WO2023028104A2 - Utilisation de lectines microbiennes humaines pour traiter une maladie - Google Patents
Utilisation de lectines microbiennes humaines pour traiter une maladie Download PDFInfo
- Publication number
- WO2023028104A2 WO2023028104A2 PCT/US2022/041315 US2022041315W WO2023028104A2 WO 2023028104 A2 WO2023028104 A2 WO 2023028104A2 US 2022041315 W US2022041315 W US 2022041315W WO 2023028104 A2 WO2023028104 A2 WO 2023028104A2
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- lectin
- composition
- subject
- cells
- cbeg5
- Prior art date
Links
- 108090001090 Lectins Proteins 0.000 title claims abstract description 186
- 239000002523 lectin Substances 0.000 title claims abstract description 178
- 102000004856 Lectins Human genes 0.000 title claims abstract description 163
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 title description 36
- 201000010099 disease Diseases 0.000 title description 20
- 230000000813 microbial effect Effects 0.000 title description 6
- 238000000034 method Methods 0.000 claims abstract description 101
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 81
- 239000000203 mixture Substances 0.000 claims abstract description 73
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 60
- 108010038196 saccharide-binding proteins Proteins 0.000 claims abstract description 43
- 210000001035 gastrointestinal tract Anatomy 0.000 claims abstract description 30
- 208000027866 inflammatory disease Diseases 0.000 claims abstract description 23
- 230000002255 enzymatic effect Effects 0.000 claims abstract description 15
- 210000004027 cell Anatomy 0.000 claims description 217
- 210000001616 monocyte Anatomy 0.000 claims description 40
- 239000002671 adjuvant Substances 0.000 claims description 33
- 230000000694 effects Effects 0.000 claims description 29
- 239000000427 antigen Substances 0.000 claims description 25
- 239000000872 buffer Substances 0.000 claims description 24
- 108010076504 Protein Sorting Signals Proteins 0.000 claims description 22
- 210000001072 colon Anatomy 0.000 claims description 22
- 108091007433 antigens Proteins 0.000 claims description 20
- 102000036639 antigens Human genes 0.000 claims description 20
- 210000002540 macrophage Anatomy 0.000 claims description 20
- 241000588724 Escherichia coli Species 0.000 claims description 17
- 206010028980 Neoplasm Diseases 0.000 claims description 15
- 201000011510 cancer Diseases 0.000 claims description 15
- 210000002865 immune cell Anatomy 0.000 claims description 14
- 230000000968 intestinal effect Effects 0.000 claims description 14
- 210000000066 myeloid cell Anatomy 0.000 claims description 14
- 230000001580 bacterial effect Effects 0.000 claims description 13
- 239000000546 pharmaceutical excipient Substances 0.000 claims description 13
- 239000003937 drug carrier Substances 0.000 claims description 12
- 239000003085 diluting agent Substances 0.000 claims description 10
- 230000001717 pathogenic effect Effects 0.000 claims description 10
- 102100026122 High affinity immunoglobulin gamma Fc receptor I Human genes 0.000 claims description 9
- 101000913074 Homo sapiens High affinity immunoglobulin gamma Fc receptor I Proteins 0.000 claims description 9
- 239000002775 capsule Substances 0.000 claims description 8
- 230000001225 therapeutic effect Effects 0.000 claims description 8
- 241000725643 Respiratory syncytial virus Species 0.000 claims description 7
- 208000009956 adenocarcinoma Diseases 0.000 claims description 7
- 230000009851 immunogenic response Effects 0.000 claims description 7
- 229960005486 vaccine Drugs 0.000 claims description 7
- 201000005010 Streptococcus pneumonia Diseases 0.000 claims description 6
- 241000193998 Streptococcus pneumoniae Species 0.000 claims description 6
- 208000025721 COVID-19 Diseases 0.000 claims description 5
- 206010009944 Colon cancer Diseases 0.000 claims description 5
- 201000005505 Measles Diseases 0.000 claims description 5
- 208000005647 Mumps Diseases 0.000 claims description 5
- 206010061902 Pancreatic neoplasm Diseases 0.000 claims description 5
- 241000702670 Rotavirus Species 0.000 claims description 5
- 230000002708 enhancing effect Effects 0.000 claims description 5
- 208000005252 hepatitis A Diseases 0.000 claims description 5
- 208000002672 hepatitis B Diseases 0.000 claims description 5
- 206010022000 influenza Diseases 0.000 claims description 5
- 208000015486 malignant pancreatic neoplasm Diseases 0.000 claims description 5
- 208000010805 mumps infectious disease Diseases 0.000 claims description 5
- 201000011519 neuroendocrine tumor Diseases 0.000 claims description 5
- 201000002528 pancreatic cancer Diseases 0.000 claims description 5
- 208000008443 pancreatic carcinoma Diseases 0.000 claims description 5
- 201000005404 rubella Diseases 0.000 claims description 5
- 206010073373 small intestine adenocarcinoma Diseases 0.000 claims description 5
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 claims description 4
- 206010009900 Colitis ulcerative Diseases 0.000 claims description 4
- 208000022559 Inflammatory bowel disease Diseases 0.000 claims description 4
- 201000006704 Ulcerative Colitis Diseases 0.000 claims description 4
- 210000002429 large intestine Anatomy 0.000 claims description 4
- 208000008275 microscopic colitis Diseases 0.000 claims description 4
- 208000015943 Coeliac disease Diseases 0.000 claims description 3
- 208000011231 Crohn disease Diseases 0.000 claims description 3
- 206010052399 Neuroendocrine tumour Diseases 0.000 claims description 3
- 208000015634 Rectal Neoplasms Diseases 0.000 claims description 3
- 208000029742 colonic neoplasm Diseases 0.000 claims description 3
- 208000002551 irritable bowel syndrome Diseases 0.000 claims description 3
- 208000016065 neuroendocrine neoplasm Diseases 0.000 claims description 3
- 206010038038 rectal cancer Diseases 0.000 claims description 3
- 201000001275 rectum cancer Diseases 0.000 claims description 3
- 206010017993 Gastrointestinal neoplasms Diseases 0.000 claims description 2
- 102100022297 Integrin alpha-X Human genes 0.000 claims description 2
- 238000005516 engineering process Methods 0.000 abstract description 3
- 235000018102 proteins Nutrition 0.000 description 55
- 101000946889 Homo sapiens Monocyte differentiation antigen CD14 Proteins 0.000 description 31
- 102100035877 Monocyte differentiation antigen CD14 Human genes 0.000 description 30
- 102000004127 Cytokines Human genes 0.000 description 28
- 108090000695 Cytokines Proteins 0.000 description 28
- 238000004458 analytical method Methods 0.000 description 28
- 108091033319 polynucleotide Proteins 0.000 description 26
- 102000040430 polynucleotide Human genes 0.000 description 26
- 239000002157 polynucleotide Substances 0.000 description 26
- 241000699666 Mus <mouse, genus> Species 0.000 description 23
- 238000011282 treatment Methods 0.000 description 23
- 108090000765 processed proteins & peptides Proteins 0.000 description 22
- 230000004044 response Effects 0.000 description 20
- 241000699670 Mus sp. Species 0.000 description 19
- 244000005702 human microbiome Species 0.000 description 19
- 210000003819 peripheral blood mononuclear cell Anatomy 0.000 description 19
- 102000004196 processed proteins & peptides Human genes 0.000 description 18
- 150000004676 glycans Chemical class 0.000 description 17
- 208000035475 disorder Diseases 0.000 description 16
- 210000000265 leukocyte Anatomy 0.000 description 16
- 241000894006 Bacteria Species 0.000 description 14
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 14
- 238000002474 experimental method Methods 0.000 description 13
- 229920001184 polypeptide Polymers 0.000 description 13
- 210000004980 monocyte derived macrophage Anatomy 0.000 description 12
- 150000007523 nucleic acids Chemical class 0.000 description 12
- 230000001965 increasing effect Effects 0.000 description 11
- 241001465754 Metazoa Species 0.000 description 10
- 210000004369 blood Anatomy 0.000 description 10
- 239000008280 blood Substances 0.000 description 10
- 230000006870 function Effects 0.000 description 10
- 239000002773 nucleotide Substances 0.000 description 10
- 125000003729 nucleotide group Chemical group 0.000 description 10
- 239000000243 solution Substances 0.000 description 10
- 210000001519 tissue Anatomy 0.000 description 10
- 230000016396 cytokine production Effects 0.000 description 9
- 238000001943 fluorescence-activated cell sorting Methods 0.000 description 9
- 230000006698 induction Effects 0.000 description 9
- 239000000523 sample Substances 0.000 description 9
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 8
- 210000004443 dendritic cell Anatomy 0.000 description 8
- 230000036541 health Effects 0.000 description 8
- 244000005700 microbiome Species 0.000 description 8
- 102000039446 nucleic acids Human genes 0.000 description 8
- 108020004707 nucleic acids Proteins 0.000 description 8
- 239000008188 pellet Substances 0.000 description 8
- 230000032258 transport Effects 0.000 description 8
- 102100022119 Lipoprotein lipase Human genes 0.000 description 7
- 241000700159 Rattus Species 0.000 description 7
- 210000001744 T-lymphocyte Anatomy 0.000 description 7
- 239000006228 supernatant Substances 0.000 description 7
- 102000019034 Chemokines Human genes 0.000 description 6
- 108010012236 Chemokines Proteins 0.000 description 6
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 6
- 206010061218 Inflammation Diseases 0.000 description 6
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 6
- 239000003814 drug Substances 0.000 description 6
- 238000000684 flow cytometry Methods 0.000 description 6
- 239000005090 green fluorescent protein Substances 0.000 description 6
- RAXXELZNTBOGNW-UHFFFAOYSA-N imidazole Natural products C1=CNC=N1 RAXXELZNTBOGNW-UHFFFAOYSA-N 0.000 description 6
- 230000004054 inflammatory process Effects 0.000 description 6
- 210000003734 kidney Anatomy 0.000 description 6
- 238000004519 manufacturing process Methods 0.000 description 6
- 239000013610 patient sample Substances 0.000 description 6
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 description 5
- 241000606125 Bacteroides Species 0.000 description 5
- 102100037362 Fibronectin Human genes 0.000 description 5
- 108010067306 Fibronectins Proteins 0.000 description 5
- 108010043121 Green Fluorescent Proteins Proteins 0.000 description 5
- 102000004144 Green Fluorescent Proteins Human genes 0.000 description 5
- 239000007995 HEPES buffer Substances 0.000 description 5
- 101000917858 Homo sapiens Low affinity immunoglobulin gamma Fc region receptor III-A Proteins 0.000 description 5
- 101000917839 Homo sapiens Low affinity immunoglobulin gamma Fc region receptor III-B Proteins 0.000 description 5
- 102100029185 Low affinity immunoglobulin gamma Fc region receptor III-B Human genes 0.000 description 5
- 206010025323 Lymphomas Diseases 0.000 description 5
- 230000004913 activation Effects 0.000 description 5
- 150000001413 amino acids Chemical class 0.000 description 5
- 244000052616 bacterial pathogen Species 0.000 description 5
- BLFLLBZGZJTVJG-UHFFFAOYSA-N benzocaine Chemical compound CCOC(=O)C1=CC=C(N)C=C1 BLFLLBZGZJTVJG-UHFFFAOYSA-N 0.000 description 5
- 239000003651 drinking water Substances 0.000 description 5
- 235000020188 drinking water Nutrition 0.000 description 5
- 229940079593 drug Drugs 0.000 description 5
- 239000003102 growth factor Substances 0.000 description 5
- 206010073071 hepatocellular carcinoma Diseases 0.000 description 5
- 210000000936 intestine Anatomy 0.000 description 5
- 244000052769 pathogen Species 0.000 description 5
- 230000002265 prevention Effects 0.000 description 5
- 230000028327 secretion Effects 0.000 description 5
- 238000012360 testing method Methods 0.000 description 5
- 239000013598 vector Substances 0.000 description 5
- 239000012981 Hank's balanced salt solution Substances 0.000 description 4
- 108090000174 Interleukin-10 Proteins 0.000 description 4
- 208000002606 Paramyxoviridae Infections Diseases 0.000 description 4
- 208000005718 Stomach Neoplasms Diseases 0.000 description 4
- 108060008682 Tumor Necrosis Factor Proteins 0.000 description 4
- 102100040247 Tumor necrosis factor Human genes 0.000 description 4
- 238000003556 assay Methods 0.000 description 4
- 230000008901 benefit Effects 0.000 description 4
- 230000004071 biological effect Effects 0.000 description 4
- 150000001720 carbohydrates Chemical class 0.000 description 4
- 235000014633 carbohydrates Nutrition 0.000 description 4
- 230000024245 cell differentiation Effects 0.000 description 4
- 150000001875 compounds Chemical class 0.000 description 4
- 108020001507 fusion proteins Proteins 0.000 description 4
- 102000037865 fusion proteins Human genes 0.000 description 4
- 206010017758 gastric cancer Diseases 0.000 description 4
- 230000002962 histologic effect Effects 0.000 description 4
- 208000037797 influenza A Diseases 0.000 description 4
- 238000002955 isolation Methods 0.000 description 4
- 229930027917 kanamycin Natural products 0.000 description 4
- 229960000318 kanamycin Drugs 0.000 description 4
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 description 4
- 229930182823 kanamycin A Natural products 0.000 description 4
- 108020004999 messenger RNA Proteins 0.000 description 4
- 239000013612 plasmid Substances 0.000 description 4
- 238000012163 sequencing technique Methods 0.000 description 4
- 238000010186 staining Methods 0.000 description 4
- 201000011549 stomach cancer Diseases 0.000 description 4
- 239000000126 substance Substances 0.000 description 4
- 208000024891 symptom Diseases 0.000 description 4
- OZFAFGSSMRRTDW-UHFFFAOYSA-N (2,4-dichlorophenyl) benzenesulfonate Chemical compound ClC1=CC(Cl)=CC=C1OS(=O)(=O)C1=CC=CC=C1 OZFAFGSSMRRTDW-UHFFFAOYSA-N 0.000 description 3
- 201000009030 Carcinoma Diseases 0.000 description 3
- 239000012591 Dulbecco’s Phosphate Buffered Saline Substances 0.000 description 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- 239000007836 KH2PO4 Substances 0.000 description 3
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 description 3
- 229930182816 L-glutamine Natural products 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 229930040373 Paraformaldehyde Natural products 0.000 description 3
- 108010089430 Phosphoproteins Proteins 0.000 description 3
- 102000007982 Phosphoproteins Human genes 0.000 description 3
- 206010035226 Plasma cell myeloma Diseases 0.000 description 3
- 206010035664 Pneumonia Diseases 0.000 description 3
- 239000006146 Roswell Park Memorial Institute medium Substances 0.000 description 3
