −7 through day 21 post-transplant; the target trough level is 5 to 12 ng/ml. Concomitant anti-inflammatory therapy is with i) α-IL-6R (tocilizumab, Actemra®) at 10 mg/kg IV on days −7, 0, 7, 14, and 21, and ii) sTNFR (etanercept, Enbrel®) at 1 mg/kg IV on days −7 and 0 and 0.5 mg/kg subcutaneously ondays - The primary efficacy outcome is the proportion of transplanted non-human primate with rejection-free allograft survival (confirmed by histopathology) at day 365 post-transplant. Accordingly, follow-up is to day 365 or graft failure, whichever occurs first. The group experimental group that received ADLs coupled to CD40 binding peptides is expected to exhibit enhanced rejection-free allograft survival compared to the control group that received only the short-term immunosuppression and anti-inflammatory therapies.
Claims (20)
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US17/380,303 US20220048948A1 (en) | 2018-11-13 | 2021-07-20 | Cd40 targeted peptides and uses thereof |
Applications Claiming Priority (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US201862760796P | 2018-11-13 | 2018-11-13 | |
PCT/US2019/061325 WO2020102454A1 (en) | 2018-11-13 | 2019-11-13 | Cd40 targeted peptides and uses thereof |
US17/089,463 US11130783B2 (en) | 2018-11-13 | 2020-11-04 | CD40 targeted peptides and uses thereof |
US17/380,303 US20220048948A1 (en) | 2018-11-13 | 2021-07-20 | Cd40 targeted peptides and uses thereof |
Related Parent Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US17/089,463 Continuation US11130783B2 (en) | 2018-11-13 | 2020-11-04 | CD40 targeted peptides and uses thereof |
Publications (1)
Publication Number | Publication Date |
---|---|
US20220048948A1 true US20220048948A1 (en) | 2022-02-17 |
Family
ID=70731952
Family Applications (2)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US17/089,463 Active US11130783B2 (en) | 2018-11-13 | 2020-11-04 | CD40 targeted peptides and uses thereof |
US17/380,303 Pending US20220048948A1 (en) | 2018-11-13 | 2021-07-20 | Cd40 targeted peptides and uses thereof |
Family Applications Before (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US17/089,463 Active US11130783B2 (en) | 2018-11-13 | 2020-11-04 | CD40 targeted peptides and uses thereof |
Country Status (2)
Country | Link |
---|---|
US (2) | US11130783B2 (en) |
WO (1) | WO2020102454A1 (en) |
Families Citing this family (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN112119092A (en) | 2018-02-12 | 2020-12-22 | 无糖尿病公司 | Improved antagonistic anti-human CD40 monoclonal antibodies |
WO2020102454A1 (en) | 2018-11-13 | 2020-05-22 | Regents Of The University Of Minnesota | Cd40 targeted peptides and uses thereof |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20070041971A1 (en) * | 2003-07-07 | 2007-02-22 | Wagner David H | Methods for predicting development of auto-immune diseases and treatment of same |
US11130783B2 (en) * | 2018-11-13 | 2021-09-28 | Regents Of The University Of Minnesota | CD40 targeted peptides and uses thereof |
Family Cites Families (62)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US3536809A (en) | 1969-02-17 | 1970-10-27 | Alza Corp | Medication method |
US3598123A (en) | 1969-04-01 | 1971-08-10 | Alza Corp | Bandage for administering drugs |
US3845770A (en) | 1972-06-05 | 1974-11-05 | Alza Corp | Osmatic dispensing device for releasing beneficial agent |
US3916899A (en) | 1973-04-25 | 1975-11-04 | Alza Corp | Osmotic dispensing device with maximum and minimum sizes for the passageway |
US4008719A (en) | 1976-02-02 | 1977-02-22 | Alza Corporation | Osmotic system having laminar arrangement for programming delivery of active agent |
US4747825A (en) | 1984-06-29 | 1988-05-31 | Ferring Laboratories, Inc. | Apparatus and methodology for pulsed administration of growth promoting agents |
