WO2021155212A1

WO2021155212A1 - Novel antagonists for treatment of gi tract conditions

Info

Publication number: WO2021155212A1
Application number: PCT/US2021/015795
Authority: WO
Inventors: Malcolm Hill; Ingrid Swanson Pultz; Adam Simpson
Original assignee: Aim Biologics, Inc.
Priority date: 2020-01-30
Filing date: 2021-01-29
Publication date: 2021-08-05

Abstract

The present invention relates to novel antagonist compositions and methods for their use. The novel antagonists include novel endopeptidases and novel binding proteins that reduce the activity of gastrointestinal (GI) tract targets.

Description

NOVEL ANTAGONISTS FOR TREATMENT OF GI TRACT CONDITIONS

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U. S. Provisional Application No. 62/968,034, filed January 30, 2020, and U.S. Provisional Application No. 62/968,049, filed January 30, 2020, each of which is incorporated by reference herein in its entirety.

BACKGROUND OF THE INVENTION

[0002] During gastrointestinal tract infection or dysbiosis, microbes including exogenous pathogens, indigenous pathobionts, and commensals, produce proteins that have wide-ranging effects on the host. The result can be GI discomfort, severe GI disease, morbidities relating to other organs, and death. Broadly targeted strategies for balancing microbiome composition include the administration of probiotics and fecal transplantation. Anti-microbial therapies (e.g., antibiotics), have been used to reduce or eliminate groups of pathogens. Therapeutic strategies that specifically target unwanted GI tract organisms are needed.

[0003] Clostridium difficile is a Gram-positive, spore-forming bacterium that can colonize the gastrointestinal tract, often leading to severe colitis and death. It is spread by bacterial spores present in feces and is the most common healthcare-associated infection in U.S. hospitals. Infection is typically treated by orally-administered antibiotics, with frequent disease recurrence. Furthermore, the development of drug-resistant strains and the recent emergence of hypervirulent strains have heightened the need for superior treatment strategies for infection by C. diff. and other GI tract organisms.

SUMMARY OF THE INVENTION

[0004] The present invention provides novel antagonists that reduce the activity of gastrointestinal (GI) tract targets. These novel antagonists are non-naturally occurring proteins designed to specifically inhibit a GI tract target. The novel antagonist can be a novel binding protein or novel endopeptidase that disrupts binding of a GI tract target to its GI tract target binding partner, either through binding to a target binding site or cleavage of a target cleavage site present on the GI tract target.

[0005] GI tract targets include not only binding and virulence factors produced by organisms known to be pathogenic, but those produced by an organisms that contribute to disease while constitutively inhabiting the GI tract. In embodiments, the invention includes novel antagonist compositions and methods for the prevention or treatment of disease in a subject, by reducing an activity of a GI tract target produced by one or more commensal or constitutive GI tract organisms.

[0006] In certain embodiments, the present invention includes a novel antagonist that disrupts one or both of the major Clostridium difficile (C. diff.) toxin proteins, TcdA and TcdB, at a selected target site, e.g., a target novel endopeptidase cleavage site or a target novel binding protein binding site, with high specificity, high activity, high efficiency, high stability, or a combination thereof. This disruption results in a reduction in the cytopathicity of the C. diff bacterium, e.g., by reducing TcdA and/or TcdB cell receptor binding, membrane translocation, endosome escape/autoprocessing, and/or glucosyltransferase activity. The invention further provides methods of using the novel antagonists, including use in the treatment of C. diff infection.

[0007] The present invention includes a novel antagonist that reduces an activity of a gastrointestinal (GI) tract target. In embodiments, the novel antagonist is a novel binding protein comprising at least one binding epitope that specifically binds to a target binding site of a GI tract target that is involved in specific binding of the GI tract target to a GI tract target binding partner, wherein binding of the novel binding protein to the target binding site results in reduction of an activity of the GI tract target. In embodiments, the novel antagonist is a novel binding protein comprising a non-antibody binding protein. In embodiments, the novel antagonist is not derived from an antibody. In embodiments, the novel antagonist is a novel binding protein comprising an antibody fragment. In embodiments, the novel antagonist is a novel binding protein, wherein the target binding site on the GI tract target is located at a binding interface of the GI tract target and the GI tract target binding partner. In embodiments, the novel antagonist is a novel binding protein, wherein the target binding site of the GI tract target is not located at a binding interface of the GI tract target and the GI tract target binding partner. In embodiments, the novel antagonist is a novel binding protein, wherein when bound to the target binding site of the GI tract target, the novel binding protein blocks binding of the GI tract target to the GI tract target binding partner. In embodiments, the novel antagonist is a novel binding protein, wherein when bound to the target binding site of the GI tract target, the novel binding protein prevents binding of the GI tract target to the GI tract target binding partner by inducing conformational change of the GI tract target. In embodiments, the novel antagonist is a novel endopeptidase that cleaves a target cleavage site of the GI tract target, wherein the cleavage results in a reduced activity of the GI tract target. In embodiments, the activity of the GI tract target is selected from: receptor binding, enzyme activity, membrane translocation, cytopathicity, and any combination thereof. In embodiments, the GI tract target is from a microbe, wherein the microbe is a commensal microbe, an exogenous pathogen, or an indigenous pathobiont. In embodiments, the microbe is a bacterium, virus, fungus, archaeon, or protozoan. In embodiments, the microbe is selected from: Aeromonas, Aspergillus , Bacteroides, Bilophila , Campylobacter , Clostridioides , Coccidiosis, Crytosporidia, Enterobacter , Enterococcus , Escherichia , Firmicutes , Helicobacter , Lactobacillus , Listeria , Peptostreptococcus , Pleisiomonas , Prevote llaceae, Pseudomonas , Salmonella , Sarcina, SFB, Shigella , Staphylococcus , Streptococcus , Veillonella , Vibrio , Yersinia , a secretory antibody- coated bacterium, Candida , Mycobacterium , Mycoplasma, Rotavirus, Calicivirus, Norwalk-like viruses, adenoviruses, astroviruses, sapporo-like viruses, toroviruses, coronaviruses, picomaviruses, herpes viruses, noroviruses, Proteus, Entamoeba , Giardia , and Strongy loides, single-celled parasites, multi-celled parasites, and amoebae. In embodiments, the microbe is drug-resistant or hypervirulent. In embodiments, the microbe is selected from: Bacteroides fragilis, Bilophila wadsworthia, Campylobacter jejuni, Campylobacter coli, Clostridioides difficile, Clostridioides sordelli, Enterobacter cloacae, Enterococcus faecalis, Enterococcus faecium, Escherichia coli, enterotoxigenic Escherichia coli, Helicobacter pylori, Helicobacter sp. Flexispira, Listeria monocytogenes, Pleisiomonas shigelloides, UC Prevotellaceae, Pseudomonas aeruginosa, Salmonella enterica, Salmonella bongori, Salmonella enterica subsp. enterica serotype Typhimurium, Shigella dysenteriae, Staphylococcus aureus, Streptococcus anginosus, Streptococcus thermophilus, Vibrio cholera, Vibrio parahaemolyticus, Yersinia enterocolitica, Candida albicans, Entamoeba histolytica, Giardia lamblia, and Strongyloides stercoralis. In embodiments, the GI tract target is selected from: a microbial toxin protein, a microbial virulence factor, and a microbial adhesin protein. In embodiments, the microbe is Clostridioides difficile, wherein the GI tract target is a microbial toxin protein, wherein the microbial toxin protein is TcdA or TcdB. In embodiments, the activity of the GI tract target is selected from: receptor binding, glucosyl transferase activity, membrane translocation, cytopathicity, and any combination thereof. In embodiments, the novel antagonist is a novel binding protein, wherein the specific epitope binding to the TcdA or TcdB protein reduces the activity of the respective TcdA or TcdB. In embodiments, the novel antagonist is a novel endopeptidase, and cleavage of the target cleavage site of the TcdA or TcdB protein reduces the activity of the respective TcdA or TcdB. In embodiments, the novel antagonist is a novel binding protein that specifically binds to the C-terminal combined repetitive oligopeptide (CROP) region in the receptor binding domain (RBD) of the TcdA or TcdB. In embodiments, the novel antagonist is a novel endopeptidase, wherein the cleavage of the target cleavage site occurs in the C-terminal combined repetitive oligopeptide (CROP) region in the receptor binding domain (RBD) of the respective TcdA or TcdB. In embodiments, the novel antagonist is a novel binding protein wherein the binding epitope binds to the GI tract target with high specificity, high affinity, or both. In embodiments, the novel antagonist is a novel endopeptidase that cleaves the GI tract target with high specificity, high activity, or both. In embodiments, the novel antagonist reduces the activity of the GI tract target by about 80% to about 100%, in comparison with a control. In embodiments, the novel antagonist is non-natural, or not native to a known biological species. In embodiments, the novel antagonist is a novel binding protein, wherein the at least one binding epitope comprises a binding domain of a receptor specific for the GI tract target.

[0008] The invention also relates to a method for reducing an activity of a GI tract target, comprising: contacting the GI tract target with a novel antagonist as described, under conditions that allow interaction between the novel antagonist and the GI tract target; wherein the interaction between the novel antagonist and the GI tract target results in reduction of an activity of the GI tract target. In embodiments, the activity of the GI tract target is selected from: receptor binding, enzyme activity, membrane translocation, cytopathicity, and any combination thereof. In embodiments, the reduction in the activity of the GI tract target contacted with the novel antagonist is about 80% to about 100%, in comparison with a control. In embodiments, the novel antagonist is a novel binding protein comprising at least one binding epitope that specifically binds to a GI tract target at a binding site on the GI tract target that is involved in specific binding of the GI tract target to a GI tract target binding partner, wherein binding of the novel binding protein to the target binding site results in reduction of an activity of the GI tract target. In embodiments, the novel antagonist is a novel binding protein, wherein the GI tract target is Clostridioides difficile TcdA or TcdB, wherein the specific epitope binding to the TcdA or TcdB protein reduces an activity of the respective TcdA or TcdB. In embodiments, the novel antagonist is a novel endopeptidase capable of a cleaving a target cleavage site of the GI tract target, wherein the cleavage results in a reduced activity of the GI tract target. In embodiments, the novel antagonist is a novel endopeptidase, wherein the GI tract target is Clostridioides difficile TcdA or TcdB, wherein the cleavage reduces an activity of the respective TcdA or TcdB.

[0009] In embodiments, the invention includes a composition comprising the novel antagonist as described. In embodiments, the composition is formulated for oral or rectal administration.

In embodiments, the composition is formulated for delivery to a site in the GI tract. In embodiments, the site in the GI tract is located in the esophagus, the stomach, the small intestine, the large intestine, and the rectum, or any combination thereof. In embodiments, the site in the GI tract is located in the duodenum, the jejunum, the ileum, the caecum, the ascending colon, the transverse colon, the descending colon, the sigmoid colon, the rectum, or any combination thereof. In embodiments, the composition is formulated for oral administration and comprises an enteric coating. In embodiments, the composition comprises at least one other active agent, is administered in combination with at least one other active agent, or both. In embodiments, the at least one other active agent is a prebiotic or probiotic agent.

[0010] The invention includes a method for treating a condition in a subject in need thereof, comprising administering to the subject a novel antagonist composition as described. In embodiments, the subject has a condition resulting from production of a GI tract target. In embodiments, the GI tract target is from a microbe that is a bacterium, virus, fungus, or protozoan. In embodiments, the microbe is a bacterium selected from: Aeromonas, Aspergillus , Bacteroides, Bilophila , Campylobacter , Clostridioides , Coccidiosis, Crytosporidia,

Enterobacter , Enterococcus , Escherichia , Firmicutes , Helicobacter , Lactobacillus , Listeria , Peptostreptococcus , Pleisiomonas , Prevote llaceae, Pseudomonas , Salmonella , Sarcina, SFB, Shigella , Staphylococcus , Streptococcus , Veillonella , Vibrio , Yersinia , a secretory antibody- coated bacterium, Candida , Mycobacterium , Mycoplasma, Rotavirus, Calicivirus, Norwalk-like viruses, adenoviruses, astroviruses, sapporo-like viruses, toroviruses, coronaviruses, picomaviruses, herpes viruses, noroviruses, Proteus, Entamoeba , Giardia , and Strongy loides, single-celled parasites, multi-celled parasites, and amoebae. In embodiments, the microbe is drug-resistant or hypervirulent. In embodiments, the microbe is selected from: Bacteroides fragilis, Bilophila wadsworthia, Campylobacter jejuni, Campylobacter coli, Clostridioides difficile, Clostridioides sordelli, Enterobacter cloacae, Enterococcus faecalis, Enterococcus faecium, Escherichia coli, enterotoxigenic Escherichia coli, Helicobacter pylori, Helicobacter sp. Flexispira, Listeria monocytogenes, Pleisiomonas shigelloides, UC Prevotellaceae, Pseudomonas aeruginosa, Salmonella enterica, Salmonella bongori, Salmonella enterica subsp. enterica serotype Typhimurium, Shigella dysenteriae, Staphylococcus aureus, Streptococcus anginosus, Streptococcus thermophilus, Vibrio cholera, Vibrio parahaemolyticus, Yersinia enterocolitica, Candida albicans, Entamoeba histolytica, Giardia lamblia, and Strongyloides stercoralis. In embodiments, the microbe is Clostridioides difficile, and the GI tract target is TcdA or TcdB. In embodiments, the condition is selected from: a GI tract ulcer, gastritis, a GI tract cancer, an inflammatory or autoimmune condition, C. diff. infection, secretory antibody- coated bacteria infection, H. pylori infection, Campylobacter jejuni infection, Campylobacter coli infection, Toxigenic Escherichia coli infection, Staphylococcus aureus infection, Bacteroides fragilis infection, and Vibrio cholera infection. In embodiments, disease activity in the subject is measured before, during, or at anytime after treatment. In embodiments, the disease activity measured following treatment is substantially reduced compared to the disease activity measured prior to treatment.

[0011] The present invention also includes a method for cleaving at least one C. difficile toxin protein, comprising: contacting the at least one C. difficile toxin protein with a novel endopeptidase that cleaves the at least one C. difficile toxin protein at a target site, under conditions suitable for the target site cleavage of the at least one C. difficile toxin protein by the novel endopeptidase to occur; wherein the at least one C. difficile toxin protein is a TcdA protein, a TcdB protein, or both; and wherein the target site cleavage results in a reduction in cytopathicity of the C. difficile toxin protein in comparison with the same at least one C. difficile toxin protein that has not been cleaved. In certain embodiments, the at least one C. difficile toxin protein is a TcdA protein. In some related embodiments, the target site cleavage reduces binding of the TcdA protein to the C. difficile toxin protein receptor, thereby reducing the cytopathicity. In some embodiments, the target site cleavage of the TcdA protein removes a part of or the full receptor binding domain (RBD) of the TcdA protein. In some embodiments, the target site cleavage removes at least one C-terminal combined repetitive oligopeptide (CROP) in the RBD. In some embodiments, the target site cleavage removes all CROPs in the RBD. In some embodiments, the target site cleavage reduces membrane translocation of the TcdA protein into a target cell, thereby reducing the cytopathicity. In some embodiments, the target site cleavage reduces autocatalytic cleavage of the TcdA protein, thereby reducing membrane translocation of the TcdA protein into the target cell. In some embodiments, the target site cleavage reduces endosome escape of the TcdA protein, thereby reducing the cytopathicity. In some embodiments, the target site cleavage reduces glucosyltransferase activity of the TcdA protein, thereby reducing the cytopathicity. In some embodiments, the at least one C. difficile toxin protein is a TcdB protein. In some related embodiments, the target site cleavage reduces binding of the cleaved C. difficile TcdB protein to the C. difficile toxin protein receptor, thereby reducing the cytopathicity. In some embodiments, the target site cleavage of the TcdB protein removes a part of or the full RBD of the TcdB protein. In some embodiments, the target site cleavage removes at least one CROP in the RBD. In some embodiments, the target site cleavage removes all CROPs in the RBD. In some embodiments, the target site cleavage reduces membrane translocation of the TcdB protein into a target cell, thereby reducing the cytopathicity. In some embodiments, the target site cleavage reduces autocatalytic cleavage of the TcdB protein, thereby reducing membrane translocation of the TcdB protein into the target cell. In some embodiments, the target site cleavage reduces endosome escape of the TcdB protein, thereby reducing the cytopathicity. In some embodiments, the target site cleavage reduces glucosyltransferase activity of the TcdB protein, thereby reducing the cytopathicity. In some embodiments, the reduction in binding of the at least one C. difficile toxin protein to the cell receptor is represented by a reduction in binding affinity, the novel endopeptidase cleaves the at least one toxin protein with a high specificity, the novel endopeptidase cleaves the at least one toxin protein with a high activity, or a combination thereof. In some embodiments, the reduction in binding of the at least one C. difficile toxin protein to the cell receptor is about 80% to about 100%, in comparison with the at least one C. difficile toxin protein that has not been cleaved. In some embodiments, the reduction in membrane translocation is about 80% to about 100%, in comparison with the at least one C. difficile toxin protein that has not been cleaved. In some embodiments, the reduction in endosome escape is about 80% to about 100%, in comparison with the at least one C. difficile toxin protein that has not been cleaved. In some embodiments, the reduction in glucosyltransferase activity is about 80% to about 100%, in comparison with the at least one C. difficile toxin protein that has not been cleaved. In some embodiments, the reduction in cytopathicity is about 80% to about 100%.

