CA2282888A1 - Anti-inflammatory, tolerogenic and immunoinhibiting properties of carbohydrate binding peptides - Google Patents
Anti-inflammatory, tolerogenic and immunoinhibiting properties of carbohydrate binding peptides Download PDFInfo
- Publication number
- CA2282888A1 CA2282888A1 CA002282888A CA2282888A CA2282888A1 CA 2282888 A1 CA2282888 A1 CA 2282888A1 CA 002282888 A CA002282888 A CA 002282888A CA 2282888 A CA2282888 A CA 2282888A CA 2282888 A1 CA2282888 A1 CA 2282888A1
- Authority
- CA
- Canada
- Prior art keywords
- binding
- peptide
- peptides
- seq
- sialic acid
- 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.)
- Abandoned
Links
- 108090000765 processed proteins & peptides Proteins 0.000 title claims abstract description 320
- 108010038196 saccharide-binding proteins Proteins 0.000 title claims abstract description 94
- 102000004196 processed proteins & peptides Human genes 0.000 title abstract description 176
- 230000003614 tolerogenic effect Effects 0.000 title description 4
- 230000003110 anti-inflammatory effect Effects 0.000 title description 3
- 102000004856 Lectins Human genes 0.000 claims abstract description 122
- 108090001090 Lectins Proteins 0.000 claims abstract description 122
- 239000002523 lectin Substances 0.000 claims abstract description 122
- 239000000427 antigen Substances 0.000 claims abstract description 90
- 108091007433 antigens Proteins 0.000 claims abstract description 90
- 102000036639 antigens Human genes 0.000 claims abstract description 90
- 230000028709 inflammatory response Effects 0.000 claims abstract description 35
- 206010027476 Metastases Diseases 0.000 claims abstract description 9
- 230000009401 metastasis Effects 0.000 claims abstract description 9
- 239000008194 pharmaceutical composition Substances 0.000 claims abstract description 6
- 230000001939 inductive effect Effects 0.000 claims abstract description 5
- 150000001413 amino acids Chemical class 0.000 claims description 134
- 241000124008 Mammalia Species 0.000 claims description 65
- 210000004027 cell Anatomy 0.000 claims description 46
- 230000028993 immune response Effects 0.000 claims description 43
- 230000002401 inhibitory effect Effects 0.000 claims description 28
- 206010061218 Inflammation Diseases 0.000 claims description 27
- 230000004054 inflammatory process Effects 0.000 claims description 27
- 206010053613 Type IV hypersensitivity reaction Diseases 0.000 claims description 26
- 208000027930 type IV hypersensitivity disease Diseases 0.000 claims description 26
- 230000005951 type IV hypersensitivity Effects 0.000 claims description 25
- 230000021633 leukocyte mediated immunity Effects 0.000 claims description 20
- 230000006378 damage Effects 0.000 claims description 16
- 229910052739 hydrogen Inorganic materials 0.000 claims description 15
- 102000005962 receptors Human genes 0.000 claims description 15
- 108020003175 receptors Proteins 0.000 claims description 15
- 208000027418 Wounds and injury Diseases 0.000 claims description 14
- 208000014674 injury Diseases 0.000 claims description 14
- 230000006698 induction Effects 0.000 claims description 13
- 206010028980 Neoplasm Diseases 0.000 claims description 12
- 125000003275 alpha amino acid group Chemical group 0.000 claims description 11
- 201000011510 cancer Diseases 0.000 claims description 11
- 230000000977 initiatory effect Effects 0.000 claims description 11
- 239000003795 chemical substances by application Substances 0.000 claims description 10
- 239000003053 toxin Substances 0.000 claims description 9
- 231100000765 toxin Toxicity 0.000 claims description 9
- 108700012359 toxins Proteins 0.000 claims description 9
- 206010001052 Acute respiratory distress syndrome Diseases 0.000 claims description 8
- 208000004852 Lung Injury Diseases 0.000 claims description 8
- 206010069363 Traumatic lung injury Diseases 0.000 claims description 8
- 231100000515 lung injury Toxicity 0.000 claims description 8
- 238000001356 surgical procedure Methods 0.000 claims description 8
- 238000001574 biopsy Methods 0.000 claims description 7
- 230000007774 longterm Effects 0.000 claims description 7
- 206010039073 rheumatoid arthritis Diseases 0.000 claims description 7
- 150000003839 salts Chemical class 0.000 claims description 7
- 230000001580 bacterial effect Effects 0.000 claims description 6
- 210000004881 tumor cell Anatomy 0.000 claims description 6
- 208000022559 Inflammatory bowel disease Diseases 0.000 claims description 5
- 206010035664 Pneumonia Diseases 0.000 claims description 5
- 201000004681 Psoriasis Diseases 0.000 claims description 5
- 206010063837 Reperfusion injury Diseases 0.000 claims description 5
- 230000003612 virological effect Effects 0.000 claims description 5
- 208000001034 Frostbite Diseases 0.000 claims description 4
- 239000013566 allergen Substances 0.000 claims description 4
- 208000006673 asthma Diseases 0.000 claims description 4
- 230000037396 body weight Effects 0.000 claims description 4
- 102000023852 carbohydrate binding proteins Human genes 0.000 claims description 4
- 201000006417 multiple sclerosis Diseases 0.000 claims description 4
- 201000001178 Bacterial Pneumonia Diseases 0.000 claims description 3
- 206010040070 Septic Shock Diseases 0.000 claims description 3
- 239000003937 drug carrier Substances 0.000 claims description 3
- 230000028996 humoral immune response Effects 0.000 claims description 3
- 230000036303 septic shock Effects 0.000 claims description 3
- 206010009944 Colon cancer Diseases 0.000 claims description 2
- 201000004624 Dermatitis Diseases 0.000 claims description 2
- 206010035737 Pneumonia viral Diseases 0.000 claims description 2
- 238000001990 intravenous administration Methods 0.000 claims description 2
- 201000001441 melanoma Diseases 0.000 claims description 2
- 208000009421 viral pneumonia Diseases 0.000 claims description 2
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 claims 1
- 208000015181 infectious disease Diseases 0.000 claims 1
- 230000027455 binding Effects 0.000 abstract description 164
- 238000009739 binding Methods 0.000 abstract description 158
- 238000000034 method Methods 0.000 abstract description 49
- 230000021164 cell adhesion Effects 0.000 abstract description 9
- 235000001014 amino acid Nutrition 0.000 description 128
- 229940024606 amino acid Drugs 0.000 description 128
- 108010081690 Pertussis Toxin Proteins 0.000 description 73
- 229960002685 biotin Drugs 0.000 description 69
- 239000011616 biotin Substances 0.000 description 69
- 108060002885 fetuin Proteins 0.000 description 69
- 102000013361 fetuin Human genes 0.000 description 69
- 230000005764 inhibitory process Effects 0.000 description 35
- 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 31
- 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 30
- 239000002953 phosphate buffered saline Substances 0.000 description 30
- 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 28
- 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 25
- 108010044715 asialofetuin Proteins 0.000 description 24
- 238000002474 experimental method Methods 0.000 description 24
- 230000001404 mediated effect Effects 0.000 description 22
- 108010046516 Wheat Germ Agglutinins Proteins 0.000 description 20
- 150000001720 carbohydrates Chemical class 0.000 description 20
- 241000699670 Mus sp. Species 0.000 description 18
- 230000000694 effects Effects 0.000 description 18
- 210000000265 leukocyte Anatomy 0.000 description 18
- 239000000243 solution Substances 0.000 description 18
- 238000003556 assay Methods 0.000 description 17
- 235000014633 carbohydrates Nutrition 0.000 description 17
- 108090000184 Selectins Proteins 0.000 description 16
- 102000003800 Selectins Human genes 0.000 description 16
- 235000018102 proteins Nutrition 0.000 description 15
- 102000004169 proteins and genes Human genes 0.000 description 15
- 108090000623 proteins and genes Proteins 0.000 description 15
- 230000004044 response Effects 0.000 description 15
- 235000020958 biotin Nutrition 0.000 description 14
- 239000000203 mixture Substances 0.000 description 14
- 230000008961 swelling Effects 0.000 description 14
- 230000003993 interaction Effects 0.000 description 12
- 108090001008 Avidin Proteins 0.000 description 10
- 210000004978 chinese hamster ovary cell Anatomy 0.000 description 10
- 239000012634 fragment Substances 0.000 description 10
- 210000000987 immune system Anatomy 0.000 description 10
- 230000002757 inflammatory effect Effects 0.000 description 10
- 230000000503 lectinlike effect Effects 0.000 description 10
- 230000008614 cellular interaction Effects 0.000 description 9
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 9
- 208000027866 inflammatory disease Diseases 0.000 description 9
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 8
- UCNNZELZXFXXJQ-BZSNNMDCSA-N Leu-Leu-Tyr Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 UCNNZELZXFXXJQ-BZSNNMDCSA-N 0.000 description 8
- OVRNDRQMDRJTHS-UHFFFAOYSA-N N-acelyl-D-glucosamine Natural products CC(=O)NC1C(O)OC(CO)C(O)C1O OVRNDRQMDRJTHS-UHFFFAOYSA-N 0.000 description 8
- OVRNDRQMDRJTHS-FMDGEEDCSA-N N-acetyl-beta-D-glucosamine Chemical group CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O OVRNDRQMDRJTHS-FMDGEEDCSA-N 0.000 description 8
- MBLBDJOUHNCFQT-LXGUWJNJSA-N N-acetylglucosamine Natural products CC(=O)N[C@@H](C=O)[C@@H](O)[C@H](O)[C@H](O)CO MBLBDJOUHNCFQT-LXGUWJNJSA-N 0.000 description 8
- PXIPVTKHYLBLMZ-UHFFFAOYSA-N Sodium azide Chemical compound [Na+].[N-]=[N+]=[N-] PXIPVTKHYLBLMZ-UHFFFAOYSA-N 0.000 description 8
- 239000000562 conjugate Substances 0.000 description 8
- 229950006780 n-acetylglucosamine Drugs 0.000 description 8
- 229920001542 oligosaccharide Polymers 0.000 description 8
- 238000000159 protein binding assay Methods 0.000 description 8
- 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
- 240000006028 Sambucus nigra Species 0.000 description 7
- 235000003142 Sambucus nigra Nutrition 0.000 description 7
- 239000002253 acid Substances 0.000 description 7
- 230000001419 dependent effect Effects 0.000 description 7
- 208000035475 disorder Diseases 0.000 description 7
- 235000008995 european elder Nutrition 0.000 description 7
- 230000002829 reductive effect Effects 0.000 description 7
- 238000012216 screening Methods 0.000 description 7
- 230000001629 suppression Effects 0.000 description 7
- 241000282412 Homo Species 0.000 description 6
- 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 6
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 6
- 201000000028 adult respiratory distress syndrome Diseases 0.000 description 6
- 125000000539 amino acid group Chemical group 0.000 description 6
- 238000006243 chemical reaction Methods 0.000 description 6
- 238000011161 development Methods 0.000 description 6
- 238000010790 dilution Methods 0.000 description 6
- 239000012895 dilution Substances 0.000 description 6
- 230000004968 inflammatory condition Effects 0.000 description 6
- 239000008101 lactose Substances 0.000 description 6
- 208000010125 myocardial infarction Diseases 0.000 description 6
- 230000010807 negative regulation of binding Effects 0.000 description 6
- 210000000440 neutrophil Anatomy 0.000 description 6
- 150000002482 oligosaccharides Chemical class 0.000 description 6
- 230000009467 reduction Effects 0.000 description 6
- 210000001519 tissue Anatomy 0.000 description 6
- FJQZXCPWAGYPSD-UHFFFAOYSA-N 1,3,4,6-tetrachloro-3a,6a-diphenylimidazo[4,5-d]imidazole-2,5-dione Chemical compound ClN1C(=O)N(Cl)C2(C=3C=CC=CC=3)N(Cl)C(=O)N(Cl)C12C1=CC=CC=C1 FJQZXCPWAGYPSD-UHFFFAOYSA-N 0.000 description 5
- 241000588832 Bordetella pertussis Species 0.000 description 5
- 102000003886 Glycoproteins Human genes 0.000 description 5
- 108090000288 Glycoproteins Proteins 0.000 description 5
- 102000008212 P-Selectin Human genes 0.000 description 5
- 108010035766 P-Selectin Proteins 0.000 description 5
- 241000700605 Viruses Species 0.000 description 5
- 230000018109 developmental process Effects 0.000 description 5
- 238000009472 formulation Methods 0.000 description 5
- 238000011534 incubation Methods 0.000 description 5
- -1 oligosaccharide glycosides Chemical class 0.000 description 5
- 230000008569 process Effects 0.000 description 5
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 4
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 4
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 4
- 241000699666 Mus <mouse, genus> Species 0.000 description 4
- 229920001213 Polysorbate 20 Polymers 0.000 description 4
- 208000013616 Respiratory Distress Syndrome Diseases 0.000 description 4
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 description 4
- 239000002671 adjuvant Substances 0.000 description 4
- 230000000890 antigenic effect Effects 0.000 description 4
- OHDRQQURAXLVGJ-HLVWOLMTSA-N azane;(2e)-3-ethyl-2-[(e)-(3-ethyl-6-sulfo-1,3-benzothiazol-2-ylidene)hydrazinylidene]-1,3-benzothiazole-6-sulfonic acid Chemical compound [NH4+].[NH4+].S/1C2=CC(S([O-])(=O)=O)=CC=C2N(CC)C\1=N/N=C1/SC2=CC(S([O-])(=O)=O)=CC=C2N1CC OHDRQQURAXLVGJ-HLVWOLMTSA-N 0.000 description 4
- 230000015572 biosynthetic process Effects 0.000 description 4
- 125000000837 carbohydrate group Chemical group 0.000 description 4
- 239000007979 citrate buffer Substances 0.000 description 4
- 230000009456 molecular mechanism Effects 0.000 description 4
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 4
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 4
- AAEVYOVXGOFMJO-UHFFFAOYSA-N prometryn Chemical compound CSC1=NC(NC(C)C)=NC(NC(C)C)=N1 AAEVYOVXGOFMJO-UHFFFAOYSA-N 0.000 description 4
- 230000035886 specific defense system Effects 0.000 description 4
- 239000000758 substrate Substances 0.000 description 4
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 4
- 238000005406 washing Methods 0.000 description 4
- OIZGSVFYNBZVIK-FHHHURIISA-N 3'-sialyllactose Chemical compound O1[C@@H]([C@H](O)[C@H](O)CO)[C@H](NC(=O)C)[C@@H](O)C[C@@]1(C(O)=O)O[C@@H]1[C@@H](O)[C@H](O[C@H]([C@H](O)CO)[C@H](O)[C@@H](O)C=O)O[C@H](CO)[C@@H]1O OIZGSVFYNBZVIK-FHHHURIISA-N 0.000 description 3
- 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 3
- 208000023275 Autoimmune disease Diseases 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 201000005702 Pertussis Diseases 0.000 description 3
- 108010089814 Plant Lectins Proteins 0.000 description 3
- VDVYTKZBMFADQH-AVGNSLFASA-N Ser-Gln-Tyr Chemical compound OC[C@H](N)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 VDVYTKZBMFADQH-AVGNSLFASA-N 0.000 description 3
- 108010090804 Streptavidin Proteins 0.000 description 3
- 241000607479 Yersinia pestis Species 0.000 description 3
- 208000038016 acute inflammation Diseases 0.000 description 3
- 230000006022 acute inflammation Effects 0.000 description 3
- 108091008400 carbohydrate binding proteins Proteins 0.000 description 3
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 3
- 208000037976 chronic inflammation Diseases 0.000 description 3
- 230000006020 chronic inflammation Effects 0.000 description 3
- 239000000539 dimer Substances 0.000 description 3
- 239000002552 dosage form Substances 0.000 description 3
- 239000012091 fetal bovine serum Substances 0.000 description 3
- 238000004128 high performance liquid chromatography Methods 0.000 description 3
- 239000003112 inhibitor Substances 0.000 description 3
- 210000004072 lung Anatomy 0.000 description 3
- 206010025135 lupus erythematosus Diseases 0.000 description 3
- 210000004962 mammalian cell Anatomy 0.000 description 3
- 238000006386 neutralization reaction Methods 0.000 description 3
- 230000035781 nonspecific defense system Effects 0.000 description 3
- 210000000056 organ Anatomy 0.000 description 3
- 239000000546 pharmaceutical excipient Substances 0.000 description 3
- 239000003726 plant lectin Substances 0.000 description 3
- 229920001184 polypeptide Polymers 0.000 description 3
- 230000001105 regulatory effect Effects 0.000 description 3
- 108010026333 seryl-proline Proteins 0.000 description 3
- 125000005629 sialic acid group Chemical group 0.000 description 3
- 239000012064 sodium phosphate buffer Substances 0.000 description 3
- 230000000451 tissue damage Effects 0.000 description 3
- 231100000827 tissue damage Toxicity 0.000 description 3
- 201000001320 Atherosclerosis Diseases 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 2
- 241000283690 Bos taurus Species 0.000 description 2
- 102000014914 Carrier Proteins Human genes 0.000 description 2
- 108010049048 Cholera Toxin Proteins 0.000 description 2
- 102000009016 Cholera Toxin Human genes 0.000 description 2
- 206010053567 Coagulopathies Diseases 0.000 description 2
- 206010012438 Dermatitis atopic Diseases 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- WRFOZIJRODPLIA-QWRGUYRKSA-N Gly-Tyr-Cys Chemical compound C1=CC(=CC=C1C[C@@H](C(=O)N[C@@H](CS)C(=O)O)NC(=O)CN)O WRFOZIJRODPLIA-QWRGUYRKSA-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
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 description 2
- 125000003047 N-acetyl group Chemical group 0.000 description 2
- 108010079364 N-glycylalanine Proteins 0.000 description 2
- ONIBWKKTOPOVIA-UHFFFAOYSA-N Proline Natural products OC(=O)C1CCCN1 ONIBWKKTOPOVIA-UHFFFAOYSA-N 0.000 description 2
- 229920005654 Sephadex Polymers 0.000 description 2
- 239000012507 Sephadex™ Substances 0.000 description 2
- WBAXJMCUFIXCNI-WDSKDSINSA-N Ser-Pro Chemical compound OC[C@H](N)C(=O)N1CCC[C@H]1C(O)=O WBAXJMCUFIXCNI-WDSKDSINSA-N 0.000 description 2
- 206010044541 Traumatic shock Diseases 0.000 description 2
- 238000002835 absorbance Methods 0.000 description 2
- 230000001154 acute effect Effects 0.000 description 2
- 230000002411 adverse Effects 0.000 description 2
- 230000000172 allergic effect Effects 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 230000005875 antibody response Effects 0.000 description 2
- 201000008937 atopic dermatitis Diseases 0.000 description 2
- 208000010668 atopic eczema Diseases 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 125000002915 carbonyl group Chemical group [*:2]C([*:1])=O 0.000 description 2
- 230000011712 cell development Effects 0.000 description 2
- 230000001413 cellular effect Effects 0.000 description 2
- 230000007248 cellular mechanism Effects 0.000 description 2
- 239000011248 coating agent Substances 0.000 description 2
- 238000000576 coating method Methods 0.000 description 2
- 210000003022 colostrum Anatomy 0.000 description 2
- 235000021277 colostrum Nutrition 0.000 description 2
- 230000000295 complement effect Effects 0.000 description 2
- 238000007796 conventional method Methods 0.000 description 2
- 238000001212 derivatisation Methods 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 239000012636 effector Substances 0.000 description 2
- 210000003038 endothelium Anatomy 0.000 description 2
- 230000006870 function Effects 0.000 description 2
- VPZXBVLAVMBEQI-UHFFFAOYSA-N glycyl-DL-alpha-alanine Natural products OC(=O)C(C)NC(=O)CN VPZXBVLAVMBEQI-UHFFFAOYSA-N 0.000 description 2
- 230000012010 growth Effects 0.000 description 2
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 2
- 239000001257 hydrogen Substances 0.000 description 2
- 230000008105 immune reaction Effects 0.000 description 2
- 230000003053 immunization Effects 0.000 description 2
- 238000002649 immunization Methods 0.000 description 2
- 230000009851 immunogenic response Effects 0.000 description 2
- 230000008595 infiltration Effects 0.000 description 2
- 238000001764 infiltration Methods 0.000 description 2
- 230000002452 interceptive effect Effects 0.000 description 2
- 238000006192 iodination reaction Methods 0.000 description 2
- 108010031424 isoleucyl-prolyl-proline Proteins 0.000 description 2
- 239000003446 ligand Substances 0.000 description 2
- 210000004379 membrane Anatomy 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 230000001394 metastastic effect Effects 0.000 description 2
- 206010061289 metastatic neoplasm Diseases 0.000 description 2
- 244000005700 microbiome Species 0.000 description 2
- 230000003278 mimic effect Effects 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000023578 negative regulation of cell adhesion Effects 0.000 description 2
- 201000008383 nephritis Diseases 0.000 description 2
- 230000007170 pathology Effects 0.000 description 2
- 239000000863 peptide conjugate Substances 0.000 description 2
- 230000002688 persistence Effects 0.000 description 2
- 229920000642 polymer Polymers 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 230000035939 shock Effects 0.000 description 2
- 235000021309 simple sugar Nutrition 0.000 description 2
- 239000011734 sodium Substances 0.000 description 2
- 229910052708 sodium Inorganic materials 0.000 description 2
- 125000006850 spacer group Chemical group 0.000 description 2
- 241000894007 species Species 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 235000000346 sugar Nutrition 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 230000024033 toxin binding Effects 0.000 description 2
- 125000001493 tyrosinyl group Chemical group [H]OC1=C([H])C([H])=C(C([H])=C1[H])C([H])([H])C([H])(N([H])[H])C(*)=O 0.000 description 2
- 230000002792 vascular Effects 0.000 description 2
- 210000003462 vein Anatomy 0.000 description 2
- 239000000304 virulence factor Substances 0.000 description 2
- 230000007923 virulence factor Effects 0.000 description 2
- FJXPWVIFADLJQN-PGIATKPXSA-N (4S,5R,6R)-5-[(2-aminoacetyl)amino]-2,4-dihydroxy-6-[(1R,2R)-1,2,3-trihydroxypropyl]oxane-2-carboxylic acid Chemical compound NCC(=O)N[C@@H]1[C@@H](O)CC(O)(C(O)=O)O[C@H]1[C@H](O)[C@H](O)CO FJXPWVIFADLJQN-PGIATKPXSA-N 0.000 description 1
- PPINMSZPTPRQQB-NHCYSSNCSA-N 2-[[(2s)-1-[(2s)-2-[[(2s)-2-amino-3-methylbutanoyl]amino]propanoyl]pyrrolidine-2-carbonyl]amino]acetic acid Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](C)C(=O)N1CCC[C@H]1C(=O)NCC(O)=O PPINMSZPTPRQQB-NHCYSSNCSA-N 0.000 description 1
- TVEXGJYMHHTVKP-UHFFFAOYSA-N 6-oxabicyclo[3.2.1]oct-3-en-7-one Chemical compound C1C2C(=O)OC1C=CC2 TVEXGJYMHHTVKP-UHFFFAOYSA-N 0.000 description 1
- BJOZNDRNJJZHPZ-LUWBGTNYSA-N 9-O-acetylneuraminic acid Chemical compound CC(=O)OC[C@@H](O)[C@@H](O)[C@@H]1OC(O)(C(O)=O)C[C@H](O)[C@H]1N BJOZNDRNJJZHPZ-LUWBGTNYSA-N 0.000 description 1
- 108010049290 ADP Ribose Transferases Proteins 0.000 description 1
- 102000009062 ADP Ribose Transferases Human genes 0.000 description 1
- LZRNYBIJOSKKRJ-XVYDVKMFSA-N Ala-Asp-His Chemical compound C[C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)N[C@@H](CC1=CN=CN1)C(=O)O)N LZRNYBIJOSKKRJ-XVYDVKMFSA-N 0.000 description 1
- UQJUGHFKNKGHFQ-VZFHVOOUSA-N Ala-Cys-Thr Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CS)C(=O)N[C@@H]([C@@H](C)O)C(O)=O UQJUGHFKNKGHFQ-VZFHVOOUSA-N 0.000 description 1
- ROLXPVQSRCPVGK-XDTLVQLUSA-N Ala-Glu-Tyr Chemical compound N[C@@H](C)C(=O)N[C@@H](CCC(=O)O)C(=O)N[C@@H](CC1=CC=C(C=C1)O)C(=O)O ROLXPVQSRCPVGK-XDTLVQLUSA-N 0.000 description 1
- QHASENCZLDHBGX-ONGXEEELSA-N Ala-Gly-Phe Chemical compound C[C@H](N)C(=O)NCC(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 QHASENCZLDHBGX-ONGXEEELSA-N 0.000 description 1
- DHONNEYAZPNGSG-UBHSHLNASA-N Ala-Val-Phe Chemical compound C[C@H](N)C(=O)N[C@@H](C(C)C)C(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 DHONNEYAZPNGSG-UBHSHLNASA-N 0.000 description 1
- QGZKDVFQNNGYKY-UHFFFAOYSA-O Ammonium Chemical compound [NH4+] QGZKDVFQNNGYKY-UHFFFAOYSA-O 0.000 description 1
- KMSHNDWHPWXPEC-BQBZGAKWSA-N Arg-Asp-Gly Chemical compound NC(N)=NCCC[C@H](N)C(=O)N[C@@H](CC(O)=O)C(=O)NCC(O)=O KMSHNDWHPWXPEC-BQBZGAKWSA-N 0.000 description 1
- RRGPUNYIPJXJBU-GUBZILKMSA-N Arg-Asp-Met Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CCSC)C(O)=O RRGPUNYIPJXJBU-GUBZILKMSA-N 0.000 description 1
- LVMUGODRNHFGRA-AVGNSLFASA-N Arg-Leu-Arg Chemical compound NC(N)=NCCC[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCN=C(N)N)C(O)=O LVMUGODRNHFGRA-AVGNSLFASA-N 0.000 description 1
- NIUDXSFNLBIWOB-DCAQKATOSA-N Arg-Leu-Cys Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CS)C(=O)O)NC(=O)[C@H](CCCN=C(N)N)N NIUDXSFNLBIWOB-DCAQKATOSA-N 0.000 description 1
- 239000004475 Arginine Substances 0.000 description 1
- 206010003402 Arthropod sting Diseases 0.000 description 1
- PZXPWHFYZXTFBI-YUMQZZPRSA-N Asp-Gly-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)CNC(=O)[C@@H](N)CC(O)=O PZXPWHFYZXTFBI-YUMQZZPRSA-N 0.000 description 1
- CJUKAWUWBZCTDQ-SRVKXCTJSA-N Asp-Leu-Lys Chemical compound OC(=O)C[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCCN)C(O)=O CJUKAWUWBZCTDQ-SRVKXCTJSA-N 0.000 description 1
- DCXYFEDJOCDNAF-UHFFFAOYSA-N Asparagine Natural products OC(=O)C(N)CC(N)=O DCXYFEDJOCDNAF-UHFFFAOYSA-N 0.000 description 1
- 208000035143 Bacterial infection Diseases 0.000 description 1
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 1
- 108010078791 Carrier Proteins Proteins 0.000 description 1
- 102000000844 Cell Surface Receptors Human genes 0.000 description 1
- 108010001857 Cell Surface Receptors Proteins 0.000 description 1
- 241000699802 Cricetulus griseus Species 0.000 description 1
- PRXCTTWKGJAPMT-ZLUOBGJFSA-N Cys-Ala-Ser Chemical compound [H]N[C@@H](CS)C(=O)N[C@@H](C)C(=O)N[C@@H](CO)C(O)=O PRXCTTWKGJAPMT-ZLUOBGJFSA-N 0.000 description 1
- QCUJUETWTSWPNZ-NAKRPEOUSA-N Cys-Ile-Met Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCSC)C(=O)O)NC(=O)[C@H](CS)N QCUJUETWTSWPNZ-NAKRPEOUSA-N 0.000 description 1
- MRVSLWQRNWEROS-SVSWQMSJSA-N Cys-Ile-Thr Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H]([C@@H](C)O)C(=O)O)NC(=O)[C@H](CS)N MRVSLWQRNWEROS-SVSWQMSJSA-N 0.000 description 1
- SHZGCJCMOBCMKK-UHFFFAOYSA-N D-mannomethylose Natural products CC1OC(O)C(O)C(O)C1O SHZGCJCMOBCMKK-UHFFFAOYSA-N 0.000 description 1
- 238000002965 ELISA Methods 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 206010015866 Extravasation Diseases 0.000 description 1
- PNNNRSAQSRJVSB-SLPGGIOYSA-N Fucose Natural products C[C@H](O)[C@@H](O)[C@H](O)[C@H](O)C=O PNNNRSAQSRJVSB-SLPGGIOYSA-N 0.000 description 1
- NVEASDQHBRZPSU-BQBZGAKWSA-N Gln-Gln-Gly Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCC(N)=O)C(=O)NCC(O)=O NVEASDQHBRZPSU-BQBZGAKWSA-N 0.000 description 1
- ZQPOVSJFBBETHQ-CIUDSAMLSA-N Gln-Glu-Gln Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(N)=O)C(O)=O ZQPOVSJFBBETHQ-CIUDSAMLSA-N 0.000 description 1
- NROSLUJMIQGFKS-IUCAKERBSA-N Gln-His-Gly Chemical compound C1=C(NC=N1)C[C@@H](C(=O)NCC(=O)O)NC(=O)[C@H](CCC(=O)N)N NROSLUJMIQGFKS-IUCAKERBSA-N 0.000 description 1
- ZNTDJIMJKNNSLR-RWRJDSDZSA-N Gln-Ile-Thr Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H]([C@@H](C)O)C(=O)O)NC(=O)[C@H](CCC(=O)N)N ZNTDJIMJKNNSLR-RWRJDSDZSA-N 0.000 description 1
- FNAJNWPDTIXYJN-CIUDSAMLSA-N Gln-Pro-Ala Chemical compound OC(=O)[C@H](C)NC(=O)[C@@H]1CCCN1C(=O)[C@@H](N)CCC(N)=O FNAJNWPDTIXYJN-CIUDSAMLSA-N 0.000 description 1
- CQGBSALYGOXQPE-HTUGSXCWSA-N Glu-Thr-Phe Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)O)NC(=O)[C@H](CCC(=O)O)N)O CQGBSALYGOXQPE-HTUGSXCWSA-N 0.000 description 1
- MZZSCEANQDPJER-ONGXEEELSA-N Gly-Ala-Phe Chemical compound NCC(=O)N[C@@H](C)C(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 MZZSCEANQDPJER-ONGXEEELSA-N 0.000 description 1
- QIZJOTQTCAGKPU-KWQFWETISA-N Gly-Ala-Tyr Chemical compound [NH3+]CC(=O)N[C@@H](C)C(=O)N[C@H](C([O-])=O)CC1=CC=C(O)C=C1 QIZJOTQTCAGKPU-KWQFWETISA-N 0.000 description 1
- PYUCNHJQQVSPGN-BQBZGAKWSA-N Gly-Arg-Cys Chemical compound C(C[C@@H](C(=O)N[C@@H](CS)C(=O)O)NC(=O)CN)CN=C(N)N PYUCNHJQQVSPGN-BQBZGAKWSA-N 0.000 description 1
- XUORRGAFUQIMLC-STQMWFEESA-N Gly-Arg-Tyr Chemical compound C1=CC(=CC=C1C[C@@H](C(=O)O)NC(=O)[C@H](CCCN=C(N)N)NC(=O)CN)O XUORRGAFUQIMLC-STQMWFEESA-N 0.000 description 1
- PABFFPWEJMEVEC-JGVFFNPUSA-N Gly-Gln-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CCC(=O)N)NC(=O)CN)C(=O)O PABFFPWEJMEVEC-JGVFFNPUSA-N 0.000 description 1
- IVSWQHKONQIOHA-YUMQZZPRSA-N Gly-His-Cys Chemical compound C1=C(NC=N1)C[C@@H](C(=O)N[C@@H](CS)C(=O)O)NC(=O)CN IVSWQHKONQIOHA-YUMQZZPRSA-N 0.000 description 1
- COVXELOAORHTND-LSJOCFKGSA-N Gly-Ile-Val Chemical compound NCC(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](C(C)C)C(O)=O COVXELOAORHTND-LSJOCFKGSA-N 0.000 description 1
- IGOYNRWLWHWAQO-JTQLQIEISA-N Gly-Phe-Gly Chemical compound OC(=O)CNC(=O)[C@@H](NC(=O)CN)CC1=CC=CC=C1 IGOYNRWLWHWAQO-JTQLQIEISA-N 0.000 description 1
- ABPRMMYHROQBLY-NKWVEPMBSA-N Gly-Ser-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CO)NC(=O)CN)C(=O)O ABPRMMYHROQBLY-NKWVEPMBSA-N 0.000 description 1
- 239000004471 Glycine Substances 0.000 description 1
- 229930186217 Glycolipid Natural products 0.000 description 1
- 108091006905 Human Serum Albumin Proteins 0.000 description 1
- 102000008100 Human Serum Albumin Human genes 0.000 description 1
- FQYQMFCIJNWDQZ-CYDGBPFRSA-N Ile-Pro-Pro Chemical compound CC[C@H](C)[C@H](N)C(=O)N1CCC[C@H]1C(=O)N1[C@H](C(O)=O)CCC1 FQYQMFCIJNWDQZ-CYDGBPFRSA-N 0.000 description 1
- NAFIFZNBSPWYOO-RWRJDSDZSA-N Ile-Thr-Gln Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCC(=O)N)C(=O)O)N NAFIFZNBSPWYOO-RWRJDSDZSA-N 0.000 description 1
- HQLSBZFLOUHQJK-STECZYCISA-N Ile-Tyr-Arg Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC1=CC=C(C=C1)O)C(=O)N[C@@H](CCCN=C(N)N)C(=O)O)N HQLSBZFLOUHQJK-STECZYCISA-N 0.000 description 1
- DLEBSGAVWRPTIX-PEDHHIEDSA-N Ile-Val-Ile Chemical compound CC[C@H](C)[C@H](N)C(=O)N[C@@H](C(C)C)C(=O)N[C@H](C(O)=O)[C@@H](C)CC DLEBSGAVWRPTIX-PEDHHIEDSA-N 0.000 description 1
- 206010062016 Immunosuppression Diseases 0.000 description 1
- ONIBWKKTOPOVIA-BYPYZUCNSA-N L-Proline Chemical compound OC(=O)[C@@H]1CCCN1 ONIBWKKTOPOVIA-BYPYZUCNSA-N 0.000 description 1
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 description 1
- DCXYFEDJOCDNAF-REOHCLBHSA-N L-asparagine Chemical compound OC(=O)[C@@H](N)CC(N)=O DCXYFEDJOCDNAF-REOHCLBHSA-N 0.000 description 1
- SHZGCJCMOBCMKK-DHVFOXMCSA-N L-fucopyranose Chemical compound C[C@@H]1OC(O)[C@@H](O)[C@H](O)[C@@H]1O SHZGCJCMOBCMKK-DHVFOXMCSA-N 0.000 description 1