IE58110B1 (en) | 1984-10-30 | 1993-07-14 | Elan Corp Plc | Controlled release powder and process for its preparation |
IT1178786B (en) | 1984-12-21 | 1987-09-16 | Assorenti | PARTIALLY MODIFIED BACK-REVERSE ANALOGS OF NEUROTENSIN |
US4948592A (en) | 1986-05-09 | 1990-08-14 | Alza Corporation | Pulsed drug delivery |
US4723958A (en) | 1986-05-23 | 1988-02-09 | Merck & Co., Inc. | Pulsatile drug delivery system |
US5075109A (en) | 1986-10-24 | 1991-12-24 | Southern Research Institute | Method of potentiating an immune response |
US4965251A (en) | 1987-04-03 | 1990-10-23 | The Board Of Regents Of The University Of Washington | Pulse treatment of hemoglobinopathies with erythropoietin |
US4880078A (en) | 1987-06-29 | 1989-11-14 | Honda Giken Kogyo Kabushiki Kaisha | Exhaust muffler |
US4897268A (en) | 1987-08-03 | 1990-01-30 | Southern Research Institute | Drug delivery system and method of making the same |
US5073543A (en) | 1988-07-21 | 1991-12-17 | G. D. Searle & Co. | Controlled release formulations of trophic factors in ganglioside-lipsome vehicle |
US5223409A (en) | 1988-09-02 | 1993-06-29 | Protein Engineering Corp. | Directed evolution of novel binding proteins |
AU649217B2 (en) | 1988-11-18 | 1994-05-19 | Regents Of The University Of California, The | Method for site-specifically incorporating unnatural amino acids into proteins |
US5703055A (en) | 1989-03-21 | 1997-12-30 | Wisconsin Alumni Research Foundation | Generation of antibodies through lipid mediated DNA delivery |
IT1229203B (en) | 1989-03-22 | 1991-07-25 | Bioresearch Spa | USE OF 5 METHYLTHETRAHYDROPHOLIC ACID, 5 FORMYLTHETRAHYDROPHOLIC ACID AND THEIR PHARMACEUTICALLY ACCEPTABLE SALTS FOR THE PREPARATION OF PHARMACEUTICAL COMPOSITIONS IN THE FORM OF CONTROLLED RELEASE ACTIVE IN THE THERAPY OF MENTAL AND ORGANIC DISORDERS. |
US5399346A (en) | 1989-06-14 | 1995-03-21 | The United States Of America As Represented By The Department Of Health And Human Services | Gene therapy |
US5585362A (en) | 1989-08-22 | 1996-12-17 | The Regents Of The University Of Michigan | Adenovirus vectors for gene therapy |
US5120548A (en) | 1989-11-07 | 1992-06-09 | Merck & Co., Inc. | Swelling modulated polymeric drug delivery device |
US5733566A (en) | 1990-05-15 | 1998-03-31 | Alkermes Controlled Therapeutics Inc. Ii | Controlled release of antiparasitic agents in animals |
CA2109528A1 (en) | 1991-05-01 | 1992-11-02 | Gregory A. Prince | A method for treating infectious respiratory diseases |
US5580578A (en) | 1992-01-27 | 1996-12-03 | Euro-Celtique, S.A. | Controlled release formulations coated with aqueous dispersions of acrylic polymers |
US6261569B1 (en) | 1992-08-27 | 2001-07-17 | Deakin Research Limited | Retro-, inverso- and retro-inverso synthetic peptide analogues |
US5350674A (en) | 1992-09-04 | 1994-09-27 | Becton, Dickinson And Company | Intrinsic factor - horse peroxidase conjugates and a method for increasing the stability thereof |
US5403590A (en) | 1992-12-21 | 1995-04-04 | New England Deaconess Hospital Corporation | Method of pulsatile drug infusion |
US5591767A (en) | 1993-01-25 | 1997-01-07 | Pharmetrix Corporation | Liquid reservoir transdermal patch for the administration of ketorolac |
US5934272A (en) | 1993-01-29 | 1999-08-10 | Aradigm Corporation | Device and method of creating aerosolized mist of respiratory drug |
IT1270594B (en) | 1994-07-07 | 1997-05-07 | Recordati Chem Pharm | CONTROLLED RELEASE PHARMACEUTICAL COMPOSITION OF LIQUID SUSPENSION MOGUISTEIN |
US6019968A (en) | 1995-04-14 | 2000-02-01 | Inhale Therapeutic Systems, Inc. | Dispersible antibody compositions and methods for their preparation and use |