[0012] The invention also includes a method for reducing the cytopathicity of at least one C. difficile toxin protein comprising: contacting the at least one C. difficile toxin protein with a novel endopeptidase that cleaves the at least one C. difficile toxin protein at a target site, under conditions suitable for the target site cleavage of the at least one C. difficile toxin protein by the novel endopeptidase to occur; wherein the at least one C. difficile toxin protein is a TcdA protein, a TcdB protein, or both; and wherein the cytopathicity of the cleaved C. difficile toxin protein is reduced in comparison with the same at least one C. difficile toxin protein that has not been cleaved. In embodiments, the C. difficile toxin protein is a TcdA protein. In some related embodiments, the target site cleavage of the TcdA protein reduces binding of the cleaved TcdA protein to the C. difficile toxin protein receptor, thereby reducing the cytopathicity. In some embodiments, the target site cleavage removes a part of or the full RBD of the at least one TcdA protein. In some embodiments, the target site cleavage removes at least one CROP in the RBD. In some embodiments, the target site cleavage removes all CROPs in the RBD. In some embodiments, the target site cleavage reduces membrane translocation of the TcdA protein into a target cell, thereby reducing the cytopathicity. In some embodiments, the target site cleavage reduces autocatalytic cleavage of the TcdA protein, thereby reducing the membrane translocation. In some embodiments, the target site cleavage reduces endosome escape of the TcdA protein, thereby reducing the cytopathicity. In some embodiments, the target site cleavage reduces glucosyltransferase activity of the TcdA protein, thereby reducing the cytopathicity. In some embodiments, the C. difficile toxin protein is a TcdB protein. In some related embodiments, the target site cleavage of the TcdB protein reduces binding of the cleaved C. difficile TcdB protein to the C. difficile toxin protein receptor, thereby reducing the cytopathicity. In some embodiments, the target site cleavage removes a part of or the full RBD of the TcdB protein. In some embodiments, the target site cleavage removes at least one CROP in the RBD. In some embodiments, the target site cleavage removes all CROPs in the RBD. In some embodiments, the target site cleavage reduces membrane translocation of the TcdB protein into a target cell, thereby reducing the cytopathicity. In some embodiments, the target site cleavage reduces autocatalytic cleavage of the TcdB protein, thereby reducing membrane translocation of the TcdB protein into the target cell. In some embodiments, the target site cleavage reduces endosome escape of the TcdB protein, thereby reducing the cytopathicity. In some embodiments the reduction in binding of the at least one C. difficile toxin protein to the cell receptor is represented by a reduction in binding affinity, the novel endopeptidase cleaves the at least one toxin protein with a high specificity, the novel endopeptidase cleaves the at least one toxin protein with a high activity, or a combination thereof. In some embodiments, the reduction in binding of the at least one C. difficile toxin protein to the cell receptor is about 80% to about 100%, in comparison with the at least one C. difficile toxin protein that has not been cleaved. In some embodiments, the reduction in membrane translocation is about 80% to about 100%, in comparison with the at least one C. difficile toxin protein that has not been cleaved. In some embodiments, the reduction in endosome escape is about 80% to about 100%, in comparison with the at least one C. difficile toxin protein that has not been cleaved. In some embodiments, the reduction in glucosyltransferase activity is about 80% to about 100%, in comparison with the at least one C. difficile toxin protein that has not been cleaved. In some embodiments, the reduction in cytopathicity is about 80% to about 100%.

[0013] The invention also includes a method for treating C. difficile infection in a subject, comprising: administering to the subject a therapeutically effective amount of a composition comprising a novel endopeptidase that cleaves at least one C. difficile toxin protein at a target site; wherein the at least one C. difficile toxin protein is a TcdA protein, a TcdB protein, or both; and wherein the cytopathicity of the at least one cleaved C. difficile toxin protein is reduced in comparison with the same at least one C. difficile toxin protein that has not been cleaved. In some embodiments, the C. difficile toxin protein is a TcdA protein. In some embodiments, the target site cleavage of the TcdA protein reduces binding of the cleaved C. difficile TcdA protein to the C. difficile toxin protein receptor, thereby reducing the cytopathicity. In some embodiments, the target site cleavage removes a part of or the full RBD of the TcdA protein. In some embodiments, the target site cleavage removes at least one CROP in the RBD. In some embodiments, the target site cleavage removes all CROPs in the RBD. In some embodiments, the target site cleavage reduces membrane translocation of the TcdA protein into a target cell. In some embodiments, the target site cleavage reduces autocatalytic cleavage of the TcdA protein, thereby reducing membrane translocation of the TcdA protein into the target cell. In some embodiments, the target site cleavage reduces endosome escape of the TcdA protein. In some embodiments, the target site cleavage reduces glucosyltransferase activity of the TcdA protein. In some embodiments, the C. difficile toxin protein is a TcdB protein. In some related embodiments, the target site cleavage of the TcdB protein reduces binding of the cleaved C. difficile TcdB protein to the C. difficile toxin protein receptor, thereby reducing the cytopathicity. In some embodiments, the target site cleavage removes a part of or the full RBD of the TcdB protein. In some embodiments, the target site cleavage removes at least one CROP in the RBD. In some embodiments, the target site cleavage removes all CROPs in the RBD. In some embodiments, the target site cleavage reduces membrane translocation of the TcdB protein into a target cell. In some embodiments, the target site cleavage reduces autocatalytic cleavage of the TcdB protein, thereby reducing membrane translocation of the TcdA protein into the target cell. In some embodiments, the target site cleavage reduces endosome escape of the TcdB protein. In some embodiments, the target site cleavage reduces glucosyltransferase activity of the TcdB protein. In some embodiments, the reduction in binding of the at least one C. difficile toxin protein to the cell receptor is represented by a reduction in binding affinity, the novel endopeptidase cleaves the at least one toxin protein with a high specificity, the novel endopeptidase cleaves the at least one toxin protein with a high activity, or a combination thereof. In some embodiments, the reduction in binding of the at least one C. difficile toxin protein to the cell receptor is about 80% to about 100%, in comparison with the at least one C. difficile toxin protein that has not been cleaved. In some embodiments, the reduction in membrane translocation is about 80% to about 100%, in comparison with the at least one C. difficile toxin protein that has not been cleaved. In some embodiments, the reduction in endosome escape is about 80% to about 100%, in comparison with the at least one C. difficile toxin protein that has not been cleaved. In some embodiments, the reduction in glucosyltransferase activity is about 80% to about 100%, in comparison with the at least one C. difficile toxin protein that has not been cleaved. In some embodiments, the reduction in cytopathicity is about 80% to about 100%. In some embodiments, the composition is formulated for colonic delivery. In some embodiments, the composition is formulated for oral or rectal administration. In some embodiments, the composition is formulated for oral administration and comprises an enteric coating. In some embodiments, the composition comprises at least one other active agent, is administered in combination with at least one other active agent, or both. In some embodiments, the at least one other active agent is a prebiotic or probiotic agent. [0014] The invention also includes a novel endopeptidase capable of a target site cleavage of at least one C. difficile toxin protein selected from a TcdA protein and a TcdB protein, wherein the target site cleavage results in reduced cytopathicity of the at least one C. difficile toxin protein.

In some embodiments, the C. difficile toxin protein is a TcdA protein. In some related embodiments, the target site cleavage of the TcdA protein reduces binding of the TcdA protein to the C. difficile toxin protein receptor, thereby reducing the cytopathicity. In some embodiments, the target site cleavage removes a part of or the full RBD of the TcdA protein, thereby reducing binding of the TcdA protein to a C. difficile toxin protein receptor. In some embodiments, at least one CROP in the TcdA protein RBD is removed by the target site cleavage. In some embodiments, all CROPs in the RBD are removed by the target site cleavage. In some embodiments, the target site cleavage reduces membrane translocation of the TcdA protein into a target cell. In some embodiments, the target site cleavage reduces autocatalytic cleavage of the TcdA protein, thereby reducing membrane translocation of the TcdA protein into a target cell. In some embodiments, the target site cleavage reduces endosome escape of the TcdA protein. In some embodiments, the target site cleavage reduces glucosyltransf erase activity of the TcdA protein. In some embodiments, the C. difficile toxin protein is TcdB. In some related embodiments, the target site cleavage of the TcdB protein reduces binding of the TcdB protein to the C. difficile toxin protein receptor, thereby reducing the cytopathicity. In some embodiments, the target site cleavage removes a part of or the full RBD of the TcdB protein, thereby reducing binding of the TcdB protein to a C. difficile toxin protein receptor. In some embodiments, at least one CROP in the RBD is removed by the target site cleavage. In some embodiments, all CROPs in the RBD are removed by the target site cleavage. In some embodiments, the target site cleavage reduces membrane translocation of the TcdB protein into a target cell. In some embodiments, the target site cleavage reduces autocatalytic cleavage of the TcdB protein, thereby reducing membrane translocation of the TcdB protein into a target cell. In some embodiments, the target site cleavage reduces endosome escape of the TcdB protein. In some embodiments, the target site cleavage reduces glucosyltransferase activity of the TcdB protein. In some embodiments, the reduction in binding of the at least one C. difficile toxin protein to the cell receptor is represented by a reduction in binding affinity, the novel endopeptidase cleaves the at least one toxin protein with a high specificity, the novel endopeptidase cleaves the at least one toxin protein with a high activity, or a combination thereof. In some embodiments, the reduction in binding of the at least one C. difficile toxin protein to the cell receptor is about 80% to about 100%, in comparison with the at least one C. difficile toxin protein that has not been cleaved. In some embodiments, the reduction in membrane translocation is about 80% to about 100%, in comparison with the at least one C. difficile toxin protein that has not been cleaved. In some embodiments, the reduction in endosome escape is about 80% to about 100%, in comparison with the at least one C. difficile toxin protein that has not been cleaved. In some embodiments, the reduction in glucosyltransferase activity is about 80% to about 100%, in comparison with the at least one C. difficile toxin protein that has not been cleaved. In some embodiments, the reduction in cytopathicity is about 80% to about 100%.

[0015] The invention also relates to a composition comprising a novel endopeptidase disclosed herein. In some embodiments, the composition is formulated for colonic delivery. In some embodiments, the composition is formulated for oral or rectal administration. In some embodiments, the composition is formulated for oral administration and comprises an enteric coating. In some embodiments, the composition comprises at least one other active agent, is administered in combination with at least one other active agent, or both. In some embodiments, the at least one other active agent is a prebiotic or probiotic agent. The invention further relates to a method for treating C. difficile infection in a subject, comprising administering to the subject the disclosed composition.

[0016] The invention also relates to a method for reducing the binding of at least one C. difficile toxin protein to a cell receptor specific for the at least one C. difficile toxin protein, comprising: contacting the at least one C. difficile toxin protein with a novel endopeptidase that cleaves the at least one C. difficile toxin protein at a target site, under conditions suitable for the target site cleavage of the at least one C. difficile toxin protein by the novel endopeptidase to occur; wherein the at least one C. difficile toxin protein is a TcdA protein, a TcdB protein, or both; and wherein the binding of the cleaved C. difficile toxin protein to a C. difficile toxin protein receptor is reduced in comparison with the same at least one C. difficile toxin protein that has not been cleaved. In some embodiments, the at least one C. difficile toxin protein is a TcdA protein. In some related embodiments, the target site cleavage reduces binding of the TcdA protein to the C. difficile toxin protein receptor. In some embodiments, the target site cleavage of the TcdA protein removes a part of or the full RBD of the TcdA protein. In some embodiments, the target site cleavage removes at least one CROP in the RBD. In some embodiments, the target site cleavage removes all CROPs in the RBD. In some embodiments, the at least one C. difficile toxin protein is a TcdB protein. In some related embodiments, the target site cleavage reduces binding of the cleaved C. difficile TcdB protein to the C. difficile toxin protein receptor. In some embodiments, the target site cleavage of the TcdB protein removes a part of or the full RBD of the TcdB protein. In some embodiments, the target site cleavage removes at least one CROP in the RBD. In some embodiments, the target site cleavage removes all CROPs in the RBD. In some embodiments, the reduction in binding of the at least one C. difficile toxin protein to the cell receptor is represented by a reduction in binding affinity, the novel endopeptidase cleaves the at least one toxin protein with a high specificity, the novel endopeptidase cleaves the at least one toxin protein with a high activity, or a combination thereof. In some embodiments, the reduction in binding of the at least one C. difficile toxin protein to the cell receptor is about 80% to about 100%, in comparison with the at least one C. difficile toxin protein that has not been cleaved.

[0017] The present invention also includes a method for reducing an activity of at least one C. difficile toxin protein, comprising: contacting the at least one C. difficile toxin protein with a novel binding protein comprising at least one binding epitope that specifically binds to a target binding site of the at least one C. difficile toxin protein, wherein binding of the novel binding protein to the target binding site results in reduction of an activity of the C. difficile toxin protein; wherein the at least one C. difficile toxin protein is a TcdA protein, a TcdB protein, or both. In some embodiments, binding of the novel binding protein to the target binding site results in a reduction in cytopathicity of the C. difficile toxin protein. In certain embodiments, the at least one C. difficile toxin protein is a TcdA protein. In some related embodiments, binding of the novel binding protein to the target binding site reduces binding of the TcdA protein to the C. difficile toxin protein receptor, thereby reducing the cytopathicity. In some embodiments, the novel binding protein binds to the receptor binding domain (RBD) of the TcdA protein. In some embodiments, binding of the novel binding protein to the target binding site of the TcdA protein disrupts receptor binding of the TcdA protein. In some embodiments, binding of the novel binding protein to the target binding site disrupts at least one C-terminal combined repetitive oligopeptide (CROP) in the RBD. In some embodiments, binding of the novel binding protein to the target binding site reduces membrane translocation of the TcdA protein into a target cell, thereby reducing the cytopathicity. In some embodiments, binding of the novel binding protein to the target binding site reduces autocatalytic cleavage of the TcdA protein, thereby reducing membrane translocation of the TcdA protein into the target cell. In some embodiments, binding of the novel binding protein to the target binding site reduces endosome escape of the TcdA protein, thereby reducing the cytopathicity. In some embodiments, binding of the novel binding protein to the target binding site reduces glucosyltransferase activity of the TcdA protein, thereby reducing the cytopathicity. In some embodiments, the at least one C. difficile toxin protein is a TcdB protein. In some related embodiments, binding of the novel binding protein to the target binding site reduces binding of the C. difficile TcdB protein to the C. difficile toxin protein receptor, thereby reducing the cytopathicity. In some embodiments, the novel binding protein binds to the receptor binding domain (RBD) of the TcdA protein. In some embodiments, binding of the novel binding protein to the target binding site of the TcdB protein disrupts receptor binding of the TcdB protein. In some embodiments, binding of the novel binding protein to the target binding site disrupts at least one C-terminal combined repetitive oligopeptide (CROP) in the RBD. In some embodiments, binding of the novel binding protein to the target binding site reduces membrane translocation of the TcdB protein into a target cell, thereby reducing the cytopathicity. In some embodiments, binding of the novel binding protein to the target binding site reduces autocatalytic cleavage of the TcdB protein, thereby reducing membrane translocation of the TcdB protein into the target cell. In some embodiments, binding of the novel binding protein to the target binding site reduces endosome escape of the TcdB protein, thereby reducing the cytopathicity. In some embodiments, binding of the novel binding protein to the target binding site reduces glucosyltransferase activity of the TcdB protein, thereby reducing the cytopathicity. In some embodiments, the reduction in binding of the at least one C. difficile toxin protein to the cell receptor is represented by a reduction in binding affinity, the novel binding protein binds the at least one toxin protein with a high specificity, or both. In some embodiments, the reduction in binding of the at least one C. difficile toxin protein to the cell receptor is about 80% to about 100%, in comparison with the binding in the absence of the novel binding protein. In some embodiments, the reduction in membrane translocation is about 80% to about 100%, in comparison with the membrane translocation in the absence of the novel binding protein. In some embodiments, the reduction in endosome escape is about 80% to about 100%, in comparison with the endosome escape in the absence of the novel binding protein. In some embodiments, the reduction in glucosyltransferase activity is about 80% to about 100%, in comparison with the glucosyltransferase activity in the absence of the novel binding protein. In some embodiments, the reduction in cytopathicity is about 80% to about 100%.