- AGPKZVBTJJNPAG-WHFBIAKZSA-N L-isoleucine Chemical compound CC[C@H](C)[C@H](N)C(O)=O AGPKZVBTJJNPAG-WHFBIAKZSA-N 0.000 description 1
- ROHFNLRQFUQHCH-YFKPBYRVSA-N L-leucine Chemical compound CC(C)C[C@H](N)C(O)=O ROHFNLRQFUQHCH-YFKPBYRVSA-N 0.000 description 1
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 description 1
- QIVBCDIJIAJPQS-VIFPVBQESA-N L-tryptophane Chemical compound C1=CC=C2C(C[C@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-VIFPVBQESA-N 0.000 description 1
- KZSNJWFQEVHDMF-BYPYZUCNSA-N L-valine Chemical compound CC(C)[C@H](N)C(O)=O KZSNJWFQEVHDMF-BYPYZUCNSA-N 0.000 description 1
- BQSLGJHIAGOZCD-CIUDSAMLSA-N Leu-Ala-Ser Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@@H](CO)C(O)=O BQSLGJHIAGOZCD-CIUDSAMLSA-N 0.000 description 1
- NTRAGDHVSGKUSF-AVGNSLFASA-N Leu-Arg-Arg Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](CCCN=C(N)N)C(O)=O NTRAGDHVSGKUSF-AVGNSLFASA-N 0.000 description 1
- YWKNKRAKOCLOLH-OEAJRASXSA-N Leu-Phe-Thr Chemical compound CC(C)C[C@H](N)C(=O)N[C@H](C(=O)N[C@@H]([C@@H](C)O)C(O)=O)CC1=CC=CC=C1 YWKNKRAKOCLOLH-OEAJRASXSA-N 0.000 description 1
- ROHFNLRQFUQHCH-UHFFFAOYSA-N Leucine Natural products CC(C)CC(N)C(O)=O ROHFNLRQFUQHCH-UHFFFAOYSA-N 0.000 description 1
- VSTNAUBHKQPVJX-IHRRRGAJSA-N Lys-Met-Leu Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(C)C)C(O)=O VSTNAUBHKQPVJX-IHRRRGAJSA-N 0.000 description 1
- JHNOXVASMSXSNB-WEDXCCLWSA-N Lys-Thr-Gly Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H]([C@@H](C)O)C(=O)NCC(O)=O JHNOXVASMSXSNB-WEDXCCLWSA-N 0.000 description 1
- RIPJMCFGQHGHNP-RHYQMDGZSA-N Lys-Val-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](C(C)C)NC(=O)[C@H](CCCCN)N)O RIPJMCFGQHGHNP-RHYQMDGZSA-N 0.000 description 1
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 1
- 239000004472 Lysine Substances 0.000 description 1
- 241001521394 Maackia amurensis Species 0.000 description 1
- FYYHWMGAXLPEAU-UHFFFAOYSA-N Magnesium Chemical compound [Mg] FYYHWMGAXLPEAU-UHFFFAOYSA-N 0.000 description 1
- PNHRPOWKRRJATF-IHRRRGAJSA-N Met-Tyr-Ser Chemical compound CSCC[C@H](N)C(=O)N[C@H](C(=O)N[C@@H](CO)C(O)=O)CC1=CC=C(O)C=C1 PNHRPOWKRRJATF-IHRRRGAJSA-N 0.000 description 1
- 208000034486 Multi-organ failure Diseases 0.000 description 1
- 208000010718 Multiple Organ Failure Diseases 0.000 description 1
- WYBVBIHNJWOLCJ-UHFFFAOYSA-N N-L-arginyl-L-leucine Natural products CC(C)CC(C(O)=O)NC(=O)C(N)CCCN=C(N)N WYBVBIHNJWOLCJ-UHFFFAOYSA-N 0.000 description 1
- PESQCPHRXOFIPX-UHFFFAOYSA-N N-L-methionyl-L-tyrosine Natural products CSCCC(N)C(=O)NC(C(O)=O)CC1=CC=C(O)C=C1 PESQCPHRXOFIPX-UHFFFAOYSA-N 0.000 description 1
- 206010029719 Nonspecific reaction Diseases 0.000 description 1
- 241000282320 Panthera leo Species 0.000 description 1
- 108010033276 Peptide Fragments Proteins 0.000 description 1
- 102000007079 Peptide Fragments Human genes 0.000 description 1
- 108010039918 Polylysine Proteins 0.000 description 1
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 1
- UPJGUQPLYWTISV-GUBZILKMSA-N Pro-Gln-Glu Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCC(O)=O)C(O)=O UPJGUQPLYWTISV-GUBZILKMSA-N 0.000 description 1
- PCWLNNZTBJTZRN-AVGNSLFASA-N Pro-Pro-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@@H]1CCCN1C(=O)[C@H]1NCCC1 PCWLNNZTBJTZRN-AVGNSLFASA-N 0.000 description 1
- FZXSYIPVAFVYBH-KKUMJFAQSA-N Pro-Tyr-Glu Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CCC(O)=O)C(O)=O FZXSYIPVAFVYBH-KKUMJFAQSA-N 0.000 description 1
- 208000029464 Pulmonary infiltrates Diseases 0.000 description 1
- XGQKSRGHEZNWIS-IHRRRGAJSA-N Ser-Pro-Tyr Chemical compound N[C@@H](CO)C(=O)N1CCC[C@H]1C(=O)N[C@@H](Cc1ccc(O)cc1)C(O)=O XGQKSRGHEZNWIS-IHRRRGAJSA-N 0.000 description 1
- ZSDXEKUKQAKZFE-XAVMHZPKSA-N Ser-Thr-Pro Chemical compound C[C@H]([C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@H](CO)N)O ZSDXEKUKQAKZFE-XAVMHZPKSA-N 0.000 description 1
- 229930006000 Sucrose Natural products 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
- OUUQCZGPVNCOIJ-UHFFFAOYSA-M Superoxide Chemical class [O-][O] OUUQCZGPVNCOIJ-UHFFFAOYSA-M 0.000 description 1
- 208000033809 Suppuration Diseases 0.000 description 1
- 210000001744 T-lymphocyte Anatomy 0.000 description 1
- OJRNZRROAIAHDL-LKXGYXEUSA-N Thr-Asn-Ser Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CO)C(O)=O OJRNZRROAIAHDL-LKXGYXEUSA-N 0.000 description 1
- VYEHBMMAJFVTOI-JHEQGTHGSA-N Thr-Gly-Gln Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)NCC(=O)N[C@@H](CCC(N)=O)C(O)=O VYEHBMMAJFVTOI-JHEQGTHGSA-N 0.000 description 1
- IHAPJUHCZXBPHR-WZLNRYEVSA-N Thr-Ile-Tyr Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC1=CC=C(C=C1)O)C(=O)O)NC(=O)[C@H]([C@@H](C)O)N IHAPJUHCZXBPHR-WZLNRYEVSA-N 0.000 description 1
- QYDKSNXSBXZPFK-ZJDVBMNYSA-N Thr-Thr-Arg Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O QYDKSNXSBXZPFK-ZJDVBMNYSA-N 0.000 description 1
- QJIODPFLAASXJC-JHYOHUSXSA-N Thr-Thr-Phe Chemical compound C[C@H]([C@@H](C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)O)N)O QJIODPFLAASXJC-JHYOHUSXSA-N 0.000 description 1
- AYFVYJQAPQTCCC-UHFFFAOYSA-N Threonine Natural products CC(O)C(N)C(O)=O AYFVYJQAPQTCCC-UHFFFAOYSA-N 0.000 description 1
- 239000004473 Threonine Substances 0.000 description 1
- 102000004142 Trypsin Human genes 0.000 description 1
- 108090000631 Trypsin Proteins 0.000 description 1
- QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Natural products C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 description 1
- 239000006035 Tryptophane Substances 0.000 description 1
- GAYLGYUVTDMLKC-UWJYBYFXSA-N Tyr-Asp-Ala Chemical compound OC(=O)[C@H](C)NC(=O)[C@H](CC(O)=O)NC(=O)[C@@H](N)CC1=CC=C(O)C=C1 GAYLGYUVTDMLKC-UWJYBYFXSA-N 0.000 description 1
- PMDWYLVWHRTJIW-STQMWFEESA-N Tyr-Gly-Arg Chemical compound NC(N)=NCCC[C@@H](C(O)=O)NC(=O)CNC(=O)[C@@H](N)CC1=CC=C(O)C=C1 PMDWYLVWHRTJIW-STQMWFEESA-N 0.000 description 1
- QRCBQDPRKMYTMB-IHPCNDPISA-N Tyr-Trp-Ser Chemical compound C1=CC=C2C(=C1)C(=CN2)C[C@@H](C(=O)N[C@@H](CO)C(=O)O)NC(=O)[C@H](CC3=CC=C(C=C3)O)N QRCBQDPRKMYTMB-IHPCNDPISA-N 0.000 description 1
- AGDDLOQMXUQPDY-BZSNNMDCSA-N Tyr-Tyr-Ser Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CO)C(O)=O AGDDLOQMXUQPDY-BZSNNMDCSA-N 0.000 description 1
- VMRFIKXKOFNMHW-GUBZILKMSA-N Val-Arg-Ser Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](CO)C(=O)O)N VMRFIKXKOFNMHW-GUBZILKMSA-N 0.000 description 1
- UKEVLVBHRKWECS-LSJOCFKGSA-N Val-Ile-Gly Chemical compound CC[C@H](C)[C@@H](C(=O)NCC(=O)O)NC(=O)[C@H](C(C)C)N UKEVLVBHRKWECS-LSJOCFKGSA-N 0.000 description 1
- KZSNJWFQEVHDMF-UHFFFAOYSA-N Valine Natural products CC(C)C(N)C(O)=O KZSNJWFQEVHDMF-UHFFFAOYSA-N 0.000 description 1
- 206010000269 abscess Diseases 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 235000004279 alanine Nutrition 0.000 description 1
- 208000026935 allergic disease Diseases 0.000 description 1
- 230000000735 allogeneic effect Effects 0.000 description 1
- 150000001412 amines Chemical group 0.000 description 1
- 210000004102 animal cell Anatomy 0.000 description 1
- 230000000844 anti-bacterial effect Effects 0.000 description 1
- 230000009830 antibody antigen interaction Effects 0.000 description 1
- 239000003443 antiviral agent Substances 0.000 description 1
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 1
- 108010013835 arginine glutamate Proteins 0.000 description 1
- 125000003118 aryl group Chemical group 0.000 description 1
- 229960001230 asparagine Drugs 0.000 description 1
- 235000009582 asparagine Nutrition 0.000 description 1
- 208000022362 bacterial infectious disease Diseases 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 108091008324 binding proteins Proteins 0.000 description 1
- 150000001615 biotins Chemical class 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 210000004204 blood vessel Anatomy 0.000 description 1
- 229940098773 bovine serum albumin Drugs 0.000 description 1
- 210000000481 breast Anatomy 0.000 description 1
- 239000011575 calcium Substances 0.000 description 1
- 229910052791 calcium Inorganic materials 0.000 description 1
- 231100000504 carcinogenesis Toxicity 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 230000009087 cell motility Effects 0.000 description 1
- 230000005859 cell recognition Effects 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 230000033383 cell-cell recognition Effects 0.000 description 1
- 230000036755 cellular response Effects 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 230000001684 chronic effect Effects 0.000 description 1
- 230000035602 clotting Effects 0.000 description 1
- 238000012875 competitive assay Methods 0.000 description 1
- 230000000536 complexating effect Effects 0.000 description 1
- 230000001276 controlling effect Effects 0.000 description 1
- 229920001577 copolymer Polymers 0.000 description 1
- 239000003431 cross linking reagent Substances 0.000 description 1
- 235000018417 cysteine Nutrition 0.000 description 1
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 1
- 108010016616 cysteinylglycine Proteins 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000012217 deletion Methods 0.000 description 1
- 230000037430 deletion Effects 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 230000013020 embryo development Effects 0.000 description 1
- 210000002889 endothelial cell Anatomy 0.000 description 1
- 230000003511 endothelial effect Effects 0.000 description 1
- 210000003989 endothelium vascular Anatomy 0.000 description 1
- 239000002702 enteric coating Substances 0.000 description 1
- 238000009505 enteric coating Methods 0.000 description 1
- 210000003979 eosinophil Anatomy 0.000 description 1
- 210000002919 epithelial cell Anatomy 0.000 description 1
- 210000003743 erythrocyte Anatomy 0.000 description 1
- QPADNTZLUBYNEN-UHFFFAOYSA-N etallobarbital Chemical compound C=CCC1(CC)C(=O)NC(=O)NC1=O QPADNTZLUBYNEN-UHFFFAOYSA-N 0.000 description 1
- 230000001747 exhibiting effect Effects 0.000 description 1
- 210000003722 extracellular fluid Anatomy 0.000 description 1
- 230000036251 extravasation Effects 0.000 description 1
- 230000004720 fertilization Effects 0.000 description 1
- 239000012737 fresh medium Substances 0.000 description 1
- 210000001035 gastrointestinal tract Anatomy 0.000 description 1
- 238000001502 gel electrophoresis Methods 0.000 description 1
- 238000007429 general method Methods 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- PEDCQBHIVMGVHV-UHFFFAOYSA-N glycerol group Chemical group OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 1
- 229930182470 glycoside Natural products 0.000 description 1
- 108010075431 glycyl-alanyl-phenylalanine Proteins 0.000 description 1
- 210000005205 gut mucosa Anatomy 0.000 description 1
- 150000004820 halides Chemical group 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 230000002519 immonomodulatory effect Effects 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 230000006054 immunological memory Effects 0.000 description 1
- 230000001506 immunosuppresive effect Effects 0.000 description 1
- 238000000099 in vitro assay Methods 0.000 description 1
- 239000012678 infectious agent Substances 0.000 description 1
- 230000037456 inflammatory mechanism Effects 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 230000008611 intercellular interaction Effects 0.000 description 1
- 238000007918 intramuscular administration Methods 0.000 description 1
- 230000026045 iodination Effects 0.000 description 1
- AGPKZVBTJJNPAG-UHFFFAOYSA-N isoleucine Natural products CCC(C)C(N)C(O)=O AGPKZVBTJJNPAG-UHFFFAOYSA-N 0.000 description 1
- 229960000310 isoleucine Drugs 0.000 description 1
- 108010053037 kyotorphin Proteins 0.000 description 1
- 230000033001 locomotion Effects 0.000 description 1
- 231100000516 lung damage Toxicity 0.000 description 1
- 210000004698 lymphocyte Anatomy 0.000 description 1
- 210000002540 macrophage Anatomy 0.000 description 1
- 239000011777 magnesium Substances 0.000 description 1
- 229910052749 magnesium Inorganic materials 0.000 description 1
- 229910001629 magnesium chloride Inorganic materials 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 229930182817 methionine Natural products 0.000 description 1
- 230000008747 mitogenic response Effects 0.000 description 1
- 210000005087 mononuclear cell Anatomy 0.000 description 1
- 230000000877 morphologic effect Effects 0.000 description 1
- 208000029744 multiple organ dysfunction syndrome Diseases 0.000 description 1
- 238000002703 mutagenesis Methods 0.000 description 1
- 231100000350 mutagenesis Toxicity 0.000 description 1
- 230000012177 negative regulation of immune response Effects 0.000 description 1
- 230000010309 neoplastic transformation Effects 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 239000006186 oral dosage form Substances 0.000 description 1
- 210000001672 ovary Anatomy 0.000 description 1
- 239000006201 parenteral dosage form Substances 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 230000008506 pathogenesis Effects 0.000 description 1
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 description 1
- 108010084572 phenylalanyl-valine Proteins 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 231100000614 poison Toxicity 0.000 description 1
- 229920000656 polylysine Polymers 0.000 description 1
- 239000011591 potassium Substances 0.000 description 1
- 229910052700 potassium Inorganic materials 0.000 description 1
- 230000003389 potentiating effect Effects 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
- 125000001500 prolyl group Chemical group [H]N1C([H])(C(=O)[*])C([H])([H])C([H])([H])C1([H])[H] 0.000 description 1
- 108010029020 prolylglycine Proteins 0.000 description 1
- 108010053725 prolylvaline Proteins 0.000 description 1
- 150000003214 pyranose derivatives Chemical group 0.000 description 1
- 239000011541 reaction mixture Substances 0.000 description 1
- 238000001525 receptor binding assay Methods 0.000 description 1
- 230000010410 reperfusion Effects 0.000 description 1
- 239000011347 resin Substances 0.000 description 1
- 229920005989 resin Polymers 0.000 description 1
- 210000002345 respiratory system Anatomy 0.000 description 1
- 238000004007 reversed phase HPLC Methods 0.000 description 1
- 125000003607 serino group Chemical group [H]N([H])[C@]([H])(C(=O)[*])C(O[H])([H])[H] 0.000 description 1
- 108010071207 serylmethionine Proteins 0.000 description 1
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
- 230000009870 specific binding Effects 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 150000008163 sugars Chemical class 0.000 description 1
- 230000002459 sustained effect Effects 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 239000003440 toxic substance Substances 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 230000032258 transport Effects 0.000 description 1
- 239000012588 trypsin Substances 0.000 description 1
- 229960004799 tryptophan Drugs 0.000 description 1
- 230000004614 tumor growth Effects 0.000 description 1
- 241000712461 unidentified influenza virus Species 0.000 description 1
- 229960005486 vaccine Drugs 0.000 description 1
- 239000004474 valine Substances 0.000 description 1
- 108010072644 valyl-alanyl-prolyl-glycine Proteins 0.000 description 1
Landscapes
- Peptides Or Proteins (AREA)
Abstract
The present invention is directed to methods of supressing inflammatory responses, inducing tolerance to an antigen, and suppressing cell adhesion, e.g., involved in metastasis, by the administration of lectin derived carbohydrate binding peptides or derivatives thereof, in particular, peptides capable of binding terminally linked .alpha.-sialic acid(2~6).beta.Gal-and/or .alpha.-sialic acid(2~3).beta.Gal-groups on structures or molecules comprising such groups. Pharmaceutical compositions containing such lectin derived carbohydrate binding peptides or derivatives thereof are also disclosed.
Description
ANTI-INFLAMMATORY, TOLEROGENIC AND
IMMUNOINHIBITING PROPERTIES
OF CARBOHYDRATE BINDING PEPTIDES
Background of the Invention 1. Field of the Invention.
This invention is directed to methods for inhibiting immune responses or cellular interactions in mammals by the administration thereto of one or more lectin derived carbohydrate binding peptides. In particular, this invention is directed to methods for the suppression of inflammatory responses, induction of tolerance to antigens, modulation of the induction of immune responses to antigens, and the inhibition of cell adhesion in mammals by the administration of one or more carbohydrate binding peptides. The lectin derived carbohydrate binding peptides employed herein are preferably fragments of the S2 or S3 subunits of the pertussis toxin expressed by Bordetella pertussis or functionally equivalent variants thereof.
IMMUNOINHIBITING PROPERTIES
OF CARBOHYDRATE BINDING PEPTIDES
Background of the Invention 1. Field of the Invention.
This invention is directed to methods for inhibiting immune responses or cellular interactions in mammals by the administration thereto of one or more lectin derived carbohydrate binding peptides. In particular, this invention is directed to methods for the suppression of inflammatory responses, induction of tolerance to antigens, modulation of the induction of immune responses to antigens, and the inhibition of cell adhesion in mammals by the administration of one or more carbohydrate binding peptides. The lectin derived carbohydrate binding peptides employed herein are preferably fragments of the S2 or S3 subunits of the pertussis toxin expressed by Bordetella pertussis or functionally equivalent variants thereof.
2. References.
The following references are cited in this application as superscript numbers at the relevant portion of the application:
1. Brandley, et al., J. Leukocyte Biol., 40:97-111 (1986).
2. Jacobson, Developmental Neurobiology, New York, Plenum Press, p. 5-25 (1978).
The following references are cited in this application as superscript numbers at the relevant portion of the application:
1. Brandley, et al., J. Leukocyte Biol., 40:97-111 (1986).
2. Jacobson, Developmental Neurobiology, New York, Plenum Press, p. 5-25 (1978).
3. Trinkaus, Cells into Organs, Englewood Cliffs, N.J., Prentice Hall, p. 44-68 (1984).
4. Frazier, et al., Annu. Rev. Biochenl., 4$:491 (1979).
S. Glaser, Mediator of Developmental Processes (Substency, S.
and Wessels, N.K., Eds.) New York, Academic Press, p.
79 (1980).
6. Paulson, In "The Receptors ", Vol. II (Comm., P.M., Ed.),
S. Glaser, Mediator of Developmental Processes (Substency, S.
and Wessels, N.K., Eds.) New York, Academic Press, p.
79 (1980).
6. Paulson, In "The Receptors ", Vol. II (Comm., P.M., Ed.),
5 New York Academic Press, p. 131 (1985).
7. Sharon, Lectin-Like Bacterial Adherence to Animal Cells. In "Attachment of Microorganisms to the Gut Mucosa"
(Boeheker, E.D., Ed.), Boca Raton, Florida CRC Press, p.
129 (1984).
10 8. Wassarman, Fertilization. In "Cell Interactions and Development: Molecular Mechanisms" (Yamada, K.M., Ed.), New York, John Wiley and Sons, p. 1 (1983)..
9. Schwartz, et al., Immunol. Rev., 40:153 et seq. (1978).
10. Coutinho, et al., Immunol. Rev., 78:211 et seq. (1984).
15 11. Hoffmann, et al., Eds., Membranes in Growth and Development, New York, Alan R. Liss, p. 429-442, (1982).
12. Galeotti, et al., Eds., Membranes in Tumor Growth, Amsterdam, Elsevier, p. 77-81 (1982).
13. Nicolson, et al., Invas. Metas., 5_:144 et seq. (1985).
20 14. Aplin, et al., Biochim. Biophys. Acta 694:375 et seq.
( 1982) .
15. Barondes, Developmentally Regulated Lectins. In "Cell Interactions and Development: Molecular Mechanisms"
(Yamada, D.M., Ed.) New York, John Wiley and Sons, p.
25 185 (1983).
16. Monisigny, M., Ed., Biol. Cell, SI (Special Issue), 113 et seq. (1984).
17. Springer, et al., Nature, 349:196-197 (1991).
18. Lowe, et al., Cell, 63:475-485 (1990).
19. Phillips, et al., Science, Vol. 250:1130-1132 (1990).
20. Walz, et al., Science, 250:1132 et seq. (1990).
21. Larsen, et al., Cell, (3_:467-474 (1990).
22. Bevilacqua, et al., Endothelial-Leukocyte Adhesion Molecule - l (ELAM-1): A Vascular SELECTIN That Regulates Inflammation. In "Cellular and Molecular Mechanisms of Inflammation" Vol. 2, Academic Press, p. 1-13 (1991).
23. McEver, Leukocyte Interactions Mediated by GMP-140. In "Cellular and Molecular Mechanisms of Inflammation", Vol.
2, Academic Press, p. 15-29 ( 1991 ).
24. Larsen, et al., J. Biol. Chem., 267:11104-11110 (1992).
25. Heerze, et al., Biochem and Biophys. Res. Comm.
172:1224-1229 ( 1990) .
26. Paulson, et al., International Patent Application Publication No. W091 /19502, ( 1991 ) .
27. Ippolito, et al., U. S. Patent Application Serial No.
07/714,161, filed 10 June 1991.
28. Ippolito, et al., U. S. Patent Application Serial No.
07/889,017, filed 26 May 1992.
29. McEver, International Patent Application Publication No.
W092/Ol 718 ( 1992) .
30. Heerze, et al., Biochem and Biophys. Res. Comm., 179:1464-1469 ( 1991 ) .
31. Pearce-Pratt, et al., J. Imm. Methods, 140:159-165 (1991).
32. Smith, et al., Cell. Imm., 89:20-29 (1984).
33. Munoz, Action of Pertussigen (Pertussis Toxin) on the Host Immune System. In "Pathogenesis and Immunity in Pertussis", John Wiley & Sons Ltd., p. 173-192 (1988).
34. Reuter, et al, Glycoconjugates 5_:133-135 (1988).
35. Gaeta, et al., U. S. Patent Application Serial No.
07/538,853, filed 15 June 1990.
36. Paulson, et al., U. S. Patent Application Serial No.
07/619,319, filed 28 November 1990.
37. Paulson, et al., U. S. Patent Application Serial No.
07/632,390, filed 21 December 1990.
38. Brandley, et al., International Patent Application Publication No. W092/02527 (1992).
39. Lowe, International Patent Application Publication No.
W092/07572 (1992).
40. Furie, et al., International Patent Application Publication No. W092/16612 ( 1992) .
41. Seed, et al., International Patent Application Publication No. W092/09293 (1992).
42. Karlsson, Annu. Rev. Biochem., 5$:309-350 (1989).
43. Brennan, et al., J. Biol. Chem., 26:4895-4899 (1988).
44. Armstrong, et al., J. Biol. Chem., 263:8677-8684 (1988).
45. Smith, et al., U. S. Patent Application Serial No.
07/956,043, filed 2 October 1992.
46. Shibuya, et al., J. Biol. Chem., 21:7755-7761 (1987).
47. Wang, et al., J. Biol. Chem., 26:4576-4585 (1988).
48. Gimbrone, et al., International Patent Application Publication No. W091 /08231 ( 1991 ) .
49. Ratcliffe, et al., U. S. Patent No. 5,079,353, issued 7 January 1992.
50. Hakomori, Adv. Cancer Res., 52:257-331 (1989).
51. Sialic Acids in "Cell Biology Monographs", Schauer, Editor, Vol. 10 (1982).
52. Tyrrell, et al., Infect. Immun., x:1854-1857 (1989).
53. Bhavanandan, et al., J. Biol. Chem., 2:4000-4008 (1979).
54. Nogimori, et al., Biochemistry, 25:1355-1363 (1986).
55. Sato, et al. , Infect. Immun. , 46:415-421 ( 1986).
56. Tamura, et al., J. Biol. Chem., 2:6756-6761 (1983).
57. Tuomanen, et al., J. Exp. Med., 168:267-277 (1988).
58. Tuommenen, et al., 92nd General Meeting of the American Society of Microbiology, New Orleans, LA, Poster B-16.
59. Morgan, et al., Ann. Repts. Med. Chem. , 24:243-252 (1989).
60. Chou, et al., Annu. Rev. Biochem., 47:251-276 (1978).
61. Hopp, et al., Proc. Natl. Acad. Sci. U. S.A. , x:3824-3828 (1981).
62. Wright, J. Mol. Biol., 21:635-651 (1990).
63. Armstrong, et al., Infect. Immun., 55:1294-1299 (1987).
64. Smith, et.al., Immunology, 58:245 et seq. (1986).
65. Sleyter, et al., Arch. Microbiol., 146:19 et seq. (1986).
The disclosure of all publications, patents and patent applications cited in this application are herein incorporated by reference in their entirety to the same extent as if each individual publication or patent -b-application were specifically and individually indicated to be incorporated by reference in its entirety.
3. State of the Art.
Important processes involving mammalian cells, such as growth, locomotion, morphological development, and differentiation, are partially controlled by extracellular signals acting upon the cells' surfaces'-3.
While some external stimuli reach the cell via extracellular fluids, other signals are received from neighboring or approaching cell surfaces and exert their effects through direct cell-cell contact°~s.
Evidence suggests that specific cell-surface receptors can "sense" a molecular signal of an opposing cell via specific binding, and biochemical mechanisms exist to translate that binding into a cellular response. For example, complex cell-surface interactions are believed to help direct processes such as binding of pathogens to target tissuesb~', sperm-egg binding8, interactions among cells in the immune system9~'°, and recognition of cells during embryonic development". In addition, defects in cell-cell recognition are thought to underlie the uncontrolled cell growth and motility which characterize neoplastic transformation and metastasis'z~".
Other evidence suggests that cell-recognition processes are mediated by carbohydrate chains or glycan portions of glycoconjugates'~'a-'6. For example, the binding of the surface glycoconjugates of one cell to the complementary carbohydrate-binding proteins (lectins) on another cell can result in the initiation of a specific interaction.
_7_ One important group of carbohydrate-binding proteins are selectin (LEC-CAM) proteins (Lectin + EGF + complementary Regulatory Domain-Cell Adhesion Molecules). These or functionally similar proteins or lectins are believed to play a critical role in immune responses (including inflammatory responses) through mediation of cell-cell contact and through extra-vasation of leucocytes"~'-'-. Specific carbohydrate ligands have been identified as part of the putative receptor structures for selectin proteins and other lectins"-'-'. The structures identified include oligosaccharide glycosides containing terminally linked a-sialic acid(2->6)(3Gal- and a-sialic acid(2->3)/3Gal- groups. The use of oligosaccharides and derivatives thereof having such terminally linked groups for controlling inflammation, immunosuppression, etc. by interacting with selectin proteins and/or other lectins has been disclosed26-29.33-41 Likewise, peptides derived from the seIectin GMP-140 which inhibit binding of GMP-140 and other selectins to leukocytes, presumably by interfering with the binding of the GMP-selectin protein with the carbohydrate receptor on the leukocyte, have also been disclosed as being useful in suppressing an immune response29. Similarly, other peptides are also known to be potent modulators of neutrophil functions'g Pertussis toxin (PT)°'-, a virulence factor produced by the organism Bordetella pertussis -- the etiological agent of whooping cough, is known to bind to glycoprotein receptors which terminate in sialyllactosamine sugar sequences43~°', and we have previously shown that this protein is useful in suppressing mammalian immune responses and cellular adhesions. PT's binding specificity has been shown to be similar to that of the plant lectins from Sambucus nigra (SNA) and Maac~ia amurensis _g_ (MAL)'-s, which bind with high affinity to sialic acid-containing glycoconjugates46.°' and which have also been shown as possessing immunomodulating properties°s.
However, the use of proteins or large molecular weight polypeptides in inhibiting immune responses or cellular interactions in mammals suffers from several drawbacks, including the fact that they are difficult to produce in large quantities in pure form, that they tend to produce adverse effects when repeatedly administered to a mammal, that they can contain infectious agents or toxic substances which are contraindications to mammalian administration, and that it is difficult to modify the pharmokinetic properties of such proteins to improve their efficacy.
In view of the above, the use of peptides having lectin-like binding properties for terminal aNeuSAc(2-~3)(3Gal- and aNeuSAc(2~6)/3Ga1-groups would be particularly beneficial for use in inhibiting immune responses and cellular interactions in mammals as compared to the administration of proteins such as the pertussis toxin and the lectins derived from SNA and MAL because such peptides would mitigate the problems associated with the administration of proteins and large molecular weight polypeptides to mammals.
Summar,~r of the Invention 1"his invention is directed, in part, to the discovery that the binding domains for the a-sialic acid(2->3)~3Ga1- and/or the a-sialic acid(2-~6)~3Gal- terminally linked structures are found in certain peptide fragments of lectins (e.g., proteins and polypeptides) such as pertussis toxin and that it is not necessary to employ the entire lectin to effect binding to these carbohydrates (oligosaccharides).
This invention is further directed, in part, to the discovery that when these lectin derived carbohydrate binding peptides are administered to a mammal (e.g., human) in effective amounts, they inhibit specific immune responses and cellular interactions. In particular, this invention is directed to the discovery that such lectin derived carbohydrate binding peptides may be administered to a mammal in order to inhibit inflammatory responses, modulate the induction of an immune response to an antigen, induce long term tolerance to an antigen, and suppress cell adhesion.
This invention is particularly directed to the discovery that lectin derived carbohydrate binding peptides capable of binding terminally linked a-sialic acid(2-~3)~3Ga1- and/or a-sialic acid(2-~6)~3Ga1- (e.g., aNeuSAc(2~3)/3Ga1-) groups present in molecules (e.g., oligosaccharides, glycoproteins, glycolipids, etc.) which can be found on cell surfaces (e.g., leukocytes) may be administered to a mammal as a means for inhibiting cell adhesion or cell-mediated immune responses. Cell-mediated immune responses inhibited by the peptides disclosed herein include inflammatory responses, modulation of the induction of the immune response to an antigen and the induction of long term tolerance to an antigen.
