Protease preparation for the treatment of toxins

Abstract

A method for the treatment of one or more toxins in a subject is provided, the method comprising administering to the subject a therapeutically effective amount of a serine protease that enzymatically cleaves a specific toxin. Also provided is a method for preventing the progression of disease in a subject infected with Clostridioides difficile (CD), diarrhea, or infectious colitis, the method comprising administering to the subject a therapeutically effective amount of the protease. The protease retains its activity up to 65°C. Treatment with the protease in mice infected with CD provided a 10-fold survival benefit compared to CD-infected untreated mice. TIFF2025521857000010.tif62147

Description

Technical Field

[0001] Related Applications This application claims the benefit of the filing date of U.S. Patent Application No. 63 / 356,443, filed Jun. 28, 2022, the contents of which are hereby incorporated by reference in their entirety.

[0002] Field The field of the present invention is protease treatment for degrading toxins.

Background Art

[0003] Background Clostridioides (formerly Clostridium) difficile (CD) is a major cause of hospital-associated diarrhea. Subjects with CD infection (CDI) can experience symptoms ranging from mild diarrhea to pseudomembranous colitis, toxic megacolon, and even death. The pathogenesis of CD is toxin-mediated. Toxin A (TcdA) and toxin B (TcdB) catalyze the glycosylation and inactivation of human Rho-GTPases. Inactivation of small regulatory proteins leads to cytoskeletal collapse and cell death. Rho proteins are important in maintaining epithelial tight junctions. The toxins disrupt the epithelial barrier, leading to pseudomembranous colitis.

[0004] Currently, CD treatment involves the use of antibiotics, which have a failure rate of 20-25%. In subjects in whom antibiotics repeatedly fail, the U.S. Food and Drug Administration and Health Canada have approved the use of human fecal transplants, which recolonize the human gut and often dissipate CD. However, fecal transplants require a healthy donor and intensive screening of healthy donors for infectious diseases and are labor-intensive.

[0005] CD is the cause of over 500,000 infections and >30,000 deaths annually in North America alone. It causes a range of symptoms from mild diarrhea to pseudomembranous colitis, toxic megacolon, colonic perforation, and death. CD infection (CDI) poses a major challenge to infection control because C. difficile bacteria readily form environmentally stable spores. Risk factors for CDI onset include antibiotic use, advanced age (>65 years), and exposure to healthcare facilities including long-term care. The widespread use of antibiotics and the increased cohort of elderly in nursing facilities have contributed to the large outbreak of C. difficile, as well as the increased incidence and severity of CDI over the past 20 years.

[0006] There is a need for a treatment for C. difficile that has a low failure rate and is not labor-intensive to provide. SUMMARY OF THE INVENTION

[0007] Summary In one aspect, the present invention provides a method for the treatment of one or more toxins in a subject, the method comprising administering to the subject a therapeutically effective amount of a protein comprising an HTRA-NS protein (SEQ ID NO: 3). In one aspect, the present invention provides a method for the treatment of one or more toxins in a subject, the method comprising administering to the subject a therapeutically effective amount of an HTRA protein (SEQ ID NO: 1) or an HTRA-NS protein (SEQ ID NO: 3). In one embodiment, the one or more toxins are derived from one or more pathogens. In one embodiment, the one or more pathogens are Clostridioides difficile. In one embodiment, the subject has been administered an antibiotic. In one embodiment of the method, the one or more toxins are selected from toxin Clostridioides difficile A (TcdA), toxin Clostridioides difficile B (TcdB), or toxin Clostridioides difficile binary (referred to herein as CDT). In one embodiment, the treatment method restores the gastrointestinal microbiota to a healthy gastrointestinal microbiota.

[0008] In one aspect, the present invention provides a method for preventing the progression of a disease in a subject infected with Clostridioides difficile, diarrhea, or infectious colitis, the method comprising administering to the subject a therapeutically effective amount of a protein comprising the HTRA-NS protein (SEQ ID NO: 3). In one embodiment, the protein is the HTRA protein (SEQ ID NO: 1) or the HTRA-NS protein (SEQ ID NO: 3). In one embodiment, one or more toxins are derived from one or more pathogens. In one embodiment, one or more pathogens are Clostridioides difficile. In one embodiment, the subject has been administered an antibiotic. In one embodiment, one or more toxins are selected from TcdA, TcdB, or CDT.

[0009] In one aspect, the present invention provides a method for the prevention of one or more toxins in a subject, the method comprising administering to the subject a therapeutically effective amount of a protein comprising the HTRA-NS protein (SEQ ID NO: 3). In one embodiment, the protein is the HTRA protein (SEQ ID NO: 1) or the HTRA-NS protein (SEQ ID NO: 3). In one embodiment, one or more toxins are derived from one or more pathogens. In one embodiment, one or more pathogens are Clostridioides difficile. In one embodiment, the subject has been administered an antibiotic. In one embodiment, one or more toxins are selected from TcdA and TcdB. In one embodiment of the aspects described herein, the subject is a mammal. In one embodiment, the mammal is a human. In one embodiment, the mammal is a pig, cow, horse, or dog.