- 108700012920 TNF Proteins 0.000 description 3
- 238000002869 basic local alignment search tool Methods 0.000 description 3
- 239000011324 bead Substances 0.000 description 3
- 239000003795 chemical substances by application Substances 0.000 description 3
- 206010009887 colitis Diseases 0.000 description 3
- 239000002299 complementary DNA Substances 0.000 description 3
- 210000002919 epithelial cell Anatomy 0.000 description 3
- 210000003608 fece Anatomy 0.000 description 3
- 108091006047 fluorescent proteins Proteins 0.000 description 3
- 102000034287 fluorescent proteins Human genes 0.000 description 3
- 235000013305 food Nutrition 0.000 description 3
- 235000012631 food intake Nutrition 0.000 description 3
- 238000009472 formulation Methods 0.000 description 3
- 231100000844 hepatocellular carcinoma Toxicity 0.000 description 3
- 238000000338 in vitro Methods 0.000 description 3
- 238000011534 incubation Methods 0.000 description 3
- 230000003993 interaction Effects 0.000 description 3
- 210000004185 liver Anatomy 0.000 description 3
- 239000006166 lysate Substances 0.000 description 3
- 230000001404 mediated effect Effects 0.000 description 3
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 3
- 235000019796 monopotassium phosphate Nutrition 0.000 description 3
- 201000000050 myeloid neoplasm Diseases 0.000 description 3
- 210000000440 neutrophil Anatomy 0.000 description 3
- 229920002866 paraformaldehyde Polymers 0.000 description 3
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- 210000000664 rectum Anatomy 0.000 description 3
- 230000019491 signal transduction Effects 0.000 description 3
- 239000011780 sodium chloride Substances 0.000 description 3
- 241000894007 species Species 0.000 description 3
- 229960005322 streptomycin Drugs 0.000 description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 208000023275 Autoimmune disease Diseases 0.000 description 2
- 241000186000 Bifidobacterium Species 0.000 description 2
- 238000011740 C57BL/6 mouse Methods 0.000 description 2
- 241000282465 Canis Species 0.000 description 2
- 241000282693 Cercopithecidae Species 0.000 description 2
- 108091033380 Coding strand Proteins 0.000 description 2
- 102000029816 Collagenase Human genes 0.000 description 2
- 108060005980 Collagenase Proteins 0.000 description 2
- 208000001333 Colorectal Neoplasms Diseases 0.000 description 2
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 2
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 2
- 241000196324 Embryophyta Species 0.000 description 2
- 241000194033 Enterococcus Species 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 206010051066 Gastrointestinal stromal tumour Diseases 0.000 description 2
- WZUVPPKBWHMQCE-UHFFFAOYSA-N Haematoxylin Chemical compound C12=CC(O)=C(O)C=C2CC2(O)C1C1=CC=C(O)C(O)=C1OC2 WZUVPPKBWHMQCE-UHFFFAOYSA-N 0.000 description 2
- HTTJABKRGRZYRN-UHFFFAOYSA-N Heparin Chemical compound OC1C(NC(=O)C)C(O)OC(COS(O)(=O)=O)C1OC1C(OS(O)(=O)=O)C(O)C(OC2C(C(OS(O)(=O)=O)C(OC3C(C(O)C(O)C(O3)C(O)=O)OS(O)(=O)=O)C(CO)O2)NS(O)(=O)=O)C(C(O)=O)O1 HTTJABKRGRZYRN-UHFFFAOYSA-N 0.000 description 2
- 241000282412 Homo Species 0.000 description 2
- 101000738771 Homo sapiens Receptor-type tyrosine-protein phosphatase C Proteins 0.000 description 2
- 108090001005 Interleukin-6 Proteins 0.000 description 2
- 108090001007 Interleukin-8 Proteins 0.000 description 2
- 239000006137 Luria-Bertani broth Substances 0.000 description 2
- 241000124008 Mammalia Species 0.000 description 2
- 208000001572 Mycoplasma Pneumonia Diseases 0.000 description 2
- 201000008235 Mycoplasma pneumoniae pneumonia Diseases 0.000 description 2
- 108010038807 Oligopeptides Proteins 0.000 description 2
- 102000015636 Oligopeptides Human genes 0.000 description 2
- 239000012980 RPMI-1640 medium Substances 0.000 description 2
- 102100037422 Receptor-type tyrosine-protein phosphatase C Human genes 0.000 description 2
- 239000004098 Tetracycline Substances 0.000 description 2
- 108091023040 Transcription factor Proteins 0.000 description 2
- 102000040945 Transcription factor Human genes 0.000 description 2
- 101710159648 Uncharacterized protein Proteins 0.000 description 2
- ISAKRJDGNUQOIC-UHFFFAOYSA-N Uracil Chemical compound O=C1C=CNC(=O)N1 ISAKRJDGNUQOIC-UHFFFAOYSA-N 0.000 description 2
- 241000251539 Vertebrata <Metazoa> Species 0.000 description 2
- 241000607598 Vibrio Species 0.000 description 2
- 208000027418 Wounds and injury Diseases 0.000 description 2
- 239000004480 active ingredient Substances 0.000 description 2
- 239000013543 active substance Substances 0.000 description 2
- 229960000723 ampicillin Drugs 0.000 description 2
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 2
- 238000000137 annealing Methods 0.000 description 2
- 239000012639 bacterial effector Substances 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- -1 carboxymethylester Chemical compound 0.000 description 2
- 208000002458 carcinoid tumor Diseases 0.000 description 2
- 239000000969 carrier Substances 0.000 description 2
- 230000003197 catalytic effect Effects 0.000 description 2
- 210000000182 cd11c+cd123- dc Anatomy 0.000 description 2
- 239000002458 cell surface marker Substances 0.000 description 2
- 230000005754 cellular signaling Effects 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 238000010367 cloning Methods 0.000 description 2
- 229960002424 collagenase Drugs 0.000 description 2
- 230000001332 colony forming effect Effects 0.000 description 2
- 238000007796 conventional method Methods 0.000 description 2
- OPTASPLRGRRNAP-UHFFFAOYSA-N cytosine Chemical compound NC=1C=CNC(=O)N=1 OPTASPLRGRRNAP-UHFFFAOYSA-N 0.000 description 2
- 230000006378 damage Effects 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 230000018109 developmental process Effects 0.000 description 2
- 230000029087 digestion Effects 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 238000002224 dissection Methods 0.000 description 2
- 238000009826 distribution Methods 0.000 description 2
- 229940088598 enzyme Drugs 0.000 description 2
- 230000002550 fecal effect Effects 0.000 description 2
- 210000002950 fibroblast Anatomy 0.000 description 2
- 210000003495 flagella Anatomy 0.000 description 2
- 230000037406 food intake Effects 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- 201000011243 gastrointestinal stromal tumor Diseases 0.000 description 2
- 230000000762 glandular Effects 0.000 description 2
- UYTPUPDQBNUYGX-UHFFFAOYSA-N guanine Chemical compound O=C1NC(N)=NC2=C1N=CN2 UYTPUPDQBNUYGX-UHFFFAOYSA-N 0.000 description 2
- 229920000669 heparin Polymers 0.000 description 2
- 229960002897 heparin Drugs 0.000 description 2
- 230000013632 homeostatic process Effects 0.000 description 2
- 208000026278 immune system disease Diseases 0.000 description 2
- 238000001727 in vivo Methods 0.000 description 2
- 238000002347 injection Methods 0.000 description 2
- 239000007924 injection Substances 0.000 description 2
- 208000014674 injury Diseases 0.000 description 2
- 238000005304 joining Methods 0.000 description 2
- 210000003292 kidney cell Anatomy 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 238000004895 liquid chromatography mass spectrometry Methods 0.000 description 2
- 201000007270 liver cancer Diseases 0.000 description 2
- 239000012139 lysis buffer Substances 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 201000001441 melanoma Diseases 0.000 description 2
- 238000012544 monitoring process Methods 0.000 description 2
- 210000004400 mucous membrane Anatomy 0.000 description 2
- 210000000822 natural killer cell Anatomy 0.000 description 2
- 238000010606 normalization Methods 0.000 description 2
- 201000008968 osteosarcoma Diseases 0.000 description 2
- 210000000496 pancreas Anatomy 0.000 description 2
- 208000021010 pancreatic neuroendocrine tumor Diseases 0.000 description 2
- 238000007911 parenteral administration Methods 0.000 description 2
- 230000007170 pathology Effects 0.000 description 2
- 230000002093 peripheral effect Effects 0.000 description 2
- 230000008823 permeabilization Effects 0.000 description 2
- YBYRMVIVWMBXKQ-UHFFFAOYSA-N phenylmethanesulfonyl fluoride Chemical compound FS(=O)(=O)CC1=CC=CC=C1 YBYRMVIVWMBXKQ-UHFFFAOYSA-N 0.000 description 2
- PTMHPRAIXMAOOB-UHFFFAOYSA-L phosphoramidate Chemical compound NP([O-])([O-])=O PTMHPRAIXMAOOB-UHFFFAOYSA-L 0.000 description 2
- 229920000642 polymer Polymers 0.000 description 2
- 230000001681 protective effect Effects 0.000 description 2
- 238000011084 recovery Methods 0.000 description 2
- 230000002829 reductive effect Effects 0.000 description 2
- 238000002271 resection Methods 0.000 description 2
- 239000011347 resin Substances 0.000 description 2
- 229920005989 resin Polymers 0.000 description 2
- 238000007480 sanger sequencing Methods 0.000 description 2
- 108091006024 signal transducing proteins Proteins 0.000 description 2
- 102000034285 signal transducing proteins Human genes 0.000 description 2
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- 238000001228 spectrum Methods 0.000 description 2
- 210000000130 stem cell Anatomy 0.000 description 2
- 210000002784 stomach Anatomy 0.000 description 2
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Natural products CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 description 2
- 238000007920 subcutaneous administration Methods 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 229960002180 tetracycline Drugs 0.000 description 2
- 229930101283 tetracycline Natural products 0.000 description 2
- 235000019364 tetracycline Nutrition 0.000 description 2
- 150000003522 tetracyclines Chemical class 0.000 description 2
- 230000004797 therapeutic response Effects 0.000 description 2
- RYYWUUFWQRZTIU-UHFFFAOYSA-K thiophosphate Chemical compound [O-]P([O-])([O-])=S RYYWUUFWQRZTIU-UHFFFAOYSA-K 0.000 description 2
- RWQNBRDOKXIBIV-UHFFFAOYSA-N thymine Chemical compound CC1=CNC(=O)NC1=O RWQNBRDOKXIBIV-UHFFFAOYSA-N 0.000 description 2
- 238000013518 transcription Methods 0.000 description 2
- 230000035897 transcription Effects 0.000 description 2
- 238000013519 translation Methods 0.000 description 2
- 241000701161 unidentified adenovirus Species 0.000 description 2
- DLFVBJFMPXGRIB-UHFFFAOYSA-N Acetamide Chemical compound CC(N)=O DLFVBJFMPXGRIB-UHFFFAOYSA-N 0.000 description 1
- 229930024421 Adenine Natural products 0.000 description 1
- GFFGJBXGBJISGV-UHFFFAOYSA-N Adenine Chemical compound NC1=NC=NC2=C1N=CN2 GFFGJBXGBJISGV-UHFFFAOYSA-N 0.000 description 1
- 208000036652 Adenocarcinoma of the small intestine Diseases 0.000 description 1
- 102000002260 Alkaline Phosphatase Human genes 0.000 description 1
- 108020004774 Alkaline Phosphatase Proteins 0.000 description 1
- 102100024222 B-lymphocyte antigen CD19 Human genes 0.000 description 1
- 241000193830 Bacillus <bacterium> Species 0.000 description 1
- 241000193749 Bacillus coagulans Species 0.000 description 1
- 241000194110 Bacillus sp. (in: Bacteria) Species 0.000 description 1
- 244000063299 Bacillus subtilis Species 0.000 description 1
- 235000014469 Bacillus subtilis Nutrition 0.000 description 1
- 241000606124 Bacteroides fragilis Species 0.000 description 1
- 241000606123 Bacteroides thetaiotaomicron Species 0.000 description 1
- 241000605059 Bacteroidetes Species 0.000 description 1
- 241000901050 Bifidobacterium animalis subsp. lactis Species 0.000 description 1
- 241000186016 Bifidobacterium bifidum Species 0.000 description 1
- 241001608472 Bifidobacterium longum Species 0.000 description 1
- 241000588832 Bordetella pertussis Species 0.000 description 1
- 241000283690 Bos taurus Species 0.000 description 1
- 241000589876 Campylobacter Species 0.000 description 1
- 241000589877 Campylobacter coli Species 0.000 description 1
- 241000589875 Campylobacter jejuni Species 0.000 description 1
- 241001135528 Campylobacter upsaliensis Species 0.000 description 1
- 241000282472 Canis lupus familiaris Species 0.000 description 1
- 241000283707 Capra Species 0.000 description 1
- KXDHJXZQYSOELW-UHFFFAOYSA-M Carbamate Chemical compound NC([O-])=O KXDHJXZQYSOELW-UHFFFAOYSA-M 0.000 description 1
- BVKZGUZCCUSVTD-UHFFFAOYSA-L Carbonate Chemical compound [O-]C([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-L 0.000 description 1
- 102100025470 Carcinoembryonic antigen-related cell adhesion molecule 8 Human genes 0.000 description 1
- 206010007279 Carcinoid tumour of the gastrointestinal tract Diseases 0.000 description 1
- 108010078791 Carrier Proteins Proteins 0.000 description 1
- 108010076119 Caseins Proteins 0.000 description 1
- 241000700198 Cavia Species 0.000 description 1
- 208000000362 Chlamydial Pneumonia Diseases 0.000 description 1
- 241000282552 Chlorocebus aethiops Species 0.000 description 1
- 101000653561 Clostridioides difficile Toxin A Proteins 0.000 description 1
- 101000835721 Clostridioides difficile Toxin B Proteins 0.000 description 1
- 101000653566 Clostridioides difficile Toxin B Proteins 0.000 description 1
- 241000193403 Clostridium Species 0.000 description 1
- 241000193171 Clostridium butyricum Species 0.000 description 1
- 241000711573 Coronaviridae Species 0.000 description 1
- 241000699800 Cricetinae Species 0.000 description 1
- 241000699802 Cricetulus griseus Species 0.000 description 1
- 241000223935 Cryptosporidium Species 0.000 description 1
- 241000016605 Cyclospora cayetanensis Species 0.000 description 1
- 108020004414 DNA Proteins 0.000 description 1
- 102000012410 DNA Ligases Human genes 0.000 description 1
- 108010061982 DNA Ligases Proteins 0.000 description 1
- 108010053770 Deoxyribonucleases Proteins 0.000 description 1
- 102000016911 Deoxyribonucleases Human genes 0.000 description 1
- 206010012735 Diarrhoea Diseases 0.000 description 1
- 208000012258 Diverticular disease Diseases 0.000 description 1
- 206010013554 Diverticulum Diseases 0.000 description 1
- 241000224432 Entamoeba histolytica Species 0.000 description 1
- 241000709661 Enterovirus Species 0.000 description 1
- 241000283086 Equidae Species 0.000 description 1
- 241001198387 Escherichia coli BL21(DE3) Species 0.000 description 1
- 108010037362 Extracellular Matrix Proteins Proteins 0.000 description 1
- 102000010834 Extracellular Matrix Proteins Human genes 0.000 description 1
- 241000282326 Felis catus Species 0.000 description 1
- 229920001917 Ficoll Polymers 0.000 description 1
- 102100027581 Forkhead box protein P3 Human genes 0.000 description 1
- 206010017964 Gastrointestinal infection Diseases 0.000 description 1
- 108010010803 Gelatin Proteins 0.000 description 1
- 241000224466 Giardia Species 0.000 description 1
- 101710154441 Golvesin Proteins 0.000 description 1