Preferably, the lectin derived carbohydrate binding peptides employed herein are fragments of the S2 or S3 subunits of the pertussis toxin expressed by Bordetella pertussis or functionally equivalent variants thereof. These preferred peptides have the amino acid sequences set forth in peptides of Figure 1 below.
Accordingly; in one of its method aspects, this invention is directed to a method of suppressing an inflammatory response in a mammas by the administration of an effective amount of at least one lectin derived carbohydrate binding peptide or derivative thereof capable of binding terminally linked a-siaiic acid(2--~6)~3Gal- and/or a-sialic acid(2-->3)~3Ga1-groups on structures or molecules comprising such groups.
In another of its method aspects, this invention is directed to a method for modulating the induction of an immune response to an antigen in a mammal by administering the antigen in combination with an effective amount of at least one lectin derived carbohydrate binding peptide or derivative thereof capable of binding terminally linked a-sialic acid(2-~6)(3Gal- and/or a-sialic acid(2-~3)~3Gal- groups on structures or molecules comprising such groups.
In another of its method aspects, this invention is directed to a method for inducing in a sensitized mammal long term tolerance to an antigen by exposing (challenging) the mammal with the antigen followed by the administration of an effective amount of at least one lectin derived carbohydrate binding peptide or derivative thereof capable of binding terminally linked a-sialic acid(2-~6)J3Ga1- and/or a-sialic acid(2~3)(3Ga1-groups on structures or molecules comprising such groups.
In still another one of its method aspects, this invention is directed to a method for inhibiting cell adhesion events involved in metastasis of tumor cells or in inflammation by the administration of an effective amount of at least one lectin derived carbohydrate binding peptide or derivative thereof capable of binding terminally linked a-siaiic acid(2-~6)~3Gal- and/or a-sialic acid(2-~3)~3Gal- groups on structures or molecules comprising such groups.
In yet another one of its method aspects, this invention is directed to a method for treating lung inflammation and/or lung injury in a mammal by the administration of an effective amount of one or more lectin derived carbohydrate binding peptides or derivatives thereof capable of binding terminally linked a-sialic acid(2->6)~3Ga1- and/or a-sialic acid(2-~3)~iGal- groups on structures or molecules comprising such groups.
In its composition aspects, this invention is directed to pharmaceutical compositions comprising the subject lectin derived carbohydrate binding peptides including pharmaceutically acceptable salts of such lectin derived carbohydrate binding peptides.
Brief Description of the Drawings Figure 1 illustrates the amino acid sequence of preferred lectin derived carbohydrate binding peptides.
Figure 2 illustrates the increase in footpad swelling of immunized Balb/c mice arising from a DTH inflammatory response measured 24 hours after challenge with 20 ~,g of OVA antigen wherein some of the mice have been treated at 5 hours after challenge with 100 ~g of a lectin derived carbohydrate binding peptide.
Figure 3 illustrates the long term (2 week) effects on the DTH
responses determined by footpad swelling in groups of Balb/c mice which are immunized with 100 ~cg of the OVA antigen, challenged 7 days later with OVA, and then treated about 5 hours thereafter with a lectin derived carbohydrate binding peptide.
In Figures 2 and 3, peptide 2275 is ACS2P1 (amino acids 9-23 of SEQ ID N0:3) and peptide 2283 is SPYGRC (amino acids 18-23 of SEQ
ID N0:3), both of which are illustrated in Figure 1.
Figures 4 through 7 illustrate the same test as Figures 2 and 3 for other peptides.
The amino acid residues depicted in Figure 1 and used throughout this application are designated by the conventional single letter code set forth below:
A alanine I isoleucine R arginine C cysteine K lysine S serine D aspartic L leucine T threonine acid E glutamic M methionine V valine acid phenyl alanineN asparagine W tryptophane F
G glycine P proline Y tyrosine H histidine Q glutamine Additionally, as conventionally indicated, the amino acid residue forming the H,N- terminus of the peptide is set forth on the left side of the peptide chain, and the -COOH terminus of the peptide is set forth on the right side of the peptide chain.
Detailed Description of the Preferred Embodiments This invention is directed, in part, to the discovery that certain lectin derived carbohydrate binding peptides when administered to a mammal are effective in suppressing inflammatory responses, inducing tolerance to an antigen, modulating the induction of immune responses to antigens, and inhibiting cell adhesion events, e.g., cell adhesion events involved in metastasis of tumor cells.
I. Definitions As used herein, the following terms have the meanings set forth below:
The term "inflammatory response" or "inflammatory disorder" will refer to immune reactions involving specific and non-specific defense systems. A specific defense system reaction is a specific immune system reaction to an antigen. Examples of specific defense system reactions include antibody responses to antigens such as viruses, allergens, and delayed-type hypersensitivity. A non-specific defense system reaction is an inflammatory response mediated by leukocytes generally incapable of immunological memory. Such cells include macrophages, eosinophils and neutrophils. Examples of non-specific reactions include the immediate swelling after a bee sting, and the collection of polymorphonuclear (PMN) leukocytes at sites of bacterial infection (e.g., pulmonary infiltrates in bacterial pneumonias and pus formation in abscesses).
Other "inflammatory responses" or "inflammatory disorders"
within the scope of this invention include, e.g., autoimmune disorders such as rheumatoid arthritis, lupus, multiple sclerosis, post-ischemic leukocyte mediated tissue damage (reperfusion injury), frost-bite injury or shock, acute leukocyte-mediated lung injury CARDS), asthma, traumatic shock, septic shock, nephritis, and acute and chronic inflammation including atopic dermatitis, psoriasis, and inflammatory bowel disease.
Various platelet-mediated pathologies such as atherosclerosis and clotting are also included within the definition of "inflammatory responses" or "inflammatory disorders" . In addition, "inflammatory responses" or "inflammatory disorders" may include the adhesion of circulating cancer cells, with specific examples including carcinoma of the colon and melanoma.
Without being limited to any theory, we believe that primary events in the initiation of such inflammatory responses and inflammatory disorders are the binding of leukocytes to selectins (e.g., ELAM-l, PADGEM, etc.) through carbohydrate receptors comprising a-sialic acid(2-~3)aGal- and/or a-sialic acid(2-~6)~iGal- groups'S-'° found on the surface of the leukocytes. Likewise, it has been shown that mammalian and, in particular, human cancer cells contain a-sialic acid(2~3)~3Ga1-and/or a-sialic acid(2-~6)/3Gal- groups on the surface thereof'9~5°, and it is believed that binding of such circulating cancer cells to selectins is an integral part of the metastatic process3e.". Accordingly, by interfering with the binding of such carbohydrate receptors to these selectins, suppression of the inflammatory immune responses as well as inhibition of metastatic processes is achieved.
The term "antigen" refers to any protein, peptide, carbohydrate, nucleic acid or other non-endogenous substance which when exposed to a mammal induces an immune response in that mammal.
Disease conditions believed to be caused by antigen exposure include, by way of example, psoriasis, asthma, dermatitis, rheumatoid arthritis, delayed type hypersensitivity, inflammatory bowel disease, multiple sclerosis, viral pneumonia, bacterial pneumonia, and the like.
The term "non-sensitized mammal" refers to those mammals which have yet to be educated to a particular antigen.
The term "sensitized mammal" refers to those mammals which have been previously exposed to a particular antigen, and, accordingly, their immune systems have become educated to that antigen. Typically, initial exposure of an antigen to a mammal primes or educates the mammal's immune response to later exposure to that antigen with minimal inflammation during such initial exposure.
The term "secondary immune response" refers to the effector phase of a mammal's immune response to an antigen to which it has previously been sensitized. A mammal's secondary immune response is typically accompanied by inflammation at the point of antigen exposure.
"Acute respiratory distress syndrome" or "ARDS" refers to an inflammatory condition comprising leukocyte mediated lung injury.
Without being limited to any theory, it is believed that such lung injury is exacerbated by infiltration and subsequent disruption of neutrophils into the lungs. Specifically, the disruption of the neutrophils in the lungs releases superoxides which results in severe vascular endothelial damage.
Accordingly, while this lung damage is not antigen based, the infiltration of neutrophils into the lungs requires an adhesion event.
"Reperfusion injury" refers to an inflammatory condition comprising leukocyte mediated tissue damage. Reperfusion injury commonly occurs after myocardial infarction wherein, in response to the inflammation caused by the myocardial infarction, the endothelium cells are activated and produce selectins (e.g., ELAM-1). In turn, neutrophils are then capable of binding the selectins expressed on the vascular endothelium and cause further damage.
The term "pertussis toxin" or "PT" in this application refers to the virulence factors produced by Bordetella pertussis, the etiological agent of whooping cough. The (~-oligomer of PT binds to both a-sialic acid(2-~6)~3Ga1- and a-sialic acid(2->3)~3Gai- structures. PT also has similar binding characteristics to those of wheat germ agglutinin (WGA), which can recognize terminal N-acetylglucosamine (GIcNAc) saccharide sequences in addition to siaiic acid'°~s=~s3.
PT is a classical A-B type of toxin comprised of an A subunit (designated S 1 ) that contains an ADP-ribosyltransferase enzyme activity, which is responsible for most of the bioiogical effects of PT". The lectin-like activity is found in the complex (3 oligomer of PT, which consists of four heterogeneous subunits that are arranged in a pair of dimers (S2-S4 (Dimer 1 ) and S3-S4 (Dimer 2)) joined by a smaller SS
subunit. The functioning of the (3 oligomer is in binding to host cell sialylated oligosaccharide receptors as well as providing a delivery system for the A subunit through the cytoplasmic membranes°.ss.sb. The (3 oligomer itself can induce a mitogenic response in lymphocytes and has the ability to agglutinate erythrocytes. PT may also contribute to the attachment of B. penussis to epithelial cells lining the upper respiratory tract of humans, the only known host of B. pertussiss'. In addition, the (3 oiigomer also appears to share functional homology with the selectin family of mammalian lectins that regulate leukocyte trafficking.sg The term "cell-mediated immune response" refers to those mammalian immune responses which are mediated by cell-cell interactions. Included within this term are cell-mediated inflammatory responses to an antigen including, by way of example, such responses as delayed-type hypersensitivity (DTH) responses, virus-induced pneumonia, allergic responses, and the like as well as cell-mediated inflammatory responses arising from injuries such as myocardial infarction, shock and sequelae (e.g., multiple organ failure), acute respiratory distress syndrome CARDS), and the like. Generally, the cell-mediated immune response is a leukocyte-mediated response.
The term "humoral immune response" refers to mammalian immune responses which involve antigen-antibody interactions.
The term "DTH inflammation response" or "delayed type hypersensitivity response" is a T cell mediated reaction which results in a mononuclear cell-rich inflammation and swelling which occurs after antigenic challenge.
The term "tolerance" or "immunological tolerance" refers to a reduced immunogenic response elicited in a sensitized mammal to a particular antigen upon a second or subsequent antigenic challenge in comparison to the primary immune response elicited by said antigen under equivalent conditions (e.g., dosage). In the present invention such "tolerance" will be obtained by administration of an antigen to the sensitized mammal followed by administration of one or more lectin derived carbohydrate binding peptides which bind to a-sialic acid(2-->6)~3Gal- and/or a-sialic acid(2~3)~3Gal- structures.
The term "period for maximal inflammation" refers to the period of time typically required to achieve maximal inflammation in a mammal due to a cell-mediated immune response including both cell-mediated inflammatory responses in a sensitized mammal due to antigen exposure (challenge) and cell-mediated inflammatory responses in a mammal due to injury (e.g., myocardial infarction). This period of time depends on several factors such as the specific antigen/injury afflicting the mammal, the particular mammalian species exposed to the antigen/injury, etc.
Accordingly, the period of time required to effect maximal inflammation will vary for, by way of example, rheumatoid arthritis as opposed to myocardial infarction.
Moreover, while the specific time required to effect maximal inflammation will vary somewhat in a given mammalian species, the time typically required to effect maximal inflammation for different afflictions due to either antigen exposure or injury in human and other mammals is known in the art or is readily ascertainable by the skilled artisan. For example, in the case of a DTH response in mice, maximal inflammation is typically 24 hours after antigen exposure.
The term "sialic acid" refers to all naturally occurring structures of sialic acid and analogues of sialic acid as well as derivatives thereof.
Naturally occurring structures of sialic acid include, by way of example, 5-acetamido-3,5-dideoxy-D-glycero-D-galacto-nonulopyranosylonic acid ("NeuSAc"), N-glycoyl neuraminic acid (NeuSGc) and 9-O-acetyl neuraminic acid (Neu5,9Ac,). A complete list of naturally occurring sialic acids known to date is provided by Schauers'.
Derivatives of sialic acid refers to derivatives of naturally occurring structures of sialic acid including those wherein the sialic acid unit has been chemically modified so as to introduce and/or remove one or more functionalities from such structures. For example, such modification can result in the removal of an -OH functionality, the introduction of an amine functionality, the introduction of a halide functionality, and the like.
Certain derivatives of sialic acid are known in the art and include chemically modified sialic acid derivatives such as 9-azido-NeuSAc, 9-S amino-NeuSAc, 9-deoxy-NeuSAc, 9-fluoro-NeuSAc, 9-bromo-NeuSAc, 8-deoxy-NeuSAc, 8-epi-NeuSAc, 7-deoxy-NeuSAc, 7-epi-NeuSAc, 7-8-bis-epi-NeuSAc, 4-O-methyl-NeuSAc, 4-N-acetyl-NeuSAc, 4,7-di-deoxy-NeuSAc, 4-uno-NeuSAc, 3-hydroxy-NeuSAc, 3-fluoro-NeuSAc acid as well as 6-thin analogues of NeuSAc. Methods for preparing such sialic acid derivatives are taught in commonly assigned U.S. Patent Application Serial No. 07/889,017 filed on May 26, 1992, which application is incorporated by reference in its entirety.
The nomenclature describing derivatives of sialic acid derivatives herein is as set forth by Reuter et al~°
The term "a-sialic acid(2~6)~3Ga1- structures or groups" refers to molecules comprising the terminally linked a-sialic acid(2-~6)galactose-sequence or derivatives thereof. Molecules containing such terminal structures have been identified as comprising part of the putative receptor structure for the ELAM-1 and PADGEM selectins38~°°.
The term "a-sialic acid(2-~3)~iGal- structures or groups" refers to molecules comprising the terminally linked a-sialic acid(2-i3)galactose-sequence or derivatives thereof. Molecules comprising such terminal structures have similarly been identified as comprising part of the putative receptor structures for the SLAM-1 selectins'5.~9.
-z0-The term "lectin derived carbohydrate binding peptide" refers to any peptide or derivative thereof (including pharmaceutically acceptable salts) derived from a lectin and which is capable of binding to a-sialic acid(2->6)~iGal- and/or a-sialic acid(2-~3)/3Gal- carbohydrate structures which are preferably comprised on the surface of mammalian cells. Generally, in the present application, "lectin derived carbohydrate binding peptide" will refer to such peptides which, in monomeric form, have no more than about 35 amino acids in the lectin-like domain (i.e., the part of the peptide responsible for binding to such carbohydrate structures). Suitable derivatives of lectin derived carbohydrate binding peptides include those peptides which have the NH., and/or the COOH
terminal functionalities blocked by conventionally blocking groups as well as derivatives which include modifications, deletions or derivatizations of one or more of the amino acids that yield a peptide which is capable of binding to a-sialic acid(2-~6)~3Ga1- and/or a-sialic acid(2-~3)(3Ga1-structures.
More preferably, the lectin derived carbohydrate binding peptides refer to the peptides set forth in Figure 1.
Still more preferably, the lectin derived carbohydrate binding peptides are peptides which have a high degree of homology with a lectin-like binding domain for the a-sialic acid(2->6)/3Ga1- and/or a-sialic acid(2->3)~iGal- carbohydrate structures found in the pertussis toxin.
These peptides are represented by formula I (SEQ ID NO:1):
SPX,GX,C I
where X, is selected from the group of amino acids Y, F, W, and H or peptide mimetics thereof; and X, is selected from the group consisting of amino acids Y, F, R, W, and H or peptide mimetics thereof;
or by formula II (SEQ ID N0:2):
SPX,GX~CX3X4 II
where X, is selected from the group of amino acids Y, F, W, and H or peptide mimetics thereof;
X, is selected from the group consisting of amino acids Y, F, R, W, and H or peptide mimetics thereof;
X3 is an amino acid sequence of 4-6 amino acids; and X4 is selected from the group consisting of amino acids Y, F, W, and H or peptide mimetics thereof.
Most preferably, the lectin derived carbohydrate binding peptide refers to the hexapeptide SPYGRC (amino acids 18-23 of SEQ ID N0:3).
In addition to their use as modulating agents for mammalian immune and cellular adhesion processes, the lectin derived carbohydrate binding peptides described herein can further be used in assay methods for determining the presence of a-sialic acid(2~3)/3Ga1- and/or a-sialic acid(2-,6)~3Gal- structures on molecuies and/or cell surfaces such as in suspected cancer cells, for determining the relative binding affinity of peptides and/or proteins to a-sialic acid(2--~3)(3Ga1- and/or a-sialic acid(2->6)/3Ga1- structures, and the like. When so employed, the lectin derived carbohydrate binding peptides typically will be derivatized to include a label or a label binding moiety.
-zZ-Suitable labels are well known in the art and include, by way of example, enzymes (e.g., horseradish peroxidase), radioisotopes (e.g., ''-3I), fluorescent moieties, chemiluminscent moieties, and the like. The particular label employed is not critical, and methods for attaching labels to peptides are well known in the art.
Suitable label binding moieties are also well known in the art and include, by way of example, biotin, avidin, streptavidin antibodies, etc.
A preferred label binding moiety is biotin which permits binding of up to 4 peptide/biotin adducts to avidin. The avidin can be appropriately labeled so that the resulting peptide/biotinlavidin complex can be detected.
The term "lectins" refers to carbohydrate binding proteins of non-immune origin often obtained from plants or bacterial or viral microorganisms which comprise carbohydrate binding sites. These binding proteins typically comprise the ability to agglutinate cells and to precipitate complex carbohydrates. Lectins are classified based upon their carbohydrate binding specificity and are well known in the art.
The term "lectin-like domain" refers to those fragments) of a lectin responsible for binding the carbohydrate.
The term "pharmaceutically acceptable salts" includes the pharmaceutically acceptable addition salts of lectin derived carbohydrate binding peptides capable of binding to terminally linked a-sialic acid(2-~6)~3Gal- and/or a-sialic acid(2--~3)(3Ga1- structures. Such pharmaceutically acceptable addition salts may be derived from a variety of organic and inorganic counter salts well known in the art and include, by way of example only, sodium, potassium, calcium, magnesium, ammonium, tetralkyl-ammonium, and the like.
2. Utility Without being limited to any theory, it is believed that the subject lectin derived carbohydrate binding peptides affect the immune response in a number of ways. Lectin derived carbohydrate binding peptides can inhibit a mammal from becoming "educated" about a specific antigen when the lectin derived carbohydrate binding peptide is administered simultaneously with the first exposure of the immune system to the antigen.
The lectin derived carbohydrate binding peptides can reduce cell-mediated immune responses to injury such as inflammatory responses arising from myocardial infarction, ARDS, frost-bite, etc. The lectin derived carbohydrate binding peptides can also inhibit the effector phase of a cell-mediated immune response (e.g., the inflammatory component of a DTH response) when administered to a sensitized mammal after exposure of the sensitized mammal's immune system to the antigen. In either case, in order to effect reduction in the cell-mediated immune response, it is necessary to administer the lectin derived carbohydrate binding peptides after initiation of the mammal's immune response and at or prior to one-half the period required for maximal inflammation induced by the injury or the antigen exposure.
Additionally, the subject lectin derived carbohydrate binding peptides can induce tolerance to antigens in sensitized mammals when administered at the time of second or later exposures of the immune system to the antigen when administration is conducted after initiation of the mammal's secondary immune response to the antigen and at or prior to one-half the period required for maximal inflammation induced by the antigen exposure.
Further, the administration of lectin derived carbohydrate binding peptides that bind a-sialic acid(2--~6)/3Ga1- and/or a-sialic acid(2-'3)~iGal-structures inhibits the binding of LEC-CAM proteins and other selectins to their putative receptors therefor which include both the a-sialic acid(2-~6)~3Gal-'gw and the a-sialic acid(2-->3)/3Gal-'339 structures.
Accordingly, the subject invention provides both pharmaceutical compositions containing lectin derived carbohydrate binding peptides capable of binding terminally linked a-sialic acid(2->6)/3Ga1- and the a-sialic acid(2-~3)~3Ga1- structures which are useful in inhibiting specific immune responses or cellular interactions in mammals as well as methods which include the administration of such lectin derived carbohydrate binding peptides to a mammal for inhibiting a cell-mediated immune response.
As noted above, lectin derived carbohydrate binding peptides useful for modulating a cell-mediated immune response in a mammal include any lectin derived peptide or derivative thereof capable of binding terminally linked a-sialic acid(2->6)~3Ga1- and the a-sialic acid(2->3),QGaI-structures. Suitable lectin derived carbohydrate binding peptides preferably are peptides which, in monomeric form, have no more than about 35 amino acids in the lectin-like domain (i.e., the part of the peptide responsible for binding to such carbohydrate structures).
More preferably, the lectin derived carbohydrate binding peptides refer to the peptides set forth in Figure 1.
Still more preferably, the lectin derived carbohydrate binding peptides are peptides which have a high degree of homology with a lectin-like binding domain for terminally linked a-sialic acid(2-->6)~iGal- and/or a-sialic acid(2-~3)~iGal- carbohydrate structures found in the pertussis toxin. These peptides are represented by formula I (SEQ ID NO:1):
SPX,GX,C I
where X, is selected from the group of amino acids Y, F, W, and H or peptide mimetics thereof; and X, is selected from the group consisting of amino acids Y, F, R, W, and H or peptide mimetics thereof;
or by formula II (SEQ ID N0:2):
SPX, GX=CX~X4 II
where X, is selected from the group of amino acids Y, F, W, and H or peptide mimetics thereof;
X, is selected from the group consisting of amino acids Y, F, R, W, and H or peptide mimetics thereof;
X3 is an amino acid sequence of 4-6 amino acids; and X4 is selected from the group consisting of amino acids Y, F, W, and H or peptide mimetics thereof.
The preparation of such peptides is well known in the art and includes, by way of example, standardized commercially available peptide synthesizers such as Model ABI 403A available from Applied Biosystems, Inc., Foster City, California, U.S.A. The methods for preparing such peptides do not form a part of this invention.
Peptide mimetics refers to groups which mimic an amino acid in the peptide chain. Such mimetics and their synthesis are well known in the artsy.
The lectin derived carbohydrate binding peptides can be used either in monomeric or polymeric form. Examples of suitable polymers include the attachment of biotin to the lectin derived carbohydrate binding peptide followed by complexing with avidin which results in up to a tetravalent complex. Likewise, multivalent derivatives of lectin derived carbohydrate binding peptides can be synthesized by attaching such peptides to polymers such as polylysine or an inert protein such as human serum albumin. The multivalent derivatives so formed can contain one or a mixture of different' lectin derived carbohydrate binding peptides so as to enhance efficacy.
In the case of protein peptide conjugates, they will be chemically cross-linked with the carrier protein by known cross-linking agents using art recognized methodology.
In yet another embodiment, multivalent lectin derived carbohydrate binding peptides can be generated as a copolymer wherein the peptides are linked together through a spacer arm to provide for a repeating subunit represented by the groups:
[carbohydrate binding peptide-spacer arm]
[carbohydrate binding peptide]~-spacer arm In these subunits, the lectin derived carbohydrate binding peptide can be the same or different lectin derived carbohydrate binding peptide, and the spacer arm is selected to provide for optimal distance to bind to the carbohydrate.
However, the invention is not restricted to the use of lectin derived carbohydrate binding peptides specifically exemplified in Figure 1 or to multivalent derivatives thereof, but rather embraces the use of any lectin derived peptide or derivative thereof which binds terminally linked a-sialic acid(2~6)~3Ga1- and/or a-sialic acid(2--~3)~iGal- carbohydrate structures. As noted previously, peptides capable of binding such structures, when administered to a mammal, result in the inhibition of immune responses and cellular interactions, in particular, inflammatory responses or conditions, tolerance to antigens, modulation of the immunogenic response to antigens, and the inhibition of cell adhesion events, which are involved, e.g., in metastasis and inflammation.
It is well within the level of ordinary skill in the art to identify other lectin derived peptides capable of binding terminally linked a-sialic acid(2->6)/3Ga1- and/or a-sialic acid(2-~3)~3Ga1- structures by conventional methods for assaying binding between ligands. Such methods include, e.g., competitive binding assays and receptor binding assays. The subject application, in particular, sets forth one method in the examples which illustrates rather simple assaying techniques that are capabie of determining binding to terminally linked a-sialic acid(2-->6)~iGal- and/or a-sialic acid(2-->3)(3Ga1- structures. Other methods for determining the binding of a candidate peptide with such terminally linked structures are known in the art. See, for example, Pearce-Pratt et al."
The subject invention accordingly further provides a method by which lectin derived peptides capable of inducing or suppressing various immune responses and cellular interactions, e.g., inflammation, antigenic tolerance, modulation of antigenic response, or cell adhesion, may be putatively identified on the basis of their ability to bind to terminally linked a-siaiic acid(2->6)(3Ga1- and/or a-sialic acid(2-~3)(3Gal- structures.
In regard to the above, the subject invention contemplates the attachment of labels or label binding groups to the lectin derived carbohydrate binding peptides and/or to candidate lectin derived carbohydrate binding peptides in order to facilitate the assays described above. Such labels are conventionally formed on the peptides by methods well known in the art. Suitable labels include, by way of example, enzymes (e.g., horseradish peroxidase), radioisotopes (e.g., ''-sI), fluorescent moieties, chemiluminscent moieties, and the like.
Suitable label binding moieties are also well known in the art and include, by way of example, biotin, avidin, antibodies, etc. A preferred label binding moiety is biotin which permits binding of the peptide/biotin adduct to avidin. The avidin can be appropriately labeled so that the resulting peptide/biotin/avidin complex can be detected.
The subject invention also contemplates kits for use in conducting such assays. Such kits would comprise the labelled lectin derived carbohydrate binding peptide or the lectin derived carbohydrate binding peptide attached to label binding groups.
Suitable lectin derived carbohydrate binding peptides for use herein are those which are capable of binding terminally linked a-sialic acid(2-~6)/3Gal- and/or a-sialic acid(2->3)(3Ga1- structures. However, an additional prerequisite of efficacious lectin derived carbohydrate binding peptides will include suitability for in viv administration. In particular, the lectin derived carbohydrate binding peptide should not be toxic, and should be sufficiently soluble at the required dosages, which will typically range from about 0.5-50 mg/kg of body weight. In this regard, it is art recognized that the solubility of lectin derived carbohydrate binding peptides can be enhanced by attaching hydrophilic amino acid groups and can be reduced by attaching hydrophobic amino acid groups from the carboxyl terminal and/or amino terminal positions of the peptide.
The invention further contemplates fragments or derivatives of peptides capable of binding terminally linked a-sialic acid(2-~6)aGal-and/or a-sialic acid(2-~3)(3Ga1- structures which peptides have been modified to render them non-toxic, e.g., by chemical derivatization, mutagenesis, etc. while still retaining the ability to bind such terminally linked structures.
The subject invention provides, in particular, methods for suppressing cell-mediated immune responses in mammals including cell-mediated inflammatory responses or disorders by the administration of an effective amount of one or more lectin derived carbohydrate binding peptides capable of binding terminally linked a-sialic acid(2~6)~3Gal-and/or a-sialic acid(2-~3)(3Gal- structures or moleculesJcell surfaces comprising such structures.
-.i U-The cell-mediated immune responses or disorders treatable by the subject invention include inflammatory immune reactions involving specific and non-specific defense systems. As discussed above, such conditions include antibody responses to antigens, such as viruses, allergens, delayed-type hypersensitivity, autoimmune disorders such as rheumatoid arthritis and lupus, post-ischemic leukocyte mediated tissue damage (reperfusion injury), frost-bite injury or shock-acute leukocyte-mediated lung injury (e.g., acute respiratory distress syndrome), asthma, traumatic shock, septic shock, nephritis, and acute and chronic inflammation, including atopic dermatitis, psoriasis, and inflammatory bowel disease. Further, inflammatory disorders treatable by the subject invention may include platelet-mediated pathologies such as atherosclerosis and clotting disorders.
Inflammatory conditions of special interest include delayed type hypersensitivity reactions, reperfusion, and acute leukocyte-mediated lung injury CARDS).
This invention provides a generic method by which cell-mediated immune responses such as cell-mediated inflammatory responses or disorders in mammals (e.g., humans) may be suppressed by the administration of an effective amount of one or more lectin derived carbohydrate binding peptides or fragments or derivatives thereof capable of binding terminally linked a-sialic acid(2-->6)~3Gal- and/or a-sialic acid(2-->3)/3Ga1- structures or molecules/cell surfaces comprising such structures. In a preferred embodiment, the invention provides methods by which inflammatory responses or disorders may be treated or suppressed by the administration of an effective amount of one or more peptides selected from the group of peptides set forth in Figure 1.
The subject invention further provides a general method for inhibiting immune responses and cell adhesion events in mammals by the administration of an effective amount of one or more lectin derived carbohydrate binding peptides or fragments or derivatives thereof capable S of binding terminally linked a-sialic acid(2->6)/3Ga1- and/or cx-sialic acid(2-~3)~3Gal- structures or molecules/cell surfaces comprising such structures. Such immune responses include cell mediated and humoral immune responses. As has been discussed, such immune responses include, in particular, inflammatory responses or inflammatory disorders.
The invention further provides methods for affecting the induction of immune responses to antigens comprising administering to a mammal an antigen in conjunction with one or more lectin derived carbohydrate binding peptides capable of binding terminally linked a-sialic acid(2-~6)~iGal- and/or a-sialic acid(2-~3)(3Ga1- structures or molecules/cell surfaces comprising such structures. For example, administration of an effective amount of the SPYGRC (amino acids 18-23 of SEQ ID N0:3) hexapeptide to a mammal with an antigen will modulate the induction of the immune response in the mammal to the antigen. Accordingly, the subject lectin derived carbohydrate binding peptides may comprise applicability as immune modulators, which may be administered in conjunction with vaccines, artificial organs or tissue transplants, and allogeneic organ and tissue transplants as a means for modulating the immune response to foreign antigens comprised therein.
It has further been found that the subject lectin derived carbohydrate binding peptides capable of binding terminally linked a-sialic acid(2->6)(3Ga1- and/or a-sialic acid(2->3)~3Gal- structures or molecules/cell surfaces comprising such structures, when administered in an effective amount to a mammal which has been immunized with a particular antigen, result in the induction of long term tolerance to said antigen. In this regard, administration is conducted after onset of the secondary immune response but at or prior to one-half the period required for maximal inflammation.
In particular, it has been found that administration, during the critical period set forth above, of an effective amount of the S3P9a (SEQ
ID N0:9), the ACS2P1 (2275) (amino acids 9-23 of SEQ ID N0:3), and the SPYGRC (2283) (amino acids 18-23 of SEQ ID N0:3) peptides illustrated in Figure 1 to mammals that have been immunized with an antigen results in said mammals exhibiting a reduced immune response upon subsequent challenges) with said antigen (Figure 3). Thus, the subject lectin derived carbohydrate binding peptides have applicability as tolerogens. Given this property, such lectin derived carbohydrate binding peptides or fragments or derivatives thereof may be especially suitable for use in the treatment of allergic disorders since administration of "tolerogenic" derivatized allergens is a known means for treating allergic disorders.
The subject invention further provides methods for inhibiting the adhesion of certain cell types [e.g., tumor cells and polymorphonuclear cells (PMN's)] to endothelial cells. In this regard, the art suggests that tumor metastasis involves tumor cell adhesion to selectin bearing cells. In this regard, circulating cancer cells apparently take advantage of the body's normal inflammatory mechanisms and bind to areas of blood vessel walls were the endothelium is activated and, accordingly, contains selectins. As noted previously, the putative receptors for such selectins contain terminally linked a-sialic acid(2-~6)~3Ga1- and/or a-sialic acid(2~3)~iGal- structures. Therefore, administration of lectin derived carbohydrate binding peptides capable of binding terminally linked a-sialic acid(2-~6)(3Ga1- and/or a-sialic acid(2-~3)~3Ga1- structures should provide a method for inhibiting metastasis. For example, the subject lectin derived carbohydrate binding peptides or fragments or derivatives thereof may be administered before, during or after cancer surgery or biopsy as a means for inhibiting metastasis of tumor cells which may be released into the circulatory system during surgery. In these methods, the subject lectin derived carbohydrate binding peptides are administered either prior to, at the time of surgery or biopsy, or shortly thereafter.
Prior administration is typically no more than about 5 hours prior to surgery or biopsy, and administration and subsequent administration is typically no more than about 15 hours after surgery or biopsy. In either case, administration is either continuous or intermittent, but preferably is continuous.