[0010] In one aspect, the present invention provides a pharmaceutical preparation comprising a protein comprising a therapeutically effective amount of HTRA-NS protein (SEQ ID NO: 3) and a pharmaceutically acceptable excipient. In one aspect, the present invention provides a pharmaceutical preparation comprising a therapeutically effective amount of HTRA protein (SEQ ID NO: 1) or HTRA-NS protein (SEQ ID NO: 3). In one aspect, the present invention provides a pharmaceutical preparation comprising a therapeutically effective amount of HTRA protein (SEQ ID NO: 1) or HTRA-NS protein (SEQ ID NO: 3) and a pharmaceutically acceptable excipient. In one embodiment, the pharmaceutical preparation is for the treatment of one or more toxins in a subject. In one embodiment, the one or more toxins are derived from one or more pathogens. In one embodiment, the one or more pathogens are Clostridioides difficile. In one embodiment, the one or more toxins are selected from TcdA, TcdB, or CDT. In one embodiment, the formulation is in the form of a tablet, lyophilized formulation, solution, syrup, lozenge, suppository, enema, food additive, capsule, spray, gel, cream, lotion, ointment, or foam. In one aspect, the present invention provides a composition comprising a protein comprising HTRA-NS (SEQ ID NO: 3). In one aspect, the present invention provides a composition comprising HTRA protein (SEQ ID NO: 1) or HTRA-NS (SEQ ID NO: 3) and a pharmaceutically acceptable excipient. In one embodiment, the composition is for the treatment of one or more toxins. In one embodiment, the composition further comprises one or more additional therapeutic agents. In one embodiment, the one or more additional therapeutic agents are selected from the group consisting of antiviral agents, antibiotics, antibacterial agents, and antiproteases.

[0011] In one aspect, the present invention provides a protein comprising HTRA-NS (SEQ ID NO: 3). In one embodiment, the protein is a recombinant protein. In one aspect, the present invention provides a nucleic acid encoding the protein of SEQ ID NO: 4. In one aspect, the present invention provides a pharmaceutical composition comprising this nucleic acid. In one aspect, the present invention provides a composition comprising the protein of SEQ ID NO: 4. In one aspect, the present invention provides a method comprising administering this composition. In one embodiment of the method of the treatment aspect, the treatment is a prophylactic treatment.

[0012] In one aspect, the present invention provides a protein suitable for enzymatically cleaving TcdA, TcdB, or CDT. In one aspect, the present invention provides a method for preventing disease recurrence in a subject infected with Clostridioides difficile, diarrhea, or infectious colitis.

Brief Description of the Drawings

[0013] For a deeper understanding of the present invention and to more clearly show how the present invention can be implemented, embodiments will be described by way of example with reference to the accompanying drawings.

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Mode for Carrying Out the Invention

[0014] Detailed Description of the Embodiment The pathophysiology of CD disease is complex and is accompanied by depletion of the bacterial flora in the gastrointestinal (GI) tract, which promotes GI colonization by CD bacteria. Following this colonization, Clostridium difficile toxin A ("TcdA") and Clostridium difficile toxin B ("TcdB") are produced, both of which are required for the development of CD clinical disease. CD strains that do not synthesize toxin A and toxin B do not cause human disease. Hypervirulent strains of C. difficile that further produce binary toxin (CDT) (e.g., NAP1 / BI / 027) have emerged (see Stieglitz, F. et al., Front. Microbiol. 12:725612 (2021)).

[0015] Toxin A is an enterotoxin that induces inflammation, cytokine release, and fluid secretion, causing diarrhea. Toxin B is a cytotoxin that disrupts the cytoskeletal structure of the colon and epithelial cells, thereby catalyzing the glycosylation and inactivation of human Rho-GTPase. Both toxins act synergistically and can induce cell rounding and cytoskeletal rearrangement at picomolar concentrations in cell culture models. Rho-GTPase regulates the intracellular actin dynamics and glycosylation of these toxin molecules, leading to cytoskeletal changes and cell death.

[0016] Interventions to treat CDI by restoring the disrupted GI microbiota have been investigated and shown to be effective. The use of CD-free human feces, called fecal microbiota transplantation (FMT), has recently been added as a treatment option for recurrent CDI in clinical guidelines published by the Infectious Disease Society of America. However, the widespread use of FMT is hampered by the difficulty of finding suitable donors and the variability in the performance of feces from different donors. Donor-based differences have been associated with a lack of compatibility of bacterial strains found in the feces of donors and recipients (Li, S.S. et al. Science 352, 586-589 (2016)).

[0017] A defined microbial community (DMC) called MET-1 has been developed (see Martz, S. L. et al. J Gastroenterol 52, 452-465 (2017), Petrof, E. O., et al. Benef Microbes 4, 53-65 (2013), and Petrof, E. O. et al. Microbiome 1, 3 (2013)). MET-1 is a collection of 33 bacteria isolated from the feces of healthy donors. These 33 bacterial organisms were cultured in a controlled laboratory setting. MET-1 has been extensively studied in a mouse model of CDI and is clinically used in humans with >90% efficacy (see Petrof, E. O. et al. Microbiome 1, 3, (2013), Munoz, S. et al. Gut Microbes 7, 353-363, (2016), Grady, N. G., et al. Semin Fetal Neonatal Med 21, 418-423 (2016), Martz, S. L. et al. Sci Rep 5, 16094, (2015), and Carlucci, C. et al. Sci Rep 9, 885, (2019)).