[0018] The invention includes a method for reducing the cytopathicity of at least one C. difficile toxin protein comprising: contacting the at least one C. difficile toxin protein with a novel binding protein comprising at least one binding epitope that specifically binds to a target binding site of the at least one C. difficile toxin protein, wherein the at least one C. difficile toxin protein is a TcdA protein, a TcdB protein, or both; and wherein the cytopathicity of the C. difficile toxin protein is reduced in comparison with the cytopathicity in the absence of the novel binding protein.

[0019] The invention includes a method for treating C. difficile infection in a subject, comprising: administering to the subject a therapeutically effective amount of a composition comprising a novel binding protein comprising at least one binding epitope that specifically binds to a target binding site of the at least one C. difficile toxin protein; wherein the at least one C. difficile toxin protein is a TcdA protein, a TcdB protein, or both; and wherein the cytopathicity of the C. difficile toxin protein is reduced in comparison with the cytopathicity in the absence of the novel binding protein. In some embodiments, the novel binding protein composition is formulated for colonic delivery. In some embodiments, the composition is formulated for oral or rectal administration. In some embodiments, the composition is formulated for oral administration and comprises an enteric coating. In some embodiments, the composition comprises at least one other active agent, is administered in combination with at least one other active agent, or both. In some embodiments, the at least one other active agent is a prebiotic or probiotic agent.

[0020] The invention also includes a novel binding protein comprising at least one binding epitope that binds to a target binding site of at least one C. difficile toxin protein selected from a TcdA protein, a TcdB protein, or both, wherein binding of the novel binding protein to the target site results in reduced cytopathicity of the at least one C. difficile toxin protein. In some embodiments, binding of the novel binding protein to the target binding site results in a reduction in cytopathicity of the C. difficile toxin protein. In certain embodiments, the at least one C. difficile toxin protein is a TcdA protein. In some related embodiments, binding of the novel binding protein to the target binding site reduces binding of the TcdA protein to the C. difficile toxin protein receptor, thereby reducing the cytopathicity. In some embodiments, the novel binding protein binds to the receptor binding domain (RBD) of the TcdA protein. In some embodiments, binding of the novel binding protein to the target binding site of the TcdA protein disrupts receptor binding of the TcdA protein. In some embodiments, binding of the novel binding protein to the target binding site disrupts at least one C-terminal combined repetitive oligopeptide (CROP) in the RBD. In some embodiments, binding of the novel binding protein to the target binding site reduces membrane translocation of the TcdA protein into a target cell, thereby reducing the cytopathicity. In some embodiments, binding of the novel binding protein to the target binding site reduces autocatalytic cleavage of the TcdA protein, thereby reducing membrane translocation of the TcdA protein into the target cell. In some embodiments, binding of the novel binding protein to the target binding site reduces endosome escape of the TcdA protein, thereby reducing the cytopathicity. In some embodiments, binding of the novel binding protein to the target binding site reduces glucosyltransf erase activity of the TcdA protein, thereby reducing the cytopathicity. In some embodiments, the at least one C. difficile toxin protein is a TcdB protein. In some related embodiments, binding of the novel binding protein to the target binding site reduces binding of the C. difficile TcdB protein to the C. difficile toxin protein receptor, thereby reducing the cytopathicity. In some embodiments, the novel binding protein binds to the receptor binding domain (RBD) of the TcdA protein. In some embodiments, binding of the novel binding protein to the target binding site of the TcdB protein disrupts receptor binding of the TcdB protein. In some embodiments, binding of the novel binding protein to the target binding site disrupts at least one C-terminal combined repetitive oligopeptide (CROP) in the RBD. In some embodiments, binding of the novel binding protein to the target binding site reduces membrane translocation of the TcdB protein into a target cell, thereby reducing the cytopathicity. In some embodiments, binding of the novel binding protein to the target binding site reduces autocatalytic cleavage of the TcdB protein, thereby reducing membrane translocation of the TcdB protein into the target cell. In some embodiments, binding of the novel binding protein to the target binding site reduces endosome escape of the TcdB protein, thereby reducing the cytopathicity. In some embodiments, binding of the novel binding protein to the target binding site reduces glucosyltransf erase activity of the TcdB protein, thereby reducing the cytopathicity. In some embodiments, the reduction in binding of the at least one C. difficile toxin protein to the cell receptor is represented by a reduction in binding affinity, the novel binding protein binds the at least one toxin protein with a high specificity, or both. In some embodiments, the reduction in binding of the at least one C. difficile toxin protein to the cell receptor is about 80% to about 100%, in comparison with the binding in the absence of the novel binding protein. In some embodiments, the reduction in membrane translocation is about 80% to about 100%, in comparison with the membrane translocation in the absence of the novel binding protein. In some embodiments, the reduction in endosome escape is about 80% to about 100%, in comparison with the endosome escape in the absence of the novel binding protein. In some embodiments, the reduction in glucosyltransferase activity is about 80% to about 100%, in comparison with the glucosyltransferase activity in the absence of the novel binding protein. In some embodiments, the reduction in cytopathicity is about 80% to about 100%.

[0021] The invention also relates to a composition comprising a novel binding protein disclosed herein. In some embodiments, the composition is formulated for colonic delivery. In some embodiments, the composition is formulated for oral or rectal administration. In some embodiments, the composition is formulated for oral administration and comprises an enteric coating. In some embodiments, the composition comprises at least one other active agent, is administered in combination with at least one other active agent, or both. In some embodiments, the at least one other active agent is a prebiotic or probiotic agent. The invention further relates to a method for treating C. difficile infection in a subject, comprising administering to the subject the disclosed composition. [0022] The invention also relates to a method for reducing the binding of at least one C. difficile toxin protein to a cell receptor specific for the at least one C. difficile toxin protein, comprising: contacting the at least one C. difficile toxin protein with a novel binding protein comprising at least one binding epitope that specifically binds to a target binding site of the at least one C. difficile toxin protein, wherein binding of the novel binding protein to the target binding site results in reduction of an activity of the C. difficile toxin protein; wherein the at least one C. difficile toxin protein is a TcdA protein, a TcdB protein, or both; and wherein the binding of the C. difficile toxin protein to a C. difficile toxin protein receptor is reduced in comparison with the binding in the absence of the novel binding protein.

[0023] The present invention also includes a method for reducing an activity of a GI tract target, comprising contacting the at least one GI tract target with a novel antagonist. In embodiments, the novel antagonist is a novel binding protein comprising at least one binding epitope that specifically binds to a target binding site of the GI tract target, wherein binding of the novel binding protein to the target binding site results in reduction of an activity of the GI tract target. In embodiments, the novel antagonist is a novel endopeptidase that disrupts binding of a GI tract target to its GI tract target binding partner, by cleavage of a target cleavage site present on the GI tract target.

INCORPORATION BY REFERENCE

[0024] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. Throughout the description, “in embodiments,” “in some embodiments,” “in certain embodiments,” “in particular embodiments,” “in specific embodiments,” and similar terms do not limit the invention to only that use or construction.

BRIEF DESCRIPTION OF THE DRAWINGS

[0025] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:

[0026] Fig. 1. Example of C difficile Toxin Domains. The drawing illustrates groupings of the short repeats (SRs) in the TcdA and TcdB RBD (closed black rectangles) around each LR (open white rectangles) as reported for C. diff strain VPI 10463 by Orth et al., 2014 (see Fig. 1 therein). In this exemplary drawing, 1 denotes the glucosyltransferase domain (GTD); 2 denotes the translocation/cysteine protease domain; and 3 denotes the CROP domain.

DETAILED DESCRIPTION OF THE INVENTION

[0027] Novel Antagonists

[0028] The present invention relates to novel antagonists that reduce an activity of a gastrointestinal (GI) tract target, methods for identifying and making the novel antagonists, compositions thereof, and methods for their use. In embodiments, the novel antagonist is a novel endopeptidase. In embodiments, the novel antagonist is a novel binding protein.

Reduction in the activity of a GI tract target resulting from target site cleavage by a novel endopeptidase can occur by disruption of, e.g., partial removal, full removal, or allosteric effect on, a key GI tract target locus. Reduction in the activity of a GI tract target resulting from target site binding by a novel binding protein can occur by disruption of, e.g., partial removal, full removal, or allosteric effect on, a key GI tract target locus. The invention includes methods for treating a subject with a novel antagonist, including a subject infected with or suspected of being infected with a GI tract organism. The invention further includes methods for preventing or ameliorating symptoms in, or reducing an activity of a GI tract target in, a subject infected with or suspected of being infected with a GI tract organism.

[0029] Novel Endopeptidases

[0030] The present invention relates to novel endopeptidases and compositions thereof. In embodiments, a novel endopeptidase of the present invention is capable of a target site cleavage of at least one GI tract target, resulting in a reduction of activity of the GI tract target.

[0031] A novel endopeptidase can be made by de novo design or by optimizing an existing, starting protease. A starting protease can be optimized to produce a novel endopeptidase that cleaves the GI tract target at a target site as desired, e.g., using the Rosetta Molecular Modeling Suite to model the binding pocket of the endopeptidase, as described herein. Using such methods, candidate novel endopeptidases can be constructed and evaluated for enzymatic activity, and the preferred candidate selected for further characterization. For example, after an endopeptidase is designed using Rosetta, it may be optimized by selecting the most active candidate(s) and performing additional optimization by Rosetta and/or site saturation mutagenesis at selected residues for increased performance.

[0032] A novel endopeptidase can be selected from among candidate optimized endopeptidases based on a desirable property or parameter, e.g., high specificity, high activity, and/or high efficiency of target site cleavage. In embodiments, a property is evaluated by comparing cleavage by the candidate optimized endopeptidase or a selected novel endopeptidase with cleavage by a control. In embodiments, the control is another endopeptidase, e.g., an unoptimized, starting enzyme, another candidate optimized enzyme, or another positive or negative control enzyme or protein, used on the same substrate (e.g, a desired target site of a GI tract target). In embodiments, the property is evaluated by comparing the cleavage by a candidate optimized endopeptidase or novel endopeptidase of a desired substrate (e.g, a target site of a GI tract target), with cleavage by the same endopeptidase of a nonspecific (e.g., negative control) substrate. In embodiments, the control substrate or target is an unrelated protein or a variant GI tract target. In embodiments, the variant GI tract target differs from the non-variant GI tract target in the amino acid sequence at its target site.

[0033] In embodiments, specificity (selectivity), activity, and efficiency of target site cleavage are determined by any means known in the art. Cleavage of the GI tract target at the selected target site by the novel endopeptidase can yield fragments of predicted sizes that can be characterized using any known protein analysis method. For example, the resulting peptide species can be separated and quantitated by SDS-CGE or western blot analysis. Further analysis of the fragments to confirm the expected cleavage point sequence can be carried out by any known method, e.g., mass spectrometry.

[0034] In embodiments, the selectivity of the novel endopeptidase for the substrate, e.g., the target cleavage site of the GI tract target, relative to the selectivity of the novel endopeptidase for a nonspecific substrate, is increased by about 10 fold to about 2,000 fold. In embodiments, the selectivity of the novel endopeptidase for the substrate, e.g., the target cleavage site of the GI tract target, relative to the selectivity of the novel endopeptidase for a nonspecific substrate, is increased by about 10 fold to about 20 fold, about 10 fold to about 25 fold, about 10 fold to about 50 fold, about 10 fold to about 75 fold, about 10 fold to about 100 fold, about 10 fold to about 150 fold, about 10 fold to about 250 fold, about 10 fold to about 500 fold, about 10 fold to about 750 fold, about 10 fold to about 1,000 fold, about 10 fold to about 2,000 fold, about 20 fold to about 25 fold, about 20 fold to about 50 fold, about 20 fold to about 75 fold, about 20 fold to about 100 fold, about 20 fold to about 150 fold, about 20 fold to about 250 fold, about 20 fold to about 500 fold, about 20 fold to about 750 fold, about 20 fold to about 1,000 fold, about 20 fold to about 2,000 fold, about 25 fold to about 50 fold, about 25 fold to about 75 fold, about 25 fold to about 100 fold, about 25 fold to about 150 fold, about 25 fold to about 250 fold, about 25 fold to about 500 fold, about 25 fold to about 750 fold, about 25 fold to about 1,000 fold, about 25 fold to about 2,000 fold, about 50 fold to about 75 fold, about 50 fold to about 100 fold, about 50 fold to about 150 fold, about 50 fold to about 250 fold, about 50 fold to about 500 fold, about 50 fold to about 750 fold, about 50 fold to about 1,000 fold, about 50 fold to about 2,000 fold, about 75 fold to about 100 fold, about 75 fold to about 150 fold, about 75 fold to about 250 fold, about 75 fold to about 500 fold, about 75 fold to about 750 fold, about 75 fold to about 1,000 fold, about 75 fold to about 2,000 fold, about 100 fold to about 150 fold, about 100 fold to about 250 fold, about 100 fold to about 500 fold, about 100 fold to about 750 fold, about 100 fold to about 1,000 fold, about 100 fold to about 2,000 fold, about 150 fold to about 250 fold, about 150 fold to about 500 fold, about 150 fold to about 750 fold, about 150 fold to about 1,000 fold, about 150 fold to about 2,000 fold, about 250 fold to about 500 fold, about 250 fold to about 750 fold, about 250 fold to about 1,000 fold, about 250 fold to about 2,000 fold, about 500 fold to about 750 fold, about 500 fold to about 1,000 fold, about 500 fold to about 2,000 fold, about 750 fold to about 1,000 fold, about 750 fold to about 2,000 fold, or about 1,000 fold to about 2,000 fold. In embodiments, the selectivity of the novel endopeptidase for the substrate, e.g., the target cleavage site of the GI tract target, relative to the selectivity of the novel endopeptidase for a nonspecific substrate, is increased by about 10 fold, about 20 fold, about 25 fold, about 50 fold, about 75 fold, about 100 fold, about 150 fold, about 250 fold, about 500 fold, about 750 fold, about 1,000 fold, or about 2,000 fold. In embodiments, the selectivity of the novel endopeptidase for the substrate, e.g., the target cleavage site of the GI tract target, relative to the selectivity of the novel endopeptidase for a nonspecific substrate, is increased by at least about 10 fold, about 20 fold, about 25 fold, about 50 fold, about 75 fold, about 100 fold, about 150 fold, about 250 fold, about 500 fold, about 750 fold, or about 1,000 fold. In embodiments, the selectivity of the novel endopeptidase for the substrate, e.g., the target cleavage site of the GI tract target, relative to the selectivity of the novel endopeptidase for a nonspecific substrate, is increased by at most about 20 fold, about 25 fold, about 50 fold, about 75 fold, about 100 fold, about 150 fold, about 250 fold, about 500 fold, about 750 fold, about 1,000 fold, or about 2,000 fold.