In the methods pertaining to suppression of cell-mediated inflammatory reactions or disorders arising from injury or antigen exposure, the subject lectin derived carbohydrate binding peptides or fragments or derivatives thereof are administered after initiation of the mammal's immune response but at or prior to one-half the period required for maximal inflammation to the antigen exposure or injury.
Preferably, the subject lectin derived carbohydrate binding peptides are administered about 1-10 hours after initiation of the immune response, and more preferably about 1-5 hours after initiation of the immune response. However, the specific time for administration will vary dependent upon the particular antigen/injury and the lectin derived carbohydrate binding peptide which is administered.
In the methods pertaining to modulating the induction of an immune response to an antigen in a non-sensitized mammal, an effective amount of the subject lectin derived carbohydrate binding peptides or derivatives thereof will be administered in conjunction with the antigen.
Typically, such conjunctive administration is simultaneous with antigen administration but can be up to ~3 hours from the time of antigen administration.
In the methods pertaining to induction of long term tolerance to an antigen, an effective amount of the subject lectin derived carbohydrate binding peptides or fragments or derivatives thereof will generally be administered after antigen challenge to a sensitized mammal. In particular, administration is after initiation of the mammal's secondary immune response to the antigen challenge but at or prior to one-half the period required for maximal inflammation to the antigen challenge.
Preferably, the subject lectin derived carbohydrate binding peptides are administered about 1-10 hours after initiation of the immune response to the antigen challenge, and more preferably about I-5 hours after initiation of the immune response to the antigen challenge. However, the specific time for administration will vary dependent upon the particular antigen and the lectin derived carbohydrate binding peptide which is administered.
Generally, the subject lectin derived carbohydrate binding peptides or derivatives thereof will be administered parenterally, e.g., by intramuscular or intravenous routes. However, other dosage forms should also be suitable including, e.g., oral, transdermal, rectai, intratracheal, and intranasal formulations. For example, intranasal and intratracheal formulations may be preferred if the inflammatory condition treated involves lung inflammation, e.g., acute respiratory distress syndrome CARDS). In contrast, an oral formulation would likely be preferred if the inflammatory condition treated involves the digestive tract, e.g., inflammatory bowel disease.
Pharmaceutical compositions for use in the subject invention will generally comprise an effective amount of one or more of the subject peptides or derivatives thereof capable of binding terminally linked a-sialic acid(2->6)~iGal- and/or a-sialic acid(2-~3)~3Gal- structures in combination with a pharmaceutically acceptable carrier and/or excipients.
The particular pharmaceutically acceptable carrier and excipients will vary dependent upon the dosage form. In one embodiment, several of the subject peptides or derivatives thereof are mixed into the pharmaceutical composition to form a "cocktail" having enhanced activity.
Parenteral dosage forms may contain phosphate buffered saline as a carrier, while intranasal formulations will comprise inhalants, and oral dosage forms may comprise enteric coatings. The selection of suitable carriers and excipients and formulation of different dosage forms is well within the level of ordinary skill in the pharmaceutical art.
As noted above, the subject lectin derived carbohydrate binding peptides or derivatives thereof are administered in effective amounts. An effective amount is an amount sufficient to obtain the desired therapy without causing undue toxicity to the mammal. Preferably, the subject peptides are administered at dosages ranging from about 0.5 to SOmg/kg body weight, with 5-10 mg/kg being most preferred for each of the above-cited methods. The specific dose employed is regulated by the particular cell-mediated immune response being treated as well as by the judgement of the attending clinician depending upon factors such as the severity of the adverse immune response, the age and general condition of the patient, and the like.
Generally, the methods of the present invention will involve administration of a single dose of the subject lectin derived carbohydrate binding peptides. However, the invention further contemplates repeated administration of the subject lectin derived carbohydrate binding peptides or derivatives thereof. Repeated administration of these peptides may be desirable, e.g., in the treatment of chronic or sustained inflammatory disorders such as rheumatoid arthritis, acute and chronic inflammation, psoriasis, inflammatory bowel disorders, and autoimmune disorders associated with inflammatory responses such as lupus, multiple sclerosis or rheumatoid arthritis.
It is also contemplated that the subject peptides and derivatives are useful as receptor-targeted antibacterial and anti-viral drugs wherein the bacteria, virus, or toxin produced therefrom employs a terminally linked a-sialic acid(2-~6)/3Ga1- and/or a-sialic acid(2-~3)/3Ga1- structure as the receptor site on a cell of the targeted mammalian host.
Such bacteria/virus and/or toxins include, by way of example, influenza virus, pertussis toxin, cholera toxin, and the like. Such methods are illustrated in the examples hereinbelow wherein in vitro assays demonstrate the ability of two of the subject lectin derived carbohydrate binding peptides to neutralize the effects of pertussis toxin on Chinese Hamster Ovary cells.
Accordingly, when administered in effective amounts, the subject lectin derived carbohydrate binding peptides are useful in methods for inhibiting inoculation in mammalian hosts of bacterial/viral agents and/or their toxins which employ a terminally linked a-sialic acid(2--~6)/3Ga1-S and/or a-sialic acid(2-~3)(3Ga1- structure as the receptor site on a cell of the targeted mammalian host thereby inhibiting the likelihood that the mammalian host will become afflicted with the disease produced by the bacterial/viral agent and/or its toxin.
Effective amounts of the subject lectin derived carbohydrate binding peptides or derivatives thereof will preferably be dosages ranging from about 0.5 to SOmg/kg body weight, with S-10 mg/kg being most preferred.
3. Examples In order to fully illustrate the present invention and the advantages thereof, the following specific examples are given, it being understood that these examples are intended to be illustrative only and in nowise limitative of the scope of the present invention.
In these examples as well as in the application, all sugars disclosed herein are in their D form, except for fucose which is in its L form, and all amino acids are conventional.
In these examples, unless otherwise defined below, the abbreviations employed herein have their generally accepted meaning:
ABTS = 2,2'-azino-bis(3-ethylbenzathiazoline-6-sulfonic acid) BSA = Bovine serum albumin cm = centimeter HPLC = High Performance Liquid Chromatography S MAL = Maackia amurensis mg = milligram mM = millimolar mm = millimeter ng = nanogram nm = nanometer PBS = phosphate buffered saline PT = Pertussis toxin SNA = Sambucus nigra ~g = microgram ~d = microliter ~cM = micromolar ~mol = micromole v/v = volume/volume Unless otherwise indicated, all temperatures are in degrees Celsius (°C). Also, as noted previously, all amino acid residues recited herein employ their conventional one-letter abbreviation.
General Procedures All of the reagents used in Examples 1-10 were obtained from Sigma Chemical Company, St. Louis, Missouri, U.S.A., except for pertussis toxin (PT) which was obtained from Connaught Center for Biotechnology Research, Willowdale, Ontario, Canada; SNA-, WGA- and MAL-biotin, which were obtained from Boehringer Mannheim, Dorval, Quebec, Canada; and IODO-GEN which was obtained from Pierce Chemical Co., St. Louis, Missouri, U.S.A. The acetylated and biotinylated analogs of the peptide S2P1 (amino acids 9-23 of SEQ ID
N0:3) were prepared using conventional methods. PT-biotin as well as asialo- and asialoagalactofetuin were prepared as described earlier~°~°~
Removable flat-bottomed microtiter well strips of Immunulon 2 were from Dynatech, Alexandria, Virginia, U.S.A.
x m le 1 -- Synthesis of Synthetic Peptides Peptides corresponding to amino acid sequences found in the S2 and S3 subunits of PT were synthesized using an ABI 403A peptide synthesizer (Applied Biosystems, Inc., Foster City, California, U.S.A.), then cleaved from the resin by HF and purified by reversed-phase HPLC
on a Vydac C4 semipreparative column. All synthetic peptides used in ELISA inhibition assays were > 95 % pure as judged by analytical HPLC, and their amino acid analyses were in good agreement with the theoretical compositions.
Regions in the S2 and S3 subunits of PT that correspond to the variable amino acid sequences distinguishing the S2 from the S3 subunit were secured as above. These peptide sequences were chosen, in part, based on their high index of hydrophilic ~3-turns as judged by secondary structure prediction analysis~°~6'. Upon careful examination of the PT
sequences, 20 peptides were synthesized including those containing amino acid residues 62-73 of WGA (SEQ ID NO:11). The acetylated version of S2P1 (amino acids 9-23 of SEQ ID N0:3) was also prepared in order to better mimic the native peptide backbone. The synthesized peptides are set forth in Table I below:
TABLE I
PositionSequence* SEQ ID
NO
1. S3P1 9-33 PQEQITQHGSPYGRC 3 (aa 9=_'3) =. ACS2P1-b 9-23 BIOTIN-PQEQITQHGSPYGRC-CO-NH:3 (aa 9=_'3) $ 3. S'_' (14-23)14-'_'3TQHGSPYGRC 3 (aa 14-'_'3) 4. S2-b 14-'3 BIOTIN-TQHGSPYGRC 3 (aa 14-'_'3) 5. S3P3 1-23 STPGIVIPPQEQ1TQHGSPYGRC 3
7. Sharon, Lectin-Like Bacterial Adherence to Animal Cells. In "Attachment of Microorganisms to the Gut Mucosa"
(Boeheker, E.D., Ed.), Boca Raton, Florida CRC Press, p.
129 (1984).
10 8. Wassarman, Fertilization. In "Cell Interactions and Development: Molecular Mechanisms" (Yamada, K.M., Ed.), New York, John Wiley and Sons, p. 1 (1983)..
9. Schwartz, et al., Immunol. Rev., 40:153 et seq. (1978).
10. Coutinho, et al., Immunol. Rev., 78:211 et seq. (1984).
15 11. Hoffmann, et al., Eds., Membranes in Growth and Development, New York, Alan R. Liss, p. 429-442, (1982).
12. Galeotti, et al., Eds., Membranes in Tumor Growth, Amsterdam, Elsevier, p. 77-81 (1982).
13. Nicolson, et al., Invas. Metas., 5_:144 et seq. (1985).
20 14. Aplin, et al., Biochim. Biophys. Acta 694:375 et seq.
( 1982) .
15. Barondes, Developmentally Regulated Lectins. In "Cell Interactions and Development: Molecular Mechanisms"
(Yamada, D.M., Ed.) New York, John Wiley and Sons, p.
25 185 (1983).
16. Monisigny, M., Ed., Biol. Cell, SI (Special Issue), 113 et seq. (1984).
17. Springer, et al., Nature, 349:196-197 (1991).
18. Lowe, et al., Cell, 63:475-485 (1990).
19. Phillips, et al., Science, Vol. 250:1130-1132 (1990).
20. Walz, et al., Science, 250:1132 et seq. (1990).
21. Larsen, et al., Cell, (3_:467-474 (1990).
22. Bevilacqua, et al., Endothelial-Leukocyte Adhesion Molecule - l (ELAM-1): A Vascular SELECTIN That Regulates Inflammation. In "Cellular and Molecular Mechanisms of Inflammation" Vol. 2, Academic Press, p. 1-13 (1991).
23. McEver, Leukocyte Interactions Mediated by GMP-140. In "Cellular and Molecular Mechanisms of Inflammation", Vol.
2, Academic Press, p. 15-29 ( 1991 ).
24. Larsen, et al., J. Biol. Chem., 267:11104-11110 (1992).
25. Heerze, et al., Biochem and Biophys. Res. Comm.
172:1224-1229 ( 1990) .
26. Paulson, et al., International Patent Application Publication No. W091 /19502, ( 1991 ) .
27. Ippolito, et al., U. S. Patent Application Serial No.
07/714,161, filed 10 June 1991.
28. Ippolito, et al., U. S. Patent Application Serial No.
07/889,017, filed 26 May 1992.
29. McEver, International Patent Application Publication No.
W092/Ol 718 ( 1992) .
30. Heerze, et al., Biochem and Biophys. Res. Comm., 179:1464-1469 ( 1991 ) .
31. Pearce-Pratt, et al., J. Imm. Methods, 140:159-165 (1991).
32. Smith, et al., Cell. Imm., 89:20-29 (1984).
33. Munoz, Action of Pertussigen (Pertussis Toxin) on the Host Immune System. In "Pathogenesis and Immunity in Pertussis", John Wiley & Sons Ltd., p. 173-192 (1988).
34. Reuter, et al, Glycoconjugates 5_:133-135 (1988).
35. Gaeta, et al., U. S. Patent Application Serial No.
07/538,853, filed 15 June 1990.
36. Paulson, et al., U. S. Patent Application Serial No.
07/619,319, filed 28 November 1990.
37. Paulson, et al., U. S. Patent Application Serial No.
07/632,390, filed 21 December 1990.
38. Brandley, et al., International Patent Application Publication No. W092/02527 (1992).
39. Lowe, International Patent Application Publication No.
W092/07572 (1992).
40. Furie, et al., International Patent Application Publication No. W092/16612 ( 1992) .
41. Seed, et al., International Patent Application Publication No. W092/09293 (1992).
42. Karlsson, Annu. Rev. Biochem., 5$:309-350 (1989).
43. Brennan, et al., J. Biol. Chem., 26:4895-4899 (1988).
44. Armstrong, et al., J. Biol. Chem., 263:8677-8684 (1988).
45. Smith, et al., U. S. Patent Application Serial No.
07/956,043, filed 2 October 1992.
46. Shibuya, et al., J. Biol. Chem., 21:7755-7761 (1987).
47. Wang, et al., J. Biol. Chem., 26:4576-4585 (1988).
48. Gimbrone, et al., International Patent Application Publication No. W091 /08231 ( 1991 ) .
49. Ratcliffe, et al., U. S. Patent No. 5,079,353, issued 7 January 1992.
50. Hakomori, Adv. Cancer Res., 52:257-331 (1989).
51. Sialic Acids in "Cell Biology Monographs", Schauer, Editor, Vol. 10 (1982).
52. Tyrrell, et al., Infect. Immun., x:1854-1857 (1989).
53. Bhavanandan, et al., J. Biol. Chem., 2:4000-4008 (1979).
54. Nogimori, et al., Biochemistry, 25:1355-1363 (1986).
55. Sato, et al. , Infect. Immun. , 46:415-421 ( 1986).
56. Tamura, et al., J. Biol. Chem., 2:6756-6761 (1983).
57. Tuomanen, et al., J. Exp. Med., 168:267-277 (1988).
58. Tuommenen, et al., 92nd General Meeting of the American Society of Microbiology, New Orleans, LA, Poster B-16.
59. Morgan, et al., Ann. Repts. Med. Chem. , 24:243-252 (1989).
60. Chou, et al., Annu. Rev. Biochem., 47:251-276 (1978).
61. Hopp, et al., Proc. Natl. Acad. Sci. U. S.A. , x:3824-3828 (1981).
62. Wright, J. Mol. Biol., 21:635-651 (1990).
63. Armstrong, et al., Infect. Immun., 55:1294-1299 (1987).
64. Smith, et.al., Immunology, 58:245 et seq. (1986).
65. Sleyter, et al., Arch. Microbiol., 146:19 et seq. (1986).
The disclosure of all publications, patents and patent applications cited in this application are herein incorporated by reference in their entirety to the same extent as if each individual publication or patent -b-application were specifically and individually indicated to be incorporated by reference in its entirety.
3. State of the Art.
Important processes involving mammalian cells, such as growth, locomotion, morphological development, and differentiation, are partially controlled by extracellular signals acting upon the cells' surfaces'-3.
While some external stimuli reach the cell via extracellular fluids, other signals are received from neighboring or approaching cell surfaces and exert their effects through direct cell-cell contact°~s.
Evidence suggests that specific cell-surface receptors can "sense" a molecular signal of an opposing cell via specific binding, and biochemical mechanisms exist to translate that binding into a cellular response. For example, complex cell-surface interactions are believed to help direct processes such as binding of pathogens to target tissuesb~', sperm-egg binding8, interactions among cells in the immune system9~'°, and recognition of cells during embryonic development". In addition, defects in cell-cell recognition are thought to underlie the uncontrolled cell growth and motility which characterize neoplastic transformation and metastasis'z~".
Other evidence suggests that cell-recognition processes are mediated by carbohydrate chains or glycan portions of glycoconjugates'~'a-'6. For example, the binding of the surface glycoconjugates of one cell to the complementary carbohydrate-binding proteins (lectins) on another cell can result in the initiation of a specific interaction.
_7_ One important group of carbohydrate-binding proteins are selectin (LEC-CAM) proteins (Lectin + EGF + complementary Regulatory Domain-Cell Adhesion Molecules). These or functionally similar proteins or lectins are believed to play a critical role in immune responses (including inflammatory responses) through mediation of cell-cell contact and through extra-vasation of leucocytes"~'-'-. Specific carbohydrate ligands have been identified as part of the putative receptor structures for selectin proteins and other lectins"-'-'. The structures identified include oligosaccharide glycosides containing terminally linked a-sialic acid(2->6)(3Gal- and a-sialic acid(2->3)/3Gal- groups. The use of oligosaccharides and derivatives thereof having such terminally linked groups for controlling inflammation, immunosuppression, etc. by interacting with selectin proteins and/or other lectins has been disclosed26-29.33-41 Likewise, peptides derived from the seIectin GMP-140 which inhibit binding of GMP-140 and other selectins to leukocytes, presumably by interfering with the binding of the GMP-selectin protein with the carbohydrate receptor on the leukocyte, have also been disclosed as being useful in suppressing an immune response29. Similarly, other peptides are also known to be potent modulators of neutrophil functions'g Pertussis toxin (PT)°'-, a virulence factor produced by the organism Bordetella pertussis -- the etiological agent of whooping cough, is known to bind to glycoprotein receptors which terminate in sialyllactosamine sugar sequences43~°', and we have previously shown that this protein is useful in suppressing mammalian immune responses and cellular adhesions. PT's binding specificity has been shown to be similar to that of the plant lectins from Sambucus nigra (SNA) and Maac~ia amurensis _g_ (MAL)'-s, which bind with high affinity to sialic acid-containing glycoconjugates46.°' and which have also been shown as possessing immunomodulating properties°s.
However, the use of proteins or large molecular weight polypeptides in inhibiting immune responses or cellular interactions in mammals suffers from several drawbacks, including the fact that they are difficult to produce in large quantities in pure form, that they tend to produce adverse effects when repeatedly administered to a mammal, that they can contain infectious agents or toxic substances which are contraindications to mammalian administration, and that it is difficult to modify the pharmokinetic properties of such proteins to improve their efficacy.
In view of the above, the use of peptides having lectin-like binding properties for terminal aNeuSAc(2-~3)(3Gal- and aNeuSAc(2~6)/3Ga1-groups would be particularly beneficial for use in inhibiting immune responses and cellular interactions in mammals as compared to the administration of proteins such as the pertussis toxin and the lectins derived from SNA and MAL because such peptides would mitigate the problems associated with the administration of proteins and large molecular weight polypeptides to mammals.
Summar,~r of the Invention 1"his invention is directed, in part, to the discovery that the binding domains for the a-sialic acid(2->3)~3Ga1- and/or the a-sialic acid(2-~6)~3Gal- terminally linked structures are found in certain peptide fragments of lectins (e.g., proteins and polypeptides) such as pertussis toxin and that it is not necessary to employ the entire lectin to effect binding to these carbohydrates (oligosaccharides).
This invention is further directed, in part, to the discovery that when these lectin derived carbohydrate binding peptides are administered to a mammal (e.g., human) in effective amounts, they inhibit specific immune responses and cellular interactions. In particular, this invention is directed to the discovery that such lectin derived carbohydrate binding peptides may be administered to a mammal in order to inhibit inflammatory responses, modulate the induction of an immune response to an antigen, induce long term tolerance to an antigen, and suppress cell adhesion.
This invention is particularly directed to the discovery that lectin derived carbohydrate binding peptides capable of binding terminally linked a-sialic acid(2-~3)~3Ga1- and/or a-sialic acid(2-~6)~3Ga1- (e.g., aNeuSAc(2~3)/3Ga1-) groups present in molecules (e.g., oligosaccharides, glycoproteins, glycolipids, etc.) which can be found on cell surfaces (e.g., leukocytes) may be administered to a mammal as a means for inhibiting cell adhesion or cell-mediated immune responses. Cell-mediated immune responses inhibited by the peptides disclosed herein include inflammatory responses, modulation of the induction of the immune response to an antigen and the induction of long term tolerance to an antigen.
Preferably, the lectin derived carbohydrate binding peptides employed herein are fragments of the S2 or S3 subunits of the pertussis toxin expressed by Bordetella pertussis or functionally equivalent variants thereof. These preferred peptides have the amino acid sequences set forth in peptides of Figure 1 below.
Accordingly; in one of its method aspects, this invention is directed to a method of suppressing an inflammatory response in a mammas by the administration of an effective amount of at least one lectin derived carbohydrate binding peptide or derivative thereof capable of binding terminally linked a-siaiic acid(2--~6)~3Gal- and/or a-sialic acid(2-->3)~3Ga1-groups on structures or molecules comprising such groups.
In another of its method aspects, this invention is directed to a method for modulating the induction of an immune response to an antigen in a mammal by administering the antigen in combination with an effective amount of at least one lectin derived carbohydrate binding peptide or derivative thereof capable of binding terminally linked a-sialic acid(2-~6)(3Gal- and/or a-sialic acid(2-~3)~3Gal- groups on structures or molecules comprising such groups.
In another of its method aspects, this invention is directed to a method for inducing in a sensitized mammal long term tolerance to an antigen by exposing (challenging) the mammal with the antigen followed by the administration of an effective amount of at least one lectin derived carbohydrate binding peptide or derivative thereof capable of binding terminally linked a-sialic acid(2-~6)J3Ga1- and/or a-sialic acid(2~3)(3Ga1-groups on structures or molecules comprising such groups.
In still another one of its method aspects, this invention is directed to a method for inhibiting cell adhesion events involved in metastasis of tumor cells or in inflammation by the administration of an effective amount of at least one lectin derived carbohydrate binding peptide or derivative thereof capable of binding terminally linked a-siaiic acid(2-~6)~3Gal- and/or a-sialic acid(2-~3)~3Gal- groups on structures or molecules comprising such groups.
In yet another one of its method aspects, this invention is directed to a method for treating lung inflammation and/or lung injury in a mammal by the administration of an effective amount of one or more lectin derived carbohydrate binding peptides or derivatives thereof capable of binding terminally linked a-sialic acid(2->6)~3Ga1- and/or a-sialic acid(2-~3)~iGal- groups on structures or molecules comprising such groups.
In its composition aspects, this invention is directed to pharmaceutical compositions comprising the subject lectin derived carbohydrate binding peptides including pharmaceutically acceptable salts of such lectin derived carbohydrate binding peptides.
Brief Description of the Drawings Figure 1 illustrates the amino acid sequence of preferred lectin derived carbohydrate binding peptides.
Figure 2 illustrates the increase in footpad swelling of immunized Balb/c mice arising from a DTH inflammatory response measured 24 hours after challenge with 20 ~,g of OVA antigen wherein some of the mice have been treated at 5 hours after challenge with 100 ~g of a lectin derived carbohydrate binding peptide.
Figure 3 illustrates the long term (2 week) effects on the DTH
responses determined by footpad swelling in groups of Balb/c mice which are immunized with 100 ~cg of the OVA antigen, challenged 7 days later with OVA, and then treated about 5 hours thereafter with a lectin derived carbohydrate binding peptide.
In Figures 2 and 3, peptide 2275 is ACS2P1 (amino acids 9-23 of SEQ ID N0:3) and peptide 2283 is SPYGRC (amino acids 18-23 of SEQ
ID N0:3), both of which are illustrated in Figure 1.
Figures 4 through 7 illustrate the same test as Figures 2 and 3 for other peptides.
The amino acid residues depicted in Figure 1 and used throughout this application are designated by the conventional single letter code set forth below:
A alanine I isoleucine R arginine C cysteine K lysine S serine D aspartic L leucine T threonine acid E glutamic M methionine V valine acid phenyl alanineN asparagine W tryptophane F
G glycine P proline Y tyrosine H histidine Q glutamine Additionally, as conventionally indicated, the amino acid residue forming the H,N- terminus of the peptide is set forth on the left side of the peptide chain, and the -COOH terminus of the peptide is set forth on the right side of the peptide chain.
Detailed Description of the Preferred Embodiments This invention is directed, in part, to the discovery that certain lectin derived carbohydrate binding peptides when administered to a mammal are effective in suppressing inflammatory responses, inducing tolerance to an antigen, modulating the induction of immune responses to antigens, and inhibiting cell adhesion events, e.g., cell adhesion events involved in metastasis of tumor cells.
I. Definitions As used herein, the following terms have the meanings set forth below:
The term "inflammatory response" or "inflammatory disorder" will refer to immune reactions involving specific and non-specific defense systems. A specific defense system reaction is a specific immune system reaction to an antigen. Examples of specific defense system reactions include antibody responses to antigens such as viruses, allergens, and delayed-type hypersensitivity. A non-specific defense system reaction is an inflammatory response mediated by leukocytes generally incapable of immunological memory. Such cells include macrophages, eosinophils and neutrophils. Examples of non-specific reactions include the immediate swelling after a bee sting, and the collection of polymorphonuclear (PMN) leukocytes at sites of bacterial infection (e.g., pulmonary infiltrates in bacterial pneumonias and pus formation in abscesses).
Other "inflammatory responses" or "inflammatory disorders"
within the scope of this invention include, e.g., autoimmune disorders such as rheumatoid arthritis, lupus, multiple sclerosis, post-ischemic leukocyte mediated tissue damage (reperfusion injury), frost-bite injury or shock, acute leukocyte-mediated lung injury CARDS), asthma, traumatic shock, septic shock, nephritis, and acute and chronic inflammation including atopic dermatitis, psoriasis, and inflammatory bowel disease.
Various platelet-mediated pathologies such as atherosclerosis and clotting are also included within the definition of "inflammatory responses" or "inflammatory disorders" . In addition, "inflammatory responses" or "inflammatory disorders" may include the adhesion of circulating cancer cells, with specific examples including carcinoma of the colon and melanoma.
Without being limited to any theory, we believe that primary events in the initiation of such inflammatory responses and inflammatory disorders are the binding of leukocytes to selectins (e.g., ELAM-l, PADGEM, etc.) through carbohydrate receptors comprising a-sialic acid(2-~3)aGal- and/or a-sialic acid(2-~6)~iGal- groups'S-'° found on the surface of the leukocytes. Likewise, it has been shown that mammalian and, in particular, human cancer cells contain a-sialic acid(2~3)~3Ga1-and/or a-sialic acid(2-~6)/3Gal- groups on the surface thereof'9~5°, and it is believed that binding of such circulating cancer cells to selectins is an integral part of the metastatic process3e.". Accordingly, by interfering with the binding of such carbohydrate receptors to these selectins, suppression of the inflammatory immune responses as well as inhibition of metastatic processes is achieved.
The term "antigen" refers to any protein, peptide, carbohydrate, nucleic acid or other non-endogenous substance which when exposed to a mammal induces an immune response in that mammal.
Disease conditions believed to be caused by antigen exposure include, by way of example, psoriasis, asthma, dermatitis, rheumatoid arthritis, delayed type hypersensitivity, inflammatory bowel disease, multiple sclerosis, viral pneumonia, bacterial pneumonia, and the like.
The term "non-sensitized mammal" refers to those mammals which have yet to be educated to a particular antigen.
The term "sensitized mammal" refers to those mammals which have been previously exposed to a particular antigen, and, accordingly, their immune systems have become educated to that antigen. Typically, initial exposure of an antigen to a mammal primes or educates the mammal's immune response to later exposure to that antigen with minimal inflammation during such initial exposure.
The term "secondary immune response" refers to the effector phase of a mammal's immune response to an antigen to which it has previously been sensitized. A mammal's secondary immune response is typically accompanied by inflammation at the point of antigen exposure.
"Acute respiratory distress syndrome" or "ARDS" refers to an inflammatory condition comprising leukocyte mediated lung injury.
Without being limited to any theory, it is believed that such lung injury is exacerbated by infiltration and subsequent disruption of neutrophils into the lungs. Specifically, the disruption of the neutrophils in the lungs releases superoxides which results in severe vascular endothelial damage.
Accordingly, while this lung damage is not antigen based, the infiltration of neutrophils into the lungs requires an adhesion event.
"Reperfusion injury" refers to an inflammatory condition comprising leukocyte mediated tissue damage. Reperfusion injury commonly occurs after myocardial infarction wherein, in response to the inflammation caused by the myocardial infarction, the endothelium cells are activated and produce selectins (e.g., ELAM-1). In turn, neutrophils are then capable of binding the selectins expressed on the vascular endothelium and cause further damage.
The term "pertussis toxin" or "PT" in this application refers to the virulence factors produced by Bordetella pertussis, the etiological agent of whooping cough. The (~-oligomer of PT binds to both a-sialic acid(2-~6)~3Ga1- and a-sialic acid(2->3)~3Gai- structures. PT also has similar binding characteristics to those of wheat germ agglutinin (WGA), which can recognize terminal N-acetylglucosamine (GIcNAc) saccharide sequences in addition to siaiic acid'°~s=~s3.
PT is a classical A-B type of toxin comprised of an A subunit (designated S 1 ) that contains an ADP-ribosyltransferase enzyme activity, which is responsible for most of the bioiogical effects of PT". The lectin-like activity is found in the complex (3 oligomer of PT, which consists of four heterogeneous subunits that are arranged in a pair of dimers (S2-S4 (Dimer 1 ) and S3-S4 (Dimer 2)) joined by a smaller SS
subunit. The functioning of the (3 oligomer is in binding to host cell sialylated oligosaccharide receptors as well as providing a delivery system for the A subunit through the cytoplasmic membranes°.ss.sb. The (3 oligomer itself can induce a mitogenic response in lymphocytes and has the ability to agglutinate erythrocytes. PT may also contribute to the attachment of B. penussis to epithelial cells lining the upper respiratory tract of humans, the only known host of B. pertussiss'. In addition, the (3 oiigomer also appears to share functional homology with the selectin family of mammalian lectins that regulate leukocyte trafficking.sg The term "cell-mediated immune response" refers to those mammalian immune responses which are mediated by cell-cell interactions. Included within this term are cell-mediated inflammatory responses to an antigen including, by way of example, such responses as delayed-type hypersensitivity (DTH) responses, virus-induced pneumonia, allergic responses, and the like as well as cell-mediated inflammatory responses arising from injuries such as myocardial infarction, shock and sequelae (e.g., multiple organ failure), acute respiratory distress syndrome CARDS), and the like. Generally, the cell-mediated immune response is a leukocyte-mediated response.
The term "humoral immune response" refers to mammalian immune responses which involve antigen-antibody interactions.
The term "DTH inflammation response" or "delayed type hypersensitivity response" is a T cell mediated reaction which results in a mononuclear cell-rich inflammation and swelling which occurs after antigenic challenge.
The term "tolerance" or "immunological tolerance" refers to a reduced immunogenic response elicited in a sensitized mammal to a particular antigen upon a second or subsequent antigenic challenge in comparison to the primary immune response elicited by said antigen under equivalent conditions (e.g., dosage). In the present invention such "tolerance" will be obtained by administration of an antigen to the sensitized mammal followed by administration of one or more lectin derived carbohydrate binding peptides which bind to a-sialic acid(2-->6)~3Gal- and/or a-sialic acid(2~3)~3Gal- structures.
The term "period for maximal inflammation" refers to the period of time typically required to achieve maximal inflammation in a mammal due to a cell-mediated immune response including both cell-mediated inflammatory responses in a sensitized mammal due to antigen exposure (challenge) and cell-mediated inflammatory responses in a mammal due to injury (e.g., myocardial infarction). This period of time depends on several factors such as the specific antigen/injury afflicting the mammal, the particular mammalian species exposed to the antigen/injury, etc.
Accordingly, the period of time required to effect maximal inflammation will vary for, by way of example, rheumatoid arthritis as opposed to myocardial infarction.
Moreover, while the specific time required to effect maximal inflammation will vary somewhat in a given mammalian species, the time typically required to effect maximal inflammation for different afflictions due to either antigen exposure or injury in human and other mammals is known in the art or is readily ascertainable by the skilled artisan. For example, in the case of a DTH response in mice, maximal inflammation is typically 24 hours after antigen exposure.
The term "sialic acid" refers to all naturally occurring structures of sialic acid and analogues of sialic acid as well as derivatives thereof.
Naturally occurring structures of sialic acid include, by way of example, 5-acetamido-3,5-dideoxy-D-glycero-D-galacto-nonulopyranosylonic acid ("NeuSAc"), N-glycoyl neuraminic acid (NeuSGc) and 9-O-acetyl neuraminic acid (Neu5,9Ac,). A complete list of naturally occurring sialic acids known to date is provided by Schauers'.
Derivatives of sialic acid refers to derivatives of naturally occurring structures of sialic acid including those wherein the sialic acid unit has been chemically modified so as to introduce and/or remove one or more functionalities from such structures. For example, such modification can result in the removal of an -OH functionality, the introduction of an amine functionality, the introduction of a halide functionality, and the like.
Certain derivatives of sialic acid are known in the art and include chemically modified sialic acid derivatives such as 9-azido-NeuSAc, 9-S amino-NeuSAc, 9-deoxy-NeuSAc, 9-fluoro-NeuSAc, 9-bromo-NeuSAc, 8-deoxy-NeuSAc, 8-epi-NeuSAc, 7-deoxy-NeuSAc, 7-epi-NeuSAc, 7-8-bis-epi-NeuSAc, 4-O-methyl-NeuSAc, 4-N-acetyl-NeuSAc, 4,7-di-deoxy-NeuSAc, 4-uno-NeuSAc, 3-hydroxy-NeuSAc, 3-fluoro-NeuSAc acid as well as 6-thin analogues of NeuSAc. Methods for preparing such sialic acid derivatives are taught in commonly assigned U.S. Patent Application Serial No. 07/889,017 filed on May 26, 1992, which application is incorporated by reference in its entirety.