[0018] According to one aspect of the present invention, there is provided a method for treating a subject having Clostridium difficile (Clostridioides difficile) infection (CDI) by administering to the subject one or more proteins that cause degradation of one or more toxins associated with CDI. According to embodiments, the one or more toxins are selected from TcdA, TcdB, and combinations thereof. In one embodiment, the one or more proteins are HTRA (SEQ ID NO: 1). In another embodiment, the one or more proteins include HTRA-NS (SEQ ID NO: 3). In another embodiment, the one or more proteins are HTRA-NS (SEQ ID NO: 3). According to some embodiments, treating the subject by administering the pharmaceutical composition described herein may prevent CDI recurrence in the subject. According to some embodiments, treating the subject by administering the pharmaceutical composition described herein may include prophylactic treatment. According to some embodiments, treating the subject by administering the pharmaceutical composition described herein may at least partially restore the normal gastrointestinal (GI) flora in the subject.

[0019] Another aspect of the present invention provides a composition comprising one or more proteins selected from HTRA (SEQ ID NO: 1), HTRA-NS (SEQ ID NO: 3), and combinations thereof. In some embodiments, the composition may be a pharmaceutical composition. The pharmaceutical composition may include one or more additional agents, diluents, carriers, excipients, etc., suitable for administration to a subject, and / or one or more additional therapeutic agents. The subject may be human. In some embodiments, the pharmaceutical composition may be useful for treating a subject having CDI. In some embodiments, the pharmaceutical composition may be useful for degrading one or more toxins associated with CDI. According to embodiments, the one or more toxins are selected from TcdA, TcdB, and combinations thereof. In some embodiments, the pharmaceutical composition may be useful in prophylactic treatment of a subject. In some embodiments, the pharmaceutical composition may be useful for at least partially restoring the normal gastrointestinal (GI) flora in the subject.

[0020] Parabacteroides distanosis is an anaerobic Gram-negative strain. It has a serine protease that has not been previously evaluated for its activity against CD or other toxins. HTRA was obtained from the bacterial species Parabacteroides distanosis, which can be found in the human intestine and is a member of both the MET-1 and DMC-4 bacterial communities. The gene sequence (SEQ ID NO: 2) encoding the HTRA protein (SEQ ID NO: 1) was extracted and introduced into Escherichia coli (E. coli). The HTRA protein was expressed at high levels in E. coli, and HTRA was isolated and purified. HTRA-NS (SEQ ID NO: 3) is similar to HTRA in several respects but is a different protein because the signal peptide has been removed from the gene sequence of HTRA-NS (SEQ ID NO: 4). Removal of the signal peptide allowed for better stability of the resulting protein HTRA-NS (SEQ ID NO: 3) compared to HTRA (SEQ ID NO: 1). A dose-response effect was demonstrated in which HTRA-NS (SEQ ID NO: 3) degrades both TcdA and TcdB in a concentration-dependent manner (see FIGS. 1A-1B). In response to toxin A, a 70% decrease in cell rounding was demonstrated in cultured fibroblasts, which are cells that are very sensitive to CD toxin (see details in the examples).

[0021] As described herein, experiments were conducted in which untreated feces were collected from mice infected with CD or treated with HTRA-NS after infection with CD. In mice infected with CD and treated with HTRA, CD toxin activity (TcdA, TcdB, and CDT) was neutralized compared to mice infected with CD but not treated with HTRA-NS.

[0022] Referring to FIGS. 1A - 1B, the bar graph shows that pre - incubation of TcdA and TcdB toxins with HTRA protein resulted in the degradation of both TcdA and TcdB toxins in an HTRA - NS concentration - dependent manner. The highest concentration (i.e., 30 μg) showed 80% degradation of TcdA and >95% degradation of TcdB when measured using Western blot assay. The Western blot bands showed a decrease in band intensity, indicating that the toxin was not detected when incubated with increasing concentrations of HTRA - NS (0.3 - 30 μg).

[0023] Referring to FIG. 2A, the bar graph shows that purified HTRA - NS protected fibroblasts from TcdA - mediated cytoskeletal rearrangement and cell rounding. As the concentration of HTRA increased, the protection increased, suggesting a dose - response mechanism of protection against TcdA. Cultured 3T3 mouse fibroblasts exposed to TcdA resulted in significant cytoskeletal rearrangement leading to cell rounding and death. In contrast, TcdA toxin pre - incubated with each concentration of HTRA - NS (1.0 - 100 μg) for 1 hour at 37°C and then added to cultured fibroblasts resulted in 60% less cell rounding (see the last bar). A statistically significant decrease in cell rounding (using the Mann Whitney statistical test) was observed when compared to cells exposed to TcdA alone in an HTRA - NS concentration - dependent manner.

[0024] Referring to FIG. 2B, a bar graph of the percentage of round cells (%) in feces from mice at specific days after CD infection is shown. The results showed that feces from CD + HTRA - NS mice contained less toxin pelletized than feces from CD control mice on days 1 and 2 after infection and were significantly less in mice on day 3 after infection (p = 0.0001).

[0025] Referring to FIG. 3A, a line graph showing the change in body weight (%) in a CD - infected mouse model is shown. Mice lost 20% of their body weight after CD infection, and forced oral administration of HTRA - NS (400 μg) to mice provided protection from significant CD - mediated weight loss.