[0035] In embodiments, the selectivity of a novel endopeptidase for the substrate, e.g., the target cleavage site of the GI tract target, relative to the selectivity of the unoptimized endopeptidase for the same substrate, is increased by about 10 fold to about 2,000 fold. In embodiments, the selectivity of the novel endopeptidase for the substrate, e.g., the target cleavage site of the GI tract target, relative to the selectivity of the unoptimized endopeptidase for the same substrate, is increased by about 10 fold to about 20 fold, about 10 fold to about 25 fold, about 10 fold to about 50 fold, about 10 fold to about 75 fold, about 10 fold to about 100 fold, about 10 fold to about 150 fold, about 10 fold to about 250 fold, about 10 fold to about 500 fold, about 10 fold to about 750 fold, about 10 fold to about 1,000 fold, about 10 fold to about 2,000 fold, about 20 fold to about 25 fold, about 20 fold to about 50 fold, about 20 fold to about 75 fold, about 20 fold to about 100 fold, about 20 fold to about 150 fold, about 20 fold to about 250 fold, about 20 fold to about 500 fold, about 20 fold to about 750 fold, about 20 fold to about 1,000 fold, about 20 fold to about 2,000 fold, about 25 fold to about 50 fold, about 25 fold to about 75 fold, about 25 fold to about 100 fold, about 25 fold to about 150 fold, about 25 fold to about 250 fold, about 25 fold to about 500 fold, about 25 fold to about 750 fold, about 25 fold to about 1,000 fold, about 25 fold to about 2,000 fold, about 50 fold to about 75 fold, about 50 fold to about 100 fold, about 50 fold to about 150 fold, about 50 fold to about 250 fold, about 50 fold to about 500 fold, about 50 fold to about 750 fold, about 50 fold to about 1,000 fold, about 50 fold to about 2,000 fold, about 75 fold to about 100 fold, about 75 fold to about 150 fold, about 75 fold to about 250 fold, about 75 fold to about 500 fold, about 75 fold to about 750 fold, about 75 fold to about 1,000 fold, about 75 fold to about 2,000 fold, about 100 fold to about 150 fold, about 100 fold to about 250 fold, about 100 fold to about 500 fold, about 100 fold to about 750 fold, about 100 fold to about 1,000 fold, about 100 fold to about 2,000 fold, about 150 fold to about 250 fold, about 150 fold to about 500 fold, about 150 fold to about 750 fold, about 150 fold to about 1,000 fold, about 150 fold to about 2,000 fold, about 250 fold to about 500 fold, about 250 fold to about 750 fold, about 250 fold to about 1,000 fold, about 250 fold to about 2,000 fold, about 500 fold to about 750 fold, about 500 fold to about 1,000 fold, about 500 fold to about 2,000 fold, about 750 fold to about 1,000 fold, about 750 fold to about 2,000 fold, or about 1,000 fold to about 2,000 fold. In embodiments, the selectivity of the novel endopeptidase for the substrate, e.g., the target cleavage site of the GI tract target, relative to the selectivity of the unoptimized endopeptidase for the same substrate, is increased by about 10 fold, about 20 fold, about 25 fold, about 50 fold, about 75 fold, about 100 fold, about 150 fold, about 250 fold, about 500 fold, about 750 fold, about 1,000 fold, or about 2,000 fold. In embodiments, the selectivity of the novel endopeptidase for the substrate, e.g., the target cleavage site of the GI tract target, relative to the selectivity of the unoptimized endopeptidase for the same substrate, is increased by at least about 10 fold, about 20 fold, about 25 fold, about 50 fold, about 75 fold, about 100 fold, about 150 fold, about 250 fold, about 500 fold, about 750 fold, or about 1,000 fold. In embodiments, the selectivity of the novel endopeptidase for the substrate, e.g., the target cleavage site of the GI tract target, relative to the selectivity of the unoptimized endopeptidase for the same substrate, is increased by at most about 20 fold, about 25 fold, about 50 fold, about 75 fold, about 100 fold, about 150 fold, about 250 fold, about 500 fold, about 750 fold, about 1,000 fold, or about 2,000 fold.

[0036] In embodiments, the novel endopeptidase is selected based on a cleavage efficiency (catalytic efficiency) of 75 % to 100 %, that is, 75 % to 100 % of the substrate GI tract target protein is cleaved into fragments having the predicted sizes and sequences. In embodiments, the novel endopeptidase is selected based on a GI tract target cleavage efficiency of about 75 % to about 80 %, about 75 % to about 85 %, about 75 % to about 90 %, about 75 % to about 93 %, about 75 % to about 94 %, about 75 % to about 95 %, about 75 % to about 96 %, about 75 % to about 96 %, about 75 % to about 98 %, about 75 % to about 99 %, about 75 % to about 100 %, about 80 % to about 85 %, about 80 % to about 90 %, about 80 % to about 93 %, about 80 % to about 94 %, about 80 % to about 95 %, about 80 % to about 96 %, about 80 % to about 96 %, about 80 % to about 98 %, about 80 % to about 99 %, about 80 % to about 100 %, about 85 % to about 90 %, about 85 % to about 93 %, about 85 % to about 94 %, about 85 % to about 95 %, about 85 % to about 96 %, about 85 % to about 96 %, about 85 % to about 98 %, about 85 % to about 99 %, about 85 % to about 100 %, about 90 % to about 93 %, about 90 % to about 94 %, about 90 % to about 95 %, about 90 % to about 96 %, about 90 % to about 96 %, about 90 % to about 98 %, about 90 % to about 99 %, about 90 % to about 100 %, about 93 % to about 94 %, about 93 % to about 95 %, about 93 % to about 96 %, about 93 % to about 96 %, about 93 % to about 98 %, about 93 % to about 99 %, about 93 % to about 100 %, about 94 % to about 95 %, about 94 % to about 96 %, about 94 % to about 96 %, about 94 % to about 98 %, about 94 % to about 99 %, about 94 % to about 100 %, about 95 % to about 96 %, about 95 % to about 96 %, about 95 % to about 98 %, about 95 % to about 99 %, about 95 % to about 100 %, about 96 % to about 96 %, about 96 % to about 98 %, about 96 % to about 99 %, about 96 % to about 100 %, about 96 % to about 98 %, about 96 % to about 99 %, about 96 % to about 100 %, about 98 % to about 99 %, about 98 % to about 100 %, or about 99 % to about 100 %. In embodiments, the novel endopeptidase is selected based on GI tract target cleavage efficiency of about 75 %, about 80 %, about 85 %, about 90 %, about 93 %, about 94 %, about 95 %, about 96 %, about 96 %, about 98 %, about 99 %, or about 100 %. In embodiments, the novel endopeptidase is selected based on GI tract target cleavage efficiency of at least about 75 %, about 80 %, about 85 %, about 90 %, about 93 %, about 94 %, about 95 %, about 96 %, about 96 %, about 98 %, or about 99 %. In embodiments, the novel endopeptidase is selected based on GI tract target protein cleavage efficiency of at most about 80 %, about 85 %, about 90 %, about 93 %, about 94 %, about 95 %, about 96 %, about 96 %, about 98 %, about 99 %, or about 100 %.

[0037] In embodiments, the activity of the novel endopeptidase is increased about 10-fold to about 5000-fold relative to the starting endopeptidase. In embodiments, the activity of the novel endopeptidase is increased about 10 fold to about 4,000 fold. In embodiments, the activity of the novel endopeptidase is increased about 10 fold to about 20 fold, about 10 fold to about 50 fold, about 10 fold to about 100 fold, about 10 fold to about 150 fold, about 10 fold to about 200 fold, about 10 fold to about 250 fold, about 10 fold to about 500 fold, about 10 fold to about 750 fold, about 10 fold to about 1,000 fold, about 10 fold to about 2,500 fold, about 10 fold to about 4,000 fold, about 20 fold to about 50 fold, about 20 fold to about 100 fold, about 20 fold to about 150 fold, about 20 fold to about 200 fold, about 20 fold to about 250 fold, about 20 fold to about 500 fold, about 20 fold to about 750 fold, about 20 fold to about 1,000 fold, about 20 fold to about 2,500 fold, about 20 fold to about 4,000 fold, about 50 fold to about 100 fold, about 50 fold to about 150 fold, about 50 fold to about 200 fold, about 50 fold to about 250 fold, about 50 fold to about 500 fold, about 50 fold to about 750 fold, about 50 fold to about 1,000 fold, about 50 fold to about 2,500 fold, about 50 fold to about 4,000 fold, about 100 fold to about 150 fold, about 100 fold to about 200 fold, about 100 fold to about 250 fold, about 100 fold to about 500 fold, about 100 fold to about 750 fold, about 100 fold to about 1,000 fold, about 100 fold to about 2,500 fold, about 100 fold to about 4,000 fold, about 150 fold to about 200 fold, about 150 fold to about 250 fold, about 150 fold to about 500 fold, about 150 fold to about 750 fold, about 150 fold to about 1,000 fold, about 150 fold to about 2,500 fold, about 150 fold to about 4,000 fold, about 200 fold to about 250 fold, about 200 fold to about 500 fold, about 200 fold to about 750 fold, about 200 fold to about 1,000 fold, about 200 fold to about 2,500 fold, about 200 fold to about 4,000 fold, about 250 fold to about 500 fold, about 250 fold to about 750 fold, about 250 fold to about 1,000 fold, about 250 fold to about 2,500 fold, about 250 fold to about 4,000 fold, about 500 fold to about 750 fold, about 500 fold to about 1,000 fold, about 500 fold to about 2,500 fold, about 500 fold to about 4,000 fold, about 750 fold to about 1,000 fold, about 750 fold to about 2,500 fold, about 750 fold to about 4,000 fold, about 1,000 fold to about 2,500 fold, about 1,000 fold to about 4,000 fold, or about 2,500 fold to about 4,000 fold. In embodiments, the activity of the novel endopeptidase is increased about 10 fold, about 20 fold, about 25 fold, about 50 fold, about 75 fold, about 100 fold, about 125 fold, about 150 fold, about 200 fold, about 250 fold, about 300 fold, about 400 fold, about 500 fold, about 600 fold, about 750 fold, about 1,000 fold, about 1,500 fold, about 2,500 fold, about 3000 fold, about 3,500 fold, about 4,000 fold, or about 5,000 fold. In embodiments, the activity of the novel endopeptidase is increased at least about 10 fold, about 20 fold, about 50 fold, about 100 fold, about 150 fold, about 200 fold, about 250 fold, about 500 fold, about 750 fold, about 1,000 fold, about 2,500 fold, or about 4,000 fold. In embodiments, the activity of the novel endopeptidase is increased at most about 20 fold, about 50 fold, about 100 fold, about 150 fold, about 200 fold, about 250 fold, about 500 fold, about 750 fold, about 1,000 fold, about 2,500 fold, about 4,000 fold, or about 5,000 fold.

[0038] In embodiments, a novel endopeptidase of the present invention cleaves a GI tract target at a target site, also referred to herein as a target cleavage site. A target cleavage site refers to the location of the break in the GI tract target amino acid sequence that results from cleavage by the novel endopeptidase. [0039] In embodiments, target site cleavage reduces, blocks, or otherwise alters the interaction of the GI tract target with its binding partner (e.g., a cell surface receptor or other feature of a target cell or tissue) to result in a reduced activity of the GI tract target. In embodiments, the reduced activity is a reduction in any activity of the GI tract target, including, but not limited to, receptor signaling, cytopathicity, enzyme activity, and membrane translocation. In embodiments, the novel endopeptidase does not cleave or bind to the receptor or any other structure or feature of a target cell or tissue. In embodiments, the novel endopeptidase does not induce off-target effects.

[0040] In embodiments, the present invention relates to a novel endopeptidase composition made using the methods described herein. In embodiments, the present invention relates to a method for making the described novel endopeptidase composition using the methods set forth herein.

[0041] Novel Binding Proteins

[0042] The present invention relates to novel binding proteins, and compositions thereof. In embodiments, interaction of a novel binding protein of the present invention with a target site of a GI tract target results in a reduction of activity of the GI tract target. In embodiments, a novel binding protein comprises at least one binding epitope that specifically binds to a target binding site of a GI tract target.

[0043] A novel binding protein can be made by de novo design, or by optimizing an existing, starting protein to bind to a GI tract target. In embodiments, the novel binding protein binds to a GI tract target at a site on the GI tract target that is involved in specific binding of the GI tract target to a GI tract target binding partner. In embodiments, the novel binding protein comprises at least one binding epitope that specifically binds to a target binding site of a GI tract target that is involved in specific binding of the GI tract target to a GI tract target binding partner. In embodiments, the binding epitope of the novel binding protein comprises all or part of a binding domain or region of the GI tract target binding partner. For example, a novel binding protein can be modeled, derived, or made from an amino acid sequence known to be or to mimic a site on the GI tract binding partner that binds to or otherwise interacts with the GI tract target.

[0044] The starting protein can be optimized to a novel binding protein that binds to the target site of the GI tract target as desired, e.g., using the Rosetta Molecular Modeling Suite to model a binding site of the binding protein as described herein. Candidate optimized binding proteins can be constructed and evaluated for binding activity, and the preferred candidate selected for further characterization. For example, after a binding protein is designed using Rosetta, it may be optimized by selecting the most active candidate(s) and performing additional optimization by Rosetta and/or site saturation mutagenesis at selected residues for increased performance.

[0045] A novel binding protein can be selected from among candidate optimized binding proteins based on any desirable parameter, e.g., high binding specificity, high affinity, stability, a reduction in an activity of the GI tract target, and a combination thereof. The selection can be made using any appropriate method, e.g., any protein-ligand binding analysis method known to those of skill in the art. In embodiments, the candidate optimized binding protein or a selected novel binding protein is evaluated by a binding assay with the GI tract target. In embodiments, the candidate optimized binding protein or a selected novel binding protein is evaluated by its effect on a GI tract target activity. In embodiments, the candidate optimized binding protein or novel binding protein is evaluated relative to a positive or negative control protein. In embodiments, the control protein is the unoptimized starting protein, a different candidate binding protein, or a non-binding (nonspecific) control protein. In embodiments, a binding property is evaluated by comparing binding of the candidate optimized binding protein or a selected novel binding protein to a target site of a GI tract target with its binding to a control target. In embodiments, the control target is an unrelated protein or a variant GI tract target. In embodiments, the variant GI tract target differs from the non-variant GI tract target in the amino acid sequence at its target site.