The nomenclature describing derivatives of sialic acid derivatives herein is as set forth by Reuter et al~°
The term "a-sialic acid(2~6)~3Ga1- structures or groups" refers to molecules comprising the terminally linked a-sialic acid(2-~6)galactose-sequence or derivatives thereof. Molecules containing such terminal structures have been identified as comprising part of the putative receptor structure for the ELAM-1 and PADGEM selectins38~°°.
The term "a-sialic acid(2-~3)~iGal- structures or groups" refers to molecules comprising the terminally linked a-sialic acid(2-i3)galactose-sequence or derivatives thereof. Molecules comprising such terminal structures have similarly been identified as comprising part of the putative receptor structures for the SLAM-1 selectins'5.~9.
-z0-The term "lectin derived carbohydrate binding peptide" refers to any peptide or derivative thereof (including pharmaceutically acceptable salts) derived from a lectin and which is capable of binding to a-sialic acid(2->6)~iGal- and/or a-sialic acid(2-~3)/3Gal- carbohydrate structures which are preferably comprised on the surface of mammalian cells. Generally, in the present application, "lectin derived carbohydrate binding peptide" will refer to such peptides which, in monomeric form, have no more than about 35 amino acids in the lectin-like domain (i.e., the part of the peptide responsible for binding to such carbohydrate structures). Suitable derivatives of lectin derived carbohydrate binding peptides include those peptides which have the NH., and/or the COOH
terminal functionalities blocked by conventionally blocking groups as well as derivatives which include modifications, deletions or derivatizations of one or more of the amino acids that yield a peptide which is capable of binding to a-sialic acid(2-~6)~3Ga1- and/or a-sialic acid(2-~3)(3Ga1-structures.
More preferably, the lectin derived carbohydrate binding peptides refer to the peptides set forth in Figure 1.
Still more preferably, the lectin derived carbohydrate binding peptides are peptides which have a high degree of homology with a lectin-like binding domain for the a-sialic acid(2->6)/3Ga1- and/or a-sialic acid(2->3)~iGal- carbohydrate structures found in the pertussis toxin.
These peptides are represented by formula I (SEQ ID NO:1):
SPX,GX,C I
where X, is selected from the group of amino acids Y, F, W, and H or peptide mimetics thereof; and X, is selected from the group consisting of amino acids Y, F, R, W, and H or peptide mimetics thereof;
or by formula II (SEQ ID N0:2):
SPX,GX~CX3X4 II
where X, is selected from the group of amino acids Y, F, W, and H or peptide mimetics thereof;
X, is selected from the group consisting of amino acids Y, F, R, W, and H or peptide mimetics thereof;
X3 is an amino acid sequence of 4-6 amino acids; and X4 is selected from the group consisting of amino acids Y, F, W, and H or peptide mimetics thereof.
Most preferably, the lectin derived carbohydrate binding peptide refers to the hexapeptide SPYGRC (amino acids 18-23 of SEQ ID N0:3).
In addition to their use as modulating agents for mammalian immune and cellular adhesion processes, the lectin derived carbohydrate binding peptides described herein can further be used in assay methods for determining the presence of a-sialic acid(2~3)/3Ga1- and/or a-sialic acid(2-,6)~3Gal- structures on molecuies and/or cell surfaces such as in suspected cancer cells, for determining the relative binding affinity of peptides and/or proteins to a-sialic acid(2--~3)(3Ga1- and/or a-sialic acid(2->6)/3Ga1- structures, and the like. When so employed, the lectin derived carbohydrate binding peptides typically will be derivatized to include a label or a label binding moiety.
-zZ-Suitable labels are well known in the art and include, by way of example, enzymes (e.g., horseradish peroxidase), radioisotopes (e.g., ''-3I), fluorescent moieties, chemiluminscent moieties, and the like. The particular label employed is not critical, and methods for attaching labels to peptides are well known in the art.
Suitable label binding moieties are also well known in the art and include, by way of example, biotin, avidin, streptavidin antibodies, etc.
A preferred label binding moiety is biotin which permits binding of up to 4 peptide/biotin adducts to avidin. The avidin can be appropriately labeled so that the resulting peptide/biotinlavidin complex can be detected.
The term "lectins" refers to carbohydrate binding proteins of non-immune origin often obtained from plants or bacterial or viral microorganisms which comprise carbohydrate binding sites. These binding proteins typically comprise the ability to agglutinate cells and to precipitate complex carbohydrates. Lectins are classified based upon their carbohydrate binding specificity and are well known in the art.
The term "lectin-like domain" refers to those fragments) of a lectin responsible for binding the carbohydrate.
The term "pharmaceutically acceptable salts" includes the pharmaceutically acceptable addition salts of lectin derived carbohydrate binding peptides capable of binding to terminally linked a-sialic acid(2-~6)~3Gal- and/or a-sialic acid(2--~3)(3Ga1- structures. Such pharmaceutically acceptable addition salts may be derived from a variety of organic and inorganic counter salts well known in the art and include, by way of example only, sodium, potassium, calcium, magnesium, ammonium, tetralkyl-ammonium, and the like.
2. Utility Without being limited to any theory, it is believed that the subject lectin derived carbohydrate binding peptides affect the immune response in a number of ways. Lectin derived carbohydrate binding peptides can inhibit a mammal from becoming "educated" about a specific antigen when the lectin derived carbohydrate binding peptide is administered simultaneously with the first exposure of the immune system to the antigen.
The lectin derived carbohydrate binding peptides can reduce cell-mediated immune responses to injury such as inflammatory responses arising from myocardial infarction, ARDS, frost-bite, etc. The lectin derived carbohydrate binding peptides can also inhibit the effector phase of a cell-mediated immune response (e.g., the inflammatory component of a DTH response) when administered to a sensitized mammal after exposure of the sensitized mammal's immune system to the antigen. In either case, in order to effect reduction in the cell-mediated immune response, it is necessary to administer the lectin derived carbohydrate binding peptides after initiation of the mammal's immune response and at or prior to one-half the period required for maximal inflammation induced by the injury or the antigen exposure.
Additionally, the subject lectin derived carbohydrate binding peptides can induce tolerance to antigens in sensitized mammals when administered at the time of second or later exposures of the immune system to the antigen when administration is conducted after initiation of the mammal's secondary immune response to the antigen and at or prior to one-half the period required for maximal inflammation induced by the antigen exposure.
Further, the administration of lectin derived carbohydrate binding peptides that bind a-sialic acid(2--~6)/3Ga1- and/or a-sialic acid(2-'3)~iGal-structures inhibits the binding of LEC-CAM proteins and other selectins to their putative receptors therefor which include both the a-sialic acid(2-~6)~3Gal-'gw and the a-sialic acid(2-->3)/3Gal-'339 structures.
Accordingly, the subject invention provides both pharmaceutical compositions containing lectin derived carbohydrate binding peptides capable of binding terminally linked a-sialic acid(2->6)/3Ga1- and the a-sialic acid(2-~3)~3Ga1- structures which are useful in inhibiting specific immune responses or cellular interactions in mammals as well as methods which include the administration of such lectin derived carbohydrate binding peptides to a mammal for inhibiting a cell-mediated immune response.
As noted above, lectin derived carbohydrate binding peptides useful for modulating a cell-mediated immune response in a mammal include any lectin derived peptide or derivative thereof capable of binding terminally linked a-sialic acid(2->6)~3Ga1- and the a-sialic acid(2->3),QGaI-structures. Suitable lectin derived carbohydrate binding peptides preferably are peptides which, in monomeric form, have no more than about 35 amino acids in the lectin-like domain (i.e., the part of the peptide responsible for binding to such carbohydrate structures).
More preferably, the lectin derived carbohydrate binding peptides refer to the peptides set forth in Figure 1.
Still more preferably, the lectin derived carbohydrate binding peptides are peptides which have a high degree of homology with a lectin-like binding domain for terminally linked a-sialic acid(2-->6)~iGal- and/or a-sialic acid(2-~3)~iGal- carbohydrate structures found in the pertussis toxin. These peptides are represented by formula I (SEQ ID NO:1):
SPX,GX,C I
where X, is selected from the group of amino acids Y, F, W, and H or peptide mimetics thereof; and X, is selected from the group consisting of amino acids Y, F, R, W, and H or peptide mimetics thereof;
or by formula II (SEQ ID N0:2):
SPX, GX=CX~X4 II
where X, is selected from the group of amino acids Y, F, W, and H or peptide mimetics thereof;
X, is selected from the group consisting of amino acids Y, F, R, W, and H or peptide mimetics thereof;
X3 is an amino acid sequence of 4-6 amino acids; and X4 is selected from the group consisting of amino acids Y, F, W, and H or peptide mimetics thereof.
The preparation of such peptides is well known in the art and includes, by way of example, standardized commercially available peptide synthesizers such as Model ABI 403A available from Applied Biosystems, Inc., Foster City, California, U.S.A. The methods for preparing such peptides do not form a part of this invention.
Peptide mimetics refers to groups which mimic an amino acid in the peptide chain. Such mimetics and their synthesis are well known in the artsy.
The lectin derived carbohydrate binding peptides can be used either in monomeric or polymeric form. Examples of suitable polymers include the attachment of biotin to the lectin derived carbohydrate binding peptide followed by complexing with avidin which results in up to a tetravalent complex. Likewise, multivalent derivatives of lectin derived carbohydrate binding peptides can be synthesized by attaching such peptides to polymers such as polylysine or an inert protein such as human serum albumin. The multivalent derivatives so formed can contain one or a mixture of different' lectin derived carbohydrate binding peptides so as to enhance efficacy.
In the case of protein peptide conjugates, they will be chemically cross-linked with the carrier protein by known cross-linking agents using art recognized methodology.
In yet another embodiment, multivalent lectin derived carbohydrate binding peptides can be generated as a copolymer wherein the peptides are linked together through a spacer arm to provide for a repeating subunit represented by the groups:
[carbohydrate binding peptide-spacer arm]
[carbohydrate binding peptide]~-spacer arm In these subunits, the lectin derived carbohydrate binding peptide can be the same or different lectin derived carbohydrate binding peptide, and the spacer arm is selected to provide for optimal distance to bind to the carbohydrate.
However, the invention is not restricted to the use of lectin derived carbohydrate binding peptides specifically exemplified in Figure 1 or to multivalent derivatives thereof, but rather embraces the use of any lectin derived peptide or derivative thereof which binds terminally linked a-sialic acid(2~6)~3Ga1- and/or a-sialic acid(2--~3)~iGal- carbohydrate structures. As noted previously, peptides capable of binding such structures, when administered to a mammal, result in the inhibition of immune responses and cellular interactions, in particular, inflammatory responses or conditions, tolerance to antigens, modulation of the immunogenic response to antigens, and the inhibition of cell adhesion events, which are involved, e.g., in metastasis and inflammation.
It is well within the level of ordinary skill in the art to identify other lectin derived peptides capable of binding terminally linked a-sialic acid(2->6)/3Ga1- and/or a-sialic acid(2-~3)~3Ga1- structures by conventional methods for assaying binding between ligands. Such methods include, e.g., competitive binding assays and receptor binding assays. The subject application, in particular, sets forth one method in the examples which illustrates rather simple assaying techniques that are capabie of determining binding to terminally linked a-sialic acid(2-->6)~iGal- and/or a-sialic acid(2-->3)(3Ga1- structures. Other methods for determining the binding of a candidate peptide with such terminally linked structures are known in the art. See, for example, Pearce-Pratt et al."
The subject invention accordingly further provides a method by which lectin derived peptides capable of inducing or suppressing various immune responses and cellular interactions, e.g., inflammation, antigenic tolerance, modulation of antigenic response, or cell adhesion, may be putatively identified on the basis of their ability to bind to terminally linked a-siaiic acid(2->6)(3Ga1- and/or a-sialic acid(2-~3)(3Gal- structures.
In regard to the above, the subject invention contemplates the attachment of labels or label binding groups to the lectin derived carbohydrate binding peptides and/or to candidate lectin derived carbohydrate binding peptides in order to facilitate the assays described above. Such labels are conventionally formed on the peptides by methods well known in the art. Suitable labels include, by way of example, enzymes (e.g., horseradish peroxidase), radioisotopes (e.g., ''-sI), fluorescent moieties, chemiluminscent moieties, and the like.
Suitable label binding moieties are also well known in the art and include, by way of example, biotin, avidin, antibodies, etc. A preferred label binding moiety is biotin which permits binding of the peptide/biotin adduct to avidin. The avidin can be appropriately labeled so that the resulting peptide/biotin/avidin complex can be detected.
The subject invention also contemplates kits for use in conducting such assays. Such kits would comprise the labelled lectin derived carbohydrate binding peptide or the lectin derived carbohydrate binding peptide attached to label binding groups.
Suitable lectin derived carbohydrate binding peptides for use herein are those which are capable of binding terminally linked a-sialic acid(2-~6)/3Gal- and/or a-sialic acid(2->3)(3Ga1- structures. However, an additional prerequisite of efficacious lectin derived carbohydrate binding peptides will include suitability for in viv administration. In particular, the lectin derived carbohydrate binding peptide should not be toxic, and should be sufficiently soluble at the required dosages, which will typically range from about 0.5-50 mg/kg of body weight. In this regard, it is art recognized that the solubility of lectin derived carbohydrate binding peptides can be enhanced by attaching hydrophilic amino acid groups and can be reduced by attaching hydrophobic amino acid groups from the carboxyl terminal and/or amino terminal positions of the peptide.
The invention further contemplates fragments or derivatives of peptides capable of binding terminally linked a-sialic acid(2-~6)aGal-and/or a-sialic acid(2-~3)(3Ga1- structures which peptides have been modified to render them non-toxic, e.g., by chemical derivatization, mutagenesis, etc. while still retaining the ability to bind such terminally linked structures.
The subject invention provides, in particular, methods for suppressing cell-mediated immune responses in mammals including cell-mediated inflammatory responses or disorders by the administration of an effective amount of one or more lectin derived carbohydrate binding peptides capable of binding terminally linked a-sialic acid(2~6)~3Gal-and/or a-sialic acid(2-~3)(3Gal- structures or moleculesJcell surfaces comprising such structures.
-.i U-The cell-mediated immune responses or disorders treatable by the subject invention include inflammatory immune reactions involving specific and non-specific defense systems. As discussed above, such conditions include antibody responses to antigens, such as viruses, allergens, delayed-type hypersensitivity, autoimmune disorders such as rheumatoid arthritis and lupus, post-ischemic leukocyte mediated tissue damage (reperfusion injury), frost-bite injury or shock-acute leukocyte-mediated lung injury (e.g., acute respiratory distress syndrome), asthma, traumatic shock, septic shock, nephritis, and acute and chronic inflammation, including atopic dermatitis, psoriasis, and inflammatory bowel disease. Further, inflammatory disorders treatable by the subject invention may include platelet-mediated pathologies such as atherosclerosis and clotting disorders.
Inflammatory conditions of special interest include delayed type hypersensitivity reactions, reperfusion, and acute leukocyte-mediated lung injury CARDS).
This invention provides a generic method by which cell-mediated immune responses such as cell-mediated inflammatory responses or disorders in mammals (e.g., humans) may be suppressed by the administration of an effective amount of one or more lectin derived carbohydrate binding peptides or fragments or derivatives thereof capable of binding terminally linked a-sialic acid(2-->6)~3Gal- and/or a-sialic acid(2-->3)/3Ga1- structures or molecules/cell surfaces comprising such structures. In a preferred embodiment, the invention provides methods by which inflammatory responses or disorders may be treated or suppressed by the administration of an effective amount of one or more peptides selected from the group of peptides set forth in Figure 1.
The subject invention further provides a general method for inhibiting immune responses and cell adhesion events in mammals by the administration of an effective amount of one or more lectin derived carbohydrate binding peptides or fragments or derivatives thereof capable S of binding terminally linked a-sialic acid(2->6)/3Ga1- and/or cx-sialic acid(2-~3)~3Gal- structures or molecules/cell surfaces comprising such structures. Such immune responses include cell mediated and humoral immune responses. As has been discussed, such immune responses include, in particular, inflammatory responses or inflammatory disorders.
The invention further provides methods for affecting the induction of immune responses to antigens comprising administering to a mammal an antigen in conjunction with one or more lectin derived carbohydrate binding peptides capable of binding terminally linked a-sialic acid(2-~6)~iGal- and/or a-sialic acid(2-~3)(3Ga1- structures or molecules/cell surfaces comprising such structures. For example, administration of an effective amount of the SPYGRC (amino acids 18-23 of SEQ ID N0:3) hexapeptide to a mammal with an antigen will modulate the induction of the immune response in the mammal to the antigen. Accordingly, the subject lectin derived carbohydrate binding peptides may comprise applicability as immune modulators, which may be administered in conjunction with vaccines, artificial organs or tissue transplants, and allogeneic organ and tissue transplants as a means for modulating the immune response to foreign antigens comprised therein.
It has further been found that the subject lectin derived carbohydrate binding peptides capable of binding terminally linked a-sialic acid(2->6)(3Ga1- and/or a-sialic acid(2->3)~3Gal- structures or molecules/cell surfaces comprising such structures, when administered in an effective amount to a mammal which has been immunized with a particular antigen, result in the induction of long term tolerance to said antigen. In this regard, administration is conducted after onset of the secondary immune response but at or prior to one-half the period required for maximal inflammation.
In particular, it has been found that administration, during the critical period set forth above, of an effective amount of the S3P9a (SEQ
ID N0:9), the ACS2P1 (2275) (amino acids 9-23 of SEQ ID N0:3), and the SPYGRC (2283) (amino acids 18-23 of SEQ ID N0:3) peptides illustrated in Figure 1 to mammals that have been immunized with an antigen results in said mammals exhibiting a reduced immune response upon subsequent challenges) with said antigen (Figure 3). Thus, the subject lectin derived carbohydrate binding peptides have applicability as tolerogens. Given this property, such lectin derived carbohydrate binding peptides or fragments or derivatives thereof may be especially suitable for use in the treatment of allergic disorders since administration of "tolerogenic" derivatized allergens is a known means for treating allergic disorders.
The subject invention further provides methods for inhibiting the adhesion of certain cell types [e.g., tumor cells and polymorphonuclear cells (PMN's)] to endothelial cells. In this regard, the art suggests that tumor metastasis involves tumor cell adhesion to selectin bearing cells. In this regard, circulating cancer cells apparently take advantage of the body's normal inflammatory mechanisms and bind to areas of blood vessel walls were the endothelium is activated and, accordingly, contains selectins. As noted previously, the putative receptors for such selectins contain terminally linked a-sialic acid(2-~6)~3Ga1- and/or a-sialic acid(2~3)~iGal- structures. Therefore, administration of lectin derived carbohydrate binding peptides capable of binding terminally linked a-sialic acid(2-~6)(3Ga1- and/or a-sialic acid(2-~3)~3Ga1- structures should provide a method for inhibiting metastasis. For example, the subject lectin derived carbohydrate binding peptides or fragments or derivatives thereof may be administered before, during or after cancer surgery or biopsy as a means for inhibiting metastasis of tumor cells which may be released into the circulatory system during surgery. In these methods, the subject lectin derived carbohydrate binding peptides are administered either prior to, at the time of surgery or biopsy, or shortly thereafter.
Prior administration is typically no more than about 5 hours prior to surgery or biopsy, and administration and subsequent administration is typically no more than about 15 hours after surgery or biopsy. In either case, administration is either continuous or intermittent, but preferably is continuous.
In the methods pertaining to suppression of cell-mediated inflammatory reactions or disorders arising from injury or antigen exposure, the subject lectin derived carbohydrate binding peptides or fragments or derivatives thereof are administered after initiation of the mammal's immune response but at or prior to one-half the period required for maximal inflammation to the antigen exposure or injury.
Preferably, the subject lectin derived carbohydrate binding peptides are administered about 1-10 hours after initiation of the immune response, and more preferably about 1-5 hours after initiation of the immune response. However, the specific time for administration will vary dependent upon the particular antigen/injury and the lectin derived carbohydrate binding peptide which is administered.
In the methods pertaining to modulating the induction of an immune response to an antigen in a non-sensitized mammal, an effective amount of the subject lectin derived carbohydrate binding peptides or derivatives thereof will be administered in conjunction with the antigen.
Typically, such conjunctive administration is simultaneous with antigen administration but can be up to ~3 hours from the time of antigen administration.
In the methods pertaining to induction of long term tolerance to an antigen, an effective amount of the subject lectin derived carbohydrate binding peptides or fragments or derivatives thereof will generally be administered after antigen challenge to a sensitized mammal. In particular, administration is after initiation of the mammal's secondary immune response to the antigen challenge but at or prior to one-half the period required for maximal inflammation to the antigen challenge.
Preferably, the subject lectin derived carbohydrate binding peptides are administered about 1-10 hours after initiation of the immune response to the antigen challenge, and more preferably about I-5 hours after initiation of the immune response to the antigen challenge. However, the specific time for administration will vary dependent upon the particular antigen and the lectin derived carbohydrate binding peptide which is administered.
Generally, the subject lectin derived carbohydrate binding peptides or derivatives thereof will be administered parenterally, e.g., by intramuscular or intravenous routes. However, other dosage forms should also be suitable including, e.g., oral, transdermal, rectai, intratracheal, and intranasal formulations. For example, intranasal and intratracheal formulations may be preferred if the inflammatory condition treated involves lung inflammation, e.g., acute respiratory distress syndrome CARDS). In contrast, an oral formulation would likely be preferred if the inflammatory condition treated involves the digestive tract, e.g., inflammatory bowel disease.
Pharmaceutical compositions for use in the subject invention will generally comprise an effective amount of one or more of the subject peptides or derivatives thereof capable of binding terminally linked a-sialic acid(2->6)~iGal- and/or a-sialic acid(2-~3)~3Gal- structures in combination with a pharmaceutically acceptable carrier and/or excipients.
The particular pharmaceutically acceptable carrier and excipients will vary dependent upon the dosage form. In one embodiment, several of the subject peptides or derivatives thereof are mixed into the pharmaceutical composition to form a "cocktail" having enhanced activity.
Parenteral dosage forms may contain phosphate buffered saline as a carrier, while intranasal formulations will comprise inhalants, and oral dosage forms may comprise enteric coatings. The selection of suitable carriers and excipients and formulation of different dosage forms is well within the level of ordinary skill in the pharmaceutical art.
As noted above, the subject lectin derived carbohydrate binding peptides or derivatives thereof are administered in effective amounts. An effective amount is an amount sufficient to obtain the desired therapy without causing undue toxicity to the mammal. Preferably, the subject peptides are administered at dosages ranging from about 0.5 to SOmg/kg body weight, with 5-10 mg/kg being most preferred for each of the above-cited methods. The specific dose employed is regulated by the particular cell-mediated immune response being treated as well as by the judgement of the attending clinician depending upon factors such as the severity of the adverse immune response, the age and general condition of the patient, and the like.
Generally, the methods of the present invention will involve administration of a single dose of the subject lectin derived carbohydrate binding peptides. However, the invention further contemplates repeated administration of the subject lectin derived carbohydrate binding peptides or derivatives thereof. Repeated administration of these peptides may be desirable, e.g., in the treatment of chronic or sustained inflammatory disorders such as rheumatoid arthritis, acute and chronic inflammation, psoriasis, inflammatory bowel disorders, and autoimmune disorders associated with inflammatory responses such as lupus, multiple sclerosis or rheumatoid arthritis.
It is also contemplated that the subject peptides and derivatives are useful as receptor-targeted antibacterial and anti-viral drugs wherein the bacteria, virus, or toxin produced therefrom employs a terminally linked a-sialic acid(2-~6)/3Ga1- and/or a-sialic acid(2-~3)/3Ga1- structure as the receptor site on a cell of the targeted mammalian host.
Such bacteria/virus and/or toxins include, by way of example, influenza virus, pertussis toxin, cholera toxin, and the like. Such methods are illustrated in the examples hereinbelow wherein in vitro assays demonstrate the ability of two of the subject lectin derived carbohydrate binding peptides to neutralize the effects of pertussis toxin on Chinese Hamster Ovary cells.
Accordingly, when administered in effective amounts, the subject lectin derived carbohydrate binding peptides are useful in methods for inhibiting inoculation in mammalian hosts of bacterial/viral agents and/or their toxins which employ a terminally linked a-sialic acid(2--~6)/3Ga1-S and/or a-sialic acid(2-~3)(3Ga1- structure as the receptor site on a cell of the targeted mammalian host thereby inhibiting the likelihood that the mammalian host will become afflicted with the disease produced by the bacterial/viral agent and/or its toxin.
Effective amounts of the subject lectin derived carbohydrate binding peptides or derivatives thereof will preferably be dosages ranging from about 0.5 to SOmg/kg body weight, with S-10 mg/kg being most preferred.
3. Examples In order to fully illustrate the present invention and the advantages thereof, the following specific examples are given, it being understood that these examples are intended to be illustrative only and in nowise limitative of the scope of the present invention.
In these examples as well as in the application, all sugars disclosed herein are in their D form, except for fucose which is in its L form, and all amino acids are conventional.
In these examples, unless otherwise defined below, the abbreviations employed herein have their generally accepted meaning:
ABTS = 2,2'-azino-bis(3-ethylbenzathiazoline-6-sulfonic acid) BSA = Bovine serum albumin cm = centimeter HPLC = High Performance Liquid Chromatography S MAL = Maackia amurensis mg = milligram mM = millimolar mm = millimeter ng = nanogram nm = nanometer PBS = phosphate buffered saline PT = Pertussis toxin SNA = Sambucus nigra ~g = microgram ~d = microliter ~cM = micromolar ~mol = micromole v/v = volume/volume Unless otherwise indicated, all temperatures are in degrees Celsius (°C). Also, as noted previously, all amino acid residues recited herein employ their conventional one-letter abbreviation.
General Procedures All of the reagents used in Examples 1-10 were obtained from Sigma Chemical Company, St. Louis, Missouri, U.S.A., except for pertussis toxin (PT) which was obtained from Connaught Center for Biotechnology Research, Willowdale, Ontario, Canada; SNA-, WGA- and MAL-biotin, which were obtained from Boehringer Mannheim, Dorval, Quebec, Canada; and IODO-GEN which was obtained from Pierce Chemical Co., St. Louis, Missouri, U.S.A. The acetylated and biotinylated analogs of the peptide S2P1 (amino acids 9-23 of SEQ ID
N0:3) were prepared using conventional methods. PT-biotin as well as asialo- and asialoagalactofetuin were prepared as described earlier~°~°~
Removable flat-bottomed microtiter well strips of Immunulon 2 were from Dynatech, Alexandria, Virginia, U.S.A.
x m le 1 -- Synthesis of Synthetic Peptides Peptides corresponding to amino acid sequences found in the S2 and S3 subunits of PT were synthesized using an ABI 403A peptide synthesizer (Applied Biosystems, Inc., Foster City, California, U.S.A.), then cleaved from the resin by HF and purified by reversed-phase HPLC
on a Vydac C4 semipreparative column. All synthetic peptides used in ELISA inhibition assays were > 95 % pure as judged by analytical HPLC, and their amino acid analyses were in good agreement with the theoretical compositions.
Regions in the S2 and S3 subunits of PT that correspond to the variable amino acid sequences distinguishing the S2 from the S3 subunit were secured as above. These peptide sequences were chosen, in part, based on their high index of hydrophilic ~3-turns as judged by secondary structure prediction analysis~°~6'. Upon careful examination of the PT
sequences, 20 peptides were synthesized including those containing amino acid residues 62-73 of WGA (SEQ ID NO:11). The acetylated version of S2P1 (amino acids 9-23 of SEQ ID N0:3) was also prepared in order to better mimic the native peptide backbone. The synthesized peptides are set forth in Table I below:
TABLE I
PositionSequence* SEQ ID
NO
1. S3P1 9-33 PQEQITQHGSPYGRC 3 (aa 9=_'3) =. ACS2P1-b 9-23 BIOTIN-PQEQITQHGSPYGRC-CO-NH:3 (aa 9=_'3) $ 3. S'_' (14-23)14-'_'3TQHGSPYGRC 3 (aa 14-'_'3) 4. S2-b 14-'3 BIOTIN-TQHGSPYGRC 3 (aa 14-'_'3) 5. S3P3 1-23 STPGIVIPPQEQ1TQHGSPYGRC 3
6. SPYGRC 18-.3 SPYGRC-CO-NH, 3 (aa 18-23)
7. SPYGRC-b 18-23 BIOTIN-SPYGRC-CO-NHz 3 (aa 18-23) lO 8. S2P3 78-98 GAFDLKTTFCIMTTRNTGQPA 4 9. S2P4 138-154YDGKYWSMYSRLRKMLY 5 (aa 16-3'_') 10. S2P6 133-154FVRSGQPVIGACTSPYDGKYWSMYSRLRKML5 Y
11. S3PI 9-23 PKALFTQQGGAYGRC 6 (aa 9 ~3) 12. S3P2 I--~_3VAPG1V1PPKALFTQQGGAYGRC 6 1$ 13. S3P3 87-108CITT1YKTGQPAADHYYSKVTA 7 (aa 10-31) 14. S3P4 78-108AGFIYRETFCITT1YKTGQPAADHYYSKVTA7 15. S3P5 134-154CASPYEGRYRDMYDALRRLLY g 16. S3P9a 110-1?7RLLASTNSRLCAVFVRDG 9 17. S2fWGA)k- PQEQITQHGSQYGYC 10 2O 18. S2fWGA)-b- BIOTIN-PQEQ1TQHGSQYGYC 10 19. WGA(63-73)- SQYGYCGFGAEY 11 20.WGA(62-73)-b- BIOTIN-SQYGYCGFGAEY 11 * Underlined sequences correspond to sequences found in the PT
peptide that showed homology to the sequence SQYGHC (SEQ ID
2$ N0:12) found in the binding site of WGA isolectin 2 (24).
~t PT S2P1 sequence (amino acids 9-23 of SEQ ID N0:3) inserted with the WGA hexapeptide sequence (SQYGYC) (amino acids 10-15 of SEQ ID NO:10) as amino acid b biotin The above peptides were tested for their ability to bind to terminally linked a-sialic acid(2->6)~3Ga1- and a-sialic acid(2-->3)/3Gal-structures in the examples below. In these examples, the peptides were first screened for their ability to inhibit binding of different lectins (PT, SNA, and MAL) known to bind to fetuin, a carbohydrate containing multiple copies of the aNeuSAc(2-->3)~3Ga1(1->4)~3Glc- structure.
Example 2 -- Binding Inhibition Assays (Initial Peptide Screening) Microtiter wells were coated with 100 ~cl of fetuin or asialofetuin (50 ~cg/ml) in 50 mM sodium phosphate buffer (pH 6.8) containing S mM
MgCh and 15 mM NaN3 for 16 hours at 4°C. The solution was removed by aspiration and replaced with 100 ~,1 of 1 % BSA in PBS containing 0.05 % Tween 20 (PBST). After incubation for 2-4 hours at room temperature, the microtiter wells were washed four times with 300 ~.1 of PBST. Peptides ranging in concentration from 0.5 to 4.5 mg/ml in PBS
(40 ~cl) were added to each well, and PT-biotin (IO ~,1 containing 10 ng in PBS) was then added to the microtiter wells. After incubating for 1 hour, the binding reaction was stopped by aspirating the solutions, and the plate was washed with PBST (300 ~.1). Horseradish peroxidase-conjugated avidin (100 ~cl, 1/3000 dilution in PBST to a concentration of 0.3 ~,g/ml) was then added to the wells, and the plates were incubated at ambient temperature for 1 hour. After washing the wells as described above, the substrate solution (1 mM ABTS in 5 mM citrate buffer, pH 4.2, containing 0.1 % hydrogen peroxide, v/v) was added, and the plates were incubated for 30 minutes. Color development was recorded at 405 nm using a Titertek Multiskan MC plate reader. Maximum binding was determined in the absence of peptide, and background binding was measured in wells coated with BSA only. Binding assays for each peptide were done in duplicate. Binding inhibition experiments utilizing SNA-, WGA-, and MAL-biotin were performed as described above using 10 ng of each biotinylated lectin in PBS.
The panel of 20 peptides was assayed for the ability to inhibit PT-, WGA-, MAL-, and SNA-biotin binding to fetuin or asialofetuin. The biotinylated plant lectins have proved to be useful controls, since we have previously showed that these lectins possess similar binding specificities as P'If'-~''6. The results from this example are set forth in Table II below.
These results indicate that peptides S2P3 (SEQ ID N0:4) and S2P6 (SEQ
ID N0:5) inhibited PT biotin binding by 15-20% both to fetuin as well as to asialofetuin. Two additional peptides derived from the S3 subunit [S3P3 (amino acids 10-31 of SEQ ID N0:7) and S3P5 (SEQ ID NO:B)]
were found to reduce binding of PT to asialofetuin only. The majority of the other peptides displayed in Table II exhibited either marginal inhibition or enhancement in binding of biotinylated PT to fetuin or asialofetuin. Peptides S2P1 (amino acids 9-23 of SEQ ID N0:3), ACS2P1 (amino acids 9-23 of SEQ ID N0:3) and S2P2 (SEQ ID N0:3) showed nearly a 2-fold enhancement of PT-biotin binding relative to control experiments done in the absence of peptide.