[0026] Referring to FIG. 3B, a column dot plot demonstrating the effect of CD on the mouse colon including colon shortening is shown. Mice exposed to HTRA-NS did not experience a change in their colon length despite being infected with CD. Mice infected with CD and not treated with HTRA-NS showed significant colon shortening. Each experiment was performed 3 times using 15 mice in each experiment, and in subsequent detailed studies (see FIG. 3C) including negative control (2), CD positive control (n = 5), CD + HTRA-NS (n = 5), and negative control (n = 2), it was demonstrated that the colon length was significantly more colon shortening in CD control mice than in the other groups (i.e., HTRA-NS control, CD + HTRA-NS, and negative control; p = 0.0001 vs. negative control, p = 0.009 vs. HTRA-NS control, and p = 0.04 vs. CD + HTRA-NS). The total histological score of the distal colon mucosa revealed that CD + HTRA-NS mice had a significantly lower level of inflammation than CD control mice (p = 0.03), but significantly higher than negative control and HTRA control mice (p = 0.01).

[0027] Referring to FIG. 3D, the time points for feces from CD-infected mice were 48 hours and 72 hours of incubation. Feces from mice treated with HTRA-NS did not cause fibroblast cell rounding and protected cells from cytoskeletal rearrangement and cell death as observed when feces from CD-uninfected mice were incubated on fibroblasts for 48 hours and 72 hours.

[0028] Referring to FIG. 4A, the line graph shows the effect of forced administration of different concentrations of HTRA-NS (200, 400, and 800 μg) on the body weight of mice after antibiotic treatment. Mice that received forced administration of HTRA-NS did not experience significant weight loss compared to control mice that did not receive HTRA-NS (Tris buffer control mice) (lines of 200, 400, 800 μg), demonstrating that HTRA therapy does not result in significant weight loss, a prominent feature of colitis in this mouse model.

[0029] Referring to FIG. 4B, the vertical bar graph shows that, unlike the case of CD infection (FIG. 3B), exposure to HTRA-NS at different concentrations (200, 400, and 800 μg) did not result in significant colon shortening in mice.

[0030] Referring to FIGS. 5A - 5E, the graph shows that the results of 16S rRNA sequencing of mouse feces exposed to different concentrations of HTRA-NS (200, 400, and 800 μg) did not result in significant changes in specific dominant gut bacterial populations compared to mice given only saline (control).

[0031] Referring to FIG. 6, the line graph shows the effect of temperature on the relative activity of HTRA. Importantly, this temperature stability data showed that HTRA-NS retained its activity up to 65°C.

[0032] Referring to FIG. 7, a Kaplan - Meyer survival curve showing the probability of survival when treated with HTRA-NS is shown. The probability was found to be 60%, whereas untreated mice had a 6.7% chance of surviving CDI in this model (p = 0.003 ** ). The survival study was performed three times in three independent experiments. Notably, it showed a 10 - fold survival benefit in mice (n = 15 for each treatment group).

[0033] Referring to the examples, in a mouse model of C. difficile, the HTRA-1-NS protein has been evaluated to quantify its protective ability. The protein in this mouse model was obtained using recombinant HTRA-producing live bacteria to produce HTRA (SEQ ID NO: 1). Then, HTRA was concentrated and quality controlled before use. As described herein, HTRA has been found to be protective against CD disease (see FIGS. 3A - 3D).

[0034] As described herein, an amount of the purified protein described herein in the amount of 100 - 800 μg provided effective treatment against CD and other toxin-producing bacteria. In one embodiment, the concentration was 200 - 800 μg of the purified protein described herein. This protein therapy inactivated the bacterial toxin. Without wishing to be bound by theory, the inventors suggest that the mechanism of action of this protein therapy is different from that used by antibiotics that target the bacteria themselves and are prone to resistance. Due to this difference in mechanism, this protein therapy is not adversely affected by the development of resistance.

[0035] The present invention also provides a combination therapy in which two or more therapeutic compounds, such as an HTRA protein (SEQ ID NO: 1) or an HTRA-NS protein (SEQ ID NO: 3), are administered with one or more additional therapeutic agents selected from antiviral agents, antibiotics, antibacterial agents, and / or anti-proteases. Each of the therapeutic compounds can be administered by the same route or different routes. Also, the compounds can be administered at the same time (i.e., simultaneously) or at different times. In some treatment regimens, it may be beneficial to administer one of the compounds at a higher or lower frequency than the other.

[0036] Dispersions containing therapeutic compounds can be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof, as well as in oils. Under normal conditions of storage and use, these formulations may contain preservatives to prevent the growth of microorganisms. Pharmaceutical compositions suitable for injection include sterile aqueous solutions (where water-soluble), or dispersions, and sterile powders for the immediate preparation of sterile injectable solutions or dispersions. In all cases, the composition must be sterile and must be fluid to the extent that it can be easily administered by syringe. It must be stable under the conditions of manufacture and storage and must be protected against the contaminating action of microorganisms such as bacteria and fungi. The vehicle can be, for example, a solvent or dispersion medium containing water, ethanol, polyols (such as glycerol, propylene glycol, liquid polyethylene glycols, etc.), suitable mixtures thereof, and oils (such as vegetable oils). Suitable fluidity can be maintained, for example, by the use of coatings such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

[0037] Sterile injectable solutions can be prepared by incorporating the required amount of the therapeutic compound in a suitable solvent with one or a combination of the ingredients listed above, as required, followed by filtration sterilization. Generally, dispersions are prepared by incorporating the therapeutic compound into a sterile vehicle containing a basic dispersion medium and the necessary other ingredients from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and lyophilization, which yield a powder of the active ingredient (i.e., the therapeutic compound) together with any additional desired ingredients, if any, from its previously sterile filtered solution.