AU2063197A (en) | 1996-03-04 | 1997-09-22 | Massachusetts Institute Of Technology | Materials and methods for enhancing cellular internalization |
US5855913A (en) | 1997-01-16 | 1999-01-05 | Massachusetts Instite Of Technology | Particles incorporating surfactants for pulmonary drug delivery |
US5985309A (en) | 1996-05-24 | 1999-11-16 | Massachusetts Institute Of Technology | Preparation of particles for inhalation |
US5874064A (en) | 1996-05-24 | 1999-02-23 | Massachusetts Institute Of Technology | Aerodynamically light particles for pulmonary drug delivery |
US6339069B1 (en) | 1996-10-15 | 2002-01-15 | Elan Pharmaceuticalstechnologies, Inc. | Peptide-lipid conjugates, liposomes and lipsomal drug delivery |
ATE287257T1 (en) | 1997-01-16 | 2005-02-15 | Massachusetts Inst Technology | PREPARATION OF PARTICLE-CONTAINING MEDICINAL PRODUCTS FOR INHALATION |
US6103489A (en) | 1997-03-21 | 2000-08-15 | University Of Hawaii | Cell-free protein synthesis system with protein translocation and processing |
WO1998056370A2 (en) | 1997-06-13 | 1998-12-17 | Johns Hopkins University School Of Medicine | Therapeutic nanospheres |
US6365185B1 (en) | 1998-03-26 | 2002-04-02 | University Of Cincinnati | Self-destructing, controlled release peroral drug delivery system |
JP2002518432A (en) | 1998-06-24 | 2002-06-25 | アドバンスト インハレーション リサーチ,インコーポレイテッド | Large porous particles released from an inhaler |
AU1086501A (en) | 1999-10-15 | 2001-04-30 | Carnegie Institution Of Washington | Rna interference pathway genes as tools for targeted genetic interference |
US6326193B1 (en) | 1999-11-05 | 2001-12-04 | Cambria Biosciences, Llc | Insect control agent |
AU2001275474A1 (en) | 2000-06-12 | 2001-12-24 | Akkadix Corporation | Materials and methods for the control of nematodes |
FR2840307B1 (en) | 2002-05-30 | 2006-07-07 | Centre Nat Rech Scient | NOVEL MULTIMERIC MOLECULES, PROCESS FOR THE PREPARATION THEREOF, AND USE THEREOF FOR THE PREPARATION OF MEDICAMENTS |
PT1682178E (en) | 2003-11-04 | 2010-10-21 | Novartis Vaccines & Diagnostic | Methods of therapy for cancers expressing the cd40 antigen |
US20080318837A1 (en) | 2003-12-26 | 2008-12-25 | Nastech Pharmaceutical Company Inc. | Pharmaceutical Formation For Increased Epithelial Permeability of Glucose-Regulating Peptide |
US7659252B2 (en) | 2005-09-15 | 2010-02-09 | Novomed Technologies, Inc. (Shanghai) | Transdermal delivery peptides and method of use thereof |
US20070237826A1 (en) | 2006-04-05 | 2007-10-11 | Rao Kollipara K | Polymerized solid lipid nanoparticles for oral or mucosal delivery of therapeutic proteins and peptides |
EP1854810A1 (en) | 2006-05-09 | 2007-11-14 | PanGenetics B.V. | Deimmunized antagonistic anti-human CD40 monoclonal antibody from the ch5D12 antibody |
EP2067785A1 (en) | 2007-12-03 | 2009-06-10 | Fresenius Medical Care Deutschland GmbH | Human CD154-binding synthetic peptide and uses thereof |
US8734786B2 (en) | 2009-09-16 | 2014-05-27 | Northwestern University | Use of ECDI-fixed cell tolerance as a method for preventing allograft rejection |
US9289381B2 (en) | 2009-12-03 | 2016-03-22 | The University Of North Carolina At Charlotte | Stabilization and storage of biological pharmaceutical compositions |
EP3178851B1 (en) | 2010-03-31 | 2020-04-29 | Boehringer Ingelheim International GmbH | Anti-cd40 antibodies |
EP3421487B1 (en) | 2010-10-19 | 2023-06-28 | The Regents of the University of Colorado, a body corporate | Peptides for modulating t-cell activity and uses thereof |
US20120302505A1 (en) | 2011-04-21 | 2012-11-29 | Fetzer Oliver S | Cyclodextrin-based polymers for therapeutic delivery |
KR101329411B1 (en) | 2012-05-31 | 2013-11-14 | 주식회사 엘지생활건강 | Novel transdermal peptide |
