WO2024123891A1 - Compositions de micro-organismes modifiés et leurs applications - Google Patents

Compositions de micro-organismes modifiés et leurs applications Download PDF

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WO2024123891A1
WO2024123891A1 PCT/US2023/082700 US2023082700W WO2024123891A1 WO 2024123891 A1 WO2024123891 A1 WO 2024123891A1 US 2023082700 W US2023082700 W US 2023082700W WO 2024123891 A1 WO2024123891 A1 WO 2024123891A1
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yeast cells
boulardii
ligand
fibronectin
microorganism composition
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PCT/US2023/082700
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English (en)
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Juliane Nguyen
Mairead HEAVEY
Anthony HAZELTON
Emma ETTER
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The University Of North Carolina At Chapel Hill Office Of Technology Commercialization
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Publication of WO2024123891A1 publication Critical patent/WO2024123891A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/06—Fungi, e.g. yeasts
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/37—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi
    • C07K14/39—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi from yeasts
    • C07K14/395—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi from yeasts from Saccharomyces
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material

Definitions

  • the present invention relates to engineered microorganism compositions and, in particular, to yeast cells exhibiting structure and composition for binding to inflamed sites of the gastrointestinal mucosa and secreting one or more therapeutic proteins.
  • IBD Inflammatory bowel disease
  • Crohn’s disease and ulcerative colitis are chronic, relapsing-remitting conditions that affect more than 1.6 million adults and 80,000 children in the US alone. Patients suffer from severe abdominal pain, bloody diarrhea, and systemic symptoms caused by chronic inflammation and ulcer formation in the gastrointestinal (GI) tract.
  • Current therapies are not curative, and 25% of IBD patients require hospitalization and 45% relapse.
  • Many currently available drugs have severe side-effects, particularly over the long term, and many patients eventually require surgery due to intractable disease or the development of chronic sequelae such as strictures or cancer.
  • the microorganism composition comprises yeast cells having cellular surfaces comprising at least one ligand species targeting fibronectin or collagen IV.
  • the at least one ligand species for example, can comprise an antibody, antibody fragment, or microbially-derived adhesin protein.
  • the at least one ligand species can be coupled to surfaces of the yeast cells by any mechanism not inconsistent with the technical objectives described herein.
  • the ligand species is coupled to the cellular surfaces via one or more protein binding interactions. Any protein binding interaction consistent with the technical objectives herein can be employed.
  • the ligand species for example, can be coupled to yeast cell surfaces via streptavidin-biotin interactions, in some embodiments.
  • FIG. 1 A illustrates coupling of anti-fibronectin antibody to outer surfaces of a yeast cell, according to some embodiments.
  • the yeast cells can be genetically modified to display streptavidin on cellular surfaces. Biotinylated anti-fibronectin antibody is subsequently provided for binding with the streptavidin, resulting the surface modified yeast cells.
  • FIG. IB illustrates reduction in ligand surface density as a result of yeast cell division.
  • Yeast cellular surfaces can also be modified with anti-collagen IV antibody via streptavidin-biotin interactions.
  • the yeast cells can be genetically modified to express the targeting ligand species on the cellular surfaces.
  • FIG. 2 illustrates surface expression of targeting ligand by yeast cells according to some embodiments.
  • the yeast cells exhibit surface expression of single-chain variable fragment targeting fibronectin.
  • the ligand density resulting from yeast expression does not decrease with yeast proliferation.
  • the yeast cells in additional to displaying modified or engineered outer surfaces comprising ligand species targeting fibronectin, can also secrete one or more therapeutic proteins.
  • the therapeutic proteins can be employed to treat IBD.
  • the yeast cells secrete anti-tumor necrosis factor-a (anti-TNF-a) nanobodies.
  • anti-TNF-a anti-tumor necrosis factor-a
  • the yeast cells can be located and retained at inflamed regions of the gastrointestinal mucosa via binding with fibronectin of the inflamed regions. Once located and retained at the inflamed regions, the yeast cell may secrete one or more therapeutic proteins for the treatment of IBD. Inflamed regions include ulcerated mucosa.
  • a microorganism composition described herein comprises a mixture including a first component of yeast cells having cellular surfaces modified with at least one ligand targeting fibronectin, and a second component of yeast cells having cellular surfaces modified with at least one ligand targeting collagen IV.
  • Yeast cells of one or both first and second components can also secrete one or more therapeutic proteins, including proteins for the treatment of IBD.
  • a method comprises administering to a patient a microorganism composition comprising yeast cells having cellular surfaces comprising at least one ligand species targeting fibronectin or collagen IV, and localizing and retaining the yeast cells at inflamed regions of the gastrointestinal mucosa via binding with fibronectin or collagen IV of the inflamed regions.
  • the method may further comprise secreting one or more proteins with the yeast cells, the one or more proteins having a therapeutic effect on the inflamed regions of the gastrointestinal mucosa.
  • FIG. 3 is a schematic of a method described herein, according to some embodiments.
  • the surface modified yeast cells have selective delivery to inflamed regions of the gastrointestinal mucosa via fibronectin targeting. Once located and fixed at the inflamed regions, the yeast cells can secrete one or more therapeutic proteins for the treatment of IBD, such as proteins that block or mitigate inflammation.
  • the yeast cells may also exhibit intrinsic probiotic activity.
  • the yeast cells may additionally proliferate at the inflamed regions.
  • the fibronectin ligand targeting density may decrease with proliferation or may remain static.