- 108010017213 Granulocyte-Macrophage Colony-Stimulating Factor Proteins 0.000 description 1
- 102100039620 Granulocyte-macrophage colony-stimulating factor Human genes 0.000 description 1
- 241000590002 Helicobacter pylori Species 0.000 description 1
- 241000238631 Hexapoda Species 0.000 description 1
- 101000980825 Homo sapiens B-lymphocyte antigen CD19 Proteins 0.000 description 1
- 101000914320 Homo sapiens Carcinoembryonic antigen-related cell adhesion molecule 8 Proteins 0.000 description 1
- 101000861452 Homo sapiens Forkhead box protein P3 Proteins 0.000 description 1
- 101000581981 Homo sapiens Neural cell adhesion molecule 1 Proteins 0.000 description 1
- 241000711467 Human coronavirus 229E Species 0.000 description 1
- 241000482741 Human coronavirus NL63 Species 0.000 description 1
- 241001428935 Human coronavirus OC43 Species 0.000 description 1
- 241000342334 Human metapneumovirus Species 0.000 description 1
- 241000430519 Human rhinovirus sp. Species 0.000 description 1
- 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 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
- 241000186660 Lactobacillus Species 0.000 description 1
- 240000001046 Lactobacillus acidophilus Species 0.000 description 1
- 235000013956 Lactobacillus acidophilus Nutrition 0.000 description 1
- 244000199885 Lactobacillus bulgaricus Species 0.000 description 1
- 235000013960 Lactobacillus bulgaricus Nutrition 0.000 description 1
- 244000199866 Lactobacillus casei Species 0.000 description 1
- 235000013958 Lactobacillus casei Nutrition 0.000 description 1
- 241001468157 Lactobacillus johnsonii Species 0.000 description 1
- 241000186605 Lactobacillus paracasei Species 0.000 description 1
- 240000006024 Lactobacillus plantarum Species 0.000 description 1
- 235000013965 Lactobacillus plantarum Nutrition 0.000 description 1
- 241000186604 Lactobacillus reuteri Species 0.000 description 1
- 241000218588 Lactobacillus rhamnosus Species 0.000 description 1
- 241000194036 Lactococcus Species 0.000 description 1
- 239000006142 Luria-Bertani Agar Substances 0.000 description 1
- 241000736262 Microbiota Species 0.000 description 1
- 229930191564 Monensin Natural products 0.000 description 1
- GAOZTHIDHYLHMS-UHFFFAOYSA-N Monensin A Natural products O1C(CC)(C2C(CC(O2)C2C(CC(C)C(O)(CO)O2)C)C)CCC1C(O1)(C)CCC21CC(O)C(C)C(C(C)C(OC)C(C)C(O)=O)O2 GAOZTHIDHYLHMS-UHFFFAOYSA-N 0.000 description 1
- 101000981253 Mus musculus GPI-linked NAD(P)(+)-arginine ADP-ribosyltransferase 1 Proteins 0.000 description 1
- 241000186366 Mycobacterium bovis Species 0.000 description 1
- 102100027347 Neural cell adhesion molecule 1 Human genes 0.000 description 1
- 206010029260 Neuroblastoma Diseases 0.000 description 1
- 241000532183 Norovirus GI Species 0.000 description 1
- 241000532184 Norovirus GII Species 0.000 description 1
- 101710163270 Nuclease Proteins 0.000 description 1
- TTZMPOZCBFTTPR-UHFFFAOYSA-N O=P1OCO1 Chemical compound O=P1OCO1 TTZMPOZCBFTTPR-UHFFFAOYSA-N 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 241001494479 Pecora Species 0.000 description 1
- 241000606999 Plesiomonas shigelloides Species 0.000 description 1
- 241000589516 Pseudomonas Species 0.000 description 1
- 208000019155 Radiation injury Diseases 0.000 description 1
- 241000700157 Rattus norvegicus Species 0.000 description 1
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 1
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 1
- 241001137860 Rotavirus A Species 0.000 description 1
- 241000235070 Saccharomyces Species 0.000 description 1
- 241000607142 Salmonella Species 0.000 description 1
- 241000369757 Sapovirus Species 0.000 description 1
- 108010034546 Serratia marcescens nuclease Proteins 0.000 description 1
- 108010071390 Serum Albumin Proteins 0.000 description 1
- 102000007562 Serum Albumin Human genes 0.000 description 1
- 108010079723 Shiga Toxin Proteins 0.000 description 1
- 241000607768 Shigella Species 0.000 description 1
- 241000191940 Staphylococcus Species 0.000 description 1
- 108010090804 Streptavidin Proteins 0.000 description 1
- 244000057717 Streptococcus lactis Species 0.000 description 1
- 235000014897 Streptococcus lactis Nutrition 0.000 description 1
- 241001134658 Streptococcus mitis Species 0.000 description 1
- 241000194023 Streptococcus sanguinis Species 0.000 description 1
- 241000282887 Suidae Species 0.000 description 1
- 102100024219 T-cell surface glycoprotein CD1a Human genes 0.000 description 1
- 108700019146 Transgenes Proteins 0.000 description 1
- 241000607626 Vibrio cholerae Species 0.000 description 1
- 241000607272 Vibrio parahaemolyticus Species 0.000 description 1
- 241000607265 Vibrio vulnificus Species 0.000 description 1
- 241000607734 Yersinia <bacteria> Species 0.000 description 1
- 210000000683 abdominal cavity Anatomy 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 208000038016 acute inflammation Diseases 0.000 description 1
- 230000006022 acute inflammation Effects 0.000 description 1
- 101150063416 add gene Proteins 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 229960000643 adenine Drugs 0.000 description 1
- 230000001668 ameliorated effect Effects 0.000 description 1
- 125000000539 amino acid group Chemical group 0.000 description 1
- 210000002255 anal canal Anatomy 0.000 description 1
- 238000000540 analysis of variance Methods 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- 239000004599 antimicrobial Substances 0.000 description 1
- 239000003963 antioxidant agent Substances 0.000 description 1
- 239000002216 antistatic agent Substances 0.000 description 1
- 210000000436 anus Anatomy 0.000 description 1
- 244000309743 astrovirus Species 0.000 description 1
- 210000003719 b-lymphocyte Anatomy 0.000 description 1
- 229940054340 bacillus coagulans Drugs 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 229940002008 bifidobacterium bifidum Drugs 0.000 description 1
- 229940009289 bifidobacterium lactis Drugs 0.000 description 1
- 229940009291 bifidobacterium longum Drugs 0.000 description 1
- 239000011230 binding agent Substances 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 230000008033 biological extinction Effects 0.000 description 1
- 230000031018 biological processes and functions Effects 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 238000001574 biopsy Methods 0.000 description 1
- 210000000481 breast Anatomy 0.000 description 1
- 239000006172 buffering agent Substances 0.000 description 1
- 102000023852 carbohydrate binding proteins Human genes 0.000 description 1
- 239000005018 casein Substances 0.000 description 1
- BECPQYXYKAMYBN-UHFFFAOYSA-N casein, tech. Chemical compound NCCCCC(C(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(CC(C)C)N=C(O)C(CCC(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(C(C)O)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(COP(O)(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(N)CC1=CC=CC=C1 BECPQYXYKAMYBN-UHFFFAOYSA-N 0.000 description 1
- 235000021240 caseins Nutrition 0.000 description 1
- 210000004534 cecum Anatomy 0.000 description 1
- 230000003915 cell function Effects 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 208000019065 cervical carcinoma Diseases 0.000 description 1
- 230000014564 chemokine production Effects 0.000 description 1
- 230000001684 chronic effect Effects 0.000 description 1
- 239000013599 cloning vector Substances 0.000 description 1
- 210000004922 colonic epithelial cell Anatomy 0.000 description 1
- 210000004953 colonic tissue Anatomy 0.000 description 1
- 239000003086 colorant Substances 0.000 description 1
- 230000000295 complement effect Effects 0.000 description 1
- 238000012790 confirmation Methods 0.000 description 1
- 230000021615 conjugation Effects 0.000 description 1
- 239000012228 culture supernatant Substances 0.000 description 1
- 230000009089 cytolysis Effects 0.000 description 1
- 229940104302 cytosine Drugs 0.000 description 1
- 238000007405 data analysis Methods 0.000 description 1
- 230000007123 defense Effects 0.000 description 1
- 239000008367 deionised water Substances 0.000 description 1
- 229910021641 deionized water Inorganic materials 0.000 description 1
- 239000005549 deoxyribonucleoside Substances 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 229960000633 dextran sulfate Drugs 0.000 description 1
- 238000000502 dialysis Methods 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 210000002249 digestive system Anatomy 0.000 description 1
- NAGJZTKCGNOGPW-UHFFFAOYSA-K dioxido-sulfanylidene-sulfido-$l^{5}-phosphane Chemical compound [O-]P([O-])([S-])=S NAGJZTKCGNOGPW-UHFFFAOYSA-K 0.000 description 1
- 208000037765 diseases and disorders Diseases 0.000 description 1
- KPUWHANPEXNPJT-UHFFFAOYSA-N disiloxane Chemical class [SiH3]O[SiH3] KPUWHANPEXNPJT-UHFFFAOYSA-N 0.000 description 1
- 238000010494 dissociation reaction Methods 0.000 description 1
- 230000005593 dissociations Effects 0.000 description 1
- 239000002552 dosage form Substances 0.000 description 1
- 231100000673 dose–response relationship Toxicity 0.000 description 1
- 238000001493 electron microscopy Methods 0.000 description 1
- 239000003480 eluent Substances 0.000 description 1
- 239000012149 elution buffer Substances 0.000 description 1
- 239000002158 endotoxin Substances 0.000 description 1
- 238000011013 endotoxin removal Methods 0.000 description 1
- 229940007078 entamoeba histolytica Drugs 0.000 description 1
- 230000000369 enteropathogenic effect Effects 0.000 description 1
- 230000000688 enterotoxigenic effect Effects 0.000 description 1
- 238000001976 enzyme digestion Methods 0.000 description 1
- YQGOJNYOYNNSMM-UHFFFAOYSA-N eosin Chemical compound [Na+].OC(=O)C1=CC=CC=C1C1=C2C=C(Br)C(=O)C(Br)=C2OC2=C(Br)C(O)=C(Br)C=C21 YQGOJNYOYNNSMM-UHFFFAOYSA-N 0.000 description 1
- 210000003238 esophagus Anatomy 0.000 description 1
- 230000007717 exclusion Effects 0.000 description 1
- 230000029142 excretion Effects 0.000 description 1
- 210000002744 extracellular matrix Anatomy 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 239000000945 filler Substances 0.000 description 1
- 239000000706 filtrate Substances 0.000 description 1
- 239000000796 flavoring agent Substances 0.000 description 1
- 235000013355 food flavoring agent Nutrition 0.000 description 1
- 235000003599 food sweetener Nutrition 0.000 description 1
- 230000004927 fusion Effects 0.000 description 1
- 210000000232 gallbladder Anatomy 0.000 description 1
- 230000002496 gastric effect Effects 0.000 description 1
- 201000011587 gastric lymphoma Diseases 0.000 description 1
- 201000004528 gastrointestinal lymphoma Diseases 0.000 description 1
- 238000003304 gavage Methods 0.000 description 1
- 239000008273 gelatin Substances 0.000 description 1
- 229920000159 gelatin Polymers 0.000 description 1
- 235000019322 gelatine Nutrition 0.000 description 1
- 235000011852 gelatine desserts Nutrition 0.000 description 1
- 238000012239 gene modification Methods 0.000 description 1
- 230000005017 genetic modification Effects 0.000 description 1
- 235000013617 genetically modified food Nutrition 0.000 description 1
- 208000005017 glioblastoma Diseases 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 230000035876 healing Effects 0.000 description 1
- 230000007407 health benefit Effects 0.000 description 1
- 229940037467 helicobacter pylori Drugs 0.000 description 1
- 210000003494 hepatocyte Anatomy 0.000 description 1
- 238000010231 histologic analysis Methods 0.000 description 1
- 102000046699 human CD14 Human genes 0.000 description 1
- 210000005260 human cell Anatomy 0.000 description 1
- 230000036737 immune function Effects 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 210000000987 immune system Anatomy 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 230000002458 infectious effect Effects 0.000 description 1
- 230000002757 inflammatory effect Effects 0.000 description 1
- 208000037798 influenza B Diseases 0.000 description 1
- 238000001802 infusion Methods 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 239000000138 intercalating agent Substances 0.000 description 1
- 230000008611 intercellular interaction Effects 0.000 description 1
- 238000001361 intraarterial administration Methods 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 238000007918 intramuscular administration Methods 0.000 description 1
- 238000007912 intraperitoneal administration Methods 0.000 description 1
- 238000007913 intrathecal administration Methods 0.000 description 1
- 238000001990 intravenous administration Methods 0.000 description 1
- 208000037906 ischaemic injury Diseases 0.000 description 1
- 208000028867 ischemia Diseases 0.000 description 1
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 description 1
- 229940039696 lactobacillus Drugs 0.000 description 1
- 229940039695 lactobacillus acidophilus Drugs 0.000 description 1
- 229940004208 lactobacillus bulgaricus Drugs 0.000 description 1
- 229940017800 lactobacillus casei Drugs 0.000 description 1
- 229940072205 lactobacillus plantarum Drugs 0.000 description 1
- 229940001882 lactobacillus reuteri Drugs 0.000 description 1
- 239000003446 ligand Substances 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 210000005229 liver cell Anatomy 0.000 description 1
- 208000014018 liver neoplasm Diseases 0.000 description 1
- 210000005265 lung cell Anatomy 0.000 description 1
- 210000004698 lymphocyte Anatomy 0.000 description 1
- 230000028744 lysogeny Effects 0.000 description 1
- 229920002521 macromolecule Polymers 0.000 description 1
- 230000036210 malignancy Effects 0.000 description 1
- 210000004962 mammalian cell Anatomy 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 238000012083 mass cytometry Methods 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- YACKEPLHDIMKIO-UHFFFAOYSA-N methylphosphonic acid Chemical compound CP(O)(O)=O YACKEPLHDIMKIO-UHFFFAOYSA-N 0.000 description 1
- 238000002493 microarray Methods 0.000 description 1
- 230000005012 migration Effects 0.000 description 1
- 238000013508 migration Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 229960005358 monensin Drugs 0.000 description 1
- GAOZTHIDHYLHMS-KEOBGNEYSA-N monensin A Chemical compound C([C@@](O1)(C)[C@H]2CC[C@@](O2)(CC)[C@H]2[C@H](C[C@@H](O2)[C@@H]2[C@H](C[C@@H](C)[C@](O)(CO)O2)C)C)C[C@@]21C[C@H](O)[C@@H](C)[C@@H]([C@@H](C)[C@@H](OC)[C@H](C)C(O)=O)O2 GAOZTHIDHYLHMS-KEOBGNEYSA-N 0.000 description 1
- 238000010172 mouse model Methods 0.000 description 1
- 210000000214 mouth Anatomy 0.000 description 1
- 210000004877 mucosa Anatomy 0.000 description 1
- 230000004682 mucosal barrier function Effects 0.000 description 1