Peptide S2P3 (SEQ ID N0:4) was found to inhibit binding to fetuin of all the biotinylated lectins. However, it is noted that peptides S2P1 (amino acids 9-23 of SEQ ID N0:3) and S2P2 (SEQ ID N0:3), which showed enhancement of binding of PT to fetuin, inhibited WGA
binding activity.
Table II
Percent Changes in Binding Activity peptideSEQ ID Coocea-PT/ MAL SNA/ WGA/ PT/
NO entioo feotm fetuinfetuinfeoun ~;"b.
(me/~.J feo~
S2P1 3(u9-23)1.2 +8514 +Bt4 -Sf1 -1812 +113(1) ACS2P13 (u 1.0 +8913 +1619 -311 -2216 +190123 9-23) 52 3 (u 1.0 +68 +3211 +112 +7 ND
(14-23)14-23) f f S2P2 3 1.1 +5814 +1412 -Itl -1512 +33(1) S2P3 4 0.31 2110 -1812 1314 -5618 -13(1) S2P4 3 (u 0.98 +14122+2(1) +3(1) +1311 -3(1) 16.32) 1~ S2P6 3 0.85 -286 +18116+212 +370 -18(1) S3P1 6 (u 3.2 +54 +416 -211 -9 +d3 9-23) f f f S3P2 6 3.1 +77159+12116+313 +914 +82 f S3P3 7 (u 4.3 +19167+13113+310 +bt3 -2817 10-31) S3P4 7 3.3 +1212 +7f1 +I(1) +1311 +73130 is S3P3 8 0.48 +1416 +316 0(1) +3318 -26110 S3P9r,9 1.7 +328110+1816 ~1t3 ND ND
SPYGRC3 (u 4.4 +34 -1610 212 +713 +79 18-23) f S2(WGA)10 1.0 +79115+2213 Otl -4f3 ND
WGA 11 1.0 -3215 If4 -312 -9f1 ND
(62-73) * Biotinylated lectin/glycoprotein bound to microtiter wells. Positive signs preceding the numbers in this table indicate enhancement and negative signs indicate inhibition of 25 binding.
(1) One determination only.
as amino acid ND Not determined.
-t~-Exam lp a 3 -- Binding Inhibition Assays (Determination of IC 50 Values) Peptides found to have inhibitory activity in the initial screening experiments were further analyzed in binding inhibition experiments to determine the concentration of peptide that was required to reduce binding by 50% (ICS values).
Inhibition experiments were performed by a method similar to that described in Example 2 with 2-fold dilutions of PT peptide in PBS, except that microtiter wells were coated with 3 ~,g/ml fetuin or asialofetuin.
Binding assays for each inhibitor concentration were done at least in duplicate, and the average value varied less than 15 % . The concentration of peptide required for 50% inhibition (ICso) was determined by plotting the amount of binding observed in the presence of peptide inhibitor as a percent of the maximum binding achieved without inhibitor.
Two of the peptides derived from the S2 subunit were able to inhibit PT-biotin binding to fetuin at submillimolar concentrations, but they were unable to inhibit PT-biotin binding to asialofetuin in subsequent experiments (Table III). The two peptide sequences from the S3 subunit (S3P3 [amino acids 10-31 of SEQ ID N0:7] and S3P5 [SEQ ID NO: 8]) were found to inhibit PT-biotin's interaction with asialofetuin in a concentration-dependent manner, but their ICS values were above the solubility limits of the peptide in PBS. The peptide S2P3 (SEQ ID NO:
4) was also very active at inhibiting the interaction of MAL- and WGA-biotin with fetuin. Upon closer examination this peptide proved to be non-inhibitory for SNA-biotin. Two additional peptides from the S2 subunit (S2P1 [amino acids 9-23 of SEQ ID NO: 3] and S2P2 [SEQ ID
NO: 3]) were active at inhibiting WGA-biotin binding at millimolar concentrations, suggesting that these peptide sequences may also be important for interacting with oligosaccharide in the binding site of WGA.
Table III
Concentrations of PT Peptides and WGA Peptides Resulting in 50% Inhibition of Biotinylated PT and Lectins Binding to Fetuin or Asialofetuin Biotinylated Lectin Peptide SEQ ID NO ICb(mM) (glycoproteinl PT-b (fetuin) S2P3 4 0.19+0.09 PT-b (fetuin) S2P6 5 0.'_'310.06 WGA-b (fetuin) ACS2P1 3 (aa 9 _3) 3..50.45*
WGA-b (fetuin) S2P3 3 SI
WGA-b (fetuin) S3P3 4 0.1410.03 MAL-b (fetuin) S2P3 4 0.860.0'_' PT-b (asialofetuin) S3P3 7 (aa 10-31) SI
IS PT-b (asialofetuin) S3P5 8 SI
SNA-b (fetuin) S3P9a 9 2.74+0.86 PT-b (fetuin) WGAl6''-73)I1 1.50 WGA-b (fetuin) WGA(62-73)11 3.40 SI Slightly inhibitory, but the concentration of peptide required for 50% inhibition of binding is above the solubility limit of the peptide.
* Unacetylated form of S2P1 (amino acids 9-23 of SEQ
ID NO: 3) inhibited to the same extent as the acetylated form.
as amino acid Since three of the peptides derived from the S2 subunit of PT were able to inhibit WGA binding, the amino acid sequences which constitute the sialic acid binding site of WGA were closely examined to determine if there were any homologies with the inhibitory PT S2 subunit peptide sequences. One short 6-amino acid sequence (SQYGHC) (SEQ ID NO: 12) corresponding to amino acids 62-67 in WGA isolectin 2 displayed reasonable homology with a sequence found both in the S2P1 (amino acids 9-23 of SEQ ID NO: 3) and S2P2 peptides (SPYGRC, amino acids 18-23 of SEQ ID NO: 3) from PT. This short sequence in WGA is responsible for binding with the carbonyl of the N-acetyl group of sialic acid or N-acetyl-glucosamine (i.e., serine 62) through a hydrogen bond. Non-polar interactions between the aromatic side chains of tyrosine 64 as well as histidine 66 interact with the glycerol side chain of sialic acid and the pyranose ring of sialic acid or N-acetyl-glucosamine, respectively°Z. The other inhibitory peptide, S2P3 (SEQ ID N0: 4), did not display any good homology with sequences responsible for interacting with sialic acid in WGA. This indicates that other motifs may also be functionally important for interaction with sialic acid.
Example 4 -- Binding Inhibition Studies Utilizing Biotinylated and Acetylated S2P1 (amino acids 9-23 of SEQ ID NO: 3) From the initial peptide screening results (Table II), peptides S2P1 (amino acids 9-23 of SEQ ID NO: 3) and S2P2 (SEQ ID NO: 4) showed a 2-fold enhancement in binding of PT-biotin to fetuin relative to control experiments. One possible explanation for the enhancement may be the ability of the peptide to form a bridge between PT-biotin and fetuin. In order for the peptide to act as a bridging molecule, the peptide must contain both a recognition site for fetuin as well as a sequence which binds to PT itself. To answer this question an acetylated and biotinylated form of the peptide S2P1 (amino acids 9-23 of SEQ ID NO: 3) (biotinylated at the terminal proline) was prepared to determine if we could measure direct binding of the peptide to PT and fetuin.
Binding assays were done as described in Example 2 in PBS by using ACS2P1-biotin (amino acids 9-23 of SEQ
ID NO: 3) at a concentration of 10 ug/ml in PBS.
Assays were carried out for 1 hour at room temperature, and the amount of biotinylated peptide bound to fetuin was determined by using avidin-peroxidase.
From these direct binding studies, a concentration-dependent binding both to fetuin as well as to PT immobilized in microtiter wells was observed.
Furthermore, the binding of ACS2P1-biotin (amino acids 9-23 of SEQ ID NO: 3) to PT could be inhibited by fetuin (ICso = 50 ~M; n=2) indicating that the biotinylated peptide bound at or adjacent to the fetuin binding site in PT.
In view of the above, it was concluded that because of potential bridging by the peptide between the lectin and the fetuin, any of the peptide sequences set forth in Table I which caused reduction in the binding of any of the lectins to fetuin were able to bind to terminally linked a-sialic acid(2-~6)/3Ga1- and a-sialic acid(2~3)/3Ga1- structures.
Example 5 -- Binding Inhibition Assays Using Oligosaccharides to Inhibit the Binding of ACS2P1-biotin (amino acids 9-23 of SEQ ID
NO: 3) The binding results obtained for ACS2P1-biotin (amino acids 9-23 of SEQ ID NO: 3) described in Example 4 above indicate a direct involvement of carbohydrate moiety with the lectin through a biotinylated peptide bridge.
In order to assess the binding to a-sialic acid(2~6)/3Ga1- and a-sialic acid(2-~3)~iGal- structures by this peptide as well as the hexapeptide SPYGRC
(amino acids 18-23 of SEQ ID NO: 3) which only exhibited reduction in the binding of one lectin to fetuin (Table II), inhibition experiments with simple saccharides were conducted to determine if the interaction was carbohydrate-dependent. The simple sugars chosen were sialic acid, sialyllactose from' bovine colostrum (contains a mixture of a(2~6) and a(2-~3) linked sialic acid structures), lactose, and N-acetyl-glucosamine.
Binding inhibition assays were done using sialic acid, sialyllactose, lactose, or N-acetyl-glucosamine at a concentration of 8 mM in PBS. Inhibition assays were done for 1 hour as described above (e. g., Example 2) at room temperature, and the amount of biotinylated peptide bound to fetuin (3~,g/ml) quantitated by using avidin-peroxidase. The pH of free sialic acid was carefully monitored and adjusted to physiological pH
values with dilute sodium hydroxide. The results of this example are set forth in Table IV below:
Table IV
Inhibition of Binding of ACS2P1-biotin (amino acids 9-23 of SEQ ID NO. 3) and SPYGRC-biotin (amino acids 18-23 of SEQ ID NO. 3) to Fetuin by Simple Saccharides*
Saccharide Percent Changes in Binding Activity ACS2P1-biotinSPYGRC-biotin n=3 n=3 Sialic acid -1916 +918 Sial llactose -4613 -2416 Lactose +7417 +20111 N-Acet 1 lucosamine-102 +615 Sucrose Ot3 ND
* Inhibition experiments were carried out as outlined above using O.l~Cg or 0.5ug of ACS2P1-biotin (amino acids 9-23 of SEQ ID NO. 3) and 15 SPYGRC-biotin (amino acids 18-23 of SEQ ID
N0.3) respectively.
Positive signs preceding the number in this table indicate enhancement and negative signs indicate inhibition of binding to fetuin.
ND Not determined The above results indicate that both peptides tested were able to bind to terminally linked a-sialic acid ( 2-~6 ) QGal- and a-sialic acid ( 2-~3 ) ~iGal-structures. Control experiments indicated that sialic acid was necessary for high affinity interaction since lactose failed to reduce the binding of ACS2P1-biotin (amino acids 9-23 of SEQ
ID NO. 3) on N-acetyl-glucosamine was also found to marginally inhibit the binding of biotinylated 30 peptide (10 ~ 2%, n=3) suggesting that the ACS2P1-biotin (amino acids 9-23 of SEQ ID NO. 3) may have weak affinity for the N-acetylglucosamine consistent with the finding described below, which shows enhanced binding of the peptide to asialoagalactofetuin relative to asialofetuin. This is also in good agreement with previous results which indicate PT's binding specificities to be similar to those of WGA'°~SZ.
In view of the above, it was concluded that any of the peptides which inhibit the binding of fetuin to any of the lectins set forth in Table II above are effective in binding to terminally linked a-sialic acid ( 2-~6 ) ~3Ga1- and/or a-sialic acid ( 2-~3 ) ~BGal-structures. Such peptides are set forth in Figure 1.
Example 6 -- Screening of Subunit S2 and S3 Peptides from PT for the Ability to Inhibit ACS2P1-biotin Binding to Fetuin and Asialofetuin Binding inhibition assays were conducted using subunit S2 and S3 peptides from PT in PBS as competitors for binding of ACS2P1-biotin (amino acids 9-23 of SEQ ID NO: 3 ) ( 10 ~,g/ml ) to fetuin or asialofetuin. Inhibition assays were done for 1 hour at room temperature, as described above, and the amount of biotinylated peptide bound to fetuin or asialofetuin (3 ~.g/ml) quantitated by using avidin-peroxidase.
The specificity and relative affinity of ACS2P1-biotin (amino acids 9-23 of SEQ ID NO: 3) for fetuin was determined by performing binding inhibition experiments with the peptides shown in Table I.
Binding inhibition experiments (Table V) indicated that the ACS2P1 peptide (amino acids 9-23 of SEQ ID NO: 3) bound to fetuin with high affinity (ICso = 4.1 ACM; n=2) when the unbiotinylated form of ACS2P1 (amino acids 9-23 of SEQ ID NO: 3) was used as competitor. The peptide S2P2 (SEQ ID NO: 3), an extended version of S2P1 (amino acids 9-23 of SEQ ID N0: 3), was also found to inhibit ACS2P1-biotin (amino acids 9-23 of SEQ ID
NO: 3) binding to fetuin but with a 10-fold decrease in affinity (ICso = 42.5 ~cM) . Both peptides were unable to compete for the binding of ACS2P1-biotin (amino acids 9-23 of SEQ ID NO: 3) to asialofetuin, indicating the importance of sialic acid for high affinity interaction with the peptide. Similar hexapeptide- sequences to those found within the S2P1 (amino acids 9-23 of SEQ ID
NO: 3) and S2P2 (SEQ ID NO: 3) peptides are also present in a number of the peptides from Table I (see underlined segments). Each of these peptides were analyzed for their ability to inhibit ACS2P1-biotin (amino acids 9-23 of SEQ ID NO: 3) binding to fetuin and asialofetuin at peptide concentrations 10-fold higher than the ICSa determined for the S2P1 (amino acids 9-23 of SEQ ID NO: 3) peptide. None of the other peptides examined from Table I had the ability to inhibit binding of ACS2P1-biotin (amino acids 9-23 of SEQ ID NO: 3) to fetuin or asialofetuin to the same extent indicating that the peptide sequence SPYGRC may play a crucial role for binding sialic acid.
This is further confirmed by the inability of the peptides S3P1 (amino acids 9-23 of SEQ ID NO: 6) and S3P2 (SEQ ID No. 6), which possess the strongest homologies to the sequence found in S2P1 (amino acids 9-23 of SEQ ID NO: 3), to inhibit binding even at high concentrations of peptide. The homologous sequence found in the S3 'peptides (GAYGRC) (amino acids 18-23 of SEQ ID NO: 6) lacks the serine amino acid residue, which was shown to be important for forming an important hydrogen bond with the carbonyl of the N-acetyl group in the sialic acid binding site of WGA62.
This indicates that the serine residue found in the S2P1 (amino acids 9-23 of SEQ ID NO: 3) peptide may function in an analogous manner in the binding of S2P1 (amino acids 9-23 of SEQ ID NO: 3) to sialic acid.
'J L' The results of this example are set forth in Table V below:
Table V
Screening of S2 and S3 Peptides from PT for the $ Ability to Inhibit ACS2P1-biotin (amino acids 9-23 of SEQ ID N0:3) Binding PeptideSEQ ID ConcentrationFetuin'Asialo-NO (~,~M) i fetuin' ACS2P1 3 (aa 4.1 -50 +10112 9-23) S2P2 3 42.5 -50 +6t 1 SPYGRC 3 (aa 5520 -24 -54 f 18-23) t2 3 S2P3 4 22.4 +70111 +15111 S2P4 5 (aa 6.0 -8116 +214 1Cr32) S2Pb 5 319 +495 +18116 S3P1 6 (aa 1142 +24512 +410 9-23) 8 1$ S3P2 6 934 +88172 + 12 f S3P3 7 (aa 16.1 -13116 -lO f 10-31 l l ) S3P4 7 6.4 +55126 +2612 S3P5 8 22.5 -1914 -1718 The effect of S2 and S3 PT peptides on the ZO binding of ACS2P1-biotin (amino acids 9-23 of SEQ ID NO: 3 ) ( 0. l~sg) to fetuin or asialofetuin. Negative and positive values refer to percent inhibition or enhancement respectively.
2$ as amino acid Percent Change in Binding Activity--; and Example 7--Iodination of ACS2P1-biotin (amino acids 9-23 of SEQ ID NO. 3) Previous reports have suggested the importance 30 of tyrosine amino acid residues in the binding of PT to sialylated glycoprotein receptors°°~63. These reports were based on the observation that if PT is iodinated by the conventional IODO-GEN procedure (selectively modifies tyrosine residues) without first protecting the binding site for fetuin, the binding activity of PT
was reduced.
5 To determine if the tyrosine residue found in ACS2P1-biotin (amino acids 9-23 of SEQ ID NO: 3) plays a role in the binding activity, the peptide was iodinated by the IODO-GEN procedure and its binding activity compared with the uniodinated peptide.
10 Specifically, ACS2P1-biotin (amino acids 9-23 of SEQ ID
NO: 3) (100 ~cg 0.052 ~mol) in 100 ul of PBS was placed in a 12 x 75-mm glass tube coated with IODO-GEN, and 0.1 mM solution of NaI (50 uL 5 ~mol) was added and gently mixed over a period of 10 minutes at room 15 temperature. The reaction was terminated by removing the mixture from the IODO-GEN tube and the iodinated peptide was purified on a Sephadex G-25 column (1 x 15 cm equilibrated with PBS). Individual fractions were analyzed for the presence of peptide by measuring 20 absorbance at 220 nm, and the concentration was determined by comparing the absorbance with underivatized ACS2P1-biotin (amino acids 9-23 of SEQ ID
NO: 3). A sample of iodinated and underivatized ACS2P1-biotin (amino acids 9-23 of SEQ ID NO: 3) were 25 diluted to a concentration of 10 ~g/ml and analyzed for binding to fetuin coated microtiter wells as described in Example 6. In addition, binding of ACS2P1-biotin (amino acids 9-23 of SEQ ID NO: 3) (10 ~.g/ml) to fetuin, asialofetuin, and asialoagalactofetuin (each at 30 3 ~cg/ml) was done in a similar manner.
The results of this experiment indicate that extensive iodination of ACS2P1-biotin (amino acids 9-23 of SEQ ID NO: 3) reduced binding of the peptide to fetuin by 58 ~ 3% (n=3), which suggests the importance of the tyrosine amino acid in addition to the serine for the binding activity of ACS2P1-biotin (amino acids 9-23 of SEQ ID NO: 3) to terminally linked a-sialic acid(2~6)/3Ga1- and a-sialic acid(2-~3)/3Ga1- structures.
To determine whether the amino acid sequence in the S2 subunit of PT that corresponds to the peptide S2P1 (amino acids 9-23 of SEQ ID NO: 3) plays an actual role in the lectin-like binding activity of PT, the binding specificity of the biotinylated peptide was compared with that of PT using fetuin, asialofetuin, and asialoagalactofetuin. A previous report had determined that the binding of 'z5I-PT to asialofetuin was 53 ~ 7%, while asialoagalactofetuin was 81 ~ 8%
relative to control binding to fetuin~'. This is in good agreement with the results obtained with ACS2P1-biotin (amino acids 9-23 of SEQ ID NO: 3) which showed 45 ~ 9% and 93 ~ 18% (n=3) binding to asialo-and asialoagalactofetuin, respectively, relative to fetuin. These results suggest that this amino acid sequence may contain a portion of a lectin-like binding site in the S2 subunit of PT, which is responsible for the binding results previously observed.
Example 8 Binding assays were carried out essentially as described above using microtiter wells that were coated with 50 ~1 of BSA glycoconjugate (50 ~Cg/ml) in 50 mM
sodium phosphate buffer (pH 6.8) containing 5 mM MgClz nd 15 mM NaN3 for 16 hours at 4°C. The solution was removed by aspiration and replaced with 100 ~.1 of 1%
BSA in PBS containing 0.05% Tween 20 (PEST) and incubated at room temperature for an additional 2-3 hours. The microtiter wells were washed four times with 300 ~,1 of PBST and then replaced with ACS2P1-biotin (amino acids 18-23 of SEQ ID NO. 3) (0.5 ~Cg) in 50 ~1 of PBS. After incubating for 1 hour, the binding reaction was stopped by aspirating the solutions and the plate was washed with PBST (4 X
5 300~u1). Avidin-peroxidase (100 ~C1 of a 1/3000 dilution of a 1 mg/ml solution in PBST) was added and incubated for an additional 1 hour. After washing the wells as described above, the substrate solution (1 mM ABTS in 5 mM citrate buffer, pH 4.2, 10 containing 0.1% hydrogen peroxide, v/v) was added and the plates were incubated for 30 minutes.
Binding assays for each BSA conjugate were done in triplicate, and background binding was measured in.
wells coated with BSA only. The extent of 15 biotinylated peptide binding is expressed as a percentage relative to fetuin as shown in Table VI
below:
Table VI
Binding of ACS2P1-biotin (amino acids 9-23 of SEQ
20 ID NO. 3) and SPYGRC-biotin (amino acids 18-23 of SEQ ID NO. 3) to BSA Conjugates*
Carbohydrate Structure% Binding9o BindingCommon of BSA
Conjugate RelativeRelative Name to to for Fetuin Fetuin Carbohydrat of of ACS2P1- SPYGRC- a Structure biotin biotin (n=3) (n=3) aNeuAc(2-3)(3Ga1(1-4)(3GlcNAc-BSA117112 12017 SLacNAc 25 aNeuAc(2-3)SGaI(1-3)SGIcNAc-BSA7014 97112 SLe' aNeuAc(2-6)pGal(1-4)SGIcNAc-BSA9412 12215 SLacNAc aNeuAc(2-3)(3Ga1(1-4)aGIcNAc-BSA8413 12014 SLe=
(1-3) aFuc 30 aNeuAc(2-3)(3Ga1(1-3)(3GlcNAc-HSA84114 10318 SLe'(C19.9) (I-4) aFuc *Experiments were done by coating 50 ~g/mL BSA-conjugate or fetuin and probed with 0.1 ~g ACS2P1-35 biotin (amino acids 9-23 of SEQ ID NO. 3) or 0.5 ug of SPYGRYC-biotin (amino acids 18-23 of SEQ ID NO.
3) for 1 hour at room temperature.
Example 9 -- Fonaation of a SPYGRC-Streptavidin-Biotin Conjugate Streptavidin (50 fig) in PBS was combined with $ SPYGRC-biotin (amino acids 18-23 of SEQ ID NO. 3) (50 ~1, 200 fig) and PBS (150 ~C1) and mixed for 1 hour at room temperature. The reaction mixture was loaded onto a Sephadex G-25 column (1 x 15 cm that had been equilibrated in PBS) and fractions collected. Protein containing fractions were analyzed by SDS PAGE gel electrophoresis, and the molecular weight was determined. The results were consistent with the formation of a tetravalent complex between peptide and streptavidin. Lectin binding inhibition experiments were carried out as described previously for determination of IC5°'s.
The results are set forth in Table VII below:
Table VII
The Effect of the Peptide SPYGRC or SPYGRC-biotin-Streptavidin Conjugate (amino acids 18-23 of SEQ ID
NO. 3) on the Binding of Biotinylated PT and Lectins to Fetuin*
BiotinylatedConcentration96 ChangesConcentration90 lectin of of SPYGRC (aa in BindingPeptide- Changes 23 of SEQ Activity Streptavidinin Binding ID
N0.3) Peptide Conjugate Activity (/aglml) (mg/ml) PT-b 4.4 +34 f 500 + 13 2 t 11 2$ MAL-b 4.4 -1610 1.0 -SOt3 SNA-b 4.4 -2 t 1 1.2 -50 t WGA-b 4.4 +7t3 64 -SOtI
*Positive signs preceding the numbers in this table indicate enhancement, and negative signs indicate inhibition of binding.
as amino acids Example 10 -- Neutralization of Pertussis Toxin Binding to Chinese Hamster Ovary (CHO) Cell by Synthetic Peptides Confluent monolayers of CHO cells were lifted from plastic tissue culture flasks with 0.25% trypsin and suspended at a concentration of 5 x 10° cells/ml in Ham's F12 media containing 10% fetal bovine serum (FBS). 100 ~,1 of the cell suspension was added to 96 well tissue culture plates and was allowed to establish contact with the plastic for 24 to 48 hours. The expended media was then removed and filtered-sterilized peptide 5-fold dilutions ranging in concentration from 300 to 30 femptogram per ml (80 ~1) in Ham's F12 media containing FBS were added to the CHO cells. 20 ~,1 of a PT solution (final PT concentration 2.7 ng/ml) was then added to the tissue culture wells containing peptide.
The incubation mixtures were gently mixed and incubated at 37°C for 1 hour in a COZ incubator. The incubation mixtures were then removed and replaced with fresh media. The tissue culture plates were then incubated for 24 hours at 37°C, fixed with 100% methanol, and stained with Geimsa stain. Control experiments were done in the absence of peptide alone or in the absence of PT. All determinations were done in triplicate.
Stained CHO cells were then examined for inhibition of characteristic CHO cell clumping which is mediated by pertussis toxin binding and scored as to whether there was greater than 50% inhibition of clumping of CHO
cells relative to control wells in the presence of PT
only. The results reported in Table VIII show the maximum peptide concentration required to cause a 50%
reduction in CHO cell clumping.
Table VIII
CHO Cell Neutralization Experiments Using PT S2 and S3 Peptides*
PT SEQ ID NO Maximal Inhibitory S Peptide Concentration for Greater than 50% Inhibition ACS2P1 3 (aa 9-23) 96 /ml SPYGRC 3 (aa 18-23) 480 pg/ml S3P1 6 (aa 9-23 NI
S3P9a 9 NI
* CHO cell neutralization experiments were carried out using a PT concentration of 2.4 ng/ml. CHO cell (5.4 x 104 cells/ml) were exposed to incubation mixtures containing peptide and PT for 1 hour at room temperature.
Control experiments were done in the absence of peptide.
as amino acid NI Not inhibitory at a peptide concentration of 300 ng/ml.
pg picogram--.
The above data demonstrates that at least certain of the subject peptides would be effective in inhibiting the attachment of bacterial/viral agents and/or their toxins which utilize a-sialic acid(2~6)~Gal- and/or a-sialic acid(2-~3)~BGal-structures as the attachment point on the surface of mammalian cells. Such agents/toxins include known toxins such as pertussis toxin, cholera toxin, etc., and, accordingly, administration of an effective amount of at least one of the subject peptides to a mammal would be effective in inhibiting such attachment.
Examples 11 and 12 below illustrate 'fin, vivo results for the subject peptides.
Example li -- Inhibition of DTH Inflammatory Response DTH inflammatory responses were measured using the mouse footpad swelling assay as described by Smith and Ziola~'. Briefly, groups of Balb/c mice (about 19-20 grams each) were immunized with 100 ~cg of the OVA
antigen containing 20~cg of the adjuvant (DDA --dimethyl-dioctadecylammonium bromide) which also induces a strong inflammatory DTH response. Seven days later, each group of mice was footpad-challenged with 20 ~,g of the OVA antigen (without adjuvant). The resulting inflammatory footpad swelling was measured with a Mitutoyo Engineering micrometer 24 hours after challenge.
To assess the effect of different peptides on the inflammatory DTH response, groups of mice received 100 ~.g of the following peptides: ACS2P1 (2275) (amino acids 9-23 of SEQ ID NO: 3), SPYGRC (2283) (amino acids 18-23 of SEQ ID NO: 3), and S3P9a (SEQ ID NO: 9).
These peptides were injected as a solution into the tail vein 5 hours after challenge. Control groups were left untreated or received 100 ~cL of phosphate-buffered saline (PBS). The results of this experiment are shown in Figure 2 which illustrates that the peptides employed were effective in reducing a DTH response in mice.
Example 12 -- Persistence of Suppression of the DTH
Inflammatory Response at 2 Weeks After Challenge Identical groups of mice treated with the peptides in Example 11 above were re-challenged with OVA antigen 2 weeks after primary immunization. Untreated controls responded with the usual degree of footpad swelling, whereas all other groups showed reduced footpad swelling. Specifically, these results are set forth in Figure 3 which illustrates reduction in the degree of footpad swelling in mice previously treated with the subject peptides.
In addition to providing suppression of antigen induced inflammation in a sensitized mouse, the above data demonstrate that treatment with the subject peptides as per this invention also imparts tolerance to still later challenges from the same antigen.
In view of the fact that the immune system of mice serves as a good model for the immune system of humans, the above data demonstrates that the subject peptides would be effective in suppressing cell-mediated immune responses in humans, and, when the cell-mediated immune response is to an antigen, this data also shows that the subject peptides would also impart tolerance to later challenges to the human of that antigen.
-b i -TABLE IX
Initial screening of synthetic peptides for inhibitory activity Peptide ConcentrationChanges (mg/ml) in binding activity PT/fetuin'MAL/fetuinSNA/fetuinWGA/fetuin ACS2P1 1.0 1.8910.03I.16t0.090.9710.010.7810.06 NSS2P1 2.4 1.1810.031.0310.021. l O 1.0310.02 10.02 SPYGRC 4.4 1.3410.020.8410.000.9810.021.0710.03 GSYRPC 2.2 1.4810.210.9510.160.9610.021.0910.01 SPYGYC 1.0 2.8510.161.2110.081.0710.151.0910.07 SPWGRC 1.0 1.7510.531.3910.131.0010.021.11 10.01 10 SPFGRC 1.0 1.3210.081.0610.121.0010.011.0110.01 2420 1.0 1.79' 1.43' 1.11' 2.1' ' Biotinylated lectin/glycoprotein bound to microtiter wells. Values greater than 1 in the table indicate enhancement while numbers lcss than 1 indicate inhibition of binding.
° Previously reported values.
IS ' One determination only.
Example 13 -- Binding Inhibition Assays (Initial Peptide Screening).
Microtiter wells were coated with 100 ~cl of fetuin or asialofetuin (50 ~g/ml) in 50 Mm sodium 20 phosphate buffer (pH 6.8) containing 5 mM MgCl2 and 15 mM NaN3, for 16 hours at 4°C. The solution was removed by aspiration and replaced with 100 ~.1 of 1% BSA in PBS containing 0.05% Tween 20 (PBST).
After incubation for 2-4 hours at room temperature, 25 the microtiter wells were washed four times with 300 ul of PBST. Peptides ranging in concentration from 1.0 to 4.4 mg/ml in PBS (40 ~1) were added to each well, and PT-biotin (10 ~.1 containing 10 ng in PBS) was then added to the microtiter wells. After 30 incubating for 1 hour, the binding reaction was stopped by aspirating the solutions, and the plate was washed with PBST (300 ~,1).
Horseradish peroxidase-conjugated avidin (100 ~cl, 1/3000 dilution in PEST to a concentration of 0.3 S ~cg/ml) was then added to the wells, and the plates were incubated at ambient temperature for 1 hour. After washing the wells as described above, the substrate solution (1 mM ABTS in 5 mM citrate buffer, pH 4.2, containing 0.1% hydrogen peroxide, v/v) was added, and the plates were incubated for 30 minutes. Color development was recorded at 405 nm using a Titertek Multiskan MC plate reader. Maximum binding was determined in the absence of peptide, and background binding was measured in wells coated with BSA only.
Binding assays for each peptide were done in duplicate.
Binding inhibition experiments utilizing SNA-, WGA-, and MAL-biotin were performed as described above using 10 ng of each biotinylated lectin in PBS.
The panel of 8 peptides was assayed for the ability to inhibit PT-, WGA-, MAL-, and SNA-biotin binding to fetuin or asialofetuin. The biotinylated plant lectins have proved to be useful controls, since we have previously showed that these lectins possess similar binding specificities as pT2s.ae_ The results from this example are set forth in Table IX above. These results indicate that peptides based on the SPX,GXZC (SEQ ID NO:1) motif inhibited PT
biotin binding.
-v~-TABLE X
Inhibition of biotinylated peptide binding to fetuin by simple saccharides' Saccharide Changes in binding activity SPWGRC-biotinSPFGRC-biotinSPYGYCGFGAEY-(n = 3) (n = 3) biotin (n = 3) Sialic acid 0.7110.11 1.0910.03 1.21 f0.09 Sialylactose 0.650.05 0.92 f 1.0810.03 0.04 Lactose 0.960.12 0.78 t 1.0610.00 0.05 N-Acetylglucosaminc0.8210.10 1.0710.14 1.1010.25 ' Inhibition experiments were carried out as outlined in methods using 0.5 ~cg of biotinylated peptides respectively. Binding values greater than 1 in this table indicate enhancement and values less than 1 indicate inhibition of binding to fetuin. Concentration of saccharides used were 8 mM .
Example 14 -- Binding Inhibition Assays Using Oligosaccharides In order to assess the binding to a-sialic acid ( 2-~6 ) /3Ga1- and a-sialic acid ( 2~3 ) (3Ga1-20 structures by certain peptides, inhibition experiments with simple saccharides were conducted to determine if the interaction was carbohydrate-dependent. The simple sugars chosen were sialic acid, sialyllactose from bovine colostrum (contains 25 a mixture of a(2-~6) and a(2~3) linked sialic acid structures), lactose and N-acetyl-glucosamine at a concentration of 8mM in PBS. Inhibition assays were done for 1 hour as described above (e. g., Example 2) at room temperature, and the amount of biotinylated peptide bound to fetuin (3~.g/ml) guantitated by using avidin-peroxidase. The pH of free sialic acid was carefully monitored and adjusted to physiological pH
values with dilute sodium hydroxide. The results of this example are set forth in Table X above.