TWI703161B (en) | 2014-10-31 | 2020-09-01 | 美商Ngm生物製藥公司 | Compositions and methods of use for treating metabolic disorders |
KR102656470B1 (en) | 2014-12-10 | 2024-04-09 | 리전츠 오브 더 유니버스티 오브 미네소타 | Genetically modified cells, tissues, and organs for treating disease |
SI3303395T1 (en) | 2015-05-29 | 2020-03-31 | Abbvie Inc. | Anti-cd40 antibodies and uses thereof |
US20180172683A1 (en) | 2015-06-22 | 2018-06-21 | University Of Southern California | Methods for analyzing the interaction between a target protein and a ligand |
-
2019
- 2019-11-13 WO PCT/US2019/061325 patent/WO2020102454A1/en active Application Filing
-
2020
- 2020-11-04 US US17/089,463 patent/US11130783B2/en active Active
-
2021
- 2021-07-20 US US17/380,303 patent/US20220048948A1/en active Pending
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20070041971A1 (en) * | 2003-07-07 | 2007-02-22 | Wagner David H | Methods for predicting development of auto-immune diseases and treatment of same |
US11130783B2 (en) * | 2018-11-13 | 2021-09-28 | Regents Of The University Of Minnesota | CD40 targeted peptides and uses thereof |
Non-Patent Citations (3)
Title |
---|
Leslie, Science Now, May 2012 (Year: 2012) * |
Senhaji et al. (Front. Immunol., 16 October 2015) (Year: 2015) * |
Yehuda et al. (J Crohns Colitis. 2019 Mar 26;13(3):324-329) (Year: 2019) * |
Also Published As
Publication number | Publication date |
---|---|
WO2020102454A1 (en) | 2020-05-22 |
US20210070805A1 (en) | 2021-03-11 |
US11130783B2 (en) | 2021-09-28 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US10301593B2 (en) | Synthetic membrane-receiver complexes | |
US20210180015A1 (en) | Membrane-Receiver Complex Therapeutics | |
US20170002060A1 (en) | Polynucleotides for the in vivo production of antibodies | |
EP3092250A1 (en) | Polynucleotides for the in vivo production of antibodies | |
WO2016132366A1 (en) | Combination immune therapy and cytokine control therapy for cancer treatment | |
US20220048948A1 (en) | Cd40 targeted peptides and uses thereof | |
US20220112280A1 (en) | Transplant tolerance induction with carbodiimide treated tolerizing vaccine | |
US20140288010A1 (en) | Compositions and Methods for Increasing Stem Cell Survival | |
US20210299181A1 (en) | Anucleated cells for the treatment of diseases | |
US10653760B2 (en) | Compositions and methods for reducing organ rejection by reducing heparan sulfate in donor transplants | |
JP7334249B2 (en) | Early apoptotic cells for use in treating sepsis | |
JP6649376B2 (en) | How to treat lung inflammation | |
AU2022371442A1 (en) | Hypoimmune cells | |
Blanco et al. | The Role of B-Cells and B-Cell Targeted Therapies in Lupus Nephritis | |
Khattri | The Role of B-Cells and B-Cell Targeted Therapies in Lupus Nephritis |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
AS | Assignment |
Owner name: DIABETES-FREE INC., DELAWARE Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNOR:GIRINATH, PRASHANT;REEL/FRAME:056915/0753 Effective date: 20210616 Owner name: REGENTS OF THE UNIVERSITY OF MINNESOTA, MINNESOTA Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:RAMACHANDRAN, SABRINATHAN;HERING, BERNHARD J.;SIGNING DATES FROM 20210521 TO 20210526;REEL/FRAME:056915/0716 |
|
STPP | Information on status: patent application and granting procedure in general |
Free format text: NON FINAL ACTION MAILED |
|
STPP | Information on status: patent application and granting procedure in general |
Free format text: NON FINAL ACTION MAILED |
|
STPP | Information on status: patent application and granting procedure in general |
Free format text: RESPONSE TO NON-FINAL OFFICE ACTION ENTERED AND FORWARDED TO EXAMINER |
|
STPP | Information on status: patent application and granting procedure in general |
Free format text: FINAL REJECTION MAILED |