[0046] In embodiments, the selectivity of the novel binding protein for the substrate, e.g., the target site of the GI tract target, relative to the selectivity of the novel binding protein for a nonspecific substrate, is increased by about 10 fold to about 2,000 fold. In embodiments, the selectivity of the novel binding protein for the substrate, e.g., the target site of the GI tract target, relative to the selectivity of the novel binding protein for a nonspecific substrate, is increased by about 10 fold to about 20 fold, about 10 fold to about 25 fold, about 10 fold to about 50 fold, about 10 fold to about 75 fold, about 10 fold to about 100 fold, about 10 fold to about 150 fold, about 10 fold to about 250 fold, about 10 fold to about 500 fold, about 10 fold to about 750 fold, about 10 fold to about 1,000 fold, about 10 fold to about 2,000 fold, about 20 fold to about 25 fold, about 20 fold to about 50 fold, about 20 fold to about 75 fold, about 20 fold to about 100 fold, about 20 fold to about 150 fold, about 20 fold to about 250 fold, about 20 fold to about 500 fold, about 20 fold to about 750 fold, about 20 fold to about 1,000 fold, about 20 fold to about 2,000 fold, about 25 fold to about 50 fold, about 25 fold to about 75 fold, about 25 fold to about 100 fold, about 25 fold to about 150 fold, about 25 fold to about 250 fold, about 25 fold to about 500 fold, about 25 fold to about 750 fold, about 25 fold to about 1,000 fold, about 25 fold to about 2,000 fold, about 50 fold to about 75 fold, about 50 fold to about 100 fold, about 50 fold to about 150 fold, about 50 fold to about 250 fold, about 50 fold to about 500 fold, about 50 fold to about 750 fold, about 50 fold to about 1,000 fold, about 50 fold to about 2,000 fold, about 75 fold to about 100 fold, about 75 fold to about 150 fold, about 75 fold to about 250 fold, about 75 fold to about 500 fold, about 75 fold to about 750 fold, about 75 fold to about 1,000 fold, about 75 fold to about 2,000 fold, about 100 fold to about 150 fold, about 100 fold to about 250 fold, about 100 fold to about 500 fold, about 100 fold to about 750 fold, about 100 fold to about 1,000 fold, about 100 fold to about 2,000 fold, about 150 fold to about 250 fold, about 150 fold to about 500 fold, about 150 fold to about 750 fold, about 150 fold to about 1,000 fold, about 150 fold to about 2,000 fold, about 250 fold to about 500 fold, about 250 fold to about 750 fold, about 250 fold to about 1,000 fold, about 250 fold to about 2,000 fold, about 500 fold to about 750 fold, about 500 fold to about 1,000 fold, about 500 fold to about 2,000 fold, about 750 fold to about 1,000 fold, about 750 fold to about 2,000 fold, or about 1,000 fold to about 2,000 fold. In embodiments, the selectivity of the novel binding protein for the substrate, e.g., the target site of the GI tract target, relative to the selectivity of the novel binding protein for a nonspecific substrate, is increased by about 10 fold, about 20 fold, about 25 fold, about 50 fold, about 75 fold, about 100 fold, about 150 fold, about 250 fold, about 500 fold, about 750 fold, about 1,000 fold, or about 2,000 fold. In embodiments, the selectivity of the novel binding protein for the substrate, e.g., the target site of the GI tract target, relative to the selectivity of the novel binding protein for a nonspecific substrate, is increased by at least about 10 fold, about 20 fold, about 25 fold, about 50 fold, about 75 fold, about 100 fold, about 150 fold, about 250 fold, about 500 fold, about 750 fold, or about 1,000 fold. In embodiments, the selectivity of the novel binding protein for the substrate, e.g., the target site of the GI tract target, relative to the selectivity of the novel binding protein for a nonspecific substrate, is increased by at most about 20 fold, about 25 fold, about 50 fold, about 75 fold, about 100 fold, about 150 fold, about 250 fold, about 500 fold, about 750 fold, about 1,000 fold, or about 2,000 fold.

[0047] In embodiments, the selectivity of the novel binding protein for the substrate, e.g., the target site of the GI tract target, relative to the selectivity of the novel binding protein for the same substrate, is increased by about 10 fold to about 2,000 fold. In embodiments, the selectivity of the novel binding protein for the substrate, e.g., the target site of the GI tract target, relative to the selectivity of the novel binding protein for the same substrate, is increased by about 10 fold to about 20 fold, about 10 fold to about 25 fold, about 10 fold to about 50 fold, about 10 fold to about 75 fold, about 10 fold to about 100 fold, about 10 fold to about 150 fold, about 10 fold to about 250 fold, about 10 fold to about 500 fold, about 10 fold to about 750 fold, about 10 fold to about 1,000 fold, about 10 fold to about 2,000 fold, about 20 fold to about 25 fold, about 20 fold to about 50 fold, about 20 fold to about 75 fold, about 20 fold to about 100 fold, about 20 fold to about 150 fold, about 20 fold to about 250 fold, about 20 fold to about 500 fold, about 20 fold to about 750 fold, about 20 fold to about 1,000 fold, about 20 fold to about 2,000 fold, about 25 fold to about 50 fold, about 25 fold to about 75 fold, about 25 fold to about 100 fold, about 25 fold to about 150 fold, about 25 fold to about 250 fold, about 25 fold to about 500 fold, about 25 fold to about 750 fold, about 25 fold to about 1,000 fold, about 25 fold to about 2,000 fold, about 50 fold to about 75 fold, about 50 fold to about 100 fold, about 50 fold to about 150 fold, about 50 fold to about 250 fold, about 50 fold to about 500 fold, about 50 fold to about 750 fold, about 50 fold to about 1,000 fold, about 50 fold to about 2,000 fold, about 75 fold to about 100 fold, about 75 fold to about 150 fold, about 75 fold to about 250 fold, about 75 fold to about 500 fold, about 75 fold to about 750 fold, about 75 fold to about 1,000 fold, about 75 fold to about 2,000 fold, about 100 fold to about 150 fold, about 100 fold to about 250 fold, about 100 fold to about 500 fold, about 100 fold to about 750 fold, about 100 fold to about 1,000 fold, about 100 fold to about 2,000 fold, about 150 fold to about 250 fold, about 150 fold to about 500 fold, about 150 fold to about 750 fold, about 150 fold to about 1,000 fold, about 150 fold to about 2,000 fold, about 250 fold to about 500 fold, about 250 fold to about 750 fold, about 250 fold to about 1,000 fold, about 250 fold to about 2,000 fold, about 500 fold to about 750 fold, about 500 fold to about 1,000 fold, about 500 fold to about 2,000 fold, about 750 fold to about 1,000 fold, about 750 fold to about 2,000 fold, or about 1,000 fold to about 2,000 fold. In embodiments, the selectivity of the novel binding protein for the substrate, e.g., the target site of the GI tract target, relative to the selectivity of the novel binding protein for the same substrate, is increased by about 10 fold, about 20 fold, about 25 fold, about 50 fold, about 75 fold, about 100 fold, about 150 fold, about 250 fold, about 500 fold, about 750 fold, about 1,000 fold, or about 2,000 fold.

In embodiments, the selectivity of the novel binding protein for the substrate, e.g., the target site of the GI tract target, relative to the selectivity of the unoptimized endopeptidase for the same substrate, is increased by at least about 10 fold, about 20 fold, about 25 fold, about 50 fold, about 75 fold, about 100 fold, about 150 fold, about 250 fold, about 500 fold, about 750 fold, or about 1,000 fold. In embodiments, the selectivity of the novel binding protein for the substrate, e.g., the target site of the GI tract target, relative to the selectivity of the novel binding protein for the same substrate, is increased by at most about 20 fold, about 25 fold, about 50 fold, about 75 fold, about 100 fold, about 150 fold, about 250 fold, about 500 fold, about 750 fold, about 1,000 fold, or about 2,000 fold.

[0048] In embodiments, the novel binding protein has or is selected based on an activity, e.g., a binding efficiency and/or a binding affinity. In embodiments, the binding efficiency is 75 % to 100 %, that is, 75 % to 100 % of the GI tract target proteins present (e.g., in a tested sample) are bound to the novel binding protein at their target site. In embodiments, the novel binding protein is selected based on a GI tract target binding efficiency of about 75 % to about 80 %, about 75 % to about 85 %, about 75 % to about 90 %, about 75 % to about 93 %, about 75 % to about 94 %, about 75 % to about 95 %, about 75 % to about 96 %, about 75 % to about 96 %, about 75 % to about 98 %, about 75 % to about 99 %, about 75 % to about 100 %, about 80 % to about 85 %, about 80 % to about 90 %, about 80 % to about 93 %, about 80 % to about 94 %, about 80 % to about 95 %, about 80 % to about 96 %, about 80 % to about 96 %, about 80 % to about 98 %, about 80 % to about 99 %, about 80 % to about 100 %, about 85 % to about 90 %, about 85 % to about 93 %, about 85 % to about 94 %, about 85 % to about 95 %, about 85 % to about 96 %, about 85 % to about 96 %, about 85 % to about 98 %, about 85 % to about 99 %, about 85 % to about 100 %, about 90 % to about 93 %, about 90 % to about 94 %, about 90 % to about 95 %, about 90 % to about 96 %, about 90 % to about 96 %, about 90 % to about 98 %, about 90 % to about 99 %, about 90 % to about 100 %, about 93 % to about 94 %, about 93 % to about 95 %, about 93 % to about 96 %, about 93 % to about 96 %, about 93 % to about 98 %, about 93 % to about 99 %, about 93 % to about 100 %, about 94 % to about 95 %, about 94 % to about 96 %, about 94 % to about 96 %, about 94 % to about 98 %, about 94 % to about 99 %, about 94 % to about 100 %, about 95 % to about 96 %, about 95 % to about 96 %, about 95 % to about 98 %, about 95 % to about 99 %, about 95 % to about 100 %, about 96 % to about 96 %, about 96 % to about 98 %, about 96 % to about 99 %, about 96 % to about 100 %, about 96 % to about 98 %, about 96 % to about 99 %, about 96 % to about 100 %, about 98 % to about 99 %, about 98 % to about 100 %, or about 99 % to about 100 %. In embodiments, the novel binding protein is selected based on GI tract target binding efficiency of about 75 %, about 80 %, about 85 %, about 90 %, about 93 %, about 94 %, about 95 %, about 96 %, about 96 %, about 98 %, about 99 %, or about 100 %. In embodiments, the novel binding protein is selected based on GI tract target binding efficiency of at least about 75 %, about 80 %, about 85 %, about 90 %, about 93 %, about 94 %, about 95 %, about 96 %, about 96 %, about 98 %, or about 99 %. In embodiments, the novel binding protein is selected based on GI tract target protein binding efficiency of at most about 80 %, about 85 %, about 90 %, about 93 %, about 94 %, about 95 %, about 96 %, about 96 %, about 98 %, about 99 %, or about 100 %. In embodiments, the novel binding protein has, or is selected based on, a binding affinity to a GI tract target that is greater than the binding affinity of a control to the GI tract target by about 1.5 fold to about 10 fold. In embodiments, the novel binding protein has, or is selected based on, a binding affinity to a GI tract target that is greater than the binding affinity of a negative control to the GI tract target by about 1.5 fold to about 2 fold, about 1.5 fold to about 2.5 fold, about 1.5 fold to about 3 fold, about 1.5 fold to about 3.5 fold, about 1.5 fold to about 4 fold, about 1.5 fold to about 5 fold, about 1.5 fold to about 6 fold, about 1.5 fold to about 7 fold, about 1.5 fold to about 8 fold, about 1.5 fold to about 9 fold, about 1.5 fold to about 10 fold, about 2 fold to about 2.5 fold, about 2 fold to about 3 fold, about 2 fold to about 3.5 fold, about 2 fold to about 4 fold, about 2 fold to about 5 fold, about 2 fold to about 6 fold, about 2 fold to about 7 fold, about 2 fold to about 8 fold, about 2 fold to about 9 fold, about 2 fold to about 10 fold, about 2.5 fold to about 3 fold, about 2.5 fold to about 3.5 fold, about 2.5 fold to about 4 fold, about 2.5 fold to about 5 fold, about 2.5 fold to about 6 fold, about 2.5 fold to about 7 fold, about 2.5 fold to about 8 fold, about

2.5 fold to about 9 fold, about 2.5 fold to about 10 fold, about 3 fold to about 3.5 fold, about 3 fold to about 4 fold, about 3 fold to about 5 fold, about 3 fold to about 6 fold, about 3 fold to about 7 fold, about 3 fold to about 8 fold, about 3 fold to about 9 fold, about 3 fold to about 10 fold, about 3.5 fold to about 4 fold, about 3.5 fold to about 5 fold, about 3.5 fold to about 6 fold, about 3.5 fold to about 7 fold, about 3.5 fold to about 8 fold, about 3.5 fold to about 9 fold, about

3.5 fold to about 10 fold, about 4 fold to about 5 fold, about 4 fold to about 6 fold, about 4 fold to about 7 fold, about 4 fold to about 8 fold, about 4 fold to about 9 fold, about 4 fold to about 10 fold, about 5 fold to about 6 fold, about 5 fold to about 7 fold, about 5 fold to about 8 fold, about 5 fold to about 9 fold, about 5 fold to about 10 fold, about 6 fold to about 7 fold, about 6 fold to about 8 fold, about 6 fold to about 9 fold, about 6 fold to about 10 fold, about 7 fold to about 8 fold, about 7 fold to about 9 fold, about 7 fold to about 10 fold, about 8 fold to about 9 fold, about 8 fold to about 10 fold, or about 9 fold to about 10 fold. In embodiments, the novel binding protein has, or is selected based on, a binding affinity to a GI tract target that is greater than the binding affinity of a control to the GI tract target by about 1.5 fold, about 2 fold, about

2.5 fold, about 3 fold, about 3.5 fold, about 4 fold, about 5 fold, about 6 fold, about 7 fold, about

8 fold, about 9 fold, or about 10 fold. In embodiments, the novel binding protein has, or is selected based on, a binding affinity to a GI tract target that is greater than the binding affinity of a control to the GI tract target by at least about 1.5 fold, about 2 fold, about 2.5 fold, about 3 fold, about 3.5 fold, about 4 fold, about 5 fold, about 6 fold, about 7 fold, about 8 fold, or about

9 fold. In embodiments, the novel binding protein has, or is selected based on, a binding affinity to a GI tract target that is greater than the binding affinity of a control to the GI tract target by at most about 2 fold, about 2.5 fold, about 3 fold, about 3.5 fold, about 4 fold, about 5 fold, about 6 fold, about 7 fold, about 8 fold, about 9 fold, or about 10 fold. In embodiments, the control is a nonspecific protein or peptide.

[0049] A novel binding protein can be evaluated based on an activity of the novel binding protein. The activity can be the effect on a GI tract target, e.g., following contact with the novel binding protein, or administration to a patient infected with a microbe producing the GI tract target. The activity of the novel binding protein can be selected from: binding specificity and/or affinity to the GI tract target; reduction in binding of the GI tract target to its receptor; reduction in GI tract target enzyme activity; reduction in GI tract target membrane translocation; reduction in GI tract target-induced cytopathicity; and any combination thereof. In embodiments, the GI tract target is C. diff. TcdA or TcdB, and the novel binding protein is evaluated based on an activity selected from: binding specificity and/or affinity to TcdA and/or TcdB; reduction in binding of TcdA and/or TcdB to a receptor; reduction in TcdA and/or TcdB glucosyl transferase activity; reduction in TcdA and/or TcdB membrane translocation; reduction in TcdA and/or TcdB-induced cytopathicity; and any combination thereof. In embodiments, the activity of the novel binding protein relative to that of a control, e.g., a different binding protein specific for the GI target, a nonspecific binding protein, no treatment, etc., is increased by about 1.5-fold to about 5000-fold. In embodiments, the activity of the novel binding protein is increased by about

1.5 fold to about 1,000 fold. In embodiments, the activity of the novel binding protein is increased by about 1.5 fold to about 2.5 fold, about 1.5 fold to about 5 fold, about 1.5 fold to about 10 fold, about 1.5 fold to about 25 fold, about 1.5 fold to about 50 fold, about 1.5 fold to about 75 fold, about 1.5 fold to about 100 fold, about 1.5 fold to about 250 fold, about 1.5 fold to about 500 fold, about 1.5 fold to about 750 fold, about 1.5 fold to about 1,000 fold, about 2.5 fold to about 5 fold, about 2.5 fold to about 10 fold, about 2.5 fold to about 25 fold, about 2.5 fold to about 50 fold, about 2.5 fold to about 75 fold, about 2.5 fold to about 100 fold, about 2.5 fold to about 250 fold, about 2.5 fold to about 500 fold, about 2.5 fold to about 750 fold, about