The above results indicate that some of the peptides tested were able to bind to terminally linked a-sialic acid(2~6)/3Gal- and a-sialic acid(2~3)~3Ga1-structures.
iA3L~ XI
9irrdirrg of biorirrylated pepridu ro sialylared BSA conj~garesa Carbohydrate Structure of Binding Relative Binding Relative Biading Relative BSA Conjugate to Fetuin of to Fetuin of to Fetuin of SPWGRC-biotin SPFGRC-biotin SPYGYCGFG-AEY-biotin (n ~ 3) (n = 3) (n . 3) aNeuAc(2-3)~iGal(1-4)~iGlcNAc-BSA0.49 t 0.070.63 0.270.27 0.12 aNeuAc(2-3)~Gal(1-3)~GlcNAc-BSA0.43 t O.IO0.36 t 0.25 t 0.17 0.04 ocNeuAc(2-6)~Gal(1-4)~GId~IAc-BSA0.07 0..030.29 0.050.28 t 0.11 aNeuAc(2-3)~Gal(1-4)~GlcNAc-BSA0.67 t O.I40.42 0.150.33 0.04 (1-3) ocFuc .
aNeuAc(2-3)~iGal(1-3)~iGlcNAc-BSA0.77 ~ 0.190.52 O.I80.51 0.12 (1~) ctFuc a F.~cperiment~ weze done by coating with 5.0 ~tg/ml BSA-coajugate or fetuin and probed with 0.5 ltg biotinylated peptide for lh. 'Ihe binding of biotinylatcd peptide to fetuin is set at a value of 1Ø
Example 15 Binding assays were carried out essentially as described above using microtiter wells that were coated with 50 ~1 of BSA glycoconjugate (50 ~.g/ml) in 50 mM
sodium phosphate buffer (pH 6.8) containing 5 mM MgCl, nd 15 mM NaN3 for 16 hours at 4°C. The solution was removed by aspiration and replaced with 100 ~cl of 1%
BSA in PBS containing 0.05% Tween 20 (PBST) and incubated at room temperature for an additional 2-3 hours. The microtiter wells were washed four times with 300 ~cl of PBST and then replaced with 0.5 microgram of biotinylated peptide. After incubating for 1 hour, the binding reaction was stopped by aspirating the solutions and the plate was washed with PBST (4 X 300,1). Avidin-peroxidase (100 ~.1 of a 1/3000 dilution of a 1 mg/ml solution in PEST) was added and incubated for an additional 1 hour. After washing the wells as described above, the substrate solution (1 mM
ARTS in 5 mM citrate buffer, pH 4.2, containing 0.1°s hydrogen peroxide, v/v) was added and the plates were incubated for 30 minutes. Binding assays for each BSA
conjugate were done in triplicate, and background binding was measured in wells coated with BSA only.
The extent of biotinylated peptide binding is expressed as a ratio relative to fetuin as shown in Table XI
above:
Example 16 -- Inhibition of DTH Inflammatory Response DTH inflammatory responses were measured using the mouse footpad swelling assay as described by Smith and Ziola~°. Briefly, groups of Balb/c mice (about 19-20 grams each) were immunized with 100 ~,g of the OVA
antigen containing 20~cg of the adjuvant (DDA --dimethyl-dioctadecylammonium bromide) which also induces a strong inflammatory DTH response. Seven days later, each group of mice was footpad-challenged with 20 ~.g of the OVA antigen (without adjuvant). The resulting inflammatory footpad swelling was measured with a Mitutoyo Engineering micrometer 24 hours after challenge.
To assess the effect of different peptides on the inflammatory DTH response, groups of mice received 100 ~,g of the following peptides: ACS2P1 (2275) (amino acids 9-23 of SEQ ID NO. 3), SPFGRC (2418), SPWGRC
(2416), AcSPYGYCGFGAEY-CONHZ (2420) SPYGYC(2365), SPYGRC
(amino acids 18-23 of SEQ ID. NO: 3) (2283), NSS2P1(2295), and GSYRPC(2294). These peptides were injected as a solution into the tail vein 5 hours after challenge. Control groups were left untreated or received 100 ~,L of phosphate-buffered saline (PBS).
The results of this experiment are shown in Figures 4 and 6 which illustrate that the peptides employed were effective in reducing a DTH response in mice.
Example 17 -- Persistence of Suppression of the DTH
Inflammatory Response at 2 Weeks After Challenge Identical groups of mice treated with the peptides in Example 16 above were re-challenged with OVA antigen two weeks or 15 days after primary immunization.
Untreated controls responded with the usual degree of footpad swelling, whereas all other groups showed reduced footpad swelling. Specifically, these results are set forth in Figures 5 and 7 which illustrate reduction in the degree of footpad swelling in mice previously treated with the subject peptides.
In addition to providing suppression of antigen induced inflammation in a sensitized mouse, the above data demonstrate that treatment with the subject peptides as per this invention also imparts tolerance to still later challenges from the same antigen.
In view of the fact that the immune system of mice serves as a good model for the immune system of humans, the above data demonstrates that the subject peptides would be effective in suppressing cell-mediated immune responses in humans, and, when the cell-mediated immune response is to an antigen, this data also shows that the subject peptides would also impart tolerance to later challenges to the human of that antigen.
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT:
(A) NAME: ALBERTA RESEARCH COUNCIL
(B) STREET: 250 KARL CLARK ROAD
(C) CITY: EDMONTON
(D) STATE: ALBERTA
(E) COUNTRY: CANADA
(F) ZIP: T6H 5X2 (G) TELEPHONE: 403-450-5111 (H) TELEFAX: 403-461-2651 (ii) TITLE OF INVENTION: ANTI-INFLAMMATORY, TOLEROGENIC AND
IMMUNOINHIBITING PROPERTIES OF CARBOHYDRATE
BINDING-PEPTIDES
(iii) NUMBER OF SEQUENCES: 12 (iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: BLAKE, CASSELS & GRAYDON
(B) STREET: BOX 25, COMMERCE COURT WEST
(C) CITY: TORONTO
(D) STATE: ONT
(E) COUNTRY: CANADA
(F) ZIP: M5L lA9 (v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version #1.25 (vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE:
(C) CLASSIFICATION:
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: US 07/995,503 (B) FILING DATE: 21-DEC-1992 (vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: US 07/956,043
11. S3PI 9-23 PKALFTQQGGAYGRC 6 (aa 9 ~3) 12. S3P2 I--~_3VAPG1V1PPKALFTQQGGAYGRC 6 1$ 13. S3P3 87-108CITT1YKTGQPAADHYYSKVTA 7 (aa 10-31) 14. S3P4 78-108AGFIYRETFCITT1YKTGQPAADHYYSKVTA7 15. S3P5 134-154CASPYEGRYRDMYDALRRLLY g 16. S3P9a 110-1?7RLLASTNSRLCAVFVRDG 9 17. S2fWGA)k- PQEQITQHGSQYGYC 10 2O 18. S2fWGA)-b- BIOTIN-PQEQ1TQHGSQYGYC 10 19. WGA(63-73)- SQYGYCGFGAEY 11 20.WGA(62-73)-b- BIOTIN-SQYGYCGFGAEY 11 * Underlined sequences correspond to sequences found in the PT
peptide that showed homology to the sequence SQYGHC (SEQ ID
2$ N0:12) found in the binding site of WGA isolectin 2 (24).
~t PT S2P1 sequence (amino acids 9-23 of SEQ ID N0:3) inserted with the WGA hexapeptide sequence (SQYGYC) (amino acids 10-15 of SEQ ID NO:10) as amino acid b biotin The above peptides were tested for their ability to bind to terminally linked a-sialic acid(2->6)~3Ga1- and a-sialic acid(2-->3)/3Gal-structures in the examples below. In these examples, the peptides were first screened for their ability to inhibit binding of different lectins (PT, SNA, and MAL) known to bind to fetuin, a carbohydrate containing multiple copies of the aNeuSAc(2-->3)~3Ga1(1->4)~3Glc- structure.
Example 2 -- Binding Inhibition Assays (Initial Peptide Screening) Microtiter wells were coated with 100 ~cl of fetuin or asialofetuin (50 ~cg/ml) in 50 mM sodium phosphate buffer (pH 6.8) containing S mM
MgCh and 15 mM NaN3 for 16 hours at 4°C. The solution was removed by aspiration and replaced with 100 ~,1 of 1 % BSA in PBS containing 0.05 % Tween 20 (PBST). After incubation for 2-4 hours at room temperature, the microtiter wells were washed four times with 300 ~.1 of PBST. Peptides ranging in concentration from 0.5 to 4.5 mg/ml in PBS
(40 ~cl) were added to each well, and PT-biotin (IO ~,1 containing 10 ng in PBS) was then added to the microtiter wells. After incubating for 1 hour, the binding reaction was stopped by aspirating the solutions, and the plate was washed with PBST (300 ~.1). Horseradish peroxidase-conjugated avidin (100 ~cl, 1/3000 dilution in PBST to a concentration of 0.3 ~,g/ml) was then added to the wells, and the plates were incubated at ambient temperature for 1 hour. After washing the wells as described above, the substrate solution (1 mM ABTS in 5 mM citrate buffer, pH 4.2, containing 0.1 % hydrogen peroxide, v/v) was added, and the plates were incubated for 30 minutes. Color development was recorded at 405 nm using a Titertek Multiskan MC plate reader. Maximum binding was determined in the absence of peptide, and background binding was measured in wells coated with BSA only. Binding assays for each peptide were done in duplicate. Binding inhibition experiments utilizing SNA-, WGA-, and MAL-biotin were performed as described above using 10 ng of each biotinylated lectin in PBS.
The panel of 20 peptides was assayed for the ability to inhibit PT-, WGA-, MAL-, and SNA-biotin binding to fetuin or asialofetuin. The biotinylated plant lectins have proved to be useful controls, since we have previously showed that these lectins possess similar binding specificities as P'If'-~''6. The results from this example are set forth in Table II below.
These results indicate that peptides S2P3 (SEQ ID N0:4) and S2P6 (SEQ
ID N0:5) inhibited PT biotin binding by 15-20% both to fetuin as well as to asialofetuin. Two additional peptides derived from the S3 subunit [S3P3 (amino acids 10-31 of SEQ ID N0:7) and S3P5 (SEQ ID NO:B)]
were found to reduce binding of PT to asialofetuin only. The majority of the other peptides displayed in Table II exhibited either marginal inhibition or enhancement in binding of biotinylated PT to fetuin or asialofetuin. Peptides S2P1 (amino acids 9-23 of SEQ ID N0:3), ACS2P1 (amino acids 9-23 of SEQ ID N0:3) and S2P2 (SEQ ID N0:3) showed nearly a 2-fold enhancement of PT-biotin binding relative to control experiments done in the absence of peptide.
Peptide S2P3 (SEQ ID N0:4) was found to inhibit binding to fetuin of all the biotinylated lectins. However, it is noted that peptides S2P1 (amino acids 9-23 of SEQ ID N0:3) and S2P2 (SEQ ID N0:3), which showed enhancement of binding of PT to fetuin, inhibited WGA
binding activity.
Table II
Percent Changes in Binding Activity peptideSEQ ID Coocea-PT/ MAL SNA/ WGA/ PT/
NO entioo feotm fetuinfetuinfeoun ~;"b.
(me/~.J feo~
S2P1 3(u9-23)1.2 +8514 +Bt4 -Sf1 -1812 +113(1) ACS2P13 (u 1.0 +8913 +1619 -311 -2216 +190123 9-23) 52 3 (u 1.0 +68 +3211 +112 +7 ND
(14-23)14-23) f f S2P2 3 1.1 +5814 +1412 -Itl -1512 +33(1) S2P3 4 0.31 2110 -1812 1314 -5618 -13(1) S2P4 3 (u 0.98 +14122+2(1) +3(1) +1311 -3(1) 16.32) 1~ S2P6 3 0.85 -286 +18116+212 +370 -18(1) S3P1 6 (u 3.2 +54 +416 -211 -9 +d3 9-23) f f f S3P2 6 3.1 +77159+12116+313 +914 +82 f S3P3 7 (u 4.3 +19167+13113+310 +bt3 -2817 10-31) S3P4 7 3.3 +1212 +7f1 +I(1) +1311 +73130 is S3P3 8 0.48 +1416 +316 0(1) +3318 -26110 S3P9r,9 1.7 +328110+1816 ~1t3 ND ND
SPYGRC3 (u 4.4 +34 -1610 212 +713 +79 18-23) f S2(WGA)10 1.0 +79115+2213 Otl -4f3 ND
WGA 11 1.0 -3215 If4 -312 -9f1 ND
(62-73) * Biotinylated lectin/glycoprotein bound to microtiter wells. Positive signs preceding the numbers in this table indicate enhancement and negative signs indicate inhibition of 25 binding.
(1) One determination only.
as amino acid ND Not determined.
-t~-Exam lp a 3 -- Binding Inhibition Assays (Determination of IC 50 Values) Peptides found to have inhibitory activity in the initial screening experiments were further analyzed in binding inhibition experiments to determine the concentration of peptide that was required to reduce binding by 50% (ICS values).
Inhibition experiments were performed by a method similar to that described in Example 2 with 2-fold dilutions of PT peptide in PBS, except that microtiter wells were coated with 3 ~,g/ml fetuin or asialofetuin.
Binding assays for each inhibitor concentration were done at least in duplicate, and the average value varied less than 15 % . The concentration of peptide required for 50% inhibition (ICso) was determined by plotting the amount of binding observed in the presence of peptide inhibitor as a percent of the maximum binding achieved without inhibitor.
Two of the peptides derived from the S2 subunit were able to inhibit PT-biotin binding to fetuin at submillimolar concentrations, but they were unable to inhibit PT-biotin binding to asialofetuin in subsequent experiments (Table III). The two peptide sequences from the S3 subunit (S3P3 [amino acids 10-31 of SEQ ID N0:7] and S3P5 [SEQ ID NO: 8]) were found to inhibit PT-biotin's interaction with asialofetuin in a concentration-dependent manner, but their ICS values were above the solubility limits of the peptide in PBS. The peptide S2P3 (SEQ ID NO:
4) was also very active at inhibiting the interaction of MAL- and WGA-biotin with fetuin. Upon closer examination this peptide proved to be non-inhibitory for SNA-biotin. Two additional peptides from the S2 subunit (S2P1 [amino acids 9-23 of SEQ ID NO: 3] and S2P2 [SEQ ID
NO: 3]) were active at inhibiting WGA-biotin binding at millimolar concentrations, suggesting that these peptide sequences may also be important for interacting with oligosaccharide in the binding site of WGA.
Table III
Concentrations of PT Peptides and WGA Peptides Resulting in 50% Inhibition of Biotinylated PT and Lectins Binding to Fetuin or Asialofetuin Biotinylated Lectin Peptide SEQ ID NO ICb(mM) (glycoproteinl PT-b (fetuin) S2P3 4 0.19+0.09 PT-b (fetuin) S2P6 5 0.'_'310.06 WGA-b (fetuin) ACS2P1 3 (aa 9 _3) 3..50.45*
WGA-b (fetuin) S2P3 3 SI
WGA-b (fetuin) S3P3 4 0.1410.03 MAL-b (fetuin) S2P3 4 0.860.0'_' PT-b (asialofetuin) S3P3 7 (aa 10-31) SI
IS PT-b (asialofetuin) S3P5 8 SI
SNA-b (fetuin) S3P9a 9 2.74+0.86 PT-b (fetuin) WGAl6''-73)I1 1.50 WGA-b (fetuin) WGA(62-73)11 3.40 SI Slightly inhibitory, but the concentration of peptide required for 50% inhibition of binding is above the solubility limit of the peptide.
* Unacetylated form of S2P1 (amino acids 9-23 of SEQ
ID NO: 3) inhibited to the same extent as the acetylated form.
as amino acid Since three of the peptides derived from the S2 subunit of PT were able to inhibit WGA binding, the amino acid sequences which constitute the sialic acid binding site of WGA were closely examined to determine if there were any homologies with the inhibitory PT S2 subunit peptide sequences. One short 6-amino acid sequence (SQYGHC) (SEQ ID NO: 12) corresponding to amino acids 62-67 in WGA isolectin 2 displayed reasonable homology with a sequence found both in the S2P1 (amino acids 9-23 of SEQ ID NO: 3) and S2P2 peptides (SPYGRC, amino acids 18-23 of SEQ ID NO: 3) from PT. This short sequence in WGA is responsible for binding with the carbonyl of the N-acetyl group of sialic acid or N-acetyl-glucosamine (i.e., serine 62) through a hydrogen bond. Non-polar interactions between the aromatic side chains of tyrosine 64 as well as histidine 66 interact with the glycerol side chain of sialic acid and the pyranose ring of sialic acid or N-acetyl-glucosamine, respectively°Z. The other inhibitory peptide, S2P3 (SEQ ID N0: 4), did not display any good homology with sequences responsible for interacting with sialic acid in WGA. This indicates that other motifs may also be functionally important for interaction with sialic acid.
Example 4 -- Binding Inhibition Studies Utilizing Biotinylated and Acetylated S2P1 (amino acids 9-23 of SEQ ID NO: 3) From the initial peptide screening results (Table II), peptides S2P1 (amino acids 9-23 of SEQ ID NO: 3) and S2P2 (SEQ ID NO: 4) showed a 2-fold enhancement in binding of PT-biotin to fetuin relative to control experiments. One possible explanation for the enhancement may be the ability of the peptide to form a bridge between PT-biotin and fetuin. In order for the peptide to act as a bridging molecule, the peptide must contain both a recognition site for fetuin as well as a sequence which binds to PT itself. To answer this question an acetylated and biotinylated form of the peptide S2P1 (amino acids 9-23 of SEQ ID NO: 3) (biotinylated at the terminal proline) was prepared to determine if we could measure direct binding of the peptide to PT and fetuin.
Binding assays were done as described in Example 2 in PBS by using ACS2P1-biotin (amino acids 9-23 of SEQ
ID NO: 3) at a concentration of 10 ug/ml in PBS.
Assays were carried out for 1 hour at room temperature, and the amount of biotinylated peptide bound to fetuin was determined by using avidin-peroxidase.
From these direct binding studies, a concentration-dependent binding both to fetuin as well as to PT immobilized in microtiter wells was observed.
Furthermore, the binding of ACS2P1-biotin (amino acids 9-23 of SEQ ID NO: 3) to PT could be inhibited by fetuin (ICso = 50 ~M; n=2) indicating that the biotinylated peptide bound at or adjacent to the fetuin binding site in PT.
In view of the above, it was concluded that because of potential bridging by the peptide between the lectin and the fetuin, any of the peptide sequences set forth in Table I which caused reduction in the binding of any of the lectins to fetuin were able to bind to terminally linked a-sialic acid(2-~6)/3Ga1- and a-sialic acid(2~3)/3Ga1- structures.
Example 5 -- Binding Inhibition Assays Using Oligosaccharides to Inhibit the Binding of ACS2P1-biotin (amino acids 9-23 of SEQ ID
NO: 3) The binding results obtained for ACS2P1-biotin (amino acids 9-23 of SEQ ID NO: 3) described in Example 4 above indicate a direct involvement of carbohydrate moiety with the lectin through a biotinylated peptide bridge.
In order to assess the binding to a-sialic acid(2~6)/3Ga1- and a-sialic acid(2-~3)~iGal- structures by this peptide as well as the hexapeptide SPYGRC
(amino acids 18-23 of SEQ ID NO: 3) which only exhibited reduction in the binding of one lectin to fetuin (Table II), inhibition experiments with simple saccharides were conducted to determine if the interaction was carbohydrate-dependent. The simple sugars chosen were sialic acid, sialyllactose from' bovine colostrum (contains a mixture of a(2~6) and a(2-~3) linked sialic acid structures), lactose, and N-acetyl-glucosamine.
Binding inhibition assays were done using sialic acid, sialyllactose, lactose, or N-acetyl-glucosamine at a concentration of 8 mM in PBS. Inhibition assays were done for 1 hour as described above (e. g., Example 2) at room temperature, and the amount of biotinylated peptide bound to fetuin (3~,g/ml) quantitated by using avidin-peroxidase. The pH of free sialic acid was carefully monitored and adjusted to physiological pH
values with dilute sodium hydroxide. The results of this example are set forth in Table IV below:
Table IV
Inhibition of Binding of ACS2P1-biotin (amino acids 9-23 of SEQ ID NO. 3) and SPYGRC-biotin (amino acids 18-23 of SEQ ID NO. 3) to Fetuin by Simple Saccharides*
Saccharide Percent Changes in Binding Activity ACS2P1-biotinSPYGRC-biotin n=3 n=3 Sialic acid -1916 +918 Sial llactose -4613 -2416 Lactose +7417 +20111 N-Acet 1 lucosamine-102 +615 Sucrose Ot3 ND
* Inhibition experiments were carried out as outlined above using O.l~Cg or 0.5ug of ACS2P1-biotin (amino acids 9-23 of SEQ ID NO. 3) and 15 SPYGRC-biotin (amino acids 18-23 of SEQ ID
N0.3) respectively.
Positive signs preceding the number in this table indicate enhancement and negative signs indicate inhibition of binding to fetuin.
ND Not determined The above results indicate that both peptides tested were able to bind to terminally linked a-sialic acid ( 2-~6 ) QGal- and a-sialic acid ( 2-~3 ) ~iGal-structures. Control experiments indicated that sialic acid was necessary for high affinity interaction since lactose failed to reduce the binding of ACS2P1-biotin (amino acids 9-23 of SEQ
ID NO. 3) on N-acetyl-glucosamine was also found to marginally inhibit the binding of biotinylated 30 peptide (10 ~ 2%, n=3) suggesting that the ACS2P1-biotin (amino acids 9-23 of SEQ ID NO. 3) may have weak affinity for the N-acetylglucosamine consistent with the finding described below, which shows enhanced binding of the peptide to asialoagalactofetuin relative to asialofetuin. This is also in good agreement with previous results which indicate PT's binding specificities to be similar to those of WGA'°~SZ.
In view of the above, it was concluded that any of the peptides which inhibit the binding of fetuin to any of the lectins set forth in Table II above are effective in binding to terminally linked a-sialic acid ( 2-~6 ) ~3Ga1- and/or a-sialic acid ( 2-~3 ) ~BGal-structures. Such peptides are set forth in Figure 1.
Example 6 -- Screening of Subunit S2 and S3 Peptides from PT for the Ability to Inhibit ACS2P1-biotin Binding to Fetuin and Asialofetuin Binding inhibition assays were conducted using subunit S2 and S3 peptides from PT in PBS as competitors for binding of ACS2P1-biotin (amino acids 9-23 of SEQ ID NO: 3 ) ( 10 ~,g/ml ) to fetuin or asialofetuin. Inhibition assays were done for 1 hour at room temperature, as described above, and the amount of biotinylated peptide bound to fetuin or asialofetuin (3 ~.g/ml) quantitated by using avidin-peroxidase.
The specificity and relative affinity of ACS2P1-biotin (amino acids 9-23 of SEQ ID NO: 3) for fetuin was determined by performing binding inhibition experiments with the peptides shown in Table I.
Binding inhibition experiments (Table V) indicated that the ACS2P1 peptide (amino acids 9-23 of SEQ ID NO: 3) bound to fetuin with high affinity (ICso = 4.1 ACM; n=2) when the unbiotinylated form of ACS2P1 (amino acids 9-23 of SEQ ID NO: 3) was used as competitor. The peptide S2P2 (SEQ ID NO: 3), an extended version of S2P1 (amino acids 9-23 of SEQ ID N0: 3), was also found to inhibit ACS2P1-biotin (amino acids 9-23 of SEQ ID
NO: 3) binding to fetuin but with a 10-fold decrease in affinity (ICso = 42.5 ~cM) . Both peptides were unable to compete for the binding of ACS2P1-biotin (amino acids 9-23 of SEQ ID NO: 3) to asialofetuin, indicating the importance of sialic acid for high affinity interaction with the peptide. Similar hexapeptide- sequences to those found within the S2P1 (amino acids 9-23 of SEQ ID
NO: 3) and S2P2 (SEQ ID NO: 3) peptides are also present in a number of the peptides from Table I (see underlined segments). Each of these peptides were analyzed for their ability to inhibit ACS2P1-biotin (amino acids 9-23 of SEQ ID NO: 3) binding to fetuin and asialofetuin at peptide concentrations 10-fold higher than the ICSa determined for the S2P1 (amino acids 9-23 of SEQ ID NO: 3) peptide. None of the other peptides examined from Table I had the ability to inhibit binding of ACS2P1-biotin (amino acids 9-23 of SEQ ID NO: 3) to fetuin or asialofetuin to the same extent indicating that the peptide sequence SPYGRC may play a crucial role for binding sialic acid.
This is further confirmed by the inability of the peptides S3P1 (amino acids 9-23 of SEQ ID NO: 6) and S3P2 (SEQ ID No. 6), which possess the strongest homologies to the sequence found in S2P1 (amino acids 9-23 of SEQ ID NO: 3), to inhibit binding even at high concentrations of peptide. The homologous sequence found in the S3 'peptides (GAYGRC) (amino acids 18-23 of SEQ ID NO: 6) lacks the serine amino acid residue, which was shown to be important for forming an important hydrogen bond with the carbonyl of the N-acetyl group in the sialic acid binding site of WGA62.
This indicates that the serine residue found in the S2P1 (amino acids 9-23 of SEQ ID NO: 3) peptide may function in an analogous manner in the binding of S2P1 (amino acids 9-23 of SEQ ID NO: 3) to sialic acid.
'J L' The results of this example are set forth in Table V below:
Table V
Screening of S2 and S3 Peptides from PT for the $ Ability to Inhibit ACS2P1-biotin (amino acids 9-23 of SEQ ID N0:3) Binding PeptideSEQ ID ConcentrationFetuin'Asialo-NO (~,~M) i fetuin' ACS2P1 3 (aa 4.1 -50 +10112 9-23) S2P2 3 42.5 -50 +6t 1 SPYGRC 3 (aa 5520 -24 -54 f 18-23) t2 3 S2P3 4 22.4 +70111 +15111 S2P4 5 (aa 6.0 -8116 +214 1Cr32) S2Pb 5 319 +495 +18116 S3P1 6 (aa 1142 +24512 +410 9-23) 8 1$ S3P2 6 934 +88172 + 12 f S3P3 7 (aa 16.1 -13116 -lO f 10-31 l l ) S3P4 7 6.4 +55126 +2612 S3P5 8 22.5 -1914 -1718 The effect of S2 and S3 PT peptides on the ZO binding of ACS2P1-biotin (amino acids 9-23 of SEQ ID NO: 3 ) ( 0. l~sg) to fetuin or asialofetuin. Negative and positive values refer to percent inhibition or enhancement respectively.
2$ as amino acid Percent Change in Binding Activity--; and Example 7--Iodination of ACS2P1-biotin (amino acids 9-23 of SEQ ID NO. 3) Previous reports have suggested the importance 30 of tyrosine amino acid residues in the binding of PT to sialylated glycoprotein receptors°°~63. These reports were based on the observation that if PT is iodinated by the conventional IODO-GEN procedure (selectively modifies tyrosine residues) without first protecting the binding site for fetuin, the binding activity of PT
was reduced.
5 To determine if the tyrosine residue found in ACS2P1-biotin (amino acids 9-23 of SEQ ID NO: 3) plays a role in the binding activity, the peptide was iodinated by the IODO-GEN procedure and its binding activity compared with the uniodinated peptide.
10 Specifically, ACS2P1-biotin (amino acids 9-23 of SEQ ID
NO: 3) (100 ~cg 0.052 ~mol) in 100 ul of PBS was placed in a 12 x 75-mm glass tube coated with IODO-GEN, and 0.1 mM solution of NaI (50 uL 5 ~mol) was added and gently mixed over a period of 10 minutes at room 15 temperature. The reaction was terminated by removing the mixture from the IODO-GEN tube and the iodinated peptide was purified on a Sephadex G-25 column (1 x 15 cm equilibrated with PBS). Individual fractions were analyzed for the presence of peptide by measuring 20 absorbance at 220 nm, and the concentration was determined by comparing the absorbance with underivatized ACS2P1-biotin (amino acids 9-23 of SEQ ID
NO: 3). A sample of iodinated and underivatized ACS2P1-biotin (amino acids 9-23 of SEQ ID NO: 3) were 25 diluted to a concentration of 10 ~g/ml and analyzed for binding to fetuin coated microtiter wells as described in Example 6. In addition, binding of ACS2P1-biotin (amino acids 9-23 of SEQ ID NO: 3) (10 ~.g/ml) to fetuin, asialofetuin, and asialoagalactofetuin (each at 30 3 ~cg/ml) was done in a similar manner.
The results of this experiment indicate that extensive iodination of ACS2P1-biotin (amino acids 9-23 of SEQ ID NO: 3) reduced binding of the peptide to fetuin by 58 ~ 3% (n=3), which suggests the importance of the tyrosine amino acid in addition to the serine for the binding activity of ACS2P1-biotin (amino acids 9-23 of SEQ ID NO: 3) to terminally linked a-sialic acid(2~6)/3Ga1- and a-sialic acid(2-~3)/3Ga1- structures.
To determine whether the amino acid sequence in the S2 subunit of PT that corresponds to the peptide S2P1 (amino acids 9-23 of SEQ ID NO: 3) plays an actual role in the lectin-like binding activity of PT, the binding specificity of the biotinylated peptide was compared with that of PT using fetuin, asialofetuin, and asialoagalactofetuin. A previous report had determined that the binding of 'z5I-PT to asialofetuin was 53 ~ 7%, while asialoagalactofetuin was 81 ~ 8%
relative to control binding to fetuin~'. This is in good agreement with the results obtained with ACS2P1-biotin (amino acids 9-23 of SEQ ID NO: 3) which showed 45 ~ 9% and 93 ~ 18% (n=3) binding to asialo-and asialoagalactofetuin, respectively, relative to fetuin. These results suggest that this amino acid sequence may contain a portion of a lectin-like binding site in the S2 subunit of PT, which is responsible for the binding results previously observed.
Example 8 Binding assays were carried out essentially as described above using microtiter wells that were coated with 50 ~1 of BSA glycoconjugate (50 ~Cg/ml) in 50 mM
sodium phosphate buffer (pH 6.8) containing 5 mM MgClz nd 15 mM NaN3 for 16 hours at 4°C. The solution was removed by aspiration and replaced with 100 ~.1 of 1%
BSA in PBS containing 0.05% Tween 20 (PEST) and incubated at room temperature for an additional 2-3 hours. The microtiter wells were washed four times with 300 ~,1 of PBST and then replaced with ACS2P1-biotin (amino acids 18-23 of SEQ ID NO. 3) (0.5 ~Cg) in 50 ~1 of PBS. After incubating for 1 hour, the binding reaction was stopped by aspirating the solutions and the plate was washed with PBST (4 X
5 300~u1). Avidin-peroxidase (100 ~C1 of a 1/3000 dilution of a 1 mg/ml solution in PBST) was added and incubated for an additional 1 hour. After washing the wells as described above, the substrate solution (1 mM ABTS in 5 mM citrate buffer, pH 4.2, 10 containing 0.1% hydrogen peroxide, v/v) was added and the plates were incubated for 30 minutes.
Binding assays for each BSA conjugate were done in triplicate, and background binding was measured in.
wells coated with BSA only. The extent of 15 biotinylated peptide binding is expressed as a percentage relative to fetuin as shown in Table VI
below:
Table VI
Binding of ACS2P1-biotin (amino acids 9-23 of SEQ
20 ID NO. 3) and SPYGRC-biotin (amino acids 18-23 of SEQ ID NO. 3) to BSA Conjugates*
Carbohydrate Structure% Binding9o BindingCommon of BSA
Conjugate RelativeRelative Name to to for Fetuin Fetuin Carbohydrat of of ACS2P1- SPYGRC- a Structure biotin biotin (n=3) (n=3) aNeuAc(2-3)(3Ga1(1-4)(3GlcNAc-BSA117112 12017 SLacNAc 25 aNeuAc(2-3)SGaI(1-3)SGIcNAc-BSA7014 97112 SLe' aNeuAc(2-6)pGal(1-4)SGIcNAc-BSA9412 12215 SLacNAc aNeuAc(2-3)(3Ga1(1-4)aGIcNAc-BSA8413 12014 SLe=
(1-3) aFuc 30 aNeuAc(2-3)(3Ga1(1-3)(3GlcNAc-HSA84114 10318 SLe'(C19.9) (I-4) aFuc *Experiments were done by coating 50 ~g/mL BSA-conjugate or fetuin and probed with 0.1 ~g ACS2P1-35 biotin (amino acids 9-23 of SEQ ID NO. 3) or 0.5 ug of SPYGRYC-biotin (amino acids 18-23 of SEQ ID NO.
3) for 1 hour at room temperature.
Example 9 -- Fonaation of a SPYGRC-Streptavidin-Biotin Conjugate Streptavidin (50 fig) in PBS was combined with $ SPYGRC-biotin (amino acids 18-23 of SEQ ID NO. 3) (50 ~1, 200 fig) and PBS (150 ~C1) and mixed for 1 hour at room temperature. The reaction mixture was loaded onto a Sephadex G-25 column (1 x 15 cm that had been equilibrated in PBS) and fractions collected. Protein containing fractions were analyzed by SDS PAGE gel electrophoresis, and the molecular weight was determined. The results were consistent with the formation of a tetravalent complex between peptide and streptavidin. Lectin binding inhibition experiments were carried out as described previously for determination of IC5°'s.