  • the patient is administered a mixture including a first component of yeast cells having cellular surfaces modified with at least one ligand targeting fibronectin, and a second component of yeast cells having cellular surfaces modified with at least one ligand targeting collagen IV.
  • Yeast cells of one or both first and second components can also secrete one or more therapeutic proteins, including proteins for the treatment of IBD.
  • the yeast cells secrete anti -tumor necrosis factor-a (anti-TNF-u) nanobodies.
  • the first and second components can be present in the mixture at any desired ratio. In some embodiments, the ratio of first component to second component ranges from 1 : 10 to 10: 1.
  • Yeast cells of the first and second components can have any composition, architecture, or properties described herein.
  • FIG. 1A is a schematic depiction of the coupling of an anti-fibronectin antibody to outer surfaces of a yeast cell, according to some embodiments of the present disclosure.
  • FIG. IB is a schematic depiction of the reduction in ligand surface density as a result of yeast cell division.
  • FIG. 2 is a schematic depiction of the surface expression of targeting ligand by yeast cells according to some embodiments of the present disclosure.
  • FIG. 3 is a schematic depiction of a method described herein, according to some embodiments.
  • FIG. 4A is a schematic representation of Saccharomyces boulardii (S. boulardii) displaying anti-fibronectin antibodies specifically binding to fibronectin.
  • S. boulardii genetically engineered to display streptavidin on the surface for site-specific attachment of biotinylated targeting ligands, such as anti-fibronectin antibodies, here referred to as dynamic-FN S.b.
  • a gene construct for genetic engineering of S. boulardii to display streptavidin on the surface is displayed in the lower portion of the figure.
  • FIG. 4C depicts images and a graphical representation of S. boulardii displaying anti- fibronectin antibodies specifically binding to fibronectin.
  • FIG. 4C depicts images and a graphical representation of S. boulardii displaying anti- fibronectin antibodies specifically binding to fibronectin.
  • In the left inset are representative images of green fluorescent protein (GFP)-labeled, dynamic-FN S.b. binding to fibronectin- coated plates.
  • GFP green fluorescent protein
  • N 9 with ***p ⁇ 0.001.
  • FIG. 5A is a graphical representation of S. boulardii displaying anti-fibronectin antibodies specifically binding to fibronectin.
  • Dextran sulfate sodium (DSS)-induced colitis mice show 30-fold enhanced fibronectin (Fnl) upregulation compared to healthy mice.
  • FIG. 5B displays images of S. boulardii displaying anti-fibronectin antibodies specifically binding to fibronectin.
  • Dynamic-FN S.b. binds to colon tissue of mice with DSS- induced ulcerative colitis. Negligible binding was observed with non-targeted S.b.
  • White arrows indicate locations of GFP-labeled dynamic-FN S.b. Scale bar H&E and left fluorescent images: 500 pm, right fluorescent images 100 pm.
  • FIG. 5C displays images of S. boulardii displaying anti-fibronectin antibodies specifically binding to fibronectin.
  • White arrows indicate locations of GFP-labeled dynamic-FN S.b. Scale bar H&E and left fluorescent images: 500 pm, right fluorescent images 100 pm.
  • FIG. 6A is a series of graphical displays of genetic engineering of dual-functional S. boulardii.
  • anti-TNFa secreted anti-tumor necrosis factor alpha
  • L929 murine fibroblasts were exposed to TNFa.
  • TNFa is known to cause cell death through the activation of type I receptors, and the anti-TNFa nanobodies are functional if they can prevent cell death of L929.
  • the supernatant of the S. boulardii engineered to secrete anti- TNFa nanobodies effectively prevented cell death and maintained 100% cell viability.
  • FIG. 6B is a schematic depiction of genetic engineering of dual-functional S. boulardii. A schematic is shown of a gene construct used to genetically engineer dual functional S. boulardii.
  • FIG. 6C is a schematic depiction and graphical display of genetic engineering of dualfunctional ,S'. boulardii. More than 40 dual-functional S. boulardii clones were generated with a range of ligand densities (expressed as targeting ligand display score) that also secrete anti-TNFa nanobodies over a wide dynamic range (expressed as TNFa nanobody secretion score).
  • FIG. 7A is a graphical representation of the stability and secretion rate of anti-TNFa nanobodies from a representative S. boulardii strain. Anti-TNFa nanobodies show a degradation half-life of approximately 60 min. in simulated intestinal fluid (SIF) at pH 4 (squares), 6 (circles), and 7.4 (triangles).
  • SIF simulated intestinal fluid
  • FIG. 7B is a graphical representation of the stability and secretion rate of anti-TNFa nanobodies from a representative S. boulardii strain.
  • a representative S. boulardii strain secretes anti-TNFa nanobodies at a rate of approximately 1605 ng/hr.
  • FIG. 8A is a schematic depiction of dynamic-FN S.b designed to target fibronectin displayed an approximately 100-fold greater concentration in the colon compared to non-targeted S. boulardii for at least 72h.
  • FIG. 8B is a graphical representation of dynamic-FN S.b designed to target fibronectin displayed an approximately 100-fold greater concentration in the colon compared to non-targeted S. boulardii for at least 72h.
  • Fecal concentrations of dynamic-FN S.b. (rightmost bars in groupings of three), non-treatment (leftmost bars in groupings of three), or non-targeted S.b (middle bars in groupings of three). All mice were dosed with 10 A 9 CFU S. boulardii. Data are presented as mean ⁇ sd, with ***p ⁇ 0.001 and **p ⁇ 0.01.