- 210000003097 mucus Anatomy 0.000 description 1
- 210000003098 myoblast Anatomy 0.000 description 1
- 230000002107 myocardial effect Effects 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 210000004412 neuroendocrine cell Anatomy 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
- 239000002777 nucleoside Substances 0.000 description 1
- 125000003835 nucleoside group Chemical group 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 229920002113 octoxynol Polymers 0.000 description 1
- 238000003305 oral gavage Methods 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 210000001672 ovary Anatomy 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 230000007918 pathogenicity Effects 0.000 description 1
- 230000037361 pathway Effects 0.000 description 1
- 210000005259 peripheral blood Anatomy 0.000 description 1
- 239000011886 peripheral blood Substances 0.000 description 1
- 239000008194 pharmaceutical composition Substances 0.000 description 1
- 150000004713 phosphodiesters Chemical class 0.000 description 1
- 108091005981 phosphorylated proteins Proteins 0.000 description 1
- 108060006184 phycobiliprotein Proteins 0.000 description 1
- 210000004180 plasmocyte Anatomy 0.000 description 1
- 238000007747 plating Methods 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 239000003755 preservative agent Substances 0.000 description 1
- 230000003449 preventive effect Effects 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 230000008741 proinflammatory signaling process Effects 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 230000000069 prophylactic effect Effects 0.000 description 1
- 210000002307 prostate Anatomy 0.000 description 1
- 238000000159 protein binding assay Methods 0.000 description 1
- 238000001742 protein purification Methods 0.000 description 1
- 239000012460 protein solution Substances 0.000 description 1
- 230000002685 pulmonary effect Effects 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 230000005855 radiation Effects 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 210000002345 respiratory system Anatomy 0.000 description 1
- 108091008146 restriction endonucleases Proteins 0.000 description 1
- 239000002342 ribonucleoside Substances 0.000 description 1
- 229960002181 saccharomyces boulardii Drugs 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 238000004904 shortening Methods 0.000 description 1
- 230000011664 signaling Effects 0.000 description 1
- 210000000813 small intestine Anatomy 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 238000000527 sonication Methods 0.000 description 1
- 239000003381 stabilizer Substances 0.000 description 1
- 150000003457 sulfones Chemical class 0.000 description 1
- 239000003765 sweetening agent Substances 0.000 description 1
- 230000008685 targeting Effects 0.000 description 1
- 210000004876 tela submucosa Anatomy 0.000 description 1
- 229940124597 therapeutic agent Drugs 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 150000003568 thioethers Chemical class 0.000 description 1
- 229940113082 thymine Drugs 0.000 description 1
- 238000011200 topical administration Methods 0.000 description 1
- 230000000699 topical effect Effects 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 239000003053 toxin Substances 0.000 description 1
- 231100000765 toxin Toxicity 0.000 description 1
- 108700012359 toxins Proteins 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 210000004881 tumor cell Anatomy 0.000 description 1
- 238000005199 ultracentrifugation Methods 0.000 description 1
- 241001515965 unidentified phage Species 0.000 description 1
- 229940035893 uracil Drugs 0.000 description 1
- 210000002700 urine Anatomy 0.000 description 1
- 229940124931 vaccine adjuvant Drugs 0.000 description 1
- 239000012646 vaccine adjuvant Substances 0.000 description 1
- 239000003981 vehicle Substances 0.000 description 1
- 229940118696 vibrio cholerae Drugs 0.000 description 1
- 108700026220 vif Genes Proteins 0.000 description 1
- 238000012800 visualization Methods 0.000 description 1
- 238000003260 vortexing Methods 0.000 description 1
- 239000011534 wash buffer Substances 0.000 description 1
- 230000004584 weight gain Effects 0.000 description 1
- 235000019786 weight gain Nutrition 0.000 description 1
- 108010068608 xanthan lyase Proteins 0.000 description 1
- 210000005253 yeast cell Anatomy 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/177—Receptors; Cell surface antigens; Cell surface determinants
- A61K38/178—Lectin superfamily, e.g. selectins
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/66—Microorganisms or materials therefrom
- A61K35/74—Bacteria
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Definitions
- Lectins are produced by almost all forms of life and mediate cell-cell interactions via proteins and carbohydrate ligands. Through interactions with glycans of the extracellular matrix, lectins assist an organism's ability to establish a niche, evade host defenses, and protect against pathogens. Lectins have also been developed therapeutically as antibiotics, vaccine adjuvants, and for the treatment of cancer.
- Human lectins have a broad range of functions across diverse cell types, including regulation of the microbiome, i.e., the collection of commensal organisms that colonize the body, are important to human health, and can perpetuate disease when disrupted. Despite the importance of lectins in cell signaling and microbiome interactions, microbial lectins from human commensal organisms, and their role in the microbiome, remain largely unexplored.
- the disclosure provides a method of treating an inflammatory disease or disorder of the gastrointestinal tract in a subject, the method comprising administering to the subject a composition comprising at least one of a genetically engineered cell expressing a lectin, a modified lectin gene encoding a lectin, or a lectin, wherein the lectin is a non-enzymatic protein comprising a carbohydrate-binding domain.
- the lectin is Cbeg4 or Cbeg5.
- the lectin lacks a signal peptide.
- the lectin affects the number and/or activity of cells having a myeloid cell lineage.
- the lectin can modulate the number and/or activity of macrophages in the gastrointestinal tract of the subject, and in some aspects, the macrophages express at least one of MHCII, CD 11c, CD64, or CD 11b.
- the lectin can modulate the number and/or activity of monocytes in the gastrointestinal tract of the subject, and in some aspects, the monocytes express at least one of Lyc or CD1 lb.
- the lectin affects the number and/or activity of intestinal or mucosal immune cells.
- the inflammatory disease affects the large intestine or colon of the subject.
- the inflammatory disease or disorder is inflammatory bowel disease, irritable bowel syndrome, Crohn's disease, ulcerative colitis, microscopic colitis, or Celiac disease.
- the genetically engineered cell is a non-pathogenic bacterial cell, for example, E. coli.
- the composition is administered to the subject in a therapeutically effective amount.
- the composition further comprises a pharmaceutically acceptable carrier, diluent, buffer, or excipient.
- the composition is administered to the subject as a capsule or tablet.
- the composition comprises the genetically engineered cell expressing the lectin, and the composition is administered to the subject as a live bio therapeutic.
- the disclosure provides a method of treating a cancer of the gastrointestinal tract in a subject, the method comprising administering to the subject a composition comprising at least one of a genetically engineered cell expressing a lectin, a modified lectin gene encoding a lectin, or a lectin, wherein the lectin is a non-enzymatic protein comprising a carbohydrate-binding domain.
- the lectin is Cbeg4 or Cbeg5.
- the lectin lacks a signal peptide.
- the lectin affects the number and/or activity of cells having a myeloid cell lineage.
- the lectin can modulate the number and/or activity of macrophages in the gastrointestinal tract of the subject, and in some aspects, the macrophages express at least one of MHCII, CD 11c, CD64, or CD 11b.
- the lectin can modulate the number and/or activity of monocytes in the gastrointestinal tract of the subject, and in some aspects, the monocytes express at least one of Lyc or CD1 lb.
- the lectin affects the number and/or activity of intestinal or mucosal immune cells.
- the cancer is an adenocarcinoma of the gastrointestinal tract.
- the cancer is colon or rectal cancer, small bowel adenocarcinoma, gastric cancer, hepatocellular carcinoma, pancreatic cancer, or a neuroendocrine tumor.
- the genetically engineered cell is a non-pathogenic bacterial cell, for example, E. coli.
- the composition is administered to the subject in a therapeutically effective amount.
- the composition further comprises a pharmaceutically acceptable carrier, diluent, buffer, or excipient.
- the composition is administered to the subject as a capsule or tablet.
- the composition comprises the genetically engineered cell expressing the lectin, and the composition is administered to the subject as a live bio therapeutic.
- the disclosure provides a genetically engineered cell expressing a lectin, wherein the lectin is a non-enzymatic protein comprising a carbohydrate-binding domain.
- the lectin is Cbeg4 or Cbeg5.
- Cbeg4 can be identified by Genbank accession no. KT336269.1.
- the lectin is Cbeg4 having the carbohydrate -binding domain is identified by SEQ ID NO: 2.
- Cbeg5 can be identified by Genbank accession no. KT336270.1.
- the lectin is Cbeg5 having the carbohydrate-binding domain is identified by SEQ ID NO: 1.
- the genetically engineered cell is a non-pathogenic bacterial cell, for example, E. coli.
- composition comprising a genetically engineered cell as described above or disclosed herein.
- composition further comprises a pharmaceutically acceptable carrier, diluent, buffer, or excipient.
- the composition is a live bio therapeutic.
- the composition is a capsule or a tablet.
- the disclosure provides a method of eliciting or enhancing an immunogenic response to an antigen, the method comprising administering to a subject the antigen and an adjuvant, wherein the adjuvant comprises a genetically engineered cell as described above or disclosed herein.
- the adjuvant is administered to the subject orally, subcutaneously, or intranasally, and/or the adjuvant is administered simultaneously with the antigen, sequentially with the antigen, or as an adjuvant-antigen conjugate.
- the adjuvant is administered to the subject with a vaccine selected from the group consisting of influenza, respiratory syncytial virus, rotavirus, hepatitis A, hepatitis B, measles, mumps, rubella, streptococcus pneumonia, and COVID- 19.
- a vaccine selected from the group consisting of influenza, respiratory syncytial virus, rotavirus, hepatitis A, hepatitis B, measles, mumps, rubella, streptococcus pneumonia, and COVID- 19.
- the disclosure provides a composition comprising a lectin, wherein the lectin is a non-enzymatic protein comprising a carbohydrate-binding domain and lacking a signal peptide.
- the lectin is Cbeg4 or Cbeg5.
- Cbeg4 can be identified by Genbank accession no. KT336269.1.
- the lectin is Cbeg4 having the carbohydrate -binding domain is identified by SEQ ID NO: 2.
- Cbeg5 can be identified by Genbank accession no. KT336270.1.
- the lectin is Cbeg5 having the carbohydrate-binding domain is identified by SEQ ID NO: 1.
- composition further comprises a pharmaceutically acceptable carrier, diluent, buffer, or excipient.
- the composition is a capsule or a tablet.
- the disclosure provides a method of eliciting or enhancing an immunogenic response to an antigen, the method comprising administering to a subject the antigen and an adjuvant, wherein the adjuvant comprises the composition as described above or disclosed herein.
- the adjuvant is administered to the subject orally, subcutaneously, or intranasally, and/or the adjuvant is administered simultaneously with the antigen, sequentially with the antigen, or as an adjuvant-antigen conjugate.
- the adjuvant is administered to the subject with a vaccine selected from the group consisting of influenza, respiratory syncytial virus, rotavirus, hepatitis A, hepatitis B, measles, mumps, rubella, streptococcus pneumonia, and COVID- 19.
- a vaccine selected from the group consisting of influenza, respiratory syncytial virus, rotavirus, hepatitis A, hepatitis B, measles, mumps, rubella, streptococcus pneumonia, and COVID- 19.
- FIG. 1 illustrates the functional domains of lectin Cbeg5.
- FIG. 2 illustrates Cbeg4 and Cbeg5 expression constructs.
- FIGS. 3A & 3B are dot plots of CBM4 and CBM5 glycan binding.
- FIG. 3C is a dot plot of normalized CBM4 and CBM5 glycan binding.
- FIG. 3D illustrates the structure of the top bound glycan of FIGS 3A - 3C.
- the dotted line indicates a conserved binding motif (glycopattem).
- FIG. 3E reflects glycomics datasets for samples containing the conserved binding motif of FIG. 3D.
- FIG. 4A depicts immune cell populations A - K distinguished by a CyTOF panel of cell surface markers.
- FIGS. 4B - 4E are bar graphs of percent positive cells for various cytokines in cell populations following exposure to Cbeg5 or the Fn5.
- FIG. 4F illustrates fold induction of 10 cytokines across cell populations A - K of FIG. 4A following exposure to full-length Cbeg5, CBM5, or Fn5.
- FIGS. 5A - 5O illustrate CyTOF analysis of PBMCs using a panel of cell surface markers.
- FIG. 6A depicts immune cell populations A - K distinguished by a CyTOF panel of cell surface markers.
- FIGS. 6B - 6F are bar graphs of percent positive cells for various cytokines in cell populations following exposure to Cbeg5 or the Fn5.
- FIG. 6G illustrates fold induction of 8 phosphorylated signaling proteins in cell populations A - K of FIG. 6A following exposure to full-length Cbeg5, CBM5, or Fn5.
- FIG. 7A is a SPADE plot for Cbeg5-induced TNF-a production from intestinal leukocytes.
- FIGS. 7B & 7C are bar graphs of Cbeg5-induced cytokine production in CD14 + CD4 + (FIG. 7B) and CD14 + CDlc + (FIG. 7C) cells.
- FIG. 7D is a heat map of cytokine production in CD14 + monocytes, monocyte-derived macrophages (MDMs), and monocyte-derived dendritic cells DC (moDC) following exposure to Cbeg5 or Fn5.
- MDMs monocyte-derived macrophages
- moDC monocyte-derived dendritic cells
- FIGS. 7E & 7F are bar graphs of Cbeg5-induced cytokine/chemokine or adhesion molecule production in CD14 + monocytes, MDMs, and moDCs.