2.5 fold to about 1,000 fold, about 5 fold to about 10 fold, about 5 fold to about 25 fold, about 5 fold to about 50 fold, about 5 fold to about 75 fold, about 5 fold to about 100 fold, about 5 fold to about 250 fold, about 5 fold to about 500 fold, about 5 fold to about 750 fold, about 5 fold to about 1,000 fold, about 10 fold to about 25 fold, about 10 fold to about 50 fold, about 10 fold to about 75 fold, about 10 fold to about 100 fold, about 10 fold to about 250 fold, about 10 fold to about 500 fold, about 10 fold to about 750 fold, about 10 fold to about 1,000 fold, about 25 fold to about 50 fold, about 25 fold to about 75 fold, about 25 fold to about 100 fold, about 25 fold to about 250 fold, about 25 fold to about 500 fold, about 25 fold to about 750 fold, about 25 fold to about 1,000 fold, about 50 fold to about 75 fold, about 50 fold to about 100 fold, about 50 fold to about 250 fold, about 50 fold to about 500 fold, about 50 fold to about 750 fold, about 50 fold to about 1,000 fold, about 75 fold to about 100 fold, about 75 fold to about 250 fold, about 75 fold to about 500 fold, about 75 fold to about 750 fold, about 75 fold to about 1,000 fold, about 100 fold to about 250 fold, about 100 fold to about 500 fold, about 100 fold to about 750 fold, about 100 fold to about 1,000 fold, about 250 fold to about 500 fold, about 250 fold to about 750 fold, about 250 fold to about 1,000 fold, about 500 fold to about 750 fold, about 500 fold to about 1,000 fold, or about 750 fold to about 1,000 fold. In embodiments, the activity of the novel binding protein is increased by about 1.5 fold, about 2.5 fold, about 5 fold, about 10 fold, about 25 fold, about 50 fold, about 75 fold, about 100 fold, about 250 fold, about 500 fold, about 750 fold, or about 1,000 fold. In embodiments, the activity of the novel binding protein is increased by about at least about 1.5 fold, about 2.5 fold, about 5 fold, about 10 fold, about 25 fold, about 50 fold, about 75 fold, about 100 fold, about 250 fold, about 500 fold, or about 750 fold. In embodiments, the activity of the novel binding protein is increased by about at most about 2.5 fold, about 5 fold, about 10 fold, about 25 fold, about 50 fold, about 75 fold, about 100 fold, about 250 fold, about 500 fold, about 750 fold, or about 1,000 fold. In embodiments, the activity of the novel binding protein is increased about 10 fold to about 5,000 fold. In embodiments, the activity of the novel binding protein is increased about 10 fold to about 20 fold, about 10 fold to about 50 fold, about 10 fold to about 100 fold, about 10 fold to about 150 fold, about 10 fold to about 200 fold, about 10 fold to about 250 fold, about 10 fold to about 500 fold, about 10 fold to about 750 fold, about 10 fold to about 1,000 fold, about 10 fold to about 2,500 fold, about 10 fold to about 4,000 fold, about 20 fold to about 50 fold, about 20 fold to about 100 fold, about 20 fold to about 150 fold, about 20 fold to about 200 fold, about 20 fold to about 250 fold, about 20 fold to about 500 fold, about 20 fold to about 750 fold, about 20 fold to about 1,000 fold, about 20 fold to about 2,500 fold, about 20 fold to about 4,000 fold, about 50 fold to about 100 fold, about 50 fold to about 150 fold, about 50 fold to about 200 fold, about 50 fold to about 250 fold, about 50 fold to about 500 fold, about 50 fold to about 750 fold, about 50 fold to about 1,000 fold, about 50 fold to about 2,500 fold, about 50 fold to about 4,000 fold, about 100 fold to about 150 fold, about 100 fold to about 200 fold, about 100 fold to about 250 fold, about 100 fold to about 500 fold, about 100 fold to about 750 fold, about 100 fold to about 1,000 fold, about 100 fold to about 2,500 fold, about 100 fold to about 4,000 fold, about 150 fold to about 200 fold, about 150 fold to about 250 fold, about 150 fold to about 500 fold, about 150 fold to about 750 fold, about 150 fold to about 1,000 fold, about 150 fold to about 2,500 fold, about 150 fold to about 4,000 fold, about 200 fold to about 250 fold, about 200 fold to about 500 fold, about 200 fold to about 750 fold, about 200 fold to about 1,000 fold, about 200 fold to about 2,500 fold, about 200 fold to about 4,000 fold, about 250 fold to about 500 fold, about 250 fold to about 750 fold, about 250 fold to about 1,000 fold, about 250 fold to about 2,500 fold, about 250 fold to about 4,000 fold, about 500 fold to about 750 fold, about 500 fold to about 1,000 fold, about 500 fold to about 2,500 fold, about 500 fold to about 4,000 fold, about 750 fold to about 1,000 fold, about 750 fold to about 2,500 fold, about 750 fold to about 4,000 fold, about 1,000 fold to about 2,500 fold, about 1,000 fold to about 4,000 fold, or about 2,500 fold to about 4,000 fold. In embodiments, the activity of the novel binding protein is increased about 10 fold, about 20 fold, about 25 fold, about 50 fold, about 75 fold, about 100 fold, about 125 fold, about 150 fold, about 200 fold, about 250 fold, about 300 fold, about 400 fold, about 500 fold, about 600 fold, about 750 fold, about 1,000 fold, about 1,500 fold, about 2,500 fold, about 3000 fold, about 3,500 fold, about 4,000 fold, or about 5,000 fold. In embodiments, the activity of the novel binding protein is increased at least about 10 fold, about 20 fold, about 50 fold, about 100 fold, about 150 fold, about 200 fold, about 250 fold, about 500 fold, about 750 fold, about 1,000 fold, about 2,500 fold, or about 4,000 fold. In embodiments, the activity of the novel binding protein is increased at most about 20 fold, about 50 fold, about 100 fold, about 150 fold, about 200 fold, about 250 fold, about 500 fold, about 750 fold, about 1,000 fold, about 2,500 fold, about 4,000 fold, or about 5,000 fold.

[0050] In embodiments, a novel binding protein of the present invention binds to a GI tract target at a target site, also referred to herein as a target binding site. A target binding site refers to, e.g., the amino acid sequence or motif with which the novel binding protein interacts. In embodiments, the novel binding protein interacts with the target binding site covalently or noncovalently.

[0051] In embodiments, the target site binding reduces, blocks, or otherwise alters the interaction of the GI tract target with its binding partner (e.g., a cell surface receptor or other feature of a target cell or tissue) to result in a reduced activity of the GI tract target. In embodiments, binding of the novel binding protein to the target binding site induces conformational change of the GI tract target that results in a reduced activity of the GI tract target. In embodiments, the reduced activity is a reduction in any activity of the GI tract target, including, but not limited to, receptor signaling, cytopathicity, enzyme activity, and membrane translocation. In embodiments, the novel binding protein mimics the receptor active site. In embodiments, the novel binding protein does not itself bind to the receptor or any other structure or feature of a target cell or tissue. In embodiments, the novel binding protein does not induce off-target effects. In embodiments, a novel binding protein and novel endopeptidase are used together to target different target binding or cleavage sites of a GI tract target.

[0052] As described, a novel antagonist of the present invention, e.g., a novel endopeptidase or novel binding protein, can reduce the activity of a GI tract target as described herein. In embodiments, the GI tract target activity is: binding of the GI tract target to its receptor; reduction in GI tract target enzyme activity; reduction in GI tract target membrane translocation; reduction in GI tract target-induced cytopathicity; or any combination thereof. In embodiments, the GI tract target is C. diff. TcdA or TcdB, and the GI tract target activity is: reduction in binding of TcdA and/or TcdB to a receptor; reduction in TcdA and/or TcdB glucosyl transferase activity; reduction in TcdA and/or TcdB membrane translocation; reduction in TcdA and/or TcdB-induced cytopathicity; or any combination thereof. [0053] The novel antagonist can reduce the activity of the GI tract target by about 25% to about 100%. In any embodiment described herein, the novel antagonist can reduce the activity of the GI tract target by about 25% to about 100%. In any embodiment described herein, the novel antagonist can reduce the activity of the GI tract target by about 25% to about 40%, about 25% to about 50%, about 25% to about 60%, about 25% to about 65%, about 25% to about 70%, about 25% to about 75%, about 25% to about 80%, about 25% to about 85%, about 25% to about 90%, about 25% to about 95%, about 25% to about 100%, about 40% to about 50%, about 40% to about 60%, about 40% to about 65%, about 40% to about 70%, about 40% to about 75%, about 40% to about 80%, about 40% to about 85%, about 40% to about 90%, about 40% to about 95%, about 40% to about 100%, about 50% to about 60%, about 50% to about 65%, about 50% to about 70%, about 50% to about 75%, about 50% to about 80%, about 50% to about 85%, about 50% to about 90%, about 50% to about 95%, about 50% to about 100%, about 60% to about 65%, about 60% to about 70%, about 60% to about 75%, about 60% to about 80%, about 60% to about 85%, about 60% to about 90%, about 60% to about 95%, about 60% to about 100%, about 65% to about 70%, about 65% to about 75%, about 65% to about 80%, about 65% to about 85%, about 65% to about 90%, about 65% to about 95%, about 65% to about 100%, about 70% to about 75%, about 70% to about 80%, about 70% to about 85%, about 70% to about 90%, about 70% to about 95%, about 70% to about 100%, about 75% to about 80%, about 75% to about 85%, about 75% to about 90%, about 75% to about 95%, about 75% to about 100%, about 80% to about 85%, about 80% to about 90%, about 80% to about 95%, about 80% to about 100%, about 85% to about 90%, about 85% to about 95%, about 85% to about 100%, about 90% to about 95%, about 90% to about 100%, or about 95% to about 100%. In any embodiment described herein, the novel antagonist can reduce the activity of the GI tract target by about 25%, about 40%, about 50%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%. In any embodiment described herein, the novel antagonist can reduce the activity of the GI tract target by at least about 25%, about 40%, about 50%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95%. In any embodiment described herein, the novel antagonist can reduce the activity of the GI tract target by at most about 40%, about 50%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%.

[0054] In any embodiment described herein, the reduction in the activity of the GI tract target by the novel antagonist is determined by comparison to a control, including but not limited to, a nonspecific antagonist or any available existing antagonist of the GI tract target, or to the absence of the novel antagonist. A control as described herein is understood to include any suitable positive or negative control. In embodiments, the control is another novel antagonist. In embodiments, a control compared with a novel endopeptidase of the invention is another endopeptidase, e.g., an unoptimized, starting enzyme, another candidate optimized enzyme, or another positive or negative control enzyme or protein, used on the same substrate (e.g, a desired target site of a GI tract target). In embodiments, a control compared with a novel binding protein of the invention is another binding protein, e.g., an unoptimized, starting binding protein, another candidate binding protein, or another positive or negative control binding protein, used on the same substrate (e.g, a desired target site of a GI tract target). In embodiments, the control is a comparison of the activity of a novel antagonist of the invention on a nonspecific substrate. In embodiments, the control substrate or target is an unrelated protein or a variant GI tract target. In embodiments, the control is no treatment.

[0055] In embodiments, the present invention relates to a novel binding protein composition made using the methods described herein. In embodiments, the present invention relates to a method for making the described novel binding protein composition using the methods set forth herein.

[0056] Novel Antagonist Identification

[0057] Computational design can be used to generate candidate antagonists for evaluation as novel antagonists of the invention. For example, theoretical mutations of an existing protein can be generated using protein design software Rosetta Molecular Modeling Suite. Candidates can be selected based on a reduction in the overall energy of the new endopeptidase or binding protein-target sequence substrate complex relative to the starting endopeptidase or binding protein’s native substrate, e.g., based on an increase of not more than 1-5 Rosetta energy units. Methods for optimizing a protein, e.g., an enzyme, using such methods, as well as for evaluating its activity and specificity, are described, e.g., by Wolf et al., 2015 Oct 14, “Engineering of Kuma030: A Gliadin Peptidase That Rapidly Degrades Immunogenic Gliadin Peptides in Gastric Conditions,” J Am Chem Soc. 137(40): 13106-13. doi: 10.1021/jacs.5b08325, Epub 2015 Sep 29. PMID: 26374198; PMCID: PMC4958374, U.S. Pat. No. 10,793,846, “Compositions and methods for treating celiac sprue disease,” U.S. Pat. No. 10,149,892, “Compositions and methods for treating celiac sprue disease,” Bender, et al., 2016, “Protocols for Molecular Modeling with Rosetta3 and RosettaScripts,” Biochemistry 55: 4748-4763, and Kaufmann, et al., 2010, “Practically Useful: What the ROSETTA Protein Modeling Suite Can Do for You,” Biochemistry 49: 2987-2998, each incorporated by reference herein.

[0058] In embodiments, a novel antagonist of the invention is identified by de novo protein design. Methods can be used to generate small stable proteins with shapes customized to bind therapeutic targets, as described in the literature, e.g., in U.S. Pat. No. 9,771,395, “De novo designed hemagglutinin binding proteins,” and by Chevalier, A., et al., 2017, “Massively parallel de novo protein design for targeted therapeutics,” Nature 550(7674): 74-79, each incorporated by reference herein in its entirety. These publications report a massively parallel approach for designing, manufacturing and screening de novo mini-protein binders using Rosetta in an integrated computational and experimental approach. As described therein, protein scaffolds of varying shapes are designed, docked onto the target, and the residues at the interface for high affinity binding are optimized and screened. Computational design strategy for high-affinity binding proteins is further described, e.g., by Fleishman, S. J. et al., 2011, May 13 “Computational design of proteins targeting the conserved stem region of influenza hemagglutinin,” Science 332(6031): 816-821, incorporated herein by reference. The authors report engineering high-affinity interactions and high shape complementarity into scaffold proteins in two steps: (i) disembodied amino-acid residues are computationally docked or positioned against the target surface to identify energetically favorable configurations with the target surface; and (ii) shape-complementary configurations of scaffold proteins are computed that incorporate the key residues.

[0059] In embodiments, the structure of the target site of the GI tract target is modeled using Rosetta, to further assist with the production of candidates for evaluation as novel antagonists.

[0060] Amino Acid Substitutions

[0061] In embodiments, the present invention includes novel antagonists identified as described herein wherein any amino acid, including any amino acid in an active site of the antagonist, is conservatively substituted, alternatively substituted, or preserved. In embodiments, one or more of the amino acids in the active site (responsible for proteolysis) are preserved. In embodiments, all amino acids in the active site are preserved. Conservative substitutions and alternative substitutions, include, but are not limited to, any known to those of skill in the art, e.g., those described herein. In embodiments, the novel antagonist comprising such an amino acid substitution retains substantially the same level of activity as the unsubstituted novel antagonist.

[0062] Amino acids can be classified based on chemical and structural properties of their sidechains, for example, naturally-occurring amino acids can be classified as hydrophobic (norleucine, Met, Ala, Val, Leu, and He), neutral hydrophilic (Cys, Ser, Thr, Asn, and Gin), acidic (Asp and Glu), basic (His, Lys, and Arg), chain orienting (Gly and Pro), and aromatic (Trp, Tyr, and Phe).

[0063] In some embodiments, a conservative amino acid substitution is made by substituting an amino acid of one of the above classes with a different member of that class. In some embodiments, conservative substitutions encompass non-naturally occurring amino acid residues, including peptidomimetics and other reversed or inverted forms of amino acid moieties.

[0064] In some embodiments, a non-conservative substitution is made by substituting an amino acid of one of the above classes with a member of a different class.

[0065] In some embodiments, substitution takes into account the hydropathic index of an amino acid (see, e.g., Kyte et al., 1982, J. Mol. Biol. 157:105-131, incorporated herein by reference). The hydropathic profile of a peptide can be calculated by giving each amino acid a numerical value, or hydropathy index, and repetitively averaging these values along the peptide chain. In such embodiments, each amino acid is assigned a hydropathic index based on hydrophobicity and charge characteristics. In some embodiments, the hydropathic indices used are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5). In some embodiments, an amino acid is substituted with a different amino acid having a hydropathic index within 0.1 to 0.5 of the original amino acid. In some embodiments, the hydropathic index is within 0.1, 0.2, 0.3, 0.4, or 0.5 of the original amino acid.

[0066] In some embodiments, amino acid substitutions are be made based on hydrophilicity. In some embodiments, the hydrophilicity values used are: arginine (+3.0); lysine (+3.0); aspartate (+3.0.+-.1); glutamate (+3.0.+-.1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5.+-.1); alanine (-0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (- 2.5) and tryptophan (-3.4).