  • FIG. 8C is a graphical representation of dynamic-FN S.b designed to target fibronectin displayed an approximately 100-fold greater concentration in the colon compared to non-targeted S. boulardii for at least 72h.
  • Colonic concentrations of dynamic-FN S.b. (rightmost bars in groupings of three), non-treatment (leftmost bars in groupings of three), or non-targeted S.b (middle bars in groupings of three). All mice were dosed with 10 A 9 CFU S. boulardii. Data are presented as mean ⁇ sd, with ***p ⁇ 0.001 and **p ⁇ 0.01.
  • FIG. 9A is a schematic representation of dynamic-FN S.b. having greater therapeutic efficacy compared with non-targeted S.b. as measured by clinical score and colon length. DSS induced colitis model. Dosing schematics. Top timeline represents DSS only, middle timeline represents non-targeted S.b., and lower timeline represents dynamic-FN S.b.
  • FIG. 9B is an image series and graphical display of dynamic-FN S.b. having greater therapeutic efficacy compared with non-targeted S.b. as measured by clinical score and colon length.
  • C57BIJ6J mice treated with dynamic-FN S.b show lowest clinical score in fecal matter.
  • Left images show positive (top) and negative (bottom) for occult blood.
  • Right analysis shows clinical scores for dynamic-FN S.b. (rightmost bars in groupings of three), DSS only (leftmost bars in groupings of three), or non-targeted S.b (middle bars in groupings of three).
  • FIG. 9C is a graphical display of dynamic-FN S.b. having greater therapeutic efficacy compared with non-targeted S.b. as measured by clinical score and colon length.
  • Body weight analysis for dynamic-FN S.b., DSS only, or non-targeted S.b. Mice were dosed with 10 A 9 CFU S. boulardii. N 3 to 5 with *p ⁇ 0.05. **p ⁇ 0.01, ***p ⁇ 0.001. ****p ⁇ 0.0001 by one-way ANOVA with post-hoc Tukey.
  • FIG. 9D is a graphical display of dynamic-FN S.b. having greater therapeutic efficacy compared with non-targeted S.b. as measured by clinical score and colon length.
  • Dynamic-FN S.b. restores colon length to healthy levels.
  • C57BL/6J mice treated with dynamic-FN S.b. displayed significantly longer colon length than non-treated mice (DSS only) or non-targeted S.b. 72 hours post-treatment.
  • FIG. 10A is a graphical representation showing that dynamic FN S.b. decreases pro- inflammatory markers and increases anti-inflammatory markers.
  • Dynamic-FN S.b robustly decreased the pro-inflammatory marker IFN-y compared to non-targeted S.b.
  • C, D Dynamic- FN S.b substantially increased the anti-inflammatory marker IL-10 and IL-6 compared to nontargeted S.b.
  • IL-6 is a cytokine with pleiotropic effects
  • FIG. IOC is a graphical representation showing that dynamic FN S.b. decreases pro- inflammatory markers and increases anti-inflammatory markers.
  • FIG. 10D is a graphical representation showing that dynamic FN S.b. decreases pro- inflammatory markers and increases anti-inflammatory markers.
  • FIG. 11 A is a schematic depiction of antibody-labeling enabling S. boulardii to bind to corresponding extracellular matrix proteins. Schematic of various biotinylated antibodies specific to extracellular matrix proteins attaching to the S. boulardii cell surface via biotin-streptavidin interactions.
  • FIG. 11C is a series of images showing antibody-labeling enabling S. boulardii to bind to corresponding extracellular matrix proteins.
  • Middle anti -fibrinogen antibody-labeled S. boulardii (S.b. FB, top) or nonspecific antibody-labeled S. boulardii (S.b.
  • FIG. 12A is a schematic representation of engineered S. boulardii retaining probiotic mechanisms of action in vitro. Schematic of probiotic mechanisms of action elicited by S. boulardii.
  • FIG. 12B is a graphical representation of engineered S. boulardii retaining probiotic mechanisms of action in vitro. Percent viability after 4-hour incubations of S. cerevisiae (S.c.), non-engineered . boulardii S.b.), S. boulardii displaying mSA (S.b. mSA), and S. b. mSA with biotinylated anti-fibronectin antibodies attached to the cell surface (S.b. FN) in media at pH 2.5, pH 4.0, or containing 0.3% bile salts, or 0.6% bile salts.
  • S.b. FN biotinylated anti-fibronectin antibodies attached to the cell surface
  • FIG. 12C is a graphical representation of engineered S. boulardii retaining probiotic mechanisms of action in vitro.
  • FIG. 12D is a graphical representation of engineered S. boulardii retaining probiotic mechanisms of action in vitro.
  • Concentration of murine interleukin- 10 in the cell culture supernatant following an 18-hour co-incubation of murine bone marrow-derived dendritic cells with phosphate buffered saline, S.c., lipopolysaccharide, S.b., S.b. mSA, or S.b. FN. Bars represent mean, error shown as standard deviation, significance assessed using ordinary one-way ANOVA with multiple comparisons, a 0.05, * p ⁇ 0.05, ** p ⁇ 0.01, *** p ⁇ 0.001, **** p ⁇ 0.0001.
  • FIG. 13A is a schematic representation of S. boulardii growth, surface labeling, and attachment efficiency in simulated intestinal fluid. Schematic depicting the growth of antibody- labeled 5. boulardii, resulting in the dilution of the number of antibodies remaining on each cell surface, and subsequent reduction in the capacity to bind to the corresponding ECM protein.