- FIGS. 8A & 8B are SPADE plots for Cbeg-5 induced TNF-a and IL- 10 from LPLs isolated from a first patient.
- FIGS. 8C & 8D are SPADE plots for Cbeg-5 induced TNF-a and IL- 10 from LPLs isolated from a second patient.
- FIGS. 8E & 8F are control (PBS) SPADE plots from LPLs isolated from the second patient.
- FIGS. 8G & 8H are bar graphs of Cbeg5-induced cytokine production from CDlc (FIG. 8G) and CD14 (FIG. 8H) cell clusters.
- FIGS. 9A & 9B are representative images of H&E stained colon sections from mice colonized with E. coli expressing cbeg5 (EC:Cbeg5; FIG. 9A) or controls (EC:Con; FIG. 9B).
- FIG. 9C illustrates histologic scoring of colon inflammation in EC:Cbeg5 mice or controls.
- FIG. 10A is a graph depicting CFU in EC:Cbeg5 mice or controls.
- FIG. 10B is a graph depicting food consumption in EC:Cbeg5 mice or controls.
- FIG. 10C is a graph depicting water consumption in EC:Cbeg5 mice or controls.
- FIG. 10D is a graph depicting weight change in EC:Cbeg5 mice or controls.
- FIG. 11A is a flow cytometry gating schema for identification of macrophages MHCII + CDl lc + CD64 + CDl lb + and monocytes MHCirLyc + CDl lb + .
- FIGS. 11B & 11C are graphs of myeloid cell populations in EC:Cbeg5 mice or controls from the flow cytometry of FIG. 11A.
- FIG. 12A is a flow cytometry gating schema for identification of T cell populations.
- FIGS. 12B - 12E are graphs of T cell populations (based on transcription factor expression) in EC:Cbeg5 mice or controls from the flow cytometry of FIG. 12A.
- FIG. 13A is a graph illustrating previously characterized lectins and Cbeg4/5 in patent stool samples.
- FIG. 13B is a circle chart showing phylum distribution of identified and unidentified lectins.
- FIG. 14A is a graph illustrating the co-occurrence of a carbohydrate-binding domain and a secondary domain in a human-microbial-lectin dataset.
- FIG. 14B illustrates domain architectures containing the Cbeg4/5 carbohydrate-binding domain (IPR338O3) and the Bacteroidetes- Associated Carbohydrate -binding Often N-terminal domain (IPR024361).
- FIG. 15 is a graph illustrating the prevalence of human-microbial-lectins in patient samples from the Human Microbiome Project.
- FIG. 16A illustrates histograms of human-microbial-lectins aligned to reference genome sequences from bacteria isolated different sites in the human microbiome.
- FIGS. 16B & 16C are bar graphs of the mean number of human-microbial-lectin genes per genome presented per body site and phylum.
- FIG. 17 illustrates overlap of lectin genes between five body sites.
- FIG. 18 is a bar graph depicting the variability of domain architecture in lectin protein sequences from patient samples.
- FIGS. 19A & 19B are graphs depicting rarefication analysis of human-microbial-lectin genes for various body sites based on the number of samples from the Human Microbiome Project.
- FIGS. 20A & 20B are graphs depecting the effect of bacteria-delivered Cbeg5 against dextran sulfate dosium (DSS)-induced colitis in mouse models.
- DSS dextran sulfate dosium
- the present disclosure relates to lectins, compositions thereof, and methods of using lectins to treat a disease or disorder.
- lectin refers to a carbohydrate-binding protein of nonimmune origin.
- the lectins of the present disclosure are non-enzymatic proteins having a carbohydrate-binding domain.
- Lectin can refer to a full-length protein or at least one domain thereof.
- the disclosure provides polynucleotides encoding at least one lectin domain.
- domain is inclusive of signal peptides for protein secretion and trafficking.
- polynucleotide as used herein is defined as a chain of nucleotides.
- nucleic acids are polymers of nucleotides.
- nucleic acids and polynucleotides as used herein are interchangeable.
- nucleic acids are polynucleotides, which can be hydrolyzed into the monomeric nucleotides. The monomeric nucleotides can be hydrolyzed into nucleosides.
- polynucleotides include, but are not limited to, all nucleic acid sequences which are obtained by any means available in the art, including, without limitation, recombinant means, i.e., the cloning of nucleic acid sequences from a recombinant library or a cell genome, using ordinary cloning technology and PCR, and the like, and by synthetic means.
- recombinant means i.e., the cloning of nucleic acid sequences from a recombinant library or a cell genome, using ordinary cloning technology and PCR, and the like, and by synthetic means.
- nucleic acid refers to any nucleic acid, and is inclusive of deoxyribonucleosides and/or ribonucleosides composed of phosphodiester linkages or modified linkages such as phosphotriester, phosphoramidate, siloxane, carbonate, carboxymethylester, acetamidate, carbamate, thioether, bridged phosphoramidate, bridged methylene phosphonate, phosphorothioate, methylphosphonate, phosphorodithioate, bridged phosphorothioate or sulfone linkages, and combinations of such linkages.
- Nucleic acid also includes nucleic acids composed of bases other than the five biologically occurring bases (adenine, guanine, thymine, cytosine, and uracil).
- peptide As used herein, "peptide,” “polypeptide,” and “protein” are used interchangeably, and refer to a compound comprised of amino acid residues covalently linked by peptide bonds.
- a protein or peptide must contain at least two amino acids, and no limitation is placed on the maximum number of amino acids that can comprise a protein or peptide sequence.
- Polypeptides include any peptide or protein comprising two or more amino acids joined to each other by peptide bonds.
- the term refers to both short chains, which also commonly are referred to in the art as peptides, oligopeptides, and oligomers, for example, and to longer chains, which generally are referred to in the art as proteins, of which there are many types.
- Polypeptides include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of poly- peptides, modified polypeptides, derivatives, analogs, fusion proteins, among others.
- the polypeptides include natural peptides, recombinant peptides, synthetic peptides, or a combination thereof.
- Encode refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom.
- a gene encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system.
- Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and the non-coding strand, used as the template for transcnptlOn of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA.
- the terms "expressing” or “expression” as used herein is defined as the transcription and/or translation of a particular nucleotide sequence driven by its promoter.
- the polynucleotides of the present disclosure encode a full-length lectin comprising a signal peptide, a fibronectin (Fn) domain, and a carbohydrate -binding module (CBM) domain.
- Fn fibronectin
- CBM carbohydrate -binding module
- the polynucleotides encode a polypeptide having a signal peptide and a lectin CBM domain, or a secretion signal peptide and a lectin Fn domain.
- the polynucleotides encode a polypeptide with a lectin CBM domain or a lectin Fn domain but lack a signal peptide.
- polynucleotides include genes from the commensal bacterial effector gene (cbeg) family, and more specifically cbeg4 and cbeg5, which express a Cbeg lectin (e.g., Cbeg4 or Cbeg5).
- the polynucleotide expresses the CBM domain of Cbeg4 (CBM4) or Cbeg5 (CBM5), with or without a signal peptide.
- the polynucleotide expresses CBM4 and an Fn domain or CBM5 and an Fn domain, with or without a signal peptide.
- Polynucleotides expressing a lectin CBM domain without a signal peptide can be referred to herein as "modified lectin genes".
- the polynucleotide expresses the CBM domain of Cbeg4 or Cbeg5 and a fibronectin (Fn) type 3 domain, with or without a signal peptide.
- Sequences of exemplary lectins (i.e., polypeptides) of the present disclosure are shown in Table 1.
- Table 1 Amino Acid Sequence of Lectin CBM Domain
- Polypeptides can be conjugated to a detectable agent or label such that the protein can be detected.
- Labels include, without limitation, a chromophore, a fluorophore, a fluorescent protein, a phosphorescent dye, a tandem dye, a particle, an enzyme, and a radioisotope.
- fluorescent proteins may be conjugated to the lectin as a label. Examples of fluorescent proteins include green fluorescent protein (GFP) and the phycobiliproteins and derivatives thereof.
- the polypeptides disclosed herein can be produced as fusion proteins containing a domain (e.g., GFP) that facilitates its detection.
- Techniques for making fusion proteins are well known. Essentially, the joining of various nucleic acid fragments coding for different polypeptide sequences is performed in accordance with conventional techniques, employing blunt-ended or stagger-ended termini for ligation, restriction enzyme digestion to provide for appropriate termini, filling-in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and enzymatic ligation.
- the fusion gene can be synthesized by conventional techniques, including automated DNA synthesizers. Alternatively, PCR amplification of gene fragments can be carried out using anchor primers, giving rise to complementary overhangs between two consecutive nucleic acid fragments, which can subsequently be annealed to generate a chimeric gene sequence. Genetically Modified Cells
- the disclosure provides genetically modified cells expressing a polynucleotide as disclosed herein.
- the genetically modified cell can be constructed from any suitable prokaryotic, eukaryotic, plant, or animal host cell.
- Suitable eukaryotic host cells include a human cell (including a stem cell), a non-human mammalian cell, a non-mammalian vertebrate cell, an invertebrate cell, an insect cell, a plant cell, a yeast cell, and a single-cell eukaryotic organism.
- the genetically modified cell is a cell line.
- Such cell lines are known to one of ordinary skill and include, but are not limited to, Chinese hamster ovary (CHO) cells, baby hamster kidney (BHK) cells; mouse myeloma NS0 cells, mouse embryonic fibroblast 3T3 cells (NIH3T3), mouse B lymphoma A20 cells; mouse melanoma B16 cells; mouse myoblast C2C12 cells; mouse myeloma SP2/0 cells; mouse embryonic mesenchymal C3H-10T1/2 cells; mouse carcinoma CT26 cells, mouse prostate DuCuP cells; mouse breast EMT6 cells; mouse hepatoma Hepalclc7 cells; mouse myeloma 15582 cells; mouse epithelial MTD-1A cells; mouse myocardial MyEnd cells; mouse renal RenCa cells; mouse pancreatic RIN-5F cells; mouse melanoma X64 cells; mouse lymphoma YAC-1 cells;
- the genetically engineered cell is a non-pathogenic bacterial cell.
- Non- pathogenic bacteria refer to bacteria that are not capable of causing disease or harmful responses in a host. In some aspects, non-pathogenic bacteria are commensal bacteria.
- non- pathogenic bacteria examples include, but are not limited to, Bacillus, Bacteroides, Bifidobacterium, Brevibacteria, Clostridium, Enterococcus, Escherichia coli, Lactobacillus, Lactococcus, Saccharomyces, and Staphylococcus, e.g., Bacillus coagulans, Bacillus subtilis, Bacteroides fragilis, Bacteroides subtilis, Bacteroides thetaiotaomicron, Bifidobacterium bifidum, Bifidobacterium in/antis, Bifidobacterium lactis, Bifidobacterium longum, Clostridium butyricum, Enterococcus f aecium, Lactobacillus acidophilus, Lactobacillus bulgaricus, Lactobacillus casei, Lactobacillus johnsonii, Lactobacillus paracasei, Lactobacillus plantarum
- Endogenously pathogenic bacteria can be genetically engineered to provide reduced or eliminated pathogenicity.
- Non-pathogenic bacteria can be genetically engineered to enhance or improve desired biological properties (e.g., surviv ability).
- the genetically modified cell is E. coli.
- Methods for genetic modification of cells are known to one of ordinary skill, and generally comprise exposing the cell to a gene transfer vector comprising a polynucleotide as disclosed herein and a promoter, such that the polynucleotide is introduced into the cell under conditions appropriate for its expression.
- a genetically modified cell expresses a full-length lectin comprising a secretion signal peptide, a fibronectin (Fn) domain, and a carbohydrate-binding module (CBM) domain.
- a genetically modified cell expresses a polypeptide having a signal peptide and a lectin CBM domain.
- a genetically modified cell expresses one of the following: cbeg4, cbeg5, a polynucleotide having a signal peptide and CBM4, or a polynucleotide having a signal peptide and CBM5.
- the genetically modified cell expresses a polynucleotide having a signal peptide, Fn type 3 domain, and CBM4.
- the genetically modified cell expresses a polynucleotide having a signal peptide, Fn type 3 domain, and CBM5.
- compositions in another aspect, the disclosure provides compositions. It will be understood that "composition”, “pharmaceutical composition”, and “therapeutic composition” can be used interchangeably.
- a composition comprises a genetically modified cell expressing a lectin as disclosed herein.
- a composition comprises a polynucleotide (e.g., lectin gene or modified lectin gene) as disclosed herein.
- a composition comprises a lectin as disclosed herein.
- the above compositions can be formulated with a pharmaceutically acceptable carrier, excipient, diluent, buffer, or stabilizer.
- compositions are suitable for administration to a human, or a non-human mammal or animal, via any one or more route of administration using methods known in the art.
- the route and/or mode of administration will vary depending upon the desired results.
- pharmaceutically acceptable carrier means one or more non-toxic materials that do not interfere with the effectiveness of the biological activity of the active ingredients.
- Such preparations may routinely contain salts, buffering agents, preservatives, compatible carriers, and optionally other therapeutic agents.
- Such pharmaceutically acceptable preparations may also contain compatible solid or liquid fillers, diluents or encapsulating substances, which are suitable for administration to a human.
- contemplated carriers, excipients, and/or additives which may be utilized in the formulations described herein include, for example, flavoring agents, antimicrobial agents, sweeteners, antioxidants, antistatic agents, lipids, protein excipients such as serum albumin, gelatin, casein, salt-forming counter-ions such as sodium and the like.
- These and additional known pharmaceutical carriers, excipients and/or additives suitable for use in the formulations described herein are known in the art, e.g., as listed in “Remington: The Science & Practice of Pharmacy", 21st ed., Lippincott Williams & Wilkins, (2005), and in the "Physician's Desk Reference", 60th ed., Medical Economics, Montvale, N.J. (2005).
- Pharmaceutically acceptable carriers can be selected that are suitable for the mode of administration, solubility and/or stability desired or required.
- compositions of the present disclosure comprise active agents (i.e., a genetically engineered cell expressing a lectin, a modified lectin gene encoding a lectin, or a lectin, wherein the lectin is a non-enzymatic protein comprising a carbohydrate-binding domain) in a concentration resulting in a w/v appropriate for a desired dose.
- active agents i.e., a genetically engineered cell expressing a lectin, a modified lectin gene encoding a lectin, or a lectin, wherein the lectin is a non-enzymatic protein comprising a carbohydrate-binding domain
- the active agent is present in an "effective amount” or a "therapeutically effective amount”.
- effective amount or “therapeutically effective amount” refer to an amount or dosage level of an active ingredient that is effective in achieving a desired therapeutic response (e.g., treating a disease or disorder) for a particular subject, composition, and mode of administration, without being toxic to the subject.
- the amount will depend upon a variety of pharmacokinetic factors, including the activity of the particular compositions employed, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.
- compositions can be formulated for particular routes of administration, such as oral, nasal, pulmonary, topical (including buccal and sublingual), rectal, vaginal and/or parenteral administration.
- parenteral administration and “administered parenterally” as used herein refer to modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrastemal injection and infusion.
- Formulations i.e., active and inactive agents
- the route of administration is oral, intranasal, or subcutaneous.
- the disclosure provides live biotherapeutic compositions comprising a genetically engineered cell as described herein.
- a biotherapeutic refers to a compound or substance produced from biological sources, such as a living organism, rather than through chemical synthesis.
- Live biotherapeutic refers to a living organism that when administered to a subject confers a health benefit to the subject. More specifically, “live biotherapeutic” as used herein refers to a living organism that when administered to a subject is applicable to the prevention, treatment, and/or cure of a disorder and/or disease.
- the live biotherapeutic disclosed herein i.e., a genetically engineered cell expressing a lectin
- a lectin i.e., non-enzymatic protein comprising a carbohydrate-binding domain
- the lectin is synthesized by the genetically engineered cells and then released into the GI tract of the subject.
- the disclosure provides a method of treating and/or preventing an inflammatory disease or disorder of the gastrointestinal tract in a subject by administering a composition comprising a genetically modified cell expressing a lectin, a polynucleotide (e.g., lectin gene or modified lectin gene), or a lectin as disclosed herein.
- a "disease" as used herein is a state of health of a subject wherein the subject cannot maintain homeostasis, and wherein if the disease is not ameliorated, the subject's health continues to deteriorate.
- a “disorder” is a state of health in which the subject is able to maintain homeostasis, but in which the subject's state of health is less favorable than it would be in the absence of the disorder.
- a disorder does not necessarily cause a further decrease in the subject's state of health if left untreated.
- a disease or disorder is "alleviated” if the severity of a sign or symptom of the disease or disorder, the frequency with which such a sign or symptom is experienced by a subject, or both, is reduced.
- the terms “subject”, “individual”, and “patient” are interchangeable, and relate to vertebrates, preferably mammals.
- mammals in the context of the disclosure are humans, non-human primates, domesticated animals such as dogs, cats, sheep, cattle, goats, pigs, horses, etc., laboratory animals such as mice, rats, rabbits, guinea pigs, etc., as well as animals in captivity such as animals in zoos.
- the term “animal” as used herein includes humans.
- the term “subject” may also include a patient, i.e., an animal, having a disease or disorder.
- inflammatory disease or "inflammatory disorder” of the gastrointestinal (GI) tract refer to diseases and disorders characterized by chronic (e.g., months or years) or acute inflammation of the GI tract as mediated predominately by macrophages, lymphocytes, and/or plasma cells and include, but are not limited to, GI autoimmune diseases and disorders, radiation injury, infectious injury, and ischemic injury.
- GI autoimmune diseases and disorders include, but are not limited to, GI autoimmune diseases and disorders, radiation injury, infectious injury, and ischemic injury.
- “Gastrointestinal tract” or “GI tract” refers to the collective organs of the digestive system, specifically, the mouth, esophagus, stomach, small intestine, large intestine, pancreas, liver, gallbladder, rectum, and anus.
- the colon will be understood as being one segment of the large intestine, the others being the cecum, rectum, and anal canal.
- Exemplary inflammatory diseases or disorders of the disclosed methods include inflammatory bowel disease, irritable bowel syndrome, Crohn's disease, ulcerative colitis, microscopic colitis, or Celiac disease.
- treat refers to administering a genetically modified cell expressing a lectin, a polynucleotide (e.g., lectin gene or modified lectin gene), or a lectin as disclosed herein to a subject in order to eliminate or reduce the clinical signs of the disease or disorder in the subject; arrest, inhibit, or slow the progression of the disease or disorder in the subject; and/or decrease the number, frequency, or severity of clinical symptoms and/or recurrence of disease or disorder in the subject who currently has or who previously had the disease or disorder.
- a genetically modified cell expressing a lectin, a polynucleotide e.g., lectin gene or modified lectin gene
- treatment of a disease includes curing, shortening the duration, ameliorating, slowing down, or inhibiting progression or worsening, or delaying the onset of clinical symptoms in a subject who has the disease or disorder.
- prophylactic, proventive, preventing, and “prevention” relate to the prevention of the occurrence of a disease or disorder or prevention of the progression of a multi-stage disease or disorder from a less severe to a more severe stage.
- the disclosure provides a method of treating and/or preventing a cancer of the GI tract in a subject by administering a composition comprising a genetically modified cell expressing a lectin, a polynucleotide (e.g., lectin gene or modified lectin gene), or a lectin as disclosed herein.
- a composition comprising a genetically modified cell expressing a lectin, a polynucleotide (e.g., lectin gene or modified lectin gene), or a lectin as disclosed herein.
- the cancer is colon cancer or rectal cancer (i.e., colorectal cancer), small bowel adenocarcinoma, gastric cancer, hepatocellular carcinoma, pancreatic cancer, or a neuroendocrine tumor.
- rectal cancer i.e., colorectal cancer
- small bowel adenocarcinoma gastric cancer
- hepatocellular carcinoma pancreatic cancer
- neuroendocrine tumor a neuroendocrine tumor.
- Colorectal cancer includes adenocarcinoma of the colon or rectum, carcinoid tumors, gastrointestinal stromal tumors, and lymphoma.
- Adenocarcinoma refers to a malignant neoplasm of epithelial cells, and, more specifically, a carcinoma derived from glandular tissue or in which the tumor cells form a glandular structure.
- Adenocarcinoma includes four subcategories: acinar, papillary, bronchoalveolar, and solid carcinoma with mucus formation.
- Small bowel adenocarcinoma refers to adenocarcinoma of the small intestine.
- Gastric cancer or “stomach cancer” refers to cancer originating from the stomach and includes adenocarcinoma, gastric lymphoma, gastrointestinal stromal tumors, and carcinoid tumors.
- Hepatocellular carcinoma will be understood to mean primary liver cancer (i.e., originating in hepatocytes), and is distinct from secondary liver cancer (i.e., a cancer that originates in another tissue and spreads to the liver).
- Pancreatic cancer refers to cancer originating in the pancreas and includes exocrine pancreatic cancer (e.g., adenocarcinoma) and pancreatic neuroendocrine tumors.
- exocrine pancreatic cancer e.g., adenocarcinoma
- pancreatic neuroendocrine tumors e.g., adenocarcinoma
- Neuroendocrine tumors refer to cancer arising from neuroendocrine cells.
- exemplary neuroendocrine tumors include carcinoid tumors and pancreatic neuroendocrine tumors.
- the methods disclosed herein provide a therapeutic response by at least one of the following: a) modulating (e.g., increasing) the number and/or activity of intestinal or mucosal immune cells; b) modulating (e.g., increasing) the number and/or activity of cells having a myeloid cell lineage; c) modulating (e.g., increasing) the number and/or activity of macrophages in the GI tract of a subject; d) modulating (e.g., increasing) the number and/or activity of macrophages expressing at least one of MHCII, CD11c, CD64, or CD1 lb in the GI tract of a subject
- Means of increasing cell number include proliferation, differentiation, and migration in response to therapeutically-induced trafficking signals.
- Increased cellular activity refers to an increase in one or more cellular or immune functions of a cell population.
- the disclosure provides a method of eliciting or enhancing an immunogenic response to an antigen by administering to a subject the antigen and an adjuvant, wherein the adjuvant comprises a genetically engineered cell or a lectin as disclosed herein.
- adjuvant refers to an agent, substance, vehicle, or therapy that enhances antigenicity.
- the adjuvant can be administered orally or intranasally to protect against a GI infection.
- the adjuvant can be administered subcutaneously.
- the adjuvant can be co-administered with an antigen.
- the adjuvant can be administered sequentially with an antigen.
- the adjuvant can be administered as an adjuvant-antigen conjugate.
- the adjuvant can be administered with a vaccine.
- exemplary vaccines include, but are not limited to, influenza, respiratory syncytial virus (RSV), rotavirus, Hepatitis A/B, measles, mumps, rubella, streptococcus pneumonia, and COVID- 19.
- RSV respiratory syncytial virus
- rotavirus rotavirus
- Hepatitis A/B Hepatitis A/B
- measles measles
- mumps rubella
- streptococcus pneumonia and COVID- 19.
- an adjuvant of the present disclosure can replace existing adjuvants used for established vaccines.
- the adjuvant, genetically engineered cell, and/or lectin of the as described herein can be used to elicit or enhance an immunogenic response to various pathogens of the GI tract including, Campylobacter species (Campylobacter jejuni/Campylobacter coli/Campylobacter upsaliensis), Clostridioides difficile toxin A/B, Plesiomonas shigelloides, Salmonella species, Vibrio species (Vibrio parahaemolyticus, Vibrio vulnificus, Vibrio cholerae), Vibrio cholerae- Yersinia species, Entero aggregative Escherichia coli (EAEC), Enteropathogenic E coli (EPEC), Enterotoxigenic E coli (ETEC), Shiga toxin, E coli 0157, Shigella/Enteroinvasive E coli (EIEC), Cryptosporidium species, Cyclospora cayetanensis
- the adjuvant, genetically engineered cell, and/or lectin of the as described herein can be used to elicit or enhance an immunogenic response to various pathogens of the respiratory tract including, Adenovirus Bordetella pertussis, Chlamydophila pneumonia, Coronavirus HKU1, Coronavirus NL63, Coronavirus 229E, Coronavirus OC43, Human metapneumovirus, Human rhino virus/entero virus, Influenza A, Influenza A/Hl, Influenza A/Hl-2009, Influenza A/H3, Influenza B, Mycoplasma pneumonia, Parainfluenza 1, Parainfluenza 2, Parainfluenza 3, Parainfluenza 4, RSV, Mycoplasmoides pneumonia, Streptococcus pneumonia, Mycoplasma pneumonia, Pseudomonas pneumonia, and Staphlococcus aureus pneumonia.
- Adenovirus Bordetella pertussis Chlamydophila pneumonia
- Coronavirus HKU1 Coronavirus NL63
- Coronavirus 229E
- Example 1 Generation of Cbeg5 and Fn5 Expression Constructs (Subcloning pET28c-Hise- Cbeg5 21 957 Cbeg5 and Fn5)
- Expression constructs of full-length cbeg5 or the cbeg5 fibronectin (Fn) domain alone (Fn5) were generated by first subcloning primers with overhanging Ndel and Xhol sequences to amplify the nucleotide sequence for each construct from a pJWCl-Cbeg5 (4L05) template vector following a standard PCR protocol for Q5 High-Fidelity DNA Polymerase (New England Biolabs).
- the annealing temperature was 67°C and the extension time was 90 sec.
- Fn5 the annealing temperature was 68°C and the extension time was 30 sec.
- Amplified gene sequences were digested with Ndel and Xhol (New England Biolabs), ligated into a Ndel- and Xhol-digested pET28c using T4 DNA ligase (New England Biolabs), and transformed into electrocompetent E. coli EC100 cells. Lysogeny broth (LB) media (50 mL with 50 pg mb 1 kanamycin) was inoculated with single colonies. Following incubation, the cultures were miniprepped, and the identity of the plasmid confirmed by Sanger sequencing.
- LB Lysogeny broth
- Example 2 Generation of Cbeg4-CBM and Cbeg5-CBM Expression Constructs (Subcloning pET28c-His6-GFP Cbeg4-CBM and Cbeg5-CBM)
- CBM carbohydrate-binding module domain
- cbeg5-CBM carbohydrate-binding module domain
- cbeg4-CBM carbohydrate-binding module domain
- first subcloning primers with overhanging Notl and Xhol sequences to amplify the CBM nucleotide sequence of cbeg5 from a pJWCl-cbeg5 (4L05) template and the CBM domain of cbeg4 from the pJWCl-cbeg4 (33g04) template.
- Templates were amplified following a standard PCR protocol for Q5 High-Fidelity DNA Polymerase (New England Biolabs).
- Amplified gene sequences were digested with Notl and Xhol (New England Biolabs), ligated into a NdeI and Xhol-digested vector pET His6 GFP TEV LIC cloning vector (Addgene), and transformed into electrocompetent E. coli EC 100 cells.
- LB media 50 mL with 50 p ⁇ mL' 1 kanamycin was inoculated with single colonies. Following incubation, the cultures were miniprepped, and the identity of the plasmid confirmed by Sanger sequencing.
- Expression constructs/plasmids were transformed into E. coli BL21(DE3) T7 Express cells (New England Biolabs), and glycerol freezer stocks were prepared from single colonies. Freezer stocks were inoculated into 50 mL LB with 50 ⁇ g mL -1 kanamycin, and cultured overnight at 37°C, 200 rpm. Overnight cultures were sub-inoculated (1:100 dilution, 10 mL) into 1 L LB media with 50 pg mL' 1 kanamycin.
- the resulting cultures were incubated at 37°C, 200 rpm until reaching an OD600 of approximately 0.6, induced with 0.5 mM IPTG, and incubated at 18°C, 200 rpm for 20 h.
- the cells were pelleted at 4,200 x g for 20 min at 4°C, resuspended with 50 mL LB, and transferred to 50 mL Falcon tubes. The cells were pelleted again, the supernatant discarded, and stored at -80°C.
- the weight of each wet cell pellet was approximately 2 g.
- Protein purifications were conducted at 2 L scales by combining two 1 L cell pellets per preparation. All steps were conducted in a cold room or on ice. Cells were thawed on ice and resuspended with 100 mL lysis buffer containing 50 mM KH2PO4 pH 7.2, 300 mM NaCl, 1 mM PMSF, 0.5% Triton-X, and 25 U Benzonase Nuclease (Millipore Sigma). Cells were lysed on ice by probe sonication using a Misonix Sonicator 3000 or French press using an Avestin Emulsiflex C5.
- the column was washed with 20 CV of wash buffer (50 mM KH2PO4 pH 7.2, 300 mM NaCl, and 10 mM imidazole), and the bound protein was eluted with 5 CV of elute buffer (50 mM KH2PO4 pH 7.2, 300 mM NaCl, and 250 mM imidazole) and collected.
- the protein 25 mL was dialyzed using Spectra/Por 12-14 kDa dialysis tubing (Spectrum Labs) against 500 mL PBS pH 7.2, the buffer was replaced 4 times over 72 h.
- the dialysate was transferred to a 50 mL Falcon tube and centrifuged (4,200 x g, 30 min at 4°C) to pellet insoluble protein precipitate, and the soluble fraction was filtered through a 0.22- pm syringe filter.
- the protein (25 mL) was concentrated using an Amicon 3K or 30K MWCO spin concentrator (Millipore Sigma), depending on the molecular weight of the protein, to 3 mL.
- the protein was further purified on an AKTA Purifier FPLC system (GE Healthcare Life Sciences) using an ENrich SEC 650 10 x 300 mm column (Bio-Rad Laboratories) housed at 4°C. A 0.5 mL volume of protein was injected onto the column.
- the elution buffer was PBS, and the flow through was 1.0 mL min 1 . Eluent fractions (0.5 mL) were collected, and those containing purified protein were identified by SDS-PAGE. LC-MS was used for final protein confirmation.
- CBM4 and Cbeg5 were sent to the Consortium for Functional Glycomics for screening against a panel of 609 target glycans. Proteins were assayed at 50 ⁇ g mL -1 and 5 ⁇ g mL -1 in 6 replicates. Streptavidin was used as a positive control. The screen output was entered into the Glycopattern program for identification of conserved binding motifs. 55 The top glycan binding motif common to both CBM4 and CBM5 was used to query diverse glycomics datasets using Glyconnect to identify structures from sample tissues, cells, or organisms containing the binding motif. The Glyconnect database was queried to identify structures that are N glycans with the conserved Gaipi-3GlcNac01 determinant.
- PBMCs peripheral blood mononuclear cells
- the plasma and PBMC layers were separated, transferred to a new 50 mL conical tube, and centrifuged for 10 min, 1600 rpm at 4°C. The supernatant was then decanted and the PBMC pellet resuspended in 5 mL PBS. Cells were counted on a CelloMeter by adding 20 pl of AOPI to 20 pl of PBMC solution. Cells were centrifuged for 10 min, 1600 rpm at 4°C and resuspended in cold culture media (RPMI 1650 with 10% FBS, 1% penicillin-streptomycin, 10 mM L-glutamine, and 20 mM HEPES) at 1 million cells perl 80 pl of media.
- cold culture media RPMI 1650 with 10% FBS, 1% penicillin-streptomycin, 10 mM L-glutamine, and 20 mM HEPES
- LPL lamina propia leukocytes
- the mucosa and submucosa were removed, and placed into a 15 mL conical tube filled with 10 mL of dissociation media (HBSS w/o Ca ++ Mg ++ , IM HEPES, 0.5M EDTA) to remove the epithelial cells.
- the tube was incubated for 15 min, 100 rpm at 37°C. The solution was then manually vortexed for 30 sec and passed through a 70 pM cell strainer.
- the solution was transferred to a 15 mL conical tube filled with 10 mL of digestion media (HBSS w/o Ca ++ Mg ++ , 2% FCS, DNasel 0.5 mg mL 1 , collagenase IV 1 mg mL 1 ).
- the solution was incubated for 40 min, 100 rpm at 37°C.
- the solution was filtered through a 70 pM cell strainer and the filtrate transferred to a 15 mL conical tube with FACS buffer (DPBS w 3% FBS and 1 mM EDTA). The cells were then centrifuged for 8 min, 1800 rpm at 4°C.
- the supernatant was decanted and cells resuspended in 1 mL of RBC lysis solution for 1 min. 11 mL of FACS buffer was added and cells again centrifuged for 8 min, 1800 rpm at 4°C. The supernatant was decanted and the cells processed using the Dead Cell Removal Kit per protocol (Miltenyi Biotec). The final cell pellet was resuspended in 500 pl of DPBS and cells counted by Tryptan blue exclusion.
- PBMCs or colon LPL cells were plated at 1 million cells per well in a 96-well plate in 180 pl of media (RPMI 1650 with 10% FBS, 1% penicillin- streptomycin, 10 mM L-glutamine, and 20 mM HEPES). Treatment proteins were prepared in lOx solutions, diluted in 20 pl PBS according to a desired final assay concentration, and transferred to wells.
- PBMCs and treatments (Cbeg5, Fn5, CBM5, or PBS) were co-incubated for 20 min at 5% CO 2 , 37°C along with Rhl03 antibody (Fluidigm).
- Cells were washed twice with FACS buffer (DPBS w 3% FBS and 1 mM EDTA) and fixed in 1.6% paraformaldehyde for 20 min at room temperature. Cells were then washed twice, diluted in FACS buffer, and placed at 4°C until staining and CyTOF analysis.
- FACS buffer DPBS w 3% FBS and 1 mM EDTA
- PBMCs or LPL cells and treatments were coincubated for 6 hours at 5% CO 2 , 37 °C along with 2 pM monensin (Biolegend). After 6 hours, Rhl03 antibody was added to the cells and co-incubated for an additional 20 min. Cells were washed with FACS buffer and fixed with 1.6% paraformaldehyde at room temperature. Cells were then washed twice in FACS buffer and placed at 4°C until staining and analysis by CyTOF. [0156] Prior to CyTOF analysis, antibody mixtures were prepared in cell staining media (CSM, Fluidigm).
- CSM Cell staining media
- Each sample was washed and resuspended in 800 pl of IX Barcode Perm Buffer (Fluidigm Inc.). Compatible Pd-barcodes were thawed, resuspended in 100 pL of IX Barcode Perm Buffer, and added to the samples. Samples were incubated on ice for 30 min, washed in CSM, and pooled together. Each barcoded set of samples (corresponding to a single treatment condition) was resuspended in 100 pL of CSM containing 100 U mL 1 heparin (Sigma) to block non-specific MaxPar antibody binding.
- a titrated surface antibody panel designed to allow identification of all major immune subsets was prepared in an additional 100 pl of CSM, filtered through a 0.1 pm spin filter (Amicon), and added directly to the sample. Samples were stained for 30 min on ice, washed with CSM, and fixed with freshly diluted 2% formaldehyde (Electron Microscopy Sciences) in PBS to cross-link and preserve all surface antibodies. The samples were then washed and permeabilized by adding 1 mL of ice-cold 100% methanol, added dropwise while vortexing. Samples were incubated on ice for 30 min, washed twice with CSM, and resuspended in 100 ul of CSM containing 100 U mL 1 heparin.
- a titrated panel of validated antibodies against phospho-protein epitopes or cytokine epitopes was prepared in an additional 100 ul of CSM, filtered through a 0.1 pm spin filter (Amicon), and added directly to the samples. Samples were incubated for 30 min on ice, washed with CSM, and incubated for 30 min in freshly-diluted 2% formaldehyde in PBS containing 0.125 nM Ir nucleic acid intercalator (Fluidigm). The samples were then washed and stored as pellets in CSM until CyTOF acquisition.
- mice were not randomized prior to colonization.
- the drinking water of 8-week old C57BL/6 mice was supplemented with 500ug/mL of ampicillin 500 pg/mL for 5-7 days to facilitate colonization.
- Drinking water was then changed to tetracycline XX, and the mice were colonized with E. coli engineered to express Cbeg5 (EC:Cbeg5) or E. coli with an empty control vector (EC:Con) by oral gavage. 10 mL of overnight cultures of each bacteria were centrifuged and resuspended in 1 mL of PBS.
- mice were gavaged with 100 pL of this culture and colonization was confirmed at day 4 by recovery of the gavaged bacteria from mouse feces by plating the feces on LB agar with and without tetracycline 15 ⁇ g mL -1 and isolating EC:Con or EC:Cbeg5 plasmids from individual colonies. There was no difference between treatment groups in the number of colony forming units in mouse feces. Endpoint experiments were performed after 7-10 days of colonization. Fecal pellets were analyzed for consistency (watery or formed) and food/water intake tracked on a per cage basis. All mice were weighed on arrival to the facility, prior to colonization, and at the end of the experiment. Drinking water and food intake were measured per cage.
- mice were euthanized by cervical dislocation. Abdominal cavities were exposed and the colon removed. 1 cm pieces of colon were aliquoted into 10% formalin for analysis by histology or into HBSS for isolation of intestinal leukocytes. Histologic analysis was performed by Histowiz including interpretation of pathology and scoring of intestinal inflammation.
- LPLs the colon tissue was placed into 10 mL of EDTA solution (HBSS with 10% FBS, 5 mM EDTA, 15 mM HEPES) and incubated for 20 min, 150 rpm at 37°C. The colon tissue was vortexed for 30 sec and minced using dissection scissors.
- the minced tissue was placed into a new conical tube with 10 mL of digestion solution (RPMI 1640, 2% FBS, 0.5 mg/mL Collagenase D, 0.5 mg/mL DNase) and incubated for 30 minutes, 150 rpm at 37°C. Contents were homogenized using an 18G needle and filtered through a 100 pM strainer. Cells were then centrifuged at 1600 rpm for 20 min. Supernatant was decanted and cells resuspended in 10 mL RPML1640. Cells were strained again through a 40 pM strainer, and pelleted by centrifugation at 1600rpm for 5 min. The cells were resuspended in RPMI 1640.
- digestion solution RPMI 1640, 2% FBS, 0.5 mg/mL Collagenase D, 0.5 mg/mL DNase
- 2.4G2 Fc blocker 100 pL was added to the cells, which were subsequently incubated at 4°C for 10 min. Cells were then centrifuged at 2000 rpm for 2 min and resuspended in FACS buffer with antibodies. Cells were stained for 30 min protected from light at 4°C. After staining, cells were fixed with 2% paraformaldehyde for 15 min at room temperature, washed with FACS buffer three times, and analyzed on the LSRII. For intracellular stains, cells were first permeabilized and fixed overnight. Cells were then washed and stained with antibodies in permeabilization buffer.
- Antibodies were co-incubated for 30 min at 4°C, washed twice in permeabilization buffer, reconstituted in FACS buffer, and analyzed on the LSRII using OneComp Beads in parallel.
- 8wk C57BL/6 mice were garaged with the Lectin producing bacteria (Cbeg) or control bacteria (pJWC) every 2 days for 1 week prior to the start of the Dextran sulfate sodium (DSS) is a sulfated polysaccharide with variable molecular weights.
- DSS causes human ulcerative colitis-like pathologies in mice, due to its toxicity to colonic epithelial cells. DSS was then administered in the drinking water for 7 days followed by 7 days of recovery. Weight was measured daily. (FIGs. 20A and 20B).
- a dataset of uncharacterized (predicted) human microbiota lectins was generated by first identifying carbohydrate -binding domains in Interpro. All protein sequences having at least one Interpro-identified carbohydrate-binding domain were downloaded. From that dataset, protein sequences having one or more of the following characteristics were removed from further analysis: a catalytic domain, a predicted function related to carbohydrate transport, a predicted function related to cell structure, a known function such as flagella, fimbriae, or adhesins. To further focus on uncharacterized proteins, protein sequences identified as "reviewed" by Uniprot were removed.
- a dataset of characterized human microbiota was downloaded from Uniprot (Unilectin), which included Cbeg4 (GenBank accession no. KT336269.1) and Cbeg5 (GenBank accession no. KT336270.1).
- the datasets of characterized and uncharacterized lectin protein sequences were used to query reference genomes from the human microbiome project (https://www.hmpdacc.org/hmp/HMRGD/) and the gene index from patient metagenomic samples across different body sites (https://www.hmpdacc.org/hmp/HMGF).
- BLAST Basic Local Alignment Search Tool
- a predicted domain analysis of cbeg4 and cbeg5 revealed both genes encode proteins having a secretion signal peptide, a fibronectin type 3 domain (Fn; IPR003961), and a carbohydrate-binding module domain (CBM, CBM6-CBM35-CBM36_like_2, IPRO338O3) (FIG. 1).
- CBM carbohydrate-binding module domain
- FIG. 1 The presence of a secretion signal and a carbohydrate-binding module domain (CBM), in conjunction with the absence of an enzymatic domain, suggests the proteins encoded by cbeg4 and cbeg5 impart biological activity by binding to an extracellular carbohydrate (i.e., a secreted lectin).
- CBM4 and Cbeg5 were assessed in a binding assay against a panel of 609 glycans (Functional Glycomics Consortium).
- CBM5 and CBM4 were assayed at 5 ⁇ g mL 1 and 50 ⁇ g mL -1 in 6 replicates, and glycan-binding quantified as relative fluorescent units (RFU).
- REU relative fluorescent units
- FIG. 3A & 3B are dot plots of CBM4 and CBM5 glycan- binding screens at a concentration of 5 ⁇ g mL -1 .
- Graphs mean +/- s.e.m.
- CBM4 and CBM5 exhibit similar glycan-binding patterns, with binding glycan structures sharing a common Ga1 ⁇ 1-3GlcNac ⁇ 31-2Manal-3Man motif binding CBM4 and/or CBM5. Further, CBM4 and CBM5 exhibited the highest signal for the same glycan.
- the structure of the top bound glycan (represented as a dot in the graphs of FIGS. 3A - 3C) is illustrated in FIG. 3D with the glycopattem of the conserved binding motif (Ga1 ⁇ 1-3GlcNac01- 2Manal-3Man) shown via a dotted line.
- Glyconnect search of glycomics datasets for samples containing the conserved glycan motif was performed (FIG. 3E). Glycan structures containing the conserved binding motif were most commonly identified in human leukocyte datasets (Glyconnect datasets generated from peripheral blood mononuclear). 24-27 Four glycan structures were shared by human leukocyte, urine, and kidney datasets. One glycan structure was exclusive to a kidney dataset. Together, these data suggest Cbeg4 and Cbeg5 are lectins that bind leukocyte-associated glycan motifs.
- PBMCs isolated from healthy patients. PBMCs were exposed to full-length Cbeg5, CBM5, or the Fn domain of Cbeg5 (Fn5). PBMCs were cultured for 6 hours with each recombinant protein and analyzed by mass cytometry (CyTOF) using antibodies to 16 cell surface markers and 10 cytokines. Supervised clustering of the 16 cell surface markers resolves leukocytes into major immune cell populations (A - K of FIG. 4A) that correlate to groups identified by unsupervised t-distributed stochastic neighbor embedding (viSNE).
- FIGS. 5A - 50 illustrate CyTOF results for 16 cell surface markers. Cells were gated into singlets and specific cell populations identified based on established cell surface markers.
- FIG. 4E illustrates the fold induction of 10 cytokines across cell populations A-K from FIG. 4A, which was calculated relative to PBS. Cytokine responses > 3 fold to > 100 fold are marked with grey shaded boxes. Proteins were assayed at 100 nM or 1000 nM, as indicated. For CD14 + monocytes (population "A"), CD16 + monocytes (population "B”), and cDC2 dendritic cells (population "D”), Cbeg5 and CBM5 increased IL-10, IL-6, IL-8, IL-10, and TNFa in a dosedependent manner (FIG. 4F).
- CDlc- CD14 CD16 CDl lc + myeloid cells CD3-CD19 CD56-CD66 HLADR + ), referred to herein as "myeloid CDl lc + (mCDl lc) cells” (population "C"), which may represent cDCl dendritic cells.
- FIGS. 4B - 4E show IL-6, IL-8, IL-10, and TNFa induction in CD14 + monocytes (A), CD16 + monocytes (B), mCDl 1c (C), cDC2 (D), plasma dendritic cells (plasmaDC; E), neutrophils (F), and natural killer (NK) cells (G) in response to Cbeg5 or Fn5 treatment.
- Cytokine induction in CD14 + monocytes, CD16 + monocytes, cDC2, and mCDl lc was significant, with a >100-fold increase relative to the PBS control. There was a smaller cytokine response in plasmaDCs, neutrophils, and NK cells.
- Cbeg5 or CBM5 There was no detectable response to Cbeg5 or CBM5 in T cells or B cells.
- cytokine induction of cytokines was the most robust in CD14 + monocytes, affecting > 90% of the cells even when exposed to as little as 100 nM of purified protein (FIGS. 4B - 4F).
- Fn5 did not elicit a cytokine response above control (PBS) in any cell population.
- PBMCs were exposed to Cbeg5 for 20 min, and the CyTOF experiment performed using the 16 cell surface marker panel and an additional panel of 8 antibodies targeting phosphorylated proteins (phospho-proteins) in diverse signaling pathways.
- phospho-proteins phospho-proteins
- LPL Lamina propria leukocytes isolated from fresh colon samples were exposed to full-length Cbeg5 or Fn5 alone for 6 hours and analyzed by CyTOF using the same cell surface marker antibodies and cytokines as Example 13.
- a SPADE Spanning- tree Progression Analysis of Density-normalized Events
- FIGS. 7A - 7F Cbeg5 increased TNFa, and IL-10 levels at least 3-fold relative to Fn5 treated cells in two branches on the SPADE tree.
- Cell surface markers associated with these two branches identify the LPLs collectively as myeloid cells (CD19 CD56 CD66- HLADR + ) with high expression of CDlc (CDlc hl cluster) in one branch and CD 14 (CD14 hl cluster) in the other branch.
- FIG. 7A illustrates a SPADE plot for Cbeg5-induced TNF-a production from intestinal leukocytes. The fold-induction was calculated relative to Fn5.
- Cbeg5 induced cytokine production in two myeloid cell clusters (CD19’CD56 CD66b’HLADR + ) that are CD14 hl or CDlc hl .
- FIGS. 7B & 7C cells from the CD 14 CDlc clusters were analyzed to identify CD14 + CD4 + cells (monocyte-derived macrophage markers) and CD14 + CDlc + cells (monocyte- derived dendritic cell markers).
- Cbeg5 activation of intestinal leukocytes is specific to myeloid cells.
- Blood CD14 + monocytes traffic to the intestine, where they differentiate into dendritic cells (monocyte derived dendritic cells; moDC) and macrophages (monocyte derived macrophages; MDM).
- CD14 + CDlc + (moDC markers) and CD14 + CD4 + (MDM markers) cell populations from the intestine were activated to a lesser extent than blood CD14 + monocytes.
- CD14 + blood monocytes were differentiated in vitro into MDMs and moDCs.
- CD14 + monocytes and the differentiated moDCs and MDMs were exposed to 500 nM of Cbeg5, Fn5, or PBS for 6 hours and the resulting culture supernatants analyzed by Luminex technology for cytokine/chemokine/growth factor/adhesion molecule production (Procarta PlexTM Human Inflammation Panel).
- FIGS. 7E & 7F illustrate bar graphs of cytokine (FIG. 7E) or chemokine and adhesion molecule (FIG. 7F) production for cell populations in response to Cbeg5 (bar graph colors are derived from labels used in the heat map of FIG. 7D).
- Cbeg5 increased cytokine production relative to PBS, while Fn5 was indistinguishable from the PBS control (FIG. 7D).
- Cbeg5-induced cytokine production was the greatest for CD14 + monocytes. The largest observed increases were in TNFa (168 fold) and IL-10 (28 fold).
- moDC and MDM had a more limited cytokine response to Cbeg5, with the largest increase being for IL- 6 (FIGS. 7E & 7F).
- Cbeg5 had its most significant effect on chemokine production for MDMs (MIP-la, MIP-10) (FIGS. 7E & 7F).
- Cbeg5 also selectively increased the production of the adhesion molecule sE-selectin and the growth factor GM-CSF for MDMs, and for CD14 + monocytes, respectively (FIGS. 7E & 7F). These activation patterns of CD14 + cell populations based on cellular differentiation suggest Cbeg5 differentially affects immune cell populations in the blood (CD 14 + monocytes) and in the intestine (moDCs, MDMs).
- FIGS. 8A - 8F illustrate a SPADE plot for Cbeg5- and PBS-induced TNF-a and IL- 10 from LPL (Cbeg5 - two patient samples, PBS - one patient sample).
- LPLs from the second patient were separately treated with PBS as a negative control.
- 8G & 8H illustrate bar graphs of Cbeg5-induced cytokine production from cells in the CD 14 cluster (CD 14 stain intensity inset) and CDlc cluster (CDlc stain intensity inset) (mean +/- s.e.m, two independent experiments). There was no difference in cytokine induction between Fn5 and the PBS control.
- Cbeg5 The activation of intestinal and blood myeloid cell populations by Cbeg5 suggested gastrointestinal tract bacterial expression of this human-microbial-lectin, affects the host immune system. Further, in vitro, Cbeg5 induced distinct responses in peripheral and intestinal myeloid cell populations suggesting its involvement in pro-inflammatory signaling or regulating immune cell differentiation and trafficking.
- FIGS 10B & 10C illustrate food and water consumption, which was measured from the time of colonization and analyzed on a per cage basis.
- colon samples were subsequently analyzed for changes in mucosal immune cell populations.
- LPLs were isolated from the colons of colonized mice, and analyzed by flow cytometry using antibody panels that distinguish myeloid cell populations (MHCII, CD64, Ly6C, CDl lc, CDl lb, CD45) from T cell populations (CD45, CD4, RORyt, FOXP3, GAT A3).
- FIG. 11A After gating for singlets and live cells (Aqua LIVE/DEAD), the gating schema for identification of macrophages (MHCII + CDl lc + CD64 + CDl lb + ) and monocytes (MHCIFLyc + CDl lb + ) 32 ’ 33 ’ 39_41 is shown in FIG. 11A, and the gating schema for identification of CD4 + T cell populations based on transcription factors is shown in FIG. 12A (T cells were gated into 4 populations).
- Cbeg5 can generate an environment that is conducive to the development of protective macrophages by facilitating cell trafficking and cell differentiation of monocytes/macrophages. This supports the use of Cbeg5 to regulate beneficial mucosal barrier functions that can be utilized therapeutically to induce protective immune interactions against pathogens as well as protect against or promote healing of mucosal injuries mediated by immune diseases, chemicals/radiation, or ischemia.
- lectins are encoded by a small subset of human microbiome samples, and are largely associated with pathobionts or pathogens.
- cbeg4 and cbeg5 which showed no significant sequence similarity to any previously characterized lectin genes in Unilectin, are highly prevalent across human microbiome samples, and are associated with common commensal species.
- FIG. 13A previously characterized lectins were in a small number of patient stool samples, while Cbeg4 and Cbeg5 were in a large percentage of patient stool samples.
- FIG. 13B illustrates characterized lectins are largely from proteobacterial species. No characterized lectins have been identified in Bacteroidetes species from which Cbeg4 and Cbeg5 were isolated.
- HMP Human Microbiome Project
- the carbohydrate-binding domain from Cbeg4 and Cbeg5 was present in a much smaller number of predicted lectins (16), and was associated with significantly fewer domain architectures (6).
- FIG. 14A all domains present in the human- microbial-lectin dataset were organized based on co-occurrence between a carbohydrate-binding domain and secondary domains in the same lectin.
- the bar graphs summarize the count of each domain in the dataset, and box color reflects the frequency of domain co-occurrence.
- the IPRO338O3 domain was only associated with those containing the Cbeg4- and Cbeg5-like domain architectures.
- IPR033803 -containing proteins deposited in NCBI revealed Cbeg4- and Cbeg5-like domain architectures were only encoded by common commensal Bacteroides species (data not shown).
- FIG. 14B lectin domain architectures containing the Cbeg4/5 carbohydrate-binding domain (IPR338O3) and the Bacteroidetes-Associated Carbohydrate -binding Often N-terminal domain (IPR024361) are shown.
- IPR0244361 is the most common domain in the human-microbial-lectin dataset and present in 891 lectins. The most prevalent lectins (top 10% for each body site) containing these domains are shown. For IPR338O3 only the Cbeg4/5 domain architecture was identified in abundance in patient samples.
- FIG. 15 shows the prevalence of human- microbial-lectins in patient samples from the HMP (red bar is mean +/- s.e.m. Summary of the average number of human-microbial-lectins per person for each body site is shown. Color of circle is proportional to the mean lectin count with stool set as 100%). Uncharacterized human- microbial-lectins were highly prevalent among patients, found in every sampled body site and present in 99% of reference genomes.
- FIG 16A shows a human-microbial-lectin dataset aligned to a database of reference genome sequences generated from bacteria isolated at different sites in the human microbiome. The number of lectin genes per genome for each bacterial species was determined and plotted as a histogram. Data is presented based on body site from where the bacterium was isolated and is colored by the bacterial phylum).
- FIG. 16B presents the mean number of human-microbial-lectin genes per genome per body site and phylum. Bar graphs are mean +/- s.e.m.
- the stool metagenome had the largest number of uncharacterized lectin sequences (2,278), with each patient's stool containing on average 323 unique lectin genes.
- body sites from the GI tract had more unique lectin sequences then body sites from the urogenital tract or skin. Only 18% of predicted lectin sequences were shared between two or more microbiome sites.
- FIG. 17 shows the overlap of lectin genes between five body sites. Oral microbiome sites shared a large percentage of lectin sequences, whereas the stool and the posterior fornix were composed almost entirely of lectins specific to those sites (95% stool, 88% posterior fornix).
- FIGS. 19A & 19B show rarefaction analysis of human-microbial-lectin genes for each body site based on the number of samples from the HMP. Stool is pictured on a separate plot due to the large number of lectin sequences.
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Pharmacology & Pharmacy (AREA)
- Epidemiology (AREA)
- Veterinary Medicine (AREA)
- Chemical & Material Sciences (AREA)
- Public Health (AREA)
- General Health & Medical Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Medicinal Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Cell Biology (AREA)
- Immunology (AREA)
- Zoology (AREA)
- Engineering & Computer Science (AREA)
- Gastroenterology & Hepatology (AREA)
- Molecular Biology (AREA)
- Microbiology (AREA)
- Mycology (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
La technologie présentement revendiquée et décrite concerne des méthodes de traitement d'une maladie ou d'un trouble inflammatoire du tractus gastro-intestinal chez un sujet par l'administration d'une cellule génétiquement modifiée exprimant une lectine, d'un gène de lectine modifiée codant une lectine, d'une lectine, ou des compositions associée, la lectine étant une protéine non enzymatique comprenant un domaine de liaison aux glucides.
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US202163236756P | 2021-08-25 | 2021-08-25 | |
US63/236,756 | 2021-08-25 |
Publications (2)
Publication Number | Publication Date |
---|---|
WO2023028104A2 true WO2023028104A2 (fr) | 2023-03-02 |
WO2023028104A3 WO2023028104A3 (fr) | 2023-08-10 |
Family
ID=85323418
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/US2022/041315 WO2023028104A2 (fr) | 2021-08-25 | 2022-08-24 | Utilisation de lectines microbiennes humaines pour traiter une maladie |
Country Status (1)
Country | Link |
---|---|
WO (1) | WO2023028104A2 (fr) |
Family Cites Families (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CA2869023A1 (fr) * | 2012-04-05 | 2013-10-10 | Gottfried Himmler | Complexe d'immunoglobuline secretoire |
-
2022
- 2022-08-24 WO PCT/US2022/041315 patent/WO2023028104A2/fr unknown
Also Published As
Publication number | Publication date |
---|---|
WO2023028104A3 (fr) | 2023-08-10 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
JP7391015B2 (ja) | P53がん特異的変異に対して抗原特異性を有するt細胞を単離する方法 | |
US11084856B2 (en) | Interleukin-4 receptor-binding fusion proteins and uses thereof | |
US8574903B2 (en) | Method for expanding monocytes | |
BR112020004347A2 (pt) | vesículas extracelulares de prevotella | |
JP6479662B2 (ja) | 免疫調節ミニ細胞および使用方法 | |
Eriksson et al. | Tumor specific phage particles promote tumor regression in a mouse melanoma model | |
EP2275572A3 (fr) | Identification d'antigènes de surface pour le diagnostique et la thérapie de tumeurs | |
TW200944228A (en) | Non-pathogenic and/or attenuated bacteria capable of inducing apoptosis in macrophages, process of manufacturing and uses thereof | |
KR20210003096A (ko) | 박테리아성 균주를 포함하는 조성물 | |
WO2022262130A1 (fr) | Récepteur antigénique chimérique spécifique du macrophage, monocyte/macrophage polarisé contrôlable exprimant le récepteur, procédé de préparation correspondant et utilisation associée | |
KR20200111183A (ko) | 암을 치료하거나 예방하기 위한 조합 치료법 | |
Breyner et al. | Oral delivery of pancreatitis‐associated protein by Lactococcus lactis displays protective effects in dinitro‐benzenesulfonic‐acid‐induced colitis model and is able to modulate the composition of the microbiota | |
TW202228653A (zh) | 細菌之固體劑型 | |
Schreiber et al. | Impact of TCR diversity on the development of transplanted or chemically induced tumors | |
WO2023028104A2 (fr) | Utilisation de lectines microbiennes humaines pour traiter une maladie | |
AU2019322090A1 (en) | Compositions comprising bacterial strains | |
WO2012020842A1 (fr) | Complexe ayant un effet de vaccin antitumoral et son utilisation | |
Leiba et al. | Dynamics of macrophage polarization support Salmonella persistence in a whole living organism | |
US12011466B2 (en) | Modified Escherichia coli strain Nissle and treatment of gastrointestinal disorder | |
Petrovskaya et al. | Alternative scaffold proteins | |
KR20200110342A (ko) | 암을 치료하거나 예방하기 위한 조합 치료법 | |
KR20200110341A (ko) | 암을 치료하거나 예방하기 위한 조합 치료법 | |
KR20200110343A (ko) | 암을 치료하거나 예방하기 위한 조합 치료법 | |
EP4170037A1 (fr) | Souche de salmonella pour le traitement du cancer et utilisation associée | |
CN117384809A (zh) | 一种原位分泌蛋白的肿瘤靶向性工程菌及其构建方法与应用 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 22862015 Country of ref document: EP Kind code of ref document: A2 |
|
NENP | Non-entry into the national phase |
Ref country code: DE |