[0067] In some embodiments, an amino acid is substituted with a different amino acid having a hydrophilicity value within 0.1 to 0.5 of the original amino acid. In some embodiments, the hydrophilicity value is within 0.1, 0.2, 0.3, 0.4, or 0.5 of the original amino acid.

[0068] In some embodiments, an amino acid is substituted as shown in the table below. In some embodiments, an amino acid is replaced with a conservative substitution as set forth in Table 2(1), or a derivative (also referred to as an analog herein) of a conservative substitution. In some embodiments, an amino acid is replaced with an alternative substitution as set forth in Table 2(11), showing each full list of alternatives for each amino acid, or a derivative of an alternative substitution. [0069] Table 2. Amino Acid Substitutions

[0070] Identification and Evaluation of Novel Antagonists

[0071] As described, a novel antagonist of the invention can be designed from an existing original or starting protein, e.g., a starting endopeptidase or starting binding protein, using computational modeling tools to optimize it toward the desired oligopeptide specificity. A starting protease or binding protein may have one or more characteristics desired in the novel endopeptidase or novel binding protein. In embodiments, a novel antagonist of the invention has optimal activity in the GI tract. In embodiments, the novel antagonist, starting protease or starting binding protein is active at a pH characteristic of a region of the GI tract. In embodiments, the novel antagonist, starting protease or starting binding protein is active at a pH at which the GI tract target is active. In embodiments, the novel antagonist, starting protease or starting binding protein is active at physiological temperature. In embodiments, the novel antagonist is active at a pH characteristic of a region of the GI tract, is active at a pH at which the GI tract target is active, is active at physiological temperature, or any combination thereof. The novel antagonist, e.g., novel endopeptidase or novel binding protein, can be identified as active based on its ability to carry out an intended function, for example, at a desired level relative to a control. In embodiments, a novel endopeptidase is identified as active based on: GI tract target cleavage by the novel endopeptidase; reduction in binding of a GI tract target to a receptor; reduction in GI tract target enzyme activity; reduction in GI tract target membrane translocation; reduction in GI tract target-induced cytopathicity; and any combination thereof.

In embodiments, the GI tract target is C. diff. TcdA or TcdB, and the novel endopeptidase is identified as active based on: TcdA or TcdB cleavage by the novel endopeptidase; reduction in binding of TcdA or TcdB to a receptor; reduction in TcdA or TcdB glucosyl transferase activity; reduction in GI tract target membrane translocation; reduction in GI tract target-induced cytopathicity; and any combination thereof.

[0072] In embodiments, a novel binding protein is identified as active based on: its binding specificity and/or affinity to the GI tract target; reduction in binding of a GI tract target to its receptor; reduction in GI tract target enzyme activity; reduction in GI tract target membrane translocation; reduction in GI tract target-induced cytopathicity; and any combination thereof.

In embodiments, the GI tract target is C. diff. TcdA or TcdB, and the novel binding protein is identified as active based on: its binding specificity and/or affinity to TcdA or TcdB; reduction in binding of TcdA or TcdB to a receptor; reduction in TcdA or TcdB glucosyl transferase activity; reduction in TcdA or TcdB membrane translocation; reduction in TcdA or TcdB- induced cytopathicity; and any combination thereof.

[0073] In embodiments, the novel antagonist is hyperstable. In embodiments, it is hyperstable to thermal denaturation, chemical denaturation, or both. Thermal or chemical denaturation can be carried out by any known method, e.g., by heating, e.g., from 25° C to 95° C, and returning to 25° C, or by incubation with any suitable denaturating agent, e.g., urea or guanidinium chloride. Secondary structure is monitored using any appropriate method, e.g., circular dichroism spectroscopy. Hyperstability can be indicated based on a threshold decrease in secondary structure under such conditions, e.g., less than 50%. In embodiments, a hyperstable novel antagonist recovers its secondary structure once the temperature or denaturant concentration is returned to the original level.

[0074] In embodiments, the novel antagonist, e.g., novel endopeptidase or novel binding protein, is about 25 to about 500 amino acids in length. In embodiments, the length of the novel antagonist, e.g., novel endopeptidase or novel binding protein, is about 25 amino acids to about 500 amino acids. In embodiments, the length of the novel antagonist, e.g., novel endopeptidase or novel binding protein, is about 25 amino acids to about 50 amino acids, about 25 amino acids to about 75 amino acids, about 25 amino acids to about 100 amino acids, about 25 amino acids to about 125 amino acids, about 25 amino acids to about 150 amino acids, about 25 amino acids to about 175 amino acids, about 25 amino acids to about 200 amino acids, about 25 amino acids to about 250 amino acids, about 25 amino acids to about 300 amino acids, about 25 amino acids to about 400 amino acids, about 25 amino acids to about 500 amino acids, about 50 amino acids to about 75 amino acids, about 50 amino acids to about 100 amino acids, about 50 amino acids to about 125 amino acids, about 50 amino acids to about 150 amino acids, about 50 amino acids to about 175 amino acids, about 50 amino acids to about 200 amino acids, about 50 amino acids to about 250 amino acids, about 50 amino acids to about 300 amino acids, about 50 amino acids to about 400 amino acids, about 50 amino acids to about 500 amino acids, about 75 amino acids to about 100 amino acids, about 75 amino acids to about 125 amino acids, about 75 amino acids to about 150 amino acids, about 75 amino acids to about 175 amino acids, about 75 amino acids to about 200 amino acids, about 75 amino acids to about 250 amino acids, about 75 amino acids to about 300 amino acids, about 75 amino acids to about 400 amino acids, about 75 amino acids to about 500 amino acids, about 100 amino acids to about 125 amino acids, about 100 amino acids to about 150 amino acids, about 100 amino acids to about 175 amino acids, about 100 amino acids to about 200 amino acids, about 100 amino acids to about 250 amino acids, about 100 amino acids to about 300 amino acids, about 100 amino acids to about 400 amino acids, about 100 amino acids to about 500 amino acids, about 125 amino acids to about 150 amino acids, about 125 amino acids to about 175 amino acids, about 125 amino acids to about 200 amino acids, about 125 amino acids to about 250 amino acids, about 125 amino acids to about 300 amino acids, about 125 amino acids to about 400 amino acids, about 125 amino acids to about 500 amino acids, about 150 amino acids to about 175 amino acids, about 150 amino acids to about 200 amino acids, about 150 amino acids to about 250 amino acids, about 150 amino acids to about 300 amino acids, about 150 amino acids to about 400 amino acids, about 150 amino acids to about 500 amino acids, about 175 amino acids to about 200 amino acids, about 175 amino acids to about 250 amino acids, about 175 amino acids to about 300 amino acids, about 175 amino acids to about 400 amino acids, about 175 amino acids to about 500 amino acids, about 200 amino acids to about 250 amino acids, about 200 amino acids to about 300 amino acids, about 200 amino acids to about 400 amino acids, about 200 amino acids to about 500 amino acids, about 250 amino acids to about 300 amino acids, about 250 amino acids to about 400 amino acids, about 250 amino acids to about 500 amino acids, about 300 amino acids to about 400 amino acids, about 300 amino acids to about 500 amino acids, or about 400 amino acids to about 500 amino acids. In embodiments, the length of the novel antagonist, e.g., novel endopeptidase or novel binding protein, is about 25 amino acids, about 50 amino acids, about 75 amino acids, about 100 amino acids, about 125 amino acids, about 150 amino acids, about 175 amino acids, about 200 amino acids, about 250 amino acids, about 300 amino acids, about 400 amino acids, or about 500 amino acids. In embodiments, the length of the novel antagonist, e.g., novel endopeptidase or novel binding protein, is at least about 25 amino acids, about 50 amino acids, about 75 amino acids, about 100 amino acids, about 125 amino acids, about 150 amino acids, about 175 amino acids, about 200 amino acids, about 250 amino acids, about 300 amino acids, or about 400 amino acids. In embodiments, the length of the novel antagonist, e.g., novel endopeptidase or novel binding protein, is at most about 50 amino acids, about 75 amino acids, about 100 amino acids, about 125 amino acids, about 150 amino acids, about 175 amino acids, about 200 amino acids, about 250 amino acids, about 300 amino acids, about 400 amino acids, or about 500 amino acids.

[0075] The present invention further relates to a nucleic acid encoding a novel antagonist of the invention, and to an expression vector comprising the nucleic acid for producing the novel antagonist.

[0076] In embodiments, the novel antagonist is easily produced. In embodiments, the produced novel antagonist is easily purified. In embodiments, the novel antagonist is manufacturable in large amounts. In embodiments, the novel antagonist is manufacturable at low cost. In embodiments, the novel antagonist is manufacturable quickly. In embodiments, the novel antagonist is manufacturable in a protein expression system. In embodiments, novel antagonist is easily produced, easily purified, manufacturable in large amounts, manufacturable at low cost, manufacturable quickly, manufacturable in a protein expression system, or any combination thereof.

[0077] In embodiments, the protein expression system is any protein expression system known in the art. In embodiments, the protein expression system is a bacteria, yeast, plant, insect, algal, cell free, or mammalian expression system. In embodiments, the protein expression system is a microbial expression system. In embodiments, the protein expression system is a prokaryotic or eukaryotic expression system. In embodiments, the protein expression system is a Gram- positive bacterial expression system. In embodiments, the bacterial expression system is E. coli, Bacillus , Lactobacillus , Streptomyces, or Pseudomonad expression system. In embodiments, the Pseudomonad expression system is a Pseudomonas expression system. In embodiments, the Pseudomonad expression system is a P.fluorescens expression system. In embodiments, the protein is expressed in Chinese hamster ovary (CHO) cells.

[0078] In embodiments, the novel antagonist, starting protease or starting binding protein is active at a pH of a GI tract compartment of a subject. In embodiments, the GI tract compartment in which the novel antagonist, starting protease or starting binding protein is active is the mouth, esophagus, stomach, small intestine, colon, rectum, or a combination thereof. In embodiments, the novel antagonist, starting protease or starting binding protein is active at a colonic pH, e.g., within a range of about pH 5 to about pH 8.

[0079] In embodiments, the novel antagonist, starting protease or starting binding protein is active at an esophageal pH of about 3.5 to about 7.4. In embodiments, the novel antagonist, starting protease or starting binding protein is active at an esophageal pH of about 3.5 to about 4, about 3.5 to about 4.5, about 3.5 to about 5, about 3.5 to about 5.5, about 3.5 to about 6, about

3.5 to about 6.5, about 3.5 to about 7, about 3.5 to about 7.1, about 3.5 to about 7.2, about 3.5 to about 7.3, about 3.5 to about 7.4, about 4 to about 4.5, about 4 to about 5, about 4 to about 5.5, about 4 to about 6, about 4 to about 6.5, about 4 to about 7, about 4 to about 7.1, about 4 to about 7.2, about 4 to about 7.3, about 4 to about 7.4, about 4.5 to about 5, about 4.5 to about 5.5, about 4.5 to about 6, about 4.5 to about 6.5, about 4.5 to about 7, about 4.5 to about 7.1, about

4.5 to about 7.2, about 4.5 to about 7.3, about 4.5 to about 7.4, about 5 to about 5.5, about 5 to about 6, about 5 to about 6.5, about 5 to about 7, about 5 to about 7.1, about 5 to about 7.2, about 5 to about 7.3, about 5 to about 7.4, about 5.5 to about 6, about 5.5 to about 6.5, about 5.5 to about 7, about 5.5 to about 7.1, about 5.5 to about 7.2, about 5.5 to about 7.3, about 5.5 to about 7.4, about 6 to about 6.5, about 6 to about 7, about 6 to about 7.1, about 6 to about 7.2, about 6 to about 7.3, about 6 to about 7.4, about 6.5 to about 7, about 6.5 to about 7.1, about 6.5 to about 7.2, about 6.5 to about 7.3, about 6.5 to about 7.4, about 7 to about 7.1, about 7 to about 7.2, about 7 to about 7.3, about 7 to about 7.4, about 7.1 to about 7.2, about 7.1 to about 7.3, about 7.1 to about 7.4, about 7.2 to about 7.3, about 7.2 to about 7.4, or about 7.3 to about 7.4. In embodiments, the novel antagonist, starting protease or starting binding protein is active at an esophageal pH of about 3.5, about 4, about 4.5, about 5, about 5.5, about 6, about 6.5, about 7, about 7.1, about 7.2, about 7.3, or about 7.4. In embodiments, the novel antagonist, starting protease or starting binding protein is active at an esophageal pH of at least about 3.5, about 4, about 4.5, about 5, about 5.5, about 6, about 6.5, about 7, about 7.1, about 7.2, or about 7.3. In embodiments, the novel antagonist, starting protease or starting binding protein is active at an esophageal pH of at most about 4, about 4.5, about 5, about 5.5, about 6, about 6.5, about 7, about 7.1, about 7.2, about 7.3, or about 7.4. In embodiments, the novel antagonist, starting protease or starting binding protein is active at a stomach pH of about 1 to about 5. In embodiments, the novel antagonist, starting protease or starting binding protein is active at a stomach pH of about 1 to about 1.5, about 1 to about 2, about 1 to about 2.5, about 1 to about 3, about 1 to about 3.5, about 1 to about 4, about 1 to about 4.5, about 1 to about 5, about 1.5 to about 2, about 1.5 to about 2.5, about 1.5 to about 3, about 1.5 to about 3.5, about 1.5 to about 4, about 1.5 to about 4.5, about 1.5 to about 5, about 2 to about 2.5, about 2 to about 3, about 2 to about 3.5, about 2 to about 4, about 2 to about 4.5, about 2 to about 5, about 2.5 to about 3, about 2.5 to about 3.5, about 2.5 to about 4, about 2.5 to about 4.5, about 2.5 to about 5, about 3 to about 3.5, about 3 to about 4, about 3 to about 4.5, about 3 to about 5, about 3.5 to about 4, about 3.5 to about 4.5, about 3.5 to about 5, about 4 to about 4.5, about 4 to about 5, or about 4.5 to about 5. In embodiments, the novel antagonist, starting protease or starting binding protein is active at a stomach pH of about 1, about 1.5, about 2, about 2.5, about 3, about 3.5, about 4, about 4.5, or about 5. In embodiments, the novel antagonist, starting protease or starting binding protein is active at a stomach pH of at least about 1, about 1.5, about 2, about 2.5, about 3, about 3.5, about 4, or about 4.5. In embodiments, the novel antagonist, starting protease or starting binding protein is active at a stomach pH of at most about 1.5, about 2, about 2.5, about 3, about 3.5, about 4, about 4.5, or about 5.

[0080] In embodiments, the starting protease and/or novel endopeptidase is active at a pH characteristic of the small intestine, e.g., starting at about pH 6 in the duodenum and increasing through the jejunum to about pH 7.4 in the terminal ileum. In embodiments, the novel antagonist, starting protease or starting binding protein is active at a small intestine pH of about 6 to about 7.4. In embodiments, the novel antagonist, starting protease or starting binding protein is active at a small intestine pH of about 6 to about 6.2, about 6 to about 6.4, about 6 to about 6.6, about 6 to about 6.8, about 6 to about 7, about 6 to about 7.2, about 6 to about 7.4, about 6.2 to about 6.4, about 6.2 to about 6.6, about 6.2 to about 6.8, about 6.2 to about 7, about 6.2 to about 7.2, about 6.2 to about 7.4, about 6.4 to about 6.6, about 6.4 to about 6.8, about 6.4 to about 7, about 6.4 to about 7.2, about 6.4 to about 7.4, about 6.6 to about 6.8, about 6.6 to about 7, about 6.6 to about 7.2, about 6.6 to about 7.4, about 6.8 to about 7, about 6.8 to about 7.2, about 6.8 to about 7.4, about 7 to about 7.2, about 7 to about 7.4, or about 7.2 to about 7.4. In embodiments, the novel antagonist, starting protease or starting binding protein is active at a small intestine pH of about 6, about 6.2, about 6.4, about 6.6, about 6.8, about 7, about 7.2, or about 7.4. In embodiments, the novel antagonist, starting protease or starting binding protein is active at a small intestine pH of at least about 6, about 6.2, about 6.4, about 6.6, about 6.8, about

7, or about 7.2. In embodiments, the novel antagonist, starting protease or starting binding protein is active at a small intestine pH of at most about 6.2, about 6.4, about 6.6, about 6.8, about 7, about 7.2, or about 7.4.