  • FIG. 13B is a graphical representation of S. boulardii growth, surface labeling, and attachment efficiency in simulated intestinal fluid.
  • Anti-fibronectin antibody -lab eled . boulardii growth squares, right y-axis
  • percent antibody remaining on the cell surface as determined by flow cytometry circles, left y-axis
  • percent mSA remaining on the cell surface as determined by flow cytometry triangles, left y-axis
  • FIG. 14A is a schematic representation of how extracellular matrix targeting enables increased gut residence time of S. boulardii and decreased inflammation markers in acute, DSS- induced model of murine colitis.
  • C57BL6/J mice were administered 2% dextran sulfate sodium (DSS) in the drinking water for 5 days, then administered regular drinking water for the remainder of the study. Mice were then dosed with 5xl0 9 colony forming units (CFU) of nontargeted (S.b. mSA), fibrinogen-targeted (S.b. FB), collagen IV-targeted (S.b. CIV), or fibronectin-targeted (S.b. FN) yeast via oral gavage. Stool was sampled every 12-24 hours post-gavage to measure viable yeast.
  • DSS dextran sulfate sodium
  • FIG. 14B is a graphical representation of how extracellular matrix targeting enables increased gut residence time of S. boulardii and decreased inflammation markers in acute, DSS- induced model of murine colitis.
  • S. boulardii CFU in the stool over time post-gavage. Bars represent mean, error shown as standard deviation, significance assessed using ordinary one-way ANOVA with multiple comparisons, a 0.05, * p ⁇ 0.05, ** p ⁇ 0.01, *** p ⁇ 0.001, **** p ⁇ 0.0001.
  • FIG. 14E is a graphical representation of how extracellular matrix targeting enables increased gut residence time of S. bcnilardii and decreased inflammation markers in acute, DSS- induced model of murine colitis.
  • Mouse colon length at study termination. Bars represent mean, error shown as standard deviation, significance assessed using ordinary one-way ANOVA with multiple comparisons, a 0.05, * p ⁇ 0.05, ** p ⁇ 0.01, *** p ⁇ 0.001, **** p ⁇ 0.0001.
  • FIG. 15A is a graphical representation of how extracellular matrix targeting enables increased gut residence time of S. boulardii and decreased inflammation markers in acute, DSS- induced model of murine colitis.
  • FIG. 15B is a series of images showing how extracellular matrix targeting enables increased gut residence time of S. boulardii and decreased inflammation markers in acute, DSS- induced model of murine colitis. Representative images of hematoxylin and eosin staining of colon Swiss rolls at study termination with black arrows indicating areas of severe inflammation/ulceration. Scale bars are 100 m.
  • FIG. 15C is a graphical representation of how extracellular matrix targeting enables increased gut residence time of S. boulardii and decreased inflammation markers in acute, DSS- induced model of murine colitis.
  • Semi-quantitative histological scores of inflammation accounting for extent of mucosal loss, hyperplasia, and erosions. Bars represent mean, error shown as standard deviation, significance assessed using ordinary one-way ANOVA with multiple comparisons, a 0.05, * p ⁇ 0.05, ** p ⁇ 0.01, *** p ⁇ 0.001, **** p ⁇ 0.0001.
  • FIG. 15D is a graphical representation of how extracellular matrix targeting enables increased gut residence time of S. boulardii and decreased inflammation markers in acute, DSS- induced model of murine colitis. Fold change in expression of fibrinogen, collagen IV, and fibronectin in the colon of DSS mice at study termination relative to healthy colon tissue. Bars represent mean, error shown as standard deviation.
  • FIG. 16A is a schematic representation of how S. boulardii targeted to collagen IV increases probiotic gut residence time and improves markers of inflammation in a repeated injury-recovery, DSS-induced murine model of colitis.
  • C57BL6/J mice were administered 2% dextran sulfate sodium (DSS) in the drinking water for 5 days (injury) followed by administration of regular drinking water for 3 days (recovery). This cycle was repeated twice more.
  • mice were administered with 5xl0 9 colony forming units (CFU) of nontargeted (S.b. mSA), fibronectin- targeted (S.b. FN), or collagen IV-targeted (S.b. CIV) yeast via oral gavage every 3 days for the remainder of the study.
  • Stool was sampled every 24 hours post-gavage to measure viable yeast.
  • FIG. 16B is a graphical representation of how S. boulardii targeted to collagen IV increases probiotic gut residence time and improves markers of inflammation in a repeated injury-recovery, DSS-induced murine model of colitis.
  • S. boulardii CFU in the stool on the 2- days post-dose timepoints (Days 11, 14, 17, 20, and 23) (left graph) and 3 -days post-dose timepoints (Days 12, 15, 18, 21, and 24) (right graph). Bars represent mean, error shown as standard deviation, significance assessed using ordinary one-way ANOVA with multiple comparisons, a 0.05, * p ⁇ 0.05, ** p ⁇ 0.01, *** p ⁇ 0.001, **** p ⁇ 0.0001.
  • FIG. 16C is a graphical representation of how S. boulardii targeted to collagen IV increases probiotic gut residence time and improves markers of inflammation in a repeated injury-recovery, DSS-induced murine model of colitis.
  • Area under the curve (AUC) analysis of CFU in the stool over time plots. Bars represent mean, error shown as standard deviation, significance assessed using ordinary one-way ANOVA with multiple comparisons, a 0.05, * p ⁇ 0.05, ** p ⁇ 0.01, *** p ⁇ 0.001, **** p ⁇ 0.0001.
  • FIG. 17B is a graphical representation of how S. boulardii targeted to collagen IV increases probiotic gut residence time and improves markers of inflammation in a repeated injury-recovery, DSS-induced murine model of colitis.
  • Mouse colon length at study termination. Bars represent mean, error shown as standard deviation, significance assessed using ordinary one-way ANOVA with multiple comparisons, a 0.05, * p ⁇ 0.05, ** p ⁇ 0.01, *** p ⁇ 0.001, **** p ⁇ 0.0001.
  • FIG. 17C is a graphical representation of how S. boulardii targeted to collagen IV increases probiotic gut residence time and improves markers of inflammation in a repeated injury-recovery, DSS-induced murine model of colitis.
  • Relative expression of pro-inflammatory (TNFoc, IFNy, IL6) and anti-inflammatory (TGF0, IL 10) cytokines as compared to the healthy control within the colon at study termination.
  • Significance determined as compared to DSS mice and assessed using ordinary one-way ANOVA with multiple comparisons, a 0.05, * p ⁇ 0.05, ** p ⁇ 0.01, *** p ⁇ 0.001, **** p ⁇ 0.0001.
  • FIG. 17D is a series of images depicting how S. boulardii targeted to collagen IV increases probiotic gut residence time and improves markers of inflammation in a repeated injury-recovery, DSS-induced murine model of colitis. Representative images of hematoxylin and eosin staining of colon Swiss rolls at study termination with black arrows indicating areas of severe inflammation/ulceration. Scale bars are 100 pm.
  • FIG. 17E is a graphical representation of how S. boulardii targeted to collagen IV increases probiotic gut residence time and improves markers of inflammation in a repeated injury-recovery, DSS-induced murine model of colitis.
  • Semi-quantitative histological scores of inflammation accounting for extent of mucosal loss, hyperplasia, and erosions. Bars represent mean, error shown as standard deviation, significance assessed using ordinary one-way ANOVA with multiple comparisons, a 0.05, * p ⁇ 0.05, ** p ⁇ 0.01, *** p ⁇ 0.001, **** p ⁇ 0.0001.
  • FIG. 17F is a graphical representation of how S. boulardii targeted to collagen IV increases probiotic gut residence time and improves markers of inflammation in a repeated injury-recovery, DSS-induced murine model of colitis. Relative expression of fibrinogen, fibronectin, and collagen IV in the colon of DSS mice at study termination relative to healthy colon tissue. Bars represent mean, error shown as standard deviation.
  • FIG. 18A is a graphical representation of . boulardii engineered to bind to fibronectin in vitro.
  • Mean fluorescence intensity (MFI) of S.b. (dark grey) and S.b. FN (light grey) binding to soluble fibronectin as measured by flow cytometry, n 3.
  • FIG. 18B is a series of images depicting 5. boulardii engineered to bind to fibronectin in vitro. Representative fluorescent images of wild type (S.b.) and engineered (S.b. FN, and S.b. FN-aTNF) strains bound to a fibronectin-coated well plate.
  • FIG. 18D is a schematic representation of 5. boulardii engineered to bind to fibronectin in vitro. Schematic depicting stability of surface expression of fibronectin binding moieties over time.
  • FIG. 18E is a graphical representation of . boulardii engineered to bind to fibronectin in vitro.
  • FIG. 19B is a graphical representation of S. boulardii engineered to secrete aTNF therapeutics and neutralize TNF in vitro.
  • aTNF nanobody concentrations in S.b. aTNF (light grey) and S.b. FN-aTNF (dark grey) cultures over time, n 3, points represent mean. Error shown as standard deviation.
  • FIG. 19D is a schematic representation and a graphical representation of S. boulardii engineered to secrete aTNF therapeutics and neutralize TNF in vitro.
  • Schematic and analysis of in vitro L929 culture assay and L929 viability with increasing culture supernatant dilutions, n 3. Samples are uncultured media (light grey), S.b. supernatant (dark grey), and S.b. aTNF supernatant (medium grey).
  • FIG. 20A is a schematic representation of dual -functional S.b. staying within the colon longer with the ability to deliver more aTNF therapeutics.
  • Germ -free, female and male 129 SvEv IL-10' ' mice, aged 8-12 weeks, were orally gavaged with a fecal microbiota transplant (FMT) consisting of a fecal slurry from conventional C57BL6/J mice and colitis was induced for 5 weeks. Thereafter, mice were orally gavaged with various treatment groups l-2x per week. Feces and serum were collected on a weekly basis, n 8-13 mice per group.
  • FMT fecal microbiota transplant
  • FIG. 20B is a graphical representation of dual -functional S.b. staying within the colon longer with the ability to deliver more aTNF therapeutics.
  • FIG. 20C is a graphical representation of dual-functional S.b. staying within the colon longer with the ability to deliver more aTNF therapeutics.
  • FIG. 20D is a graphical representation of dual -functional S.b. staying within the colon longer with the ability to deliver more aTNF therapeutics.
  • FIG. 20E is a graphical representation of dual -functional S.b. staying within the colon longer with the ability to deliver more aTNF therapeutics.
  • FIG. 21 A is a graphical representation of dual-functional S.b. demonstrating an immunomodulatory effect and subsequent impact on inflammation in murine colitis.
  • Heatmap of mean relative expression of key cytokines, chemokines, transcription factors, and barrier integrity proteins in proximal colon tissue compared to healthy controls, n 8-13 from mice described in FIG. 20. Significance indicated as compared to mean relative expression values of PBS treated, disease control. Heatmap colors and lines represent means.
  • EXAMPLE 1 -Therapeutic Yeast for Targeted Treatment of Ulcerative Colitis Fibronectin (FN)-targeted S. boulardii binds to gastrointestinal ulcers
  • the streptavidin expressed on the yeast surface acts as a handle for the attachment of a dynamic range of biotinylated targeting moi eties, allowing for both tunable ligand densities on the surface of S. boulardii as well as the flexible attachment of ligands for the optimal targeting of gastrointestinal ulcers in vivo.
  • FN fibronectin
  • GFP green fluorescent protein
  • anti-TNFa bivalent anti-tumor necrosis factor alpha
  • the murine fibroblast cell line, L929 was co-incubated with TNFa and either yeast extract-peptone-dextrose (YPD) media, unmodified 5. boulardii supernatant, or supernatant from the nanobody-secreting S. boulardii for 18 hours. The L929 cells were then assayed for cell viability via crystal violet staining. Incubation with the supernatant from the nanobody-secreting S. boidardii showed a dosedependent inhibition of TNFa induced cell killing, indicating that the secreted nanobody both effectively bound to and neutralized murine TNFa (FIG. 6A, right).
  • YPD yeast extract-peptone-dextrose
  • the anti-TNFa nanobody stability was assessed by incubating it in simulated intestinal fluid (SIF, Sigma Aldrich) containing pepsin (2000U/ml) and pancreatin (lOOU/ml) enzymes over a pH range of 4 to 7.4 for 90 minutes at 37°C (FIG. 7A).
  • SIF simulated intestinal fluid
  • pepsin 2000U/ml
  • pancreatin lOOU/ml
  • boulardii surface modification with suitable targeting ligands decreases the clearance rate and consequently increases the exposure to the probiotic.
  • Dynamic-FN S.b. demonstrate greater therapeutic efficacy in a mouse model of colitis compared with non-targeted 5.
  • Dynamic-FN S.b. has greater therapeutic efficacy compared with non-targeted S.b. as measured by clinical score (FIG. 9B), colon length (FIG. 9D), and pro- and antiinflammatory markers (FIG. 10).
  • Dynamic-FN-S.b decreased pro-inflammatory markers and increased anti-inflammatory markers
  • Analysis of gene expression levels isolated from colon tissues of mice after treatment showed that dynamic FN S.b robustly decreased the pro-inflammatory markers such as TNF-ot and IFN-y compared to non-targeted 5.
  • boulardii (FIG. 10A and FIG. 10B).
  • dynamic FN S.b substantially increased the anti-inflammatory markers IL-6 and IL-10 compared to non-targeted .
  • boulardii (FIG. 10C and FIG. 10D).
  • IL-6 is a cytokine with pleiotropic effects
  • studies have shown that IL-6 plays a crucial anti-inflammatory role in acute inflammatory responses by controlling the level of pro-inflammatory markers and that its role is synergistic to the anti-inflammatory role of IL- 10.
  • Antibody-labeling enables S. boulardii to bind to corresponding extracellular matrix proteins
  • Streptavidin was first displayed on the surface of S. boulardii to act as a handle for the attachment of a variety of biotinylated targeting ligands. Next, biotinylated anti-FN antibody, anti-fibrinogen (anti-FB) antibody, or anti-collagen IV (anti-CIV) antibody was incubated with streptavidin displaying S. boulardii (FIG. 11 A).
  • green fluorescent protein (GFP)-labeled yeast displaying streptavidin with and without the addition of biotinylated anti-FN antibody, biotinylated anti-FG antibody, or biotinylated anti-CIV antibody on its surface were incubated on a FN, FG, or CIV coated well plate, respectively, followed by thorough washing (FIG. 1 IB and FIG. 11C).
  • GFP green fluorescent protein
  • S. boulardii displaying the anti-FG antibody or anti-CIV antibody on its surface, respectively, whereas binding of non-targeted S. boulardii was negligible in both cases.
  • the engineered S. boidardii were analyzed to determine whether modifications affect its probiotic mechanisms. Probiotic mechanisms of action include resistance to physiological changes, pathogen clearance, and host immune modulation (FIG. 12 A).
  • S. cerevisiae S.c.
  • non-engineered S. boulardii S.b.
  • S. boulardii displaying mSA S.b. mSA
  • . b. mSA with biotinylated antifibronectin antibodies attached to the cell surface S.b. FN were incubated in media at pH 2.5, pH 4.0, or containing 0.3% bile salts, or 0.6% bile salts (FIG. 12B).
  • Extracellular matrix targeting enables increased gut residence time of S. boulardii and decreased inflammation markers in acute, DSS-induced model of murine colitis
  • Inflammation markers were also assessed in the DSS-induced murine colitis study.
  • Relative expression of pro-inflammatory (TNFoc, IFNy, IL6) and anti-inflammatory (TGF0, IL10) cytokines as compared to the healthy control within the colon at study termination are evaluated in FIG. 15 A.
  • representative images of colon hematoxylin and eosin staining show areas of severe inflammation/ulceration (FIG. 15B), with semi-quantitative histological scores of inflammation accounting for extent of mucosal loss, hyperplasia, and erosions in FIG. 15C.
  • fold change in expression of fibrinogen, collagen IV, and fibronectin in the colon of DSS mice at study termination relative to healthy colon tissue is shown in FIG. 15D.
  • S. boulardii targeted to collagen IV increases probiotic gut residence time and improves markers of inflammation in a repeated injury-recovery, DSS-induced murine model of colitis
  • mice were administered 2% dextran sulfate sodium (DSS) in the drinking water for 5 days (injury) followed by administration of regular drinking water for 3 days (recovery). This cycle was repeated twice more. Following the first cycle, mice were administered with 5xl0 9 colony forming units (CFU) of nontargeted (S.b. mSA), fibronectin-targeted (S.b. FN), or collagen IV-targeted (S.b. CIV) yeast via oral gavage every 3 days for the remainder of the study (FIG. 16A). Stool was sampled every 24 hours post-gavage to measure viable yeast.) S.
  • CFU colony forming units
  • boulardii CFU in the stool was measured at 2-days post-dose timepoints and 3 -days post-dose timepoints (FIG. 16B), with an area under the curve (AUC) analysis of the CFU in the stool over time provided in FIG. 16C.
  • AUC area under the curve
  • S. boulardii CFU was also evaluated in the colon (FIG. 16D), while the percent body weight of the mice was measured over the course of the study (FIG. 17A) and mouse colon length was measured at study termination (FIG. 17B).
  • FIG. 17C The relative expression of pro-inflammatory (TNFa, ZFNy, IL6) and anti-inflammatory (TGF0, IL10) cytokines as compared to the healthy control within the colon at study termination are examined in FIG. 17C. Further, representative images of colon hematoxylin and eosin staining show areas of severe inflammation/ulceration (FIG. 17D), with semi-quantitative histological scores of inflammation accounting for extent of mucosal loss, hyperplasia, and erosions in FIG. 17E. Finally, the relative expression of fibrinogen, fibronectin, and collagen IV in the colon of DSS mice at study termination relative to healthy colon tissue is shown in FIG. 17F.
  • S. boulardii can be engineered to bind to fibronectin in vitro.
  • S. boulardii-FN can bind to soluble fibronectin.
  • S.b. FN binding to soluble fibronectin displayed a dissociation constant of 38 nM, indicating a high binding affinity, compared to wild-type (WT) S. boulardii. Binding affinity was determined using mean fluorescence intensity using flow cytometry.
  • targeted S. boulardii FN and dual -functional S. boulardii FN-aTNF displays a statistically significant increase in binding to fibronectin on a stable surface. The data was quantified by fluorescence in the respective images.
  • FIG. 18D A schematic depicting ideal growth conditions of a dual -functional engineered strain is shown in FIG. 18D. While the dualfunctional S. boulardii increases in growth (top bar), expression of the fibronectin binding protein (FnBP, middle bar) and binding towards fibronectin stay stable (lower bar). Finally, S. boulardii I was shown to stably grow over the course of 48 hours in simulated intestinal fluid (SIF) while also demonstrating stable FnBP expression and binding towards fibronectin (FIG. 18E).
  • SIF simulated intestinal fluid
  • S. boulardii can be engineered secrete aTNF therapeutics and neutralize TNF in vitro.
  • the aTNF nanobodies secreted from the therapeutic strain, S.b. aTNF have a high affinity towards TNF, with a dissociation constant of 3.1 nM (FIG. 19A). These results suggest a high potential for cytokine neutralization.
  • FIG. 19B and FIG. 19C the concentration of aTNF nanobodies secreted from S.b aTNF and S.b. FN-aTNF accumulate over time while growth of these engineered strains remain unchanged when comparing to wild type S.b. These results suggest that the engineering efforts do not inhibit yeast growth.
  • FIG. 19D S.b aTNF secreted nanobodies are demonstrated to neutralize TNF in vitro. Exposing L929 murine fibroblast cells to unblocked TNF, which are is known to cause cell death via the activation of type I receptors, and then exposing them to anti-TNF nanobodies was undertaken to assess the nanobodies’ ability to prevent cell death of L929. Co-incubation with supernatants from either uncultured media or S.b. cultures resulted in minimal L929 cell viability upon TNF exposure (FIG. 19D). In contrast, incubation with titrating concentrations of supernatant from S.b. aTNF cultures resulted in dose-dependent recovery in L929 cell viability (FIG. 19D).
  • Dual -functional S.b. stays within the colon longer with the ability to deliver more aTNF therapeutics.
  • mice were bred under germ-free conditions and are exposed to foreign bacteria via a fecal microbiota transplant (FMT) to induce severe and chronic colitis.
  • FMT fecal microbiota transplant
  • This model closely resembles the dysbiotic and chronic nature of human disease and is known to form severe fibrotic lesions and ulcers within the mouse colon.
  • mice were treated lx to 2x per week, with S.b., S.b. FN, S.b. aTNF, or S.b. FN- aTNF via oral gavage for the next 5 weeks.
  • Fecal material was collected once per week to measure the yeast and nanobody concentrations (FIG. 20A).
  • Fecal material was collected at a 72- hour post-gavage timepoint, as this timepoint has been previously demonstrated for yeast clearance.
  • FIG. 20B and FIG. 20C 60% more mice treated with fibronectin targeted . boulardii have detectable amounts of yeast within their colon in comparison to wild-type S. boulardii.
  • Mice treated with S.b. aTNF had detectable levels of yeast in the feces in some mice (0-40%) at 3 out of the 5 timepoints measured.
  • the detection rate of yeast in the feces of mice treated with S.b. FN and S.b. FN-aTNF was significantly higher, ranging from 50 to 90% at all 5 timepoints measured.
  • S.b. FN and S.b. FN-aTNF treated mice had significantly higher yeast concentrations compared to the levels detected in mice treated with S.b. aTNF.
  • dual-functional yeast had higher detectable levels of aTNF nanobodies in the feces at the timepoints measured in comparison to untargeted and wild-type yeast.
  • 0-60% of mice treated with yeast-purified aTNF 10-70% of mice treated with S.b. aTNF
  • 50-90% of mice treated with S.b. FN-aTNF had detectable levels of aTNF nanobodies in the feces at the timepoints measured.
  • Plotting the detectable aTNF nanobody concentrations measured from each group indicated that mice treated with either purified aTNF or S.b.
  • aTNF had similar concentrations of aTNF nanobody in the feces and significantly higher concentrations were detected in the feces of mice treated with S.b. FN-aTNF. Together these results indicate that engineered S.b. can effectively secrete and deliver aTNF nanobodies into the gut at similar concentrations to that of purified aTNF and with slightly higher intra-group consistency. Further, targeting yeast to fibronectin not only elongates yeast gut residence time, but also increases the resulting concentrations of aTNF delivered.
  • Dual-functional S.b. demonstrates an immunomodulatory effect and subsequent impact on inflammation in murine colitis.
  • FIG. 21 A dual -functional yeast are demonstrated to decrease pro-inflammatory cytokines Mmp2 and 116, while increasing anti-inflammatory cytokines Foxp3 and Tgfb.
  • Decreased expression oT Mmp2 may indicate a decrease in tissue destruction within the colon and decreases in 116 expression levels has been suggested to decrease local inflammation.
  • Changes to Foxp3 and transforming growth factor beta expression levels indicate that treatment stimulates the expression of markers associated with regulatory immune cells, resulting in a further decrease of inflammation.
  • S.b. FN-aTNF decreases colon weight compared to PBS in murine colitis.
  • Colon weight is a metric to determine disease state in colitic mice as an increased weight is often attributed to increased colonic wall thickening due to severe inflammation. With a decrease in colon weight, inflammation with the colon can be assumed to be rescued.
  • FIG. 21C FIG. 2 ID, and FIG. 2 IE, dual -functional yeast displays the lowest distal colon score indicating lowest inflammation compared to all groups.
  • S.b. FN-aTNF results in significantly improved histological colon scores, having lowest overall summary and distal scores and sharing comparable proximal colon scores to S.b. FN.

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Abstract

L'invention concerne des compositions de micro-organismes génétiquement modifiées présentant une structure et une composition pour se lier à des sites enflammés ou à d'autres sites de la muqueuse gastro-intestinale et sécréter une ou plusieurs protéines thérapeutiques. Dans certains modes de réalisation, la composition de micro-organismes comprend des cellules de levure ayant des surfaces cellulaires comprenant au moins une espèce de ligand ciblant la fibronectine ou le collagène IV. La ou les espèces de ligand, par exemple, peuvent comprendre un anticorps, un fragment d'anticorps ou une protéine d'adhésine dérivée de manière microbienne.
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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996041236A1 (fr) * 1995-06-07 1996-12-19 The Regents Of The University Of California Microelements a usage therapeutique et leurs procedes d'obtention et d'utilisation
US20110275535A1 (en) * 2008-12-16 2011-11-10 Novartis Ag Yeast Display Systems
US20170182153A1 (en) * 2011-06-14 2017-06-29 Globeimmune, Inc. Yeast-based compositions and methods for the treatment or prevention of hepatitis delta virus infection
US20220023357A1 (en) * 2016-02-04 2022-01-27 Universiteit Gent Use of microbial communities for human and animal health

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Publication number Priority date Publication date Assignee Title
WO1996041236A1 (fr) * 1995-06-07 1996-12-19 The Regents Of The University Of California Microelements a usage therapeutique et leurs procedes d'obtention et d'utilisation
US20110275535A1 (en) * 2008-12-16 2011-11-10 Novartis Ag Yeast Display Systems
US20170182153A1 (en) * 2011-06-14 2017-06-29 Globeimmune, Inc. Yeast-based compositions and methods for the treatment or prevention of hepatitis delta virus infection
US20220023357A1 (en) * 2016-02-04 2022-01-27 Universiteit Gent Use of microbial communities for human and animal health

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CHEN KEVIN, ZHU YIXUAN, ZHANG YONGRONG, HAMZA THERWA, YU HUA, SAINT FLEUR ASHLEY, GALEN JAMES, YANG ZHIYONG, FENG HANPING: "A probiotic yeast-based immunotherapy against Clostridioides difficile infection", SCIENCE TRANSLATIONAL MEDICINE, AMERICAN ASSOCIATION FOR THE ADVANCEMENT OF SCIENCE, vol. 12, no. 567, 28 October 2020 (2020-10-28), XP055965175, ISSN: 1946-6234, DOI: 10.1126/scitranslmed.aax4905 *
VANDENBROUCKE K; DE HAARD H; BEIRNAERT E; DREIER T; LAUWEREYS M; HUYCK L; VAN HUYSSE J; DEMETTER P; STEIDLER L; REMAUT E; CUVELIER: "Orally administered L. lactis secreting an anti-TNF Nanobody demonstrate efficacy in chronic colitis", MUCOSAL IMMUNOLOGY, NATURE PUBLISHING GROUP, US, vol. 3, no. 1, 1 January 2010 (2010-01-01), US , pages 49 - 56, XP009138280, ISSN: 1935-3456 *

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