[0038] Solid dosage forms for oral administration include ingestible capsules, tablets, pills, lollipops, powders, granules, elixirs, suspensions, syrups, lozenges, cachets, oral tablets, sublingual tablets, troches, and the like. Other routes of administration include enemas or suppositories. In such solid dosage forms, the active compound is combined with at least one inert pharmaceutically acceptable excipient or diluent or assimilable edible vehicle, such as sodium citrate or dicalcium phosphate, and / or a) fillers or bulking agents, such as starch, lactose, sucrose, glucose, mannitol, and silicic acid, b) binders, such as carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose, and acacia, c) humectants, such as glycerol, d) disintegrants, such as agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate, e) dissolution retardants, such as paraffin, f) absorption promoters, such as quaternary ammonium compounds, g) wetting agents, such as cetyl alcohol and glycerol monostearate, h) absorbents, such as kaolin and bentonite clay, and i) lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate, and mixtures thereof, or is incorporated directly into the diet of the subject. In the case of capsules, tablets, and pills, the dosage form may contain buffering agents. Solid compositions of the same type can also be used as fillers in soft and hard gelatin capsules using excipients such as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like. The proportion of the therapeutic compound in the composition and formulation can, of course, vary. The amount of the therapeutic compound in such therapeutically useful compositions is an amount such that an appropriate dosage is obtained.

[0039] For oral administration, liquid dosage forms include pharmaceutically acceptable emulsions, solutions, suspensions, lozenges, syrups, and elixirs. In addition to the active compound, the liquid dosage forms may contain inert diluents commonly used in the art, such as water or other solvents, solubilizing agents and emulsifying agents, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (especially cottonseed oil, groundnut oil, germ olive oil, castor oil and sesame oil), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycol and fatty acid esters of sorbitan, and mixtures thereof. In addition to the inert diluent, the oral compositions may also contain adjuvants such as wetting agents, emulsifying agents and suspending agents, sweetening agents, flavoring agents, and perfumes.

[0040] Suspensions may contain, in addition to the active compound, suspending agents such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar, and tragacanth, and mixtures thereof.

[0041] The therapeutic compounds can be administered in a time-release or depot form in order to obtain a sustained release of the therapeutic compounds over time. The therapeutic compounds of the present invention can also be administered transdermally (e.g., by providing the therapeutic compound in the form of a patch together with a suitable vehicle).

[0042] The following disclosure should not be construed as limiting the invention in any way. Those skilled in the art will understand that numerous modifications, combinations, rearrangements, etc. are possible without exceeding the scope of the invention.

[0043] For example, in the description of various embodiments, references are made to sequences and sequence listings. Those skilled in the art will readily understand that the present invention is not limited to the specific sequences described, as many modifications are possible without departing from the invention. For example, one or more nucleotide or amino acid substitutions, mutations, deletions, and / or additions can be made or can occur without substantially affecting the functional characteristics of the embodiments described herein. Such functional equivalents can have, for example, 60%, or 70%, or 80%, or 90%, or 95%, or 99%, or more sequence identity with the sequences described herein. Such functional equivalents are intended to be included in the embodiments of the present invention.

[0044] Accordingly, while the present invention has been described with emphasis on various embodiments, it will be understood by those skilled in the art that variant forms of the disclosed embodiments can be used and that the invention can be practiced in ways other than those specifically described and / or claimed herein. The present invention is further illustrated by the following non-limiting examples.

Example

[0045] Example 1. In Vivo CD Mouse Model Female C57Bl6 / J mice from Jackson Laboratory (USA) were acclimated to the facility for 5 days prior to experimental use. The mice were freely administered a cocktail of antibiotics containing kanamycin 0.4 mg / mL (Sigma, Israel), gentamicin 0.035 mg / mL (Amresco, USA), colistin 850 U / mL (Sigma, China), metronidazole 0.215 mg / mL (Sigma, China), and vancomycin 0.045 mg / mL (Sigma, Israel) in the drinking water for 3 days. The mice were then infected with C. difficile (1×10 5 CFU / mL vegetative cells) by forced oral administration.

[0046] For phenotypic changes associated with CDI, including daily measurements of weight loss, activity levels monitored for 20 minutes per day, posture as a disease parameter, hair, fecal hardness, and eye appearance (all of which contribute to the total clinical score and CDI severity), mice were visually examined daily. Mice that experienced >10% weight loss post-infection (p.i.) or exhibited clinical signs consistent with CDI were immediately euthanized. See Figure 7 for a plot of survival rate versus days post-infection. See Figure 3D for in vivo data on the activity of HTRA-NS from mouse feces obtained from mice infected with CD and treated with HTRA-NS.

[0047] Histology: Mouse intestinal tissues, colon, and cecum were first measured to assess colon shortening and then fixed in 10% formalin followed by 70% ethanol. Fixed tissues were processed, embedded in paraffin, and 4-μm thick sections were stained with hematoxylin and eosin (H&E, Thermo Fisher Scientific, USA). Stained sections were blindly examined by a board-certified gastrointestinal pathologist using an established stepwise scoring system. The scoring system took into account (1) neutrophil migration and tissue infiltration, (2) hemorrhagic congestion, and (3) mucosal edema and epithelial cell damage. A score of 0 - 3 was assigned to each parameter, and the overall score was the sum of all scores. A score of 0 indicated no pathological damage, 1 was mild, 2 was moderate, and 3 was severe. The sum of the scores represented the total damage in the tissue. Representative images of the tissue were recorded using a microscope (Olympus BX71, USA) equipped with a digital camera (INFINITY2 or Qimagining Retiga-2000RV, USA).

[0048] Example 2. Toxin Quantification by Western Blot and ELISA Fecal pellets were collected daily from each mouse and stored according to the laboratory protocol. TcdA and TcdB levels were quantified in mouse feces collected daily and in feces collected from the colon of mice after euthanasia. The tgcBIOMICS ELISA kit (Bingen, Germany) was used to quantify feces according to the kit manufacturer's instructions. Briefly, 50 mg of feces from mice was resuspended in an Eppendorf tube containing 450 μL of kit dilution buffer and centrifuged at 2500×g for 20 minutes. 100 μL of the supernatant was added to wells pre-coated with TcdA / TcdB polyclonal antibody and incubated at room temperature for 1 hour. The plate was then washed with wash buffer (see manufacturer's instructions) (3 times) and incubated for 30 minutes with a toxin-specific secondary antibody (either anti-TcdA-HRP or TcdB-HRP conjugate). After washing with wash buffer (×3), 100 μL of substrate was added to each well and incubated at room temperature for 15 minutes. 50 μL of stop solution (see manufacturer's instructions) was added to each well, and the absorbance at 450 nm and 620 nm was measured using a microplate reader (Bio-Tek μQuant MQX200). Standard curves were generated using the TcdA and TcdB pure toxins provided in the kit to calculate sample concentrations. All test samples were tested in triplicate.

[0049] The concentrated protein was incubated with TcdA and TcdB, and toxin degradation was measured using Western blot. Protein HTRA-NS (SEQ ID NO: 3) proteolyzed both TcdA and TcdB in a dose-dependent manner. 30 μg of HTRA-NS proteolyzed 50 ng of TcdA and 50 ng of TcdB (see Figures 1A and 1B).

[0050] Example 3. Toxin activity using the cell rounding assay In the in vitro study of toxin activity using the cell rounding assay, NIH 3T3 fibroblasts (ATCC) were grown and seeded into 24-well flat-bottom tissue culture plates containing DMEM medium (GIBCO, Thermo Fischer Scientific, USA) supplemented with 10% fetal bovine serum (GIBCO, Thermo Fischer Scientific, USA) at 37 °C and 5% CO2, and incubated for 48 - 72 hours. First, at 48 hours, the fibroblasts were examined to evaluate the cell confluence, and a cell monolayer with 70% confluence was a prerequisite before being used in the cell rounding assay.

[0051] In the in vivo study of toxin activity using the cell rounding assay, the effect of CD toxin in feces was evaluated. Specifically, 50 mg of mouse feces was homogenized in 500 μl of phosphate buffered saline (pH 7.2). After centrifugation at 16,000 × g for 30 minutes, the supernatant was collected. Fibroblasts were exposed to fresh supernatants from the control group and the treatment group and incubated at 37 °C with 5% CO2 for 2 hours. After incubation, all wells were washed with phosphate buffered saline (PBS), and the cells were fixed with 10% phosphate buffered formalin (Fisher Scientific, Belgium). After incubation at room temperature for 30 minutes, the cells were rinsed twice with PBS and stained with Giemsa (Sigma Aldrich, USA). After incubating the staining solution overnight, it was washed away with PBS. Cells were imaged using a microscope (Olympus BX71) at 10× magnification with a digital camera (Qimiging, Retinga-2000RV, FAST1394). Cells were counted using Image J 1.51a software (NIH, USA). Refer to Figure 2A for the results of the in vitro neutralization ability of HTRA against TcdA.

[0052] Example 4. Protein Expression Protein expression of the gene cloned into E. coli was induced using isopropyl -β-D-1-thiogalactopyranoside (IPTG) (Hansen, L.H., et al. Curr Microbiol 36, 341 - 347, (1998)). The cell pellet was recovered, sonicated, and the clear supernatant was loaded onto a Ni-NTA (Qiagen) column. Each fraction was collected and the absorbance was measured at 280 nm. The peak fraction containing the protein was confirmed by SDS-PAGE, casein zymography gel, and Western blot analysis using an anti-6×histidine-tag antibody. The fraction containing the protein of interest was concentrated by using a 10 kD cut-off centrifugal device (Millipore, Sigma), and the buffer was exchanged to 50 mM Tris, 50 mM NaCl (pH 7.5). Aliquots of each individual protein were prepared, snap-frozen in liquid nitrogen, and stored at -80 °C.