[0081] In embodiments, the novel antagonist, starting protease or starting binding protein is active at a colon pH of about 5 to about 8. In embodiments, the novel antagonist, starting protease or starting binding protein is active at a colon pH of about 5 to about 8. In embodiments, the novel antagonist, starting protease or starting binding protein is active at a colon pH of about 5 to about 5.5, about 5 to about 5.75, about 5 to about 6, about 5 to about 6.25, about 5 to about 6.5, about 5 to about 6.75, about 5 to about 7, about 5 to about 7.25, about

5 to about 7.5, about 5 to about 7.75, about 5 to about 8, about 5.5 to about 5.75, about 5.5 to about 6, about 5.5 to about 6.25, about 5.5 to about 6.5, about 5.5 to about 6.75, about 5.5 to about 7, about 5.5 to about 7.25, about 5.5 to about 7.5, about 5.5 to about 7.75, about 5.5 to about 8, about 5.75 to about 6, about 5.75 to about 6.25, about 5.75 to about 6.5, about 5.75 to about 6.75, about 5.75 to about 7, about 5.75 to about 7.25, about 5.75 to about 7.5, about 5.75 to about 7.75, about 5.75 to about 8, about 6 to about 6.25, about 6 to about 6.5, about 6 to about

6.75, about 6 to about 7, about 6 to about 7.25, about 6 to about 7.5, about 6 to about 7.75, about

6 to about 8, about 6.25 to about 6.5, about 6.25 to about 6.75, about 6.25 to about 7, about 6.25 to about 7.25, about 6.25 to about 7.5, about 6.25 to about 7.75, about 6.25 to about 8, about 6.5 to about 6.75, about 6.5 to about 7, about 6.5 to about 7.25, about 6.5 to about 7.5, about 6.5 to about 7.75, about 6.5 to about 8, about 6.75 to about 7, about 6.75 to about 7.25, about 6.75 to about 7.5, about 6.75 to about 7.75, about 6.75 to about 8, about 7 to about 7.25, about 7 to about 7.5, about 7 to about 7.75, about 7 to about 8, about 7.25 to about 7.5, about 7.25 to about

7.75, about 7.25 to about 8, about 7.5 to about 7.75, about 7.5 to about 8, or about 7.75 to about

8. In embodiments, the novel antagonist, starting protease or starting binding protein is active at a colon pH of about 5, about 5.5, about 5.75, about 6, about 6.25, about 6.5, about 6.75, about 7, about 7.25, about 7.5, about 7.75, or about 8. In embodiments, the novel antagonist, starting protease or starting binding protein is active at a colon pH of at least about 5, about 5.5, about

5.75, about 6, about 6.25, about 6.5, about 6.75, about 7, about 7.25, about 7.5, or about 7.75. In embodiments, the novel antagonist, starting protease or starting binding protein is active at a colon pH of at most about 5.5, about 5.75, about 6, about 6.25, about 6.5, about 6.75, about 7, about 7.25, about 7.5, about 7.75, or about 8.

[0082] In embodiments, the starting protease and/or novel endopeptidase is active at a pH characteristic of the large intestine, e.g., starting at about pH 5.5 in the caecum and increasing through the colon to about pH 6.7 in the rectum. In embodiments, the novel antagonist, starting protease or starting binding protein is active at a large intestine pH of about 5.5 to about 6.7. In embodiments, the novel antagonist, starting protease or starting binding protein is active at a large intestine pH of about 5.5 to about 5.7, about 5.5 to about 5.8, about 5.5 to about 5.9, about 5.5 to about 6, about 5.5 to about 6.1, about 5.5 to about 6.2, about 5.5 to about 6.3, about 5.5 to about 6.4, about 5.5 to about 6.5, about 5.5 to about 6.6, about 5.5 to about 6.7, about 5.7 to about 5.8, about 5.7 to about 5.9, about 5.7 to about 6, about 5.7 to about 6.1, about 5.7 to about 6.2, about 5.7 to about 6.3, about 5.7 to about 6.4, about 5.7 to about 6.5, about 5.7 to about 6.6, about 5.7 to about 6.7, about 5.8 to about 5.9, about 5.8 to about 6, about 5.8 to about 6.1, about 5.8 to about 6.2, about 5.8 to about 6.3, about 5.8 to about 6.4, about 5.8 to about 6.5, about 5.8 to about 6.6, about 5.8 to about 6.7, about 5.9 to about 6, about 5.9 to about 6.1, about 5.9 to about 6.2, about 5.9 to about 6.3, about 5.9 to about 6.4, about 5.9 to about 6.5, about 5.9 to about 6.6, about 5.9 to about 6.7, about 6 to about 6.1, about 6 to about 6.2, about 6 to about 6.3, about 6 to about 6.4, about 6 to about 6.5, about 6 to about 6.6, about 6 to about 6.7, about 6.1 to about 6.2, about 6.1 to about 6.3, about 6.1 to about 6.4, about 6.1 to about 6.5, about 6.1 to about 6.6, about 6.1 to about 6.7, about 6.2 to about 6.3, about 6.2 to about 6.4, about 6.2 to about 6.5, about 6.2 to about 6.6, about 6.2 to about 6.7, about 6.3 to about 6.4, about 6.3 to about 6.5, about 6.3 to about 6.6, about 6.3 to about 6.7, about 6.4 to about 6.5, about 6.4 to about 6.6, about 6.4 to about 6.7, about 6.5 to about 6.6, about 6.5 to about 6.7, or about 6.6 to about 6.7. In embodiments, the novel antagonist, starting protease or starting binding protein is active at a large intestine pH of about 5.5, about 5.7, about 5.8, about 5.9, about 6, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, about 6.6, or about 6.7. In embodiments, the novel antagonist, starting protease or starting binding protein is active at a large intestine pH of at least about 5.5, about 5.7, about 5.8, about 5.9, about 6, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, or about 6.6. In embodiments, the novel antagonist, starting protease or starting binding protein is active at a large intestine pH of at most about 5.7, about 5.8, about 5.9, about 6, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, about 6.6, or about 6.7.

[0083] In embodiments, the novel antagonist, starting protease or starting binding protein is active at physiological temperature. In embodiments, the physiological temperature is about 33.2 °C to about 38.2 °C. In embodiments, the physiological temperature is about 33.2 °C to about 38.2 °C. In embodiments, the physiological temperature is about 33.2 °C to about 34.5 °C, about 33.2 °C to about 35 °C, about 33.2 °C to about 35.5 °C, about 33.2 °C to about 36 °C, about 33.2 °C to about 36.5 °C, about 33.2 °C to about 36.75 °C, about 33.2 °C to about 37 °C, about 33.2 °C to about 37.25 °C, about 33.2 °C to about 37.5 °C, about 33.2 °C to about 37.75

°C, about 33.2 °C to about 38.2 °C, about 34.5 °C to about 35 °C, about 34.5 °C to about 35.5 °C, about 34.5 °C to about 36 °C, about 34.5 °C to about 36.5 °C, about 34.5 °C to about 36.75 °C, about 34.5 °C to about 37 °C, about 34.5 °C to about 37.25 °C, about 34.5 °C to about 37.5 °C, about 34.5 °C to about 37.75 °C, about 34.5 °C to about 38.2 °C, about 35 °C to about 35.5 °C, about 35 °C to about 36 °C, about 35 °C to about 36.5 °C, about 35 °C to about 36.75 °C, about 35 °C to about 37 °C, about 35 °C to about 37.25 °C, about 35 °C to about 37.5 °C, about 35 °C to about 37.75 °C, about 35 °C to about 38.2 °C, about 35.5 °C to about 36 °C, about 35.5 °C to about 36.5 °C, about 35.5 °C to about 36.75 °C, about 35.5 °C to about 37 °C, about 35.5 °C to about 37.25 °C, about 35.5 °C to about 37.5 °C, about 35.5 °C to about 37.75 °C, about

35.5 °C to about 38.2 °C, about 36 °C to about 36.5 °C, about 36 °C to about 36.75 °C, about 36 °C to about 37 °C, about 36 °C to about 37.25 °C, about 36 °C to about 37.5 °C, about 36 °C to about 37.75 °C, about 36 °C to about 38.2 °C, about 36.5 °C to about 36.75 °C, about 36.5 °C to about 37 °C, about 36.5 °C to about 37.25 °C, about 36.5 °C to about 37.5 °C, about 36.5 °C to about 37.75 °C, about 36.5 °C to about 38.2 °C, about 36.75 °C to about 37 °C, about 36.75 °C to about 37.25 °C, about 36.75 °C to about 37.5 °C, about 36.75 °C to about 37.75 °C, about 36.75 °C to about 38.2 °C, about 37 °C to about 37.25 °C, about 37 °C to about 37.5 °C, about 37 °C to about 37.75 °C, about 37 °C to about 38.2 °C, about 37.25 °C to about 37.5 °C, about 37.25 °C to about 37.75 °C, about 37.25 °C to about 38.2 °C, about 37.5 °C to about 37.75 °C, about 37.5 °C to about 38.2 °C, or about 37.75 °C to about 38.2 °C. In embodiments, the physiological temperature is about 33.2 °C, about 34.5 °C, about 35 °C, about 35.5 °C, about 36 °C, about 36.5 °C, about 36.75 °C, about 37 °C, about 37.25 °C, about 37.5 °C, about 37.75 °C, or about 38.2 °C. In embodiments, the physiological temperature is at least about 33.2 °C, about

34.5 °C, about 35 °C, about 35.5 °C, about 36 °C, about 36.5 °C, about 36.75 °C, about 37 °C, about 37.25 °C, about 37.5 °C, or about 37.75 °C. In embodiments, the physiological temperature is at most about 34.5 °C, about 35 °C, about 35.5 °C, about 36 °C, about 36.5 °C, about 36.75 °C, about 37 °C, about 37.25 °C, about 37.5 °C, about 37.75 °C, or about 38.2 °C.

[0084] It is understood by those of skill in the art that these ranges can vary among individuals depending on, e.g., age, health status, and diet. GI pH is described in the literature, e.g., by Fallingborg, J., 1999 Jun, “Intraluminal pH of the human gastrointestinal tract,” Danish Medical Bulletin; 46(3): 183-96, and Koziolek, M., et ah, 2015 Sep, “Investigation of pH and Temperature Profiles in the GI Tract of Fasted Human Subjects Using the Intellicap(®)

System,” J Pharm Sci. 104(9):2855-63, each incorporated herein by reference.

[0085] In embodiments, a novel antagonist, starting protease or starting binding protein of the invention is resistant to colonic proteases, including, but not limited to reducing enzymes (e.g., nitroreductase, azoreductase, N-oxide reductase, sulfoxide reductase, and hydrogenase) and hydrolytic enzymes (e.g., esterases, amidases, glycosidases, glucuronidase, and sulfatase), as described in the literature (see, e.g., Jayaprakash and Mathew, 2012, “Colon Specific Drug Delivery Systems: A Review on Various Pharmaceutical Approaches,” J. App. Pharm. Sci.02(01): 163-169, incorporated herein by reference).

[0086] In embodiments, the reduction in cytopathicity of a GI tract target by the novel antagonist results from, e.g., a reduction in target cell receptor binding, a reduction in membrane translocation, a reduction in endosome escape/autoprocessing, a reduction in glucosyltransferase activity, a reduction or modulation of any function of the GI tract target as known in the art, or any combination thereof. Reduction in cytopathicity of a GI tract target resulting from target site cleavage by a novel endopeptidase can occur by disruption of, e.g., partial removal, full removal, or allosteric effect on, a key GI tract target locus. Reduction in cytopathicity of a GI tract target resulting from target site binding by a novel binding protein can occur by disruption of, e.g., partial removal, full removal, or allosteric effect on, a key GI tract target locus. Key GI tract target loci may include, e.g, a binding site or other feature directly or indirectly responsible for binding of the GI tract target to a target cell receptor, membrane translocation, endosome escape/autoprocessing, or glucosyltransferase activity, any feature of the GI tract target as known in the art, or any combination thereof. In embodiments, target site cleavage by a novel endopeptidase can disrupt or remove a receptor binding site of the GI tract target (a direct effect) or it can interfere allosterically with binding by changing the conformation of the receptor binding site (an indirect effect). In embodiments, target site binding by a novel endopeptidase can disrupt or block a receptor binding site of the GI tract target (a direct effect) or it can interfere allosterically with binding by changing the conformation of the receptor binding site (an indirect effect). In embodiments, the reduction in any GI tract target activity is determined by comparison with a control. In some embodiments, the control is a non-optimized enzyme or binding protein. In some embodiments, the control is any other drug or treatment for CDI known in the art. In some embodiments, the control is the absence of enzyme. In some embodiments, the control includes the use of a nonspecific target, e.g., a different cleavage or binding substrate.

[0087] Novel Antagonist Assays

[0088] Any assay known in the art can be used to evaluate a property of a novel antagonist. GI tract targets for use in assays described herein can be prepared recombinantly and purified according to known methods, or obtained commercially, e.g., from The Native Antigen Company (Oxfordshire, UK), and Abeam (Boston, MA). [0089] For example, a suitable assay for evaluating ligand binding known in the art can be used to assess binding of a GI tract target to its GI tract binding partner, following cleavage at a target cleavage site by a novel endopeptidase or binding at a target binding site by a novel binding protein. Comparison can be made to the same GI tract target not exposed to the novel antagonist, or any other appropriate control, including but not limited to the GI tract target exposed to a control antagonist, e.g., an unoptimized starting enzyme or binding protein. In embodiments, an assay used for evaluating ligand binding is: a labeled ligand-binding assay, e.g., a fluorescent ligand binding assay, a radioligand binding assays, or a bioluminescent binding assay using nanoluciferase; a label-free ligand binding assay, e.g., surface plasmon resonance (SPR), plasmon-waveguide resonance (PWR), SPR imaging for affinity -based biosensors, nanofluidic fluorescence microscopy (NFM), whispering gallery microresonator (WGM), resonant waveguide grating (RWG), or biolayer interferometry biosensor (BIB); a structure-based ligand binding assay, e.g., nuclear magnetic resonance (NMR) or X-ray crystallography; a thermodynamic binding assay, e.g., thermal denaturation (TDA) or isothermal titration calorimetry (ITC); or a whole cell ligand-binding assay, e.g., urface acoustic wave (SAW) biosensor or RWG biosensor. Ligand binding assays are described in the literature, e.g., by Yakimchuk, 2011, Mater. Methods 1 : 199, incorporated herein by reference in its entirety.

[0090] In embodiments, a receptor pull-down assay is used to evaluate GI tract target-receptor binding, e.g., as described by Chung et ah, 2018. In embodiments, membrane-bound GI tract target is measured following incubation of target cells with a GI tract target that has been exposed or not exposed to the novel antagonist, or e.g., as described by Orth et ah, 2014, J. Biol. Chem. 289(26): 18008-18021. In embodiments, a cell binding assay as described by Kroh et ah, 2017, J. Biol. Chem. 292(35): 14401-14412 is used to evaluate binding of the GI tract target to a Gl-tract binding partner. Each of these references is incorporated herein.

[0091] In embodiments, the effect of treatment of a subject with a novel antagonist is evaluated based on amelioration of a symptom or effect resulting from infection with the GI tract organism and/or the activity of the GI tract target. In embodiments, the symptom or effect of infection is any symptom or effect known in the art pertaining to the GI tract organism and/or GI tract target. In embodiments, the assay is any assay known in the art and suitable for evaluating any such symptom or effect.

[0092] Gastrointestinal Tract Targets

[0093] The present invention includes methods for reducing a function of a GI tract target. In embodiments, the GI tract target is a protein produced by a GI tract organism. In embodiments, the GI tract organism is a microbe. In embodiments, the microbe is a bacterium, virus, fungus, archaeon, or protozoan. In embