US010196453B2 ( 12) United States Patent (10 ) Patent No. : US 10 , 196 , 453 B2 Cowardin et al. ( 45) Date of Patent : Feb . 5 , 2019 ( 54 ) COMPOSITIONS AND METHODS FOR and the NF -kappaB signaling pathway ” , J. Mol . Med . Berl . Ger. 87, TREATING CLOSTRIDIUM DIFFICILE 169 - 180 ( 2009 ) . Stewart , D . B . , Berg , A . & Hegarty , J . , “ Predicting Recurrence of C . INFECTION difficile Colitis Using Bacterial Virulence Factors : Binary Toxin Is the Key ” , J . Gastrointest . Surg . 17 , 118 - 125 ( 2013 ) . (71 ) Applicant: University of Virginia Patent Hemmasi, S . et al , “ Interaction of the Clostridium difficile Binary Foundation , Charlottesville , VA (US ) Toxin CDT and its Host Cell Receptor LSR ” , J . Biol. Chem . ( 2015 ) . doi : 10 . 1074 / jbc. M115 .650523 . ( 72 ) Inventors: Carrie Adeline Cowardin , St. Louis , Papatheodorou , P . et al , “ Lipolysis - stimulated lipoprotein receptor MO (US ) ; William A . Petri, Jr . , (LSR ) is the host receptor for the binary toxin Clostridium difficile transferase ( CDT) ” , Proc . Natl . Acad . Sci. U . S . A . 108 , 16422 Charlottesville , VA (US ) 16427 ( 2011) . Buonomo, E . L . et al , “ Role of IL - 23 signaling in Clostridium ( 73 ) Assignee : University of Virginia Patent difficile Colitis ” , J . Infect . Dis . jit277 ( 2013 ) . doi: 10 . 1093 /nfdis / Foundation , Charlottesville , VA (US ) jit277 . Morgan , J . E . & Beeson , P . B . “ Experimental Observations on the ( * ) Notice : Subject to any disclaimer , the term of this Eosinopenia Induced by Acute Infection ” , Br. J . Exp . Pathol . 52 , patent is extended or adjusted under 35 214 - 220 ( 1971) . U . S . C . 154 (b ) by 83 days. Fukushima , A ., Yamaguchi, T . , Ishida , W . , Fukata , K . & Ueno , H , “ TLR2 agonist ameliorates murine experimental allergic conjunc ( 21 ) Appl. No .: 15 /597 , 384 tivitis by inducing CD4 positive T -cell apoptosis rather than by affecting the Th1/ Th2 balance ” , Biochem . Biophys . Res. Commun . May 17, 2017 339 , 1048 - 1055 (2006 ) . ( 22 ) Filed : Nawijn , M . C . et al, “ TLR - 2 Activation Induces Regulatory T Cells Prior Publication Data and Long - Term Suppression of Asthma Manifestations in Mice ” , (65 ) PLoS ONE 8 , e55307 ( 2013 ) . US 2017 / 0334994 A1 Nov . 23 , 2017 Carter , G . P . et al, “ Binary Toxin Production in Clostridium difficile Is Regulated by CdtR , a LytTR Family Response Regulator ” , J . Bacteriol. 189 , 7290 - 7301 ( 2007 ) . Related U . S . Application Data Mackin , K . E ., Carter , G . P . , Howarth , P . , Rood , J . I. & Lyras, D ., (60 ) Provisional application No . 62 /339 , 283 , filed on May “ Spo0A differentially regulates toxin production in evolutionarily 20 , 2016 . diverse strains of Clostridium difficile ” , PloS One 8 , e79666 ( 2013 ) . ( 51) Int. Ci. * cited by examiner A61K 39 /00 ( 2006 . 01 ) COOK 16 / 28 ( 2006 .01 ) Primary Examiner — Sarvamangala Devi ( 52 ) U . S . CI. ( 74 ) Attorney , Agent, or Firm — Rodney L . Sparks CPC .. CO7K 16 / 2896 ( 2013 .01 ) ; A6IK 2039 /505 ( 2013 .01 ) ; CO7K 2317 /76 ( 2013 .01 ) (57 ) ABSTRACT (58 ) Field of Classification Search Clostridium difficile is the most common hospital acquired CPC . .. .. C07K 2317 / 76 ; CO7K 16 /2803 ; CO7K pathogen in the United States , and infection is in many cases 16 / 2896 ; A61K 2039/ 505 ; A61K fatal. Toxins A and B are its major virulence factors , but 2039 /507 increasingly a third toxin may be present, known as C . See application file for complete search history . difficile transferase (CDT ) . An ADP - ribosyltransferase that causes actin cytoskeletal disruption , CDT is typically pro ( 56 ) References Cited duced by the major, hypervirulent strains and has been associated with more severe disease . It is disclosed herein U . S . PATENT DOCUMENTS that CDT enhances the virulence of two PCR - ribotype 027 strains in mice . The toxin induces pathogenic host inflam 2014 /0343256 A1 * 11/ 2014 Kirschning .. .. C07K 16 / 2896 mation via a novel Toll - like Receptor 2 ( TLR2) dependent 530 / 387 . 3 pathway , resulting in the suppression of a protective host eosinophilic response. Finally , it is disclosed that restoration OTHER PUBLICATIONS of TLR2 deficient eosinophils is sufficient for protection Unger et al. Scientific Reports 5 : Article No . 7850 , published online from a strain producing CDT. These findings offer an Jan . 19 , 2015 . * explanation for the enhanced virulence of CDT- expressing Savidge et al . Gastroenterology 125 : 413 -420 , 2003. * C . difficile and demonstrate a mechanism by which this Kuehne , S . A . et al , “ The role of toxin A and toxin B in Clostridium binary toxin subverts the host immune response . difficile infection ” , Nature 467 , 711 - 713 ( 2010 ) . Lee, J. Y . et al, “ Clostridium difficile toxin A promotes dendritic cell 9 Claims, 25 Drawing Sheets maturation and chemokine CXCL2 expression through p38 , IKK , (5 of 25 Drawing Sheet ( s ) Filed in Color) U . S . Patent Feb . 5 , 2019 Sheet 1 of 25 US 10 , 196 ,453 B2 Survival * * * * * * * * * * * * * * * * * * * * * * * ** * * * * * * * * * * * * * natin R20291 R20291 Cdta . %Survival R20291 Cdtl . Day FIG . 1A Weight Loss - R20291 R20291 Cota PercentageWeight iiiiiiiiiiiiiii * R20291 Cdtl . Day FIG . 1B atent Feb . 5 , 2019 Sheet 2 of 25 US 10 , 196 ,453 B2 Weight Loss wheth M7404 - M7404 CdtA %startingweight M7404 CdtAcomp FIG . 1C Clinical Score M7404 M7404 Cdta ClinicalScore M7404 CdtAcomp .Day 2 FIG . ID atent Feb . 5 , 2019 Sheet 3 of 25 US 10 , 196 ,453 B2 R20291 R20291 Cdte . FIG . 1E Histological Score * * * TotalHistologyScore R20291 R20291 Cdtb FIG . IF atent Feb . 5 , 2019 Sheet 4 of 25 US 10 , 196 ,453 B2 IL - 1B A pg/mLmg R20291 R20291 Abx CdtB FIG . 2A IL - 6 . * R20291 R20291 Abx Cdtl . FIG . 2B U . S . Patent Feb . 5 , 2019 Sheet 5 of 25 US 10 , 196 ,453 B2 IL - 6 (Serum ) 2000 , 3007 * * * IL-1BpgmL pg/mLig CDT - o - R20291 R20291 Abx TcdA / B FIG . 2C FIG . 2D NFKB Activation OD650 Geneexpression relativetoS14 CDT ??? Mock Mock other LPS LPS FIG . 2E FIG . 2F atent Feb . 5 , 2019 Sheet 6 of 25 US 10 , 196 ,453 B2 IL - 1B RelativeIL-1B(%ofControl). * * * mmmhmmmmhmmmmišinskimwilinment * * * Anti- CDTa . Anti -CDTD CatA / B + TcdA / B FIG . 2G R20291 R20291 Cate ???????????????????????????????????????????????? . -Vdistetenze VA ivicis. ietsthisissues . c . geriyeraprecizarrwerpen Tanzzzzzzzzzzzzzzz ,. c. dinaci . .. FIG . JA U . S . Patent Feb . 5 , 2019 Sheet 7 of 25 US 10 , 196 ,453 B2 Lamina propria cells AAAAAA R20291 A R20291 Cdte A * * * dedududududududude Abx %LiveCells . D Doord XOXOXOKOOKOOKOOKOOKOOKOVOVVVV oborodik10000 OKOOKOOKOOKOOOooo . OOOOOO Eosinophils Inflammatory Neutrophils Monocytes FIG . 3B Lamina propria cells R20291 R20291 Cdtl . om Abx 0000 AbsoluteCellNumber(x104) $ Eosinophils InflammatoryNeutrophils Monocytes FIG . 3C atent Feb . 5 , 2019 Sheet 8 of 25 US 10 , 196 ,453 B2 WeightY Y . Loss vs . Eosinophils R20291 1619 combined , %StartingWeight 0 . 0004 5 10 15 20 % Live Eosinophils FIG . 3D Survival R20291 + Isotype R20291 + asiglech VZ . : + 3 72 . : : YY www %Survival R20291 CdtB NS the Isotype . R20291 CdB . m iniingiin maSiglech Day FIG . 3E atent Feb . 5 , 2019 Sheet 9 of 25 US 10 , 196 ,453 B2 Eotaxins 1507 . R20291 R20291 Cdte . NS NS pg/mLmg f CCL11. CCL24 FIG . 4A CCR3+ Eosinophils %CCR3+Eosinophils R20291 R20291 CatB . Abx FIG . 4B atent Feb . 5 , 2019 Sheet 10 of 25 US 10 , 196 , 453 B2 Bone Marrow . Eosinophil. Progenitors . %CD45+Cells . 0, 00 R20291 R20291 CdtB - Abx FIG . 4C Bone Marrow Eosinophils %LiveCells * * * * * * * * * * * * R20291 R20291 Cdtl . Abx FIG . 4D atent Feb . 5 , 2019 Sheet 11 of 25 US 10 , 196 ,453 B2 Live Eosinophils %LiveCells R20291 R20291 Cato FIG . 4E Apoptotic Eosinophils %Eosinophils wy R20291 R20291 CatB . FIG . 4F atent Feb . 5 , 2019 Sheet 12 of 25 US 10 , 196 ,453 B2 IL . -1B 1000000 6000 B C57BL6 TLR2 4000 6 TLR4 11.1quôd$ TLRS 2000 .DDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDD .DDDD CDT CDT * TcdA / B Mock FIG . 5A IL - 1B 2500 . 2000 1500 IL-1Bpg/ml 1000 500 II . 1 . 1 NARUMv v v v v v v v v v . TcdA / B Anti- TLR2 FIG . 5B U . S . Patent Feb . 5 , 2019 Sheet 13 of 25 US 10 , 196 ,453 B2 Survival TLR2 + R20291 Cate * C57BLG - R20291 Cote * * TLR2 + R20291 %Survival more on C57BL6 + R20291 mana maneno naman sa ASEA N PARA SA ARANY FIG . 5C Eosinophils NS .. AbsoluteCellNumber Tx1003 .. B6 TLR2 % B6 TLR2 R20291 R20291 Cdib . FIG . 5D atent Feb . 5 , 2019 Sheet 14 of 25 US 10 , 196 ,453 B2 Survival solution R20291 * R20291 Cats . B6 EQ * R20291 *Survival ** * TLR2 + EQ + R20291 FIG . 5E Apoptotic Eosinophils (Live dead - Annexin V + ) NS %Eosinophils Mock CatA / B aCDT a TLR2 FIG . 5F atent Feb . 5 , 2019 Sheet 15 of 25 US 10 , 196 ,453 B2 Survival moeten R20291 Percentsurvival R20291 Cdtl Day FIG . 6A Weight Loss WeightPercentage ILIH R20291 R20291 Cdtl . 0 1 2 . 5 2 2 . 5 3 4 6 Day FIG . 6B atent Feb . 5 , 2019 Sheet 16 of 25 US 10 , 196 ,453 B2 Clinical Score ** * ** * * i R20291 ClinicalScore W R20291 Cdtl . Day 1 Day 1 . 5 Day 2 Day 2 . 5 Day 3 Day 4 Day 6 FIG . 6C Histological Score R20291 Score R20291. CotB . Cellular Mucosal Lumenal Epithelial Submucosal Infiltrate Thickening Exudate Disruption Edema FIG . 6D atent Feb . 5 , 2019 Sheet 17 of 25 US 10 , 196 ,453 B2 Toxins A and B NS 1x1005 . * * 1x1004 1x1003 R20291 R20291 Cdtl . FIG . 6E C . difficile Burden 2x10 08 NS 2x 1008 CFU/mLig * 1008 5x 1007 R20291 R20291 Cdtl . FIG . 6F atent Feb . 5 , 2019 Sheet 18 of 25 US 10 , 196 ,453 B2 Liver Bacterial Burden NS CFU/mLmg R20291 R20291 Cdtb FIG . 6G atent Feb . 5 , 2019 Sheet 19 of 25 US 10 , 196 ,453 B2 . X1 . 7 III IIIIIIIIIIIIII . 92 . 2 . 0045APC07 ' Oy . Swin. 12. 2 . .. S . A L . .. W . : . 1 . : . XXX . w . * * 2 * S * . 73 * . * . HOSE . WAXX . LiveDeadAccent FIG.7A M ielea . 11111111 * * * - - - 0D115PEO? - - -/ /- * /- /- /- /- * /- /- /- /- /- /- /- /- /- Zongulo U .S . Pateatent Feb . 5 , 2019 Sheet 20 of 25 US 10 , 196 ,453 B2 Eosinophils %LiveCells 1 R20291 Cdte Isotype R20291 Anti - Siglech FIG . 7B Eosinophils 15 NS %LiveCells - - - - - - - - - - - - - B6 TLR24 B6 TLR2 R20291 R20291 CdtB FIG . 7C atent Feb . 5 , 2019 Sheet 21 of 25 US 10 , 196 ,453 B2 Cytokines : .