[0053] Example 5. Evaluation of the in vivo toxicity of purified HTRA-NS Toxicity was evaluated in C57 / BL6 / J mice exposed to antibiotics and unexposed C57 / BL6 / J mice. Briefly, mice were either not given antibiotics or were freely given in drinking water for 3 days a cocktail of antibiotics containing kanamycin 0.4 mg / mL (Sigma, Israel), gentamicin 0.035 mg / mL (Amresco, USA), colistin 850 U / mL (Sigma, China), metronidazole 0.215 mg / mL (Sigma, China), and vancomycin 0.045 mg / mL (Sigma, Israel) (Chen, X., et al. Gastroenterology 135, 1984 - 1992, (2008)). Mice were force-fed either 0.9% saline, 200 μg, 400 μg, or 800 μg of purified HTRA once a day for 3 consecutive days (3 mice / group, 6 groups). Mice were monitored for clinical signs of discomfort / toxicity and sacrificed on the 3rd day after HTRA / saline force-feeding. Changes in body weight (Figure 4A) and histological changes including colon shortening in mice (Figure 4B) were measured to evaluate the effect of HTRA-NS. Changes in the fecal microbiota were measured over time by 16S rRNA sequencing of mouse fecal bacteria (Figure 4C).

[0054] Example 6. In vivo study of HTRA-NS The effectiveness of the protection provided by HTRA-NS was evaluated in vivo. The previously used antibiotic CDI mouse model was used to determine the effectiveness of HTRA protection in vivo (Chen, X. et al. Gastroenterology 135, 1984 - 1992, (2008)). After antibiotics, mice were force-fed orally 400 μg of purified HTRA-NS protein (4 mice / group) once a day for 3 consecutive days. The mice were rested for 24 hours and then challenged with C. difficile (1×10 5(Nutritional cells at CFU / mL) were infected. Mice that received forced oral administration of HTRA but were not accompanied by CD infection (3 mice / group; negative control), physiological saline (3 mice / group; vehicle control), and CD infection by ribotype-027 (3 mice / group; positive control) were included as controls for this experiment to show that HTRA-NS did not cause weight loss or death in mice. The positive control was euthanized 48 hours after infection because these mice progressed rapidly to clinical disease. HTRA-pretreated animals exposed to CD were monitored for clinical disease until 72 hours after CD exposure, provided that the mice did not experience significant weight loss (e.g., >15% or >25% of body weight). This experiment was repeated independently three times. At 72 hours, all mice were euthanized, intestinal tissues were processed, and scored for histological examination. Fecal pellets were assayed for TcdA and TcdB toxin concentrations, and toxin activity was evaluated using a cell rounding assay.

[0055] Example 7. Optimization of Dosage, Administration Frequency, and Administration Timing of HTRA-NS (SEQ ID NO: 3) In Vivo The safety of using HTRA was established through a dosing study in which various concentrations of HTRA were tested in mice exposed to antibiotics. Four different doses were evaluated. As previously described (Chen, X. et al. Gastroenterology 135, 1984 - 1992, (2008)), mice were given a cocktail of antibiotics in drinking water. Mice were force-fed either 0.9% physiological saline, Dose 1 (200 μg), Dose 2 (400 μg), or Dose 3 (800 μg) of purified HTRA-NS once a day for 3 consecutive days (3 mice / group, 6 groups). Then, the mice were allowed a 24-hour rest. Controls for this experiment included mice that received forced oral administration with Tris buffer (the buffer in which HTRA-NS was solubilized) (negative control). All mice were monitored for clinical disease and euthanized 72 hours later (see Figures 4A - C). Analyses of tissues, systemic inflammation, and the GI microbiota were performed as outlined in Example 5.

[0056] Example 8. Gel electrophoresis study The HTRA-NS protein purified from Parabacteroides distasonis degraded TcdA in vitro. Western blot assay of TcdA after incubating 0.3, 1, 3, 10, 30, 100 μg of the HTRA-NS protein with purified TcdA toxin (50 ng) at 37°C for 60 minutes. Degradation of TcdA was not observed at concentrations of HTRA-NS from 0.3 to 30 μg, but significant degradation of TcdA was observed at 100 μg. TcdA incubated at 37°C for 60 minutes as a control did not result in toxin degradation (Figure 1A).

[0057] The HTRA-NS protein purified from Parabacteroides distasonis degraded TcdB in vitro. Western blot assay of TcdA after incubating 0.3, 1, 3, 10, 30, 100 μg of the HTRA protein with purified TcdB toxin (50 ng) at 37°C for 60 minutes. Degradation of TcdB was not observed at concentrations of HTRA-NS from 0.3 to 1 μg. HTRA-NS at a concentration of 3 μg incubated with the TcdB protein resulted in 50% degradation, and higher concentrations (10, 30, 100 μg) of HTRA-NS resulted in a 95% decrease in detectable TcdB protein by Western blot. TcdB incubated at 37°C for 60 minutes as a control did not result in TcdB degradation (see Figure 1B).

[0058] Example 8. Temperature stability study To evaluate the effect of temperature on the protease activity of HTRA-NS, five independently generated formulations of HTRA-NS stored at -80 °C were thawed and diluted to a concentration of 1.2 μg / μL in reaction buffer (50 mM Tris, pH 7.5, 150 mM NaCl). The HTRA-NS formulations were incubated at -80, 4, 22, 37, 56, 65, 72, 80, 90, and 100 °C for 15 minutes. Next, 60 μL of the HTRA-NS reaction buffer formulation was added to a 96-well black half-area plate (CoStar), and each formulation was tested individually in triplicate wells. Then, the activity of HTRA-NS was evaluated using a FITC-casein fluorescence protease assay kit (ThermoFisher Scientific). FITC-casein was diluted to a concentration of 10 μg / mL in the reaction buffer, and 60 μL of this working solution was added to each well of the plate. The plate was then covered with tin foil and left on a shaker for 30 minutes before being read using a Spectra Max M3 plate reader (Molecular Devices) (485 / 538 nm, excitation / emission). Background fluorescence (control wells) was subtracted from all samples, and relative fluorescence units (RFU) were obtained by normalizing the fluorescence at room temperature. See Figure 6 for a graph of the relative activity of HTRA-NS versus temperature.