The results are set forth in Table VII below:
Table VII
The Effect of the Peptide SPYGRC or SPYGRC-biotin-Streptavidin Conjugate (amino acids 18-23 of SEQ ID
NO. 3) on the Binding of Biotinylated PT and Lectins to Fetuin*
BiotinylatedConcentration96 ChangesConcentration90 lectin of of SPYGRC (aa in BindingPeptide- Changes 23 of SEQ Activity Streptavidinin Binding ID
N0.3) Peptide Conjugate Activity (/aglml) (mg/ml) PT-b 4.4 +34 f 500 + 13 2 t 11 2$ MAL-b 4.4 -1610 1.0 -SOt3 SNA-b 4.4 -2 t 1 1.2 -50 t WGA-b 4.4 +7t3 64 -SOtI
*Positive signs preceding the numbers in this table indicate enhancement, and negative signs indicate inhibition of binding.
as amino acids Example 10 -- Neutralization of Pertussis Toxin Binding to Chinese Hamster Ovary (CHO) Cell by Synthetic Peptides Confluent monolayers of CHO cells were lifted from plastic tissue culture flasks with 0.25% trypsin and suspended at a concentration of 5 x 10° cells/ml in Ham's F12 media containing 10% fetal bovine serum (FBS). 100 ~,1 of the cell suspension was added to 96 well tissue culture plates and was allowed to establish contact with the plastic for 24 to 48 hours. The expended media was then removed and filtered-sterilized peptide 5-fold dilutions ranging in concentration from 300 to 30 femptogram per ml (80 ~1) in Ham's F12 media containing FBS were added to the CHO cells. 20 ~,1 of a PT solution (final PT concentration 2.7 ng/ml) was then added to the tissue culture wells containing peptide.
The incubation mixtures were gently mixed and incubated at 37°C for 1 hour in a COZ incubator. The incubation mixtures were then removed and replaced with fresh media. The tissue culture plates were then incubated for 24 hours at 37°C, fixed with 100% methanol, and stained with Geimsa stain. Control experiments were done in the absence of peptide alone or in the absence of PT. All determinations were done in triplicate.
Stained CHO cells were then examined for inhibition of characteristic CHO cell clumping which is mediated by pertussis toxin binding and scored as to whether there was greater than 50% inhibition of clumping of CHO
cells relative to control wells in the presence of PT
only. The results reported in Table VIII show the maximum peptide concentration required to cause a 50%
reduction in CHO cell clumping.
Table VIII
CHO Cell Neutralization Experiments Using PT S2 and S3 Peptides*
PT SEQ ID NO Maximal Inhibitory S Peptide Concentration for Greater than 50% Inhibition ACS2P1 3 (aa 9-23) 96 /ml SPYGRC 3 (aa 18-23) 480 pg/ml S3P1 6 (aa 9-23 NI
S3P9a 9 NI
* CHO cell neutralization experiments were carried out using a PT concentration of 2.4 ng/ml. CHO cell (5.4 x 104 cells/ml) were exposed to incubation mixtures containing peptide and PT for 1 hour at room temperature.
Control experiments were done in the absence of peptide.
as amino acid NI Not inhibitory at a peptide concentration of 300 ng/ml.
pg picogram--.
The above data demonstrates that at least certain of the subject peptides would be effective in inhibiting the attachment of bacterial/viral agents and/or their toxins which utilize a-sialic acid(2~6)~Gal- and/or a-sialic acid(2-~3)~BGal-structures as the attachment point on the surface of mammalian cells. Such agents/toxins include known toxins such as pertussis toxin, cholera toxin, etc., and, accordingly, administration of an effective amount of at least one of the subject peptides to a mammal would be effective in inhibiting such attachment.
Examples 11 and 12 below illustrate 'fin, vivo results for the subject peptides.
Example li -- Inhibition of DTH Inflammatory Response DTH inflammatory responses were measured using the mouse footpad swelling assay as described by Smith and Ziola~'. Briefly, groups of Balb/c mice (about 19-20 grams each) were immunized with 100 ~cg of the OVA
antigen containing 20~cg of the adjuvant (DDA --dimethyl-dioctadecylammonium bromide) which also induces a strong inflammatory DTH response. Seven days later, each group of mice was footpad-challenged with 20 ~,g of the OVA antigen (without adjuvant). The resulting inflammatory footpad swelling was measured with a Mitutoyo Engineering micrometer 24 hours after challenge.
To assess the effect of different peptides on the inflammatory DTH response, groups of mice received 100 ~.g of the following peptides: ACS2P1 (2275) (amino acids 9-23 of SEQ ID NO: 3), SPYGRC (2283) (amino acids 18-23 of SEQ ID NO: 3), and S3P9a (SEQ ID NO: 9).
These peptides were injected as a solution into the tail vein 5 hours after challenge. Control groups were left untreated or received 100 ~cL of phosphate-buffered saline (PBS). The results of this experiment are shown in Figure 2 which illustrates that the peptides employed were effective in reducing a DTH response in mice.
Example 12 -- Persistence of Suppression of the DTH
Inflammatory Response at 2 Weeks After Challenge Identical groups of mice treated with the peptides in Example 11 above were re-challenged with OVA antigen 2 weeks after primary immunization. Untreated controls responded with the usual degree of footpad swelling, whereas all other groups showed reduced footpad swelling. Specifically, these results are set forth in Figure 3 which illustrates reduction in the degree of footpad swelling in mice previously treated with the subject peptides.
In addition to providing suppression of antigen induced inflammation in a sensitized mouse, the above data demonstrate that treatment with the subject peptides as per this invention also imparts tolerance to still later challenges from the same antigen.
In view of the fact that the immune system of mice serves as a good model for the immune system of humans, the above data demonstrates that the subject peptides would be effective in suppressing cell-mediated immune responses in humans, and, when the cell-mediated immune response is to an antigen, this data also shows that the subject peptides would also impart tolerance to later challenges to the human of that antigen.
-b i -TABLE IX
Initial screening of synthetic peptides for inhibitory activity Peptide ConcentrationChanges (mg/ml) in binding activity PT/fetuin'MAL/fetuinSNA/fetuinWGA/fetuin ACS2P1 1.0 1.8910.03I.16t0.090.9710.010.7810.06 NSS2P1 2.4 1.1810.031.0310.021. l O 1.0310.02 10.02 SPYGRC 4.4 1.3410.020.8410.000.9810.021.0710.03 GSYRPC 2.2 1.4810.210.9510.160.9610.021.0910.01 SPYGYC 1.0 2.8510.161.2110.081.0710.151.0910.07 SPWGRC 1.0 1.7510.531.3910.131.0010.021.11 10.01 10 SPFGRC 1.0 1.3210.081.0610.121.0010.011.0110.01 2420 1.0 1.79' 1.43' 1.11' 2.1' ' Biotinylated lectin/glycoprotein bound to microtiter wells. Values greater than 1 in the table indicate enhancement while numbers lcss than 1 indicate inhibition of binding.
° Previously reported values.
IS ' One determination only.
Example 13 -- Binding Inhibition Assays (Initial Peptide Screening).
Microtiter wells were coated with 100 ~cl of fetuin or asialofetuin (50 ~g/ml) in 50 Mm sodium 20 phosphate buffer (pH 6.8) containing 5 mM MgCl2 and 15 mM NaN3, for 16 hours at 4°C. The solution was removed by aspiration and replaced with 100 ~.1 of 1% BSA in PBS containing 0.05% Tween 20 (PBST).
After incubation for 2-4 hours at room temperature, 25 the microtiter wells were washed four times with 300 ul of PBST. Peptides ranging in concentration from 1.0 to 4.4 mg/ml in PBS (40 ~1) were added to each well, and PT-biotin (10 ~.1 containing 10 ng in PBS) was then added to the microtiter wells. After 30 incubating for 1 hour, the binding reaction was stopped by aspirating the solutions, and the plate was washed with PBST (300 ~,1).
Horseradish peroxidase-conjugated avidin (100 ~cl, 1/3000 dilution in PEST to a concentration of 0.3 S ~cg/ml) was then added to the wells, and the plates were incubated at ambient temperature for 1 hour. After washing the wells as described above, the substrate solution (1 mM ABTS in 5 mM citrate buffer, pH 4.2, containing 0.1% hydrogen peroxide, v/v) was added, and the plates were incubated for 30 minutes. Color development was recorded at 405 nm using a Titertek Multiskan MC plate reader. Maximum binding was determined in the absence of peptide, and background binding was measured in wells coated with BSA only.
Binding assays for each peptide were done in duplicate.
Binding inhibition experiments utilizing SNA-, WGA-, and MAL-biotin were performed as described above using 10 ng of each biotinylated lectin in PBS.
The panel of 8 peptides was assayed for the ability to inhibit PT-, WGA-, MAL-, and SNA-biotin binding to fetuin or asialofetuin. The biotinylated plant lectins have proved to be useful controls, since we have previously showed that these lectins possess similar binding specificities as pT2s.ae_ The results from this example are set forth in Table IX above. These results indicate that peptides based on the SPX,GXZC (SEQ ID NO:1) motif inhibited PT
biotin binding.
-v~-TABLE X
Inhibition of biotinylated peptide binding to fetuin by simple saccharides' Saccharide Changes in binding activity SPWGRC-biotinSPFGRC-biotinSPYGYCGFGAEY-(n = 3) (n = 3) biotin (n = 3) Sialic acid 0.7110.11 1.0910.03 1.21 f0.09 Sialylactose 0.650.05 0.92 f 1.0810.03 0.04 Lactose 0.960.12 0.78 t 1.0610.00 0.05 N-Acetylglucosaminc0.8210.10 1.0710.14 1.1010.25 ' Inhibition experiments were carried out as outlined in methods using 0.5 ~cg of biotinylated peptides respectively. Binding values greater than 1 in this table indicate enhancement and values less than 1 indicate inhibition of binding to fetuin. Concentration of saccharides used were 8 mM .
Example 14 -- Binding Inhibition Assays Using Oligosaccharides In order to assess the binding to a-sialic acid ( 2-~6 ) /3Ga1- and a-sialic acid ( 2~3 ) (3Ga1-20 structures by certain peptides, inhibition experiments with simple saccharides were conducted to determine if the interaction was carbohydrate-dependent. The simple sugars chosen were sialic acid, sialyllactose from bovine colostrum (contains 25 a mixture of a(2-~6) and a(2~3) linked sialic acid structures), lactose and N-acetyl-glucosamine at a concentration of 8mM in PBS. Inhibition assays were done for 1 hour as described above (e. g., Example 2) at room temperature, and the amount of biotinylated peptide bound to fetuin (3~.g/ml) guantitated by using avidin-peroxidase. The pH of free sialic acid was carefully monitored and adjusted to physiological pH
values with dilute sodium hydroxide. The results of this example are set forth in Table X above.
The above results indicate that some of the peptides tested were able to bind to terminally linked a-sialic acid(2~6)/3Gal- and a-sialic acid(2~3)~3Ga1-structures.
iA3L~ XI
9irrdirrg of biorirrylated pepridu ro sialylared BSA conj~garesa Carbohydrate Structure of Binding Relative Binding Relative Biading Relative BSA Conjugate to Fetuin of to Fetuin of to Fetuin of SPWGRC-biotin SPFGRC-biotin SPYGYCGFG-AEY-biotin (n ~ 3) (n = 3) (n . 3) aNeuAc(2-3)~iGal(1-4)~iGlcNAc-BSA0.49 t 0.070.63 0.270.27 0.12 aNeuAc(2-3)~Gal(1-3)~GlcNAc-BSA0.43 t O.IO0.36 t 0.25 t 0.17 0.04 ocNeuAc(2-6)~Gal(1-4)~GId~IAc-BSA0.07 0..030.29 0.050.28 t 0.11 aNeuAc(2-3)~Gal(1-4)~GlcNAc-BSA0.67 t O.I40.42 0.150.33 0.04 (1-3) ocFuc .
aNeuAc(2-3)~iGal(1-3)~iGlcNAc-BSA0.77 ~ 0.190.52 O.I80.51 0.12 (1~) ctFuc a F.~cperiment~ weze done by coating with 5.0 ~tg/ml BSA-coajugate or fetuin and probed with 0.5 ltg biotinylated peptide for lh. 'Ihe binding of biotinylatcd peptide to fetuin is set at a value of 1Ø
Example 15 Binding assays were carried out essentially as described above using microtiter wells that were coated with 50 ~1 of BSA glycoconjugate (50 ~.g/ml) in 50 mM
sodium phosphate buffer (pH 6.8) containing 5 mM MgCl, nd 15 mM NaN3 for 16 hours at 4°C. The solution was removed by aspiration and replaced with 100 ~cl of 1%
BSA in PBS containing 0.05% Tween 20 (PBST) and incubated at room temperature for an additional 2-3 hours. The microtiter wells were washed four times with 300 ~cl of PBST and then replaced with 0.5 microgram of biotinylated peptide. After incubating for 1 hour, the binding reaction was stopped by aspirating the solutions and the plate was washed with PBST (4 X 300,1). Avidin-peroxidase (100 ~.1 of a 1/3000 dilution of a 1 mg/ml solution in PEST) was added and incubated for an additional 1 hour. After washing the wells as described above, the substrate solution (1 mM
ARTS in 5 mM citrate buffer, pH 4.2, containing 0.1°s hydrogen peroxide, v/v) was added and the plates were incubated for 30 minutes. Binding assays for each BSA
conjugate were done in triplicate, and background binding was measured in wells coated with BSA only.
The extent of biotinylated peptide binding is expressed as a ratio relative to fetuin as shown in Table XI
above:
Example 16 -- Inhibition of DTH Inflammatory Response DTH inflammatory responses were measured using the mouse footpad swelling assay as described by Smith and Ziola~°. Briefly, groups of Balb/c mice (about 19-20 grams each) were immunized with 100 ~,g of the OVA
antigen containing 20~cg of the adjuvant (DDA --dimethyl-dioctadecylammonium bromide) which also induces a strong inflammatory DTH response. Seven days later, each group of mice was footpad-challenged with 20 ~.g of the OVA antigen (without adjuvant). The resulting inflammatory footpad swelling was measured with a Mitutoyo Engineering micrometer 24 hours after challenge.
To assess the effect of different peptides on the inflammatory DTH response, groups of mice received 100 ~,g of the following peptides: ACS2P1 (2275) (amino acids 9-23 of SEQ ID NO. 3), SPFGRC (2418), SPWGRC
(2416), AcSPYGYCGFGAEY-CONHZ (2420) SPYGYC(2365), SPYGRC
(amino acids 18-23 of SEQ ID. NO: 3) (2283), NSS2P1(2295), and GSYRPC(2294). These peptides were injected as a solution into the tail vein 5 hours after challenge. Control groups were left untreated or received 100 ~,L of phosphate-buffered saline (PBS).
The results of this experiment are shown in Figures 4 and 6 which illustrate that the peptides employed were effective in reducing a DTH response in mice.
Example 17 -- Persistence of Suppression of the DTH
Inflammatory Response at 2 Weeks After Challenge Identical groups of mice treated with the peptides in Example 16 above were re-challenged with OVA antigen two weeks or 15 days after primary immunization.
Untreated controls responded with the usual degree of footpad swelling, whereas all other groups showed reduced footpad swelling. Specifically, these results are set forth in Figures 5 and 7 which illustrate reduction in the degree of footpad swelling in mice previously treated with the subject peptides.
In addition to providing suppression of antigen induced inflammation in a sensitized mouse, the above data demonstrate that treatment with the subject peptides as per this invention also imparts tolerance to still later challenges from the same antigen.
In view of the fact that the immune system of mice serves as a good model for the immune system of humans, the above data demonstrates that the subject peptides would be effective in suppressing cell-mediated immune responses in humans, and, when the cell-mediated immune response is to an antigen, this data also shows that the subject peptides would also impart tolerance to later challenges to the human of that antigen.
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT:
(A) NAME: ALBERTA RESEARCH COUNCIL
(B) STREET: 250 KARL CLARK ROAD
(C) CITY: EDMONTON
(D) STATE: ALBERTA
(E) COUNTRY: CANADA
(F) ZIP: T6H 5X2 (G) TELEPHONE: 403-450-5111 (H) TELEFAX: 403-461-2651 (ii) TITLE OF INVENTION: ANTI-INFLAMMATORY, TOLEROGENIC AND
IMMUNOINHIBITING PROPERTIES OF CARBOHYDRATE
BINDING-PEPTIDES
(iii) NUMBER OF SEQUENCES: 12 (iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: BLAKE, CASSELS & GRAYDON
(B) STREET: BOX 25, COMMERCE COURT WEST
(C) CITY: TORONTO
(D) STATE: ONT
(E) COUNTRY: CANADA
(F) ZIP: M5L lA9 (v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version #1.25 (vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE:
(C) CLASSIFICATION:
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: US 07/995,503 (B) FILING DATE: 21-DEC-1992 (vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: US 07/956,043
(8) FILING DATE: 02-OCT-1992 (viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: GRAY, BRIAN W.
(B) REGISTRATION NUMBER: 3752 (C) REFERENCE/DOCKET NUMBER:
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: 416-863-2400 (B) TELEFAX: 416-863-2653 (2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ix) FEATURE:
(A) NAME/KEY: Modified-site (B) LOCATION: 3 (D) OTHER INFORMATION: /note= "X is selected from the group consisting of amino acids Tyr, Phe, Trp and His or peptide mimetics thereof"
(ix) FEATURE:
(A) NAME/KEY: Modified-site (B) LOCATION: 5 (D) OTHER INFORMATION: /note= "X is selected from the group consisting of amino acids Tyr, Phe, Arg, Trp and His or peptide mimetics thereof"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:
Ser Pro Xaa Gly Xaa Cys (2) INFORMATION FOR SEQ ID N0:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 amino acids (8) TYPE: amino acid (D) TOPOLOGY: linear (ix) FEATURE:
(A) NAME/KEY: Modified-site (B) LOCATION: 3 (D) OTHER INFORMATION: /note= "X is selected from the group consisting of amino acids Tyr, Phe, Trp and His of peptide mimetics thereof"
(ix) FEATURE:
(A) NAME/KEY: Modified-site (B) LOCATION: 5 (D) OTHER INFORMATION: /note= "X is selected from the group consisting of amino acids Tyr, Phe, Arg, Trp, and His or peptide mimetics thereof"
(ix) FEATURE:
(A) NAME/KEY: Modified-site (H) LOCATION: 7 (D) OTHER INFORMATION: /note= "4-6 amino acids"
(ix) FEATURE:
(A) NAME/KEY: Modified-site (B) LOCATION: 8 (D) OTHER INFORMATION: /note= "X is selected from the group consisting of amino acids Tyr, Phe, Trp and His or peptide mimetics thereof"
-il-(xi) SEQUENCE DESCRIPTION: SEQ ID N0:2:
Ser Pro Xaa Gly Xaa Cys Xaa Xaa (2) INFORMATION FOR SEQ ID N0:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 23 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:3:
Ser Thr Pro Gly Ile Val Ile Pro Pro Gln Glu Gln Ile Thr Gln Hie Gly Ser Pro Tyr Gly Arg Cys (2) INFORMATION FOR SEQ ID N0:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:4:
Gly Ala Phe Asp Leu Lys Thr Thr Phe Cys Ile Met Thr Thr Arg Aan Thr Gly Gln Pro Ala (2) INFORMATION FOR SEQ ID N0:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:5:
Phe Val Arg Ser Gly Gln Pro Val Ile Gly Ala Cys Thr Ser Pro Tyr Asp Gly Lys Tyr Trp Ser Met Tyr Ser Arg Leu Arg Lys Met Leu Tyr ~7L' (2) INFORMATION FOR SEQ ID N0:6:
(i) SEQUENCE CHARACTERISTZCS:
(A) LENGTH: 23 amino acids (8) TYPE: amino acid (D) TOPOLOGY: linear ' (xi) SEQUENCE DESCRIPTION: SEQ ID N0:6:
Val Ala Pro Gly Ile Val Ile Pro Pro Lys Ala Leu Phe Thr Gln Gln Gly Gly Ala Tyr Gly Arg Cys (2) INFORMATION FOR SEQ ID N0:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 31 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:7:
Ala Gly Phe Ile Tyr Arg Glu Thr Phe Cys Ile Thr Thr Ile Tyr Lys Thr Gly Gln Pro Ala Ala Asp His Tyr Tyr Ser Lys Val Thr Ala (2) INFORMATION FOR SEQ ID NO: B:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:8:
Cys Ala Ser Pro Tyr Glu Gly Arg Tyr Arg Asp Met Tyr Asp Ala Leu Arg Arg Leu Leu Tyr (2) INFORMATION FOR SEQ ID N0:9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:9:
Arg Leu Leu Ala Ser Thr Asn Ser Arg Leu Cys Ala Val Phe Val Arg Asp Gly (2) INFORMATION FOR SEQ ID NO:10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:
Pro Gln Glu Gln Ile Thr Gln His Gly Ser Gln Tyr Gly Tyr Cys (2) INFORMATION FOR SEQ ID NO:11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:
Ser Gln Tyr Gly Tyr Cys Gly Phe Gly Ala Glu Tyr (2) INFORMATION FOR SEQ ID N0:12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino acids (8) TYPE: amino acid (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:12:
Ser Gln Tyr Gly His Cys
(A) NAME: GRAY, BRIAN W.
(B) REGISTRATION NUMBER: 3752 (C) REFERENCE/DOCKET NUMBER:
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: 416-863-2400 (B) TELEFAX: 416-863-2653 (2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ix) FEATURE:
(A) NAME/KEY: Modified-site (B) LOCATION: 3 (D) OTHER INFORMATION: /note= "X is selected from the group consisting of amino acids Tyr, Phe, Trp and His or peptide mimetics thereof"
(ix) FEATURE:
(A) NAME/KEY: Modified-site (B) LOCATION: 5 (D) OTHER INFORMATION: /note= "X is selected from the group consisting of amino acids Tyr, Phe, Arg, Trp and His or peptide mimetics thereof"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:
Ser Pro Xaa Gly Xaa Cys (2) INFORMATION FOR SEQ ID N0:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 amino acids (8) TYPE: amino acid (D) TOPOLOGY: linear (ix) FEATURE:
(A) NAME/KEY: Modified-site (B) LOCATION: 3 (D) OTHER INFORMATION: /note= "X is selected from the group consisting of amino acids Tyr, Phe, Trp and His of peptide mimetics thereof"
(ix) FEATURE:
(A) NAME/KEY: Modified-site (B) LOCATION: 5 (D) OTHER INFORMATION: /note= "X is selected from the group consisting of amino acids Tyr, Phe, Arg, Trp, and His or peptide mimetics thereof"
(ix) FEATURE:
(A) NAME/KEY: Modified-site (H) LOCATION: 7 (D) OTHER INFORMATION: /note= "4-6 amino acids"
(ix) FEATURE:
(A) NAME/KEY: Modified-site (B) LOCATION: 8 (D) OTHER INFORMATION: /note= "X is selected from the group consisting of amino acids Tyr, Phe, Trp and His or peptide mimetics thereof"
-il-(xi) SEQUENCE DESCRIPTION: SEQ ID N0:2:
Ser Pro Xaa Gly Xaa Cys Xaa Xaa (2) INFORMATION FOR SEQ ID N0:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 23 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:3:
Ser Thr Pro Gly Ile Val Ile Pro Pro Gln Glu Gln Ile Thr Gln Hie Gly Ser Pro Tyr Gly Arg Cys (2) INFORMATION FOR SEQ ID N0:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:4:
Gly Ala Phe Asp Leu Lys Thr Thr Phe Cys Ile Met Thr Thr Arg Aan Thr Gly Gln Pro Ala (2) INFORMATION FOR SEQ ID N0:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:5:
Phe Val Arg Ser Gly Gln Pro Val Ile Gly Ala Cys Thr Ser Pro Tyr Asp Gly Lys Tyr Trp Ser Met Tyr Ser Arg Leu Arg Lys Met Leu Tyr ~7L' (2) INFORMATION FOR SEQ ID N0:6:
(i) SEQUENCE CHARACTERISTZCS:
(A) LENGTH: 23 amino acids (8) TYPE: amino acid (D) TOPOLOGY: linear ' (xi) SEQUENCE DESCRIPTION: SEQ ID N0:6:
Val Ala Pro Gly Ile Val Ile Pro Pro Lys Ala Leu Phe Thr Gln Gln Gly Gly Ala Tyr Gly Arg Cys (2) INFORMATION FOR SEQ ID N0:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 31 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:7:
Ala Gly Phe Ile Tyr Arg Glu Thr Phe Cys Ile Thr Thr Ile Tyr Lys Thr Gly Gln Pro Ala Ala Asp His Tyr Tyr Ser Lys Val Thr Ala (2) INFORMATION FOR SEQ ID NO: B:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:8:
Cys Ala Ser Pro Tyr Glu Gly Arg Tyr Arg Asp Met Tyr Asp Ala Leu Arg Arg Leu Leu Tyr (2) INFORMATION FOR SEQ ID N0:9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:9:
Arg Leu Leu Ala Ser Thr Asn Ser Arg Leu Cys Ala Val Phe Val Arg Asp Gly (2) INFORMATION FOR SEQ ID NO:10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:
Pro Gln Glu Gln Ile Thr Gln His Gly Ser Gln Tyr Gly Tyr Cys (2) INFORMATION FOR SEQ ID NO:11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:
Ser Gln Tyr Gly Tyr Cys Gly Phe Gly Ala Glu Tyr (2) INFORMATION FOR SEQ ID N0:12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino acids (8) TYPE: amino acid (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:12:
Ser Gln Tyr Gly His Cys
Claims (21)
OR PRIVELEGE IS CLAIMED ARE DEFINED AS FOLLOWS:
1. A peptide comprising the amino acid sequence having the following formula II
(SEQ ID NO: 2):
where X1 is selected from the group of amino acids Y, F, W, and H or peptide mimetics there of, X2 is selected from the group consisting of amino acids Y, F, R, W, and H or peptide mimetics thereof:
X3 is an amino acid sequence of 4-6 amino acids; and X4 is selected from the group consisting of amino acids Y, F, W, and H or peptide mimetics thereof and pharmaceutically acceptable salts thereof.
(SEQ ID NO: 2):
where X1 is selected from the group of amino acids Y, F, W, and H or peptide mimetics there of, X2 is selected from the group consisting of amino acids Y, F, R, W, and H or peptide mimetics thereof:
X3 is an amino acid sequence of 4-6 amino acids; and X4 is selected from the group consisting of amino acids Y, F, W, and H or peptide mimetics thereof and pharmaceutically acceptable salts thereof.
2. The use of an effective amount of at least one peptide of Claim 1, for suppressing an inflammatory response in a mammal.
3. The use of at least one peptide of Claim 1, for modulating the induction of an immune response to an antigen in a mammal.
4. The use of an effective amount of at least one peptide of Claim 1, for inducing, in a sensitized mammal, long term tolerance to an antigen by challenging said mammal with the antigen.
5. The use of an effective amount of at least one peptide of Claim 1, for treating lung inflammation or lung injury in a mammal.
6. The use of an effective amount of at least one peptide of Claim 1, for inhibiting metastasis of tumor cells in a mammal.
7. The use of an effective amount of at least one peptide of Claim 1, for inhibiting infection in mammalian hosts by bacterial or viral agents or toxins which employ terminally linked .alpha.-sialic acid(2~6).beta.Gal- and/or .alpha. -sialic acid(2~3) .beta.Gal- structures as the receptor site on a cell of the targetted mammalian host.
8. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and the peptide of Claim 1.
9. The use of Claim 2, where said inflammatory response arises from antigen challenge in a sensitized mammal.
10. The use of Claim 9, wherein said antigen challenge is selected from the group consisting of delayed type hypersensitivity (DTH), rheumatoid arthritis, psoriasis, asthma, dermatitis, inflammatory bowel disease, multiple sclerosis, viral pneumonia, and bacterial pneumonia.
11. The use of Claim 2, wherein said inflammatory response is associated with mammalian injury.
12. The use of Claim 11, wherein said injury is selected from the group consisting of acute respiratory distress syndrome (ARDS), reperfusion injury frost bite, and septic shock.
13. The use of either of Claims 2 or 4, wherein said carbohydrate binding peptide or derivative thereof is administered after initiation of the mammal's inflammatory response but at or prior to one-half the period required for maximal inflammation.
14. The use of any of Claims 2-7, wherein said lectin derived carbohydrate binding peptide is administered at a dosage ranging form about 0.5 to 50 mg/kg body weight.
15. The use of any of Claims 2-7, wherein said lectin derived carbohydrate binding protein or derivative thereof is a parenteral, oral, intranasal, intratracheal, transdermal, intravenous or oral agent.
16. The use of Claim 3, wherein said immune response comprises a humoral or cell mediated immune response.
17. The use of either of Claims 3 or 4, wherein the antigen comprises an allergen.
18. The use of Claim 5, wherein the mammalian lung inflammation or lung injury comprises acute respiratory distress syndrome (ARDS).
19. The use of Claim 6, wherein the administration is effected in a mammal before, during, or after a cancer surgery or biopsy.
20. The use of Claim 6, wherein the administration is effected at a time ranging from about 5 hours before cancer surgery or biopsy to about 15 hours after cancer surgery or biopsy.
21. The use of Claim 6, wherein the cancer comprises colon carcinoma or melanoma.
Applications Claiming Priority (5)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US95604392A | 1992-10-02 | 1992-10-02 | |
US07/956,043 | 1992-10-02 | ||
US07/995,503 US5453272A (en) | 1992-10-02 | 1992-12-21 | Lectin derived carbohydrate binding-peptide |
US07/995,503 | 1992-12-21 | ||
CA002145881A CA2145881C (en) | 1992-10-02 | 1993-10-04 | Anti-inflammatory, tolerogenic and immunoinhibiting properties of carbohydrate binding-peptides |
Related Parent Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CA002145881A Division CA2145881C (en) | 1992-10-02 | 1993-10-04 | Anti-inflammatory, tolerogenic and immunoinhibiting properties of carbohydrate binding-peptides |
Publications (1)
Publication Number | Publication Date |
---|---|
CA2282888A1 true CA2282888A1 (en) | 1994-04-14 |
Family
ID=31720821
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CA002282888A Abandoned CA2282888A1 (en) | 1992-10-02 | 1993-10-04 | Anti-inflammatory, tolerogenic and immunoinhibiting properties of carbohydrate binding peptides |
Country Status (1)
Country | Link |
---|---|
CA (1) | CA2282888A1 (en) |
-
1993
- 1993-10-04 CA CA002282888A patent/CA2282888A1/en not_active Abandoned
Similar Documents
Publication | Publication Date | Title |
---|---|---|
EP0666758B1 (en) | Anti-inflammatory tolerogenic and immunoinhibiting properties of carbohydrate binding-peptides | |
US5453272A (en) | Lectin derived carbohydrate binding-peptide | |
Ravindranath et al. | Purification and characterization of an O-acetylsialic acid-specific lectin from a marine crab Cancer antennarius. | |
AU667813B2 (en) | Amino-acid sequence homologies between selectins and B pertussis toxin-peptides derived therefrom antibodies thereto pharmaceutical compositions | |
EA002743B1 (en) | Moraxella catarhalis outer membrane protein-106 polypeptide, dna encoding thereof, and use thereof | |
RU2461390C2 (en) | Immunologically active compounds | |
AU690421B2 (en) | Anti-inflammatory CD14 peptides | |
Heerze et al. | Investigation of the lectin-like binding domains in pertussis toxin using synthetic peptide sequences. Identification of a sialic acid binding site in the S2 subunit of the toxin. | |
EP0658115A1 (en) | Peptide medicaments for treating disease | |
AU716948B2 (en) | Anti-inflammatory tolerogenic and immunoinhibiting properties of carbohydrate binding-peptides | |
CA2282888A1 (en) | Anti-inflammatory, tolerogenic and immunoinhibiting properties of carbohydrate binding peptides | |
US5792457A (en) | Antibody recognizing endothelial cell ligand for leukocyte CR3 | |
CA2211563A1 (en) | Anti-inflammatory tolerogenic and immunoinhibiting properties of carbohydrate binding-peptides | |
Heerze et al. | Utilization of sialic acid-binding synthetic peptide sequences derived from pertussis toxin as novel anti-inflammatory agents | |
Glurich et al. | Identification of Streptococcus pyogenes proteins that bind to rabbit kidney in vitro and in vivo | |
AU665126C (en) | Anti-inflammatory, tolerogenic and immunostimulatory properties of carbohydrate binding-proteins | |
KR19990044645A (en) | Toxoplasma gondiga conjugate | |
US20040126811A1 (en) | Helicobacter pylori sialic acid binding adhesin, saba and saba-gene | |
EP2110436A1 (en) | Polypeptides having neutrophil stimulating activity | |
Buchta | Structure-function studies of the acute phase response protein: C-reactive protein | |
JPWO2004037272A1 (en) | Dengue virus infection inhibitor | |
JP2003502010A (en) | Product | |
WO2002066502A1 (en) | Helicobacter pylori sialic acid binding adhesin, saba and saba - gene | |
IE49617B1 (en) | Novel compounds of the muramyl-peptide type and medicaments containing them |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
EEER | Examination request | ||
FZDE | Dead |