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Acknowledgments Layout assistance was provided by McCann Health Medical Communications studio team. This assistance was funded by Artugen Therapeutics, Inc., Concord, MA, USA. Disclosures Michelle O’ Donnell has a patent issued: METHODS AND COMPOSITIONS FOR THE TREATMENT OF C. DIFFICILE. Brian Healy has a patent issued: METHODS AND COMPOSITIONS FOR THE TREATMENT OF C. DIFFICILE. Colin Hill has received grants and personal fees from Artugen Therapeutics Ltd.; has received grants from Janssen Pharmaceuticals, Kerry Foods, and ADARE Pharmaceuticals outside of this work; and additionally has a patent issued: METHODS AND COMPOSITIONS FOR THE TREATMENT OF C. DIFFICILE. R. Paul Ross is a paid consultant of Artugen Therapeutics Ltd.; and additionally has a patent pending: METHODS AND COMPOSITIONS FOR THE TREATMENT OF C. DIFFICILE. Mary C. Rea is a paid consultant of Artugen Therapeutics Ltd.; and additionally has a patent pending: METHODS AND COMPOSITIONS FOR THE TREATMENT OF C. DIFFICILE. Ronald Farquhar is a paid consultant of Artugen Therapeutics Ltd., in which he also holds stock; and additionally has a patent pending: METHODS AND COMPOSITIONS FOR THE TREATMENT OF C. DIFFICILE. Laurent Chesnel is an employee of Artugen Therapeutics Ltd., in which he also holds stock; and additionally has a patent issued: METHODS AND COMPOSITIONS FOR THE TREATMENT OF C. DIFFICILE. ART24, a novel live biotherapeutic product in development for the prevention of CDI, is active against a broad range of C. difficile ribotypes in vitro P1507 Artugen Therapeutics © 2020 Laurent Chesnel, PhD, Vice President Research and Development, P: +1- 978 202 4337, E: [email protected] Materials/methods • ART24 was assayed for anti-C. difficile activity against a panel of contemporary isolates using an agar well diffusion method for direct inhibition of C. difficile growth – 10 mL of fresh ART24 cells grown shaking in brain heart infusion broth (BHI) overnight was pelleted by centrifuging at 4,000 rpm for 20 minutes. The cell-free supernatant (CFS) was removed and pH-neutralized for use in well diffusion and enzyme sensitivity assays – Capsules of lyophilized formulated ART24 were resuspended in 5 mL isopropanol (IPA), vortexed for 10 minutes, centrifuged at 4,000 rpm for 20 minutes and the resulting supernatants were filtered with 0.22 µm syringe filters evaporated and resuspended in 5 mL BHI – A 1% inoculum of a C. difficile overnight culture was used to inoculate molten reinforced clostridial medium (RCM) agar which was then distributed into the wells of 6-well plates. Wells were bored into the agar and 50 µL of CFS or IPA-extract were added to the wells. The agar plates were incubated anaerobically overnight (18–24 hours). The diameters of the resulting zones of inhibition were measured. Experiments were conducted in duplicate – A total of 42 contemporary C. difficile isolates (25 distinct ribotypes from CF, CDI, and IBD patients) were tested (Figure 1) • To further characterize ART24 and the associated anti-C. difficile activity, the effect of 20 mg/mL proteinase K and gastrointestinal enzymes was investigated on CFS using well diffusion assays (WDA) – ART24 CFS were tested for resistance to gastrointestinal enzymes chymotrypsin and trypsin, and proteinase K (20 mg/mL). Each enzyme was placed in an adjacent well to the CFS. Susceptibility to the enzymes was characterized by a half-moon-shaped zone of inhibition – Dilutions of ART24 CFS were re-tested against proteinase K (20 mg/mL) to confirm resistance to the protease. The protease was placed in adjacent wells to the CFS in one assay or co-incubated in another assay – C. difficile agar plates were incubated in the anaerobic chamber overnight (18–24 hours) and analyzed for the presence of zones of inhibition Results • ART24 cell-free pH-neutralized supernatants (CFS) from freshly grown ART24 as well as IPA extracts from re-suspended lyophilized ART24 inhibited all 25 ribotypes of C. difficile tested (Table 1). The zone size/activity of the IPA extracts from the contents of the reconstituted lyophilized ART24 capsule were slightly greater than the CFS from freshly grown ART24 (Figures 2 and 3), reflecting the increased potency from