[0059] Equivalents One of ordinary skill in the art will understand that this description is made with reference to specific embodiments and that it is possible to make other embodiments that fall within the spirit and scope of the invention using the principles of the invention.

[0060] Sequence Listing SEQ ID NO:1 HTRA Protein (507 amino acids) TIFF2025521857000002.tif235147TIFF2025521857000003.tif217147TIFF2025521857000004.tif80147

[0061] SEQ ID NO:2 HTRA Gene Sequence (1521 nucleotides) TIFF2025521857000005.tif218163

[0062] Array No. 3 HTRA-NS protein (484 amino acids) TIFF2025521857000006.tif114147TIFF2025521857000007.tif235147TIFF2025521857000008.tif158147

[0063] Array No. 4 HTRA-NS gene sequence (1452 nucleotides) TIFF2025521857000009.tif210163

Claims

1. A pharmaceutical composition for treating one or more toxins in a subject, comprising a protein including the HTRA-NS protein (SEQ ID NO: 3).

2. The pharmaceutical composition according to claim 1, wherein the one or more toxins are derived from one or more pathogens.

3. The pharmaceutical composition according to claim 2, wherein one or more of the pathogens are Clostridioides difficulte.

4. The pharmaceutical composition according to any one of claims 1 to 3, wherein the subject is being administered an antibiotic.

5. The pharmaceutical composition according to any one of claims 1 to 3, wherein the one or more toxins are selected from the toxin Clostridioides difficile A (TcdA), the toxin Clostridioides difficile B (TcdB), or the toxin Clostridioides difficile binary (CDT).

6. A pharmaceutical composition for preventing disease progression in subjects infected with Clostridioides difficile, diarrhea, or infectious colitis, the pharmaceutical composition comprising a protein containing the HTRA-NS protein (SEQ ID NO: 3).

7. The pharmaceutical composition according to claim 6, wherein the one or more toxins are derived from one or more pathogens.

8. The pharmaceutical composition according to claim 7, wherein one or more of the pathogens are Clostridioides difficile.

9. The pharmaceutical composition according to any one of claims 6 to 8, wherein the subject is being administered an antibiotic.

10. The pharmaceutical composition according to any one of claims 6 to 8, wherein the one or more toxins are selected from TcdA, TcdB, or CDT.

11. A pharmaceutical composition for the prevention of one or more toxins in a target, comprising a protein containing the HTRA-NS protein (SEQ ID NO: 3).

12. The pharmaceutical composition according to claim 11, wherein the one or more toxins are derived from one or more pathogens.

13. The pharmaceutical composition according to claim 12, wherein one or more of the pathogens are Clostridioides difficile.

14. The pharmaceutical composition according to any one of claims 11 to 13, wherein the subject is being administered an antibiotic.

15. The pharmaceutical composition according to any one of claims 11 to 13, wherein one or more toxins are selected from TcdA and TcdB.

16. The pharmaceutical composition according to any one of claims 1 to 3, 6 to 8, and 11 to 13, wherein the subject is a mammal.

17. The pharmaceutical composition according to claim 16, wherein the mammal is a human.

18. The pharmaceutical composition according to claim 16, wherein the mammal is a pig, a cow, a horse, or a dog.

19. A pharmaceutical preparation comprising a therapeutically effective amount of a protein containing the HTRA-NS protein (SEQ ID NO: 3) and a pharmaceutically acceptable excipient.

20. A pharmaceutical formulation according to claim 19 for the treatment of one or more toxins in a subject.

21. The pharmaceutical preparation according to claim 20, wherein the one or more toxins are derived from one or more pathogens.

22. The pharmaceutical preparation according to claim 21, wherein one or more of the pathogens are Clostridioides difficile.

23. The pharmaceutical formulation according to any one of claims 21 to 22, wherein the one or more toxins are selected from TcdA, TcdB, or CDT.

24. The pharmaceutical preparation according to any one of claims 19 to 22, wherein the preparation is in the form of a tablet, a lyophilized preparation, a solution, a syrup, a lozenge, a suppository, an enema, a food additive, a capsule, a spray, a gel, a cream, a lotion, an ointment, or a foam.

25. A composition comprising a protein containing HTRA-NS (SEQ ID NO: 3) and a pharmaceutically acceptable excipient.

26. The composition according to claim 25 for the treatment of one or more toxins.

27. The composition according to claim 25 or 26, further comprising one or more additional therapeutic agents.

28. The composition according to claim 27, wherein the one or more additional therapeutic agents are selected from the group consisting of antiviral agents, antibiotics, antibacterial agents, and antiproteases.

29. A protein containing HTRA-NS (SEQ ID NO: 3).

30. The protein according to claim 29, which is a recombinant protein.

31. A nucleic acid (SEQ ID NO: 4) encoding the protein described in claim 29.

32. A pharmaceutical composition comprising the nucleic acid described in claim 31.

33. A composition comprising the protein described in claim 29 or 30.

34. The pharmaceutical composition according to claim 1, wherein the treatment is a preventive treatment.

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