the more concentrated IPA samples • The antimicrobial activity of ART24 CFS was found to be resistant to the high level of gastrointestinal enzymes tested (Figure 4). The levels used were 20x greater than the average normal concentration used (1 mg/mL) for testing bacteriocins. Specific re-testing with proteinase K using dilutions of ART24 CFS also showed resistance suggesting that the anti-C. difficile activity is not solely related to peptides (Figures 5 and 6) Table 1. ART24 inhibition of C. difficile isolates APC isolate number Ribotype Supernatant diameter (mm) IPA diameter (mm) 3 003 20 22–23 4 010 18–20 23–24 5 020 19–20 20–24 8 062 19–20 22–24 9 050 19–20 22–25 11 026 17–20 22–24 12 031 20–21 22–23 17 001 19–20 23 18 106 19–21 23–26 24 001 19–21 23–24 25 018 20–21 24–25 26 018 20–21 25 27 106 19–21 23–26 28 106 17–21 24 40 001 18 22–23 41 001 19–20 22–25 43 (VPI10463) 078 (VPI10463) 19–20 21 1202 017 19–20 21–23 1203 012 21 22–25 1207 001 18–19 22–24 1208 015 18–19 22 1209 106 18–19 22–23 1211 027 19–20 23–25 1212 002 19–20 21–23 1213 027 19–20 22 1214 Not typed 18–20 21–22 1398 014 18–20 21–22 1399 002 17 22 1401 126 18 21–22 1404 140 18–20 20–21 1410 015 20–22 25 1412 046 19 23 1418 039 19 23 1421 078 19 24 1424 011 19–22 23 1425 005 19 23 1427 092 19–24 22–30 1431 087 19–26 21–23 1432 356 19–20 21–22 1441* 001 19–21 22–23 1442* 015 21 23–24 1451* 027 19–20 21–23 *Thuricin-CD resistant mutants. IPA, isopropanol Figure 1. Distribution of the C. difficile ribotypes of the 42 isolates tested Ribotype 001 (6, 14%) 015 (3, 7%) 002 (2, 5%) 106 (4, 10%) 078 (2, 5%) 027 (3, 7%) 005 (1, 2%) 018 (2, 5%) 011 (1, 2%) 003 (1, 2%) 014 (1, 2%) 010 (1, 2%) 020 (1, 2%) 012 (1, 2%) 031 (1, 2%) 017 (1, 2%) 046 (1, 2%) 026 (1, 2%) 062 (1, 2%) 039 (1, 2%) 087 (1, 2%) 092 (1, 2%) 050 (1, 2%) 126 (1, 2%) 140 (1, 2%) 356 (1, 2%) Not typed (1, 2%) 14% 10% 7% 7% 5% 5% 5% 2% 2% 2% 2% 2% 2% 2% 2% 2% 2% 2% 2% 2% 2% 2% 2% 2% 2% 2% 2% Figure 3. Zone diameter distribution for the ART24 IPA extracts and cell-free supernatant against a panel of 42 contemporary C. difficile isolates 17 18 19 20 21 22 23 24 25 26 27 28 29 30 0 5 10 15 20 25 30 35 Number of isolates Zone of inhibition (mm) ART24 cell-free supernatant ART24 IPA extract IPA, isopropanol Figure 4. ART24 antimicrobials resistance to gastrointestinal enzymes C, chymotrypsin; P, proteinase K; S, cell-free supernatant; T, trypsin Thuricin CD is a peptide bacteriocin used as a positive control Figure 5. Confirmation of ART24 antimicrobials resistance to proteinase K in adjacent wells P, proteinase K; S, cell-free supernatant Thuricin CD is a peptide bacteriocin used as a positive control Figure 6. Confirmation of ART24 antimicrobials resistance to proteinase K when co-incubated M, proteinase K; S, cell-free supernatant Thuricin CD is a peptide bacteriocin used as a positive control Conclusions ART24 exhibits protease-resistant anti-C. difficile activity against a broad range of contemporary C. difficile including clinically relevant isolates of various and clinically relevant ribotypes. As such, ART24 is a promising LBP candidate in Phase 1 clinical development for the prevention of CDI recurrence. Michelle O’ Donnell, 1 Brian Healy, 1 Colin Hill, 2 R. Paul Ross, 2 Mary C. Rea, 3 Ronald Farquhar, 4 and Laurent Chesnel 4 1 University College Cork, Ireland; 2 APC Microbiome Ireland, University College Cork, Ireland; 3 Teagasc Food Research Centre, Moorepark Fermoy, Ireland; 4 Artugen Therapeutics USA, Inc. Figure 2. (A) IPA extracts of ART24 and (B) ART24 cell-free supernatant tested against C. difficile isolates APC27, APC28, APC40, APC41, APC43 (VPI10463), and APC1202 A B Background • ART24 is being investigated as a live biotherapeutic product (LBP) treatment to prevent recurrence of Clostridioides difficile infection (CDI) following successful antibacterial therapy. ART24 was isolated from a human fecal sample, identified, and purified. ART24 is a member of the B. amyloliquefaciens/B. velezensis group that was found from a screening program aimed at identifying bacteria from stool samples with direct C. difficile-killing activity. The data presented here sought to investigate the broad range of activity against contemporary clinical isolates of various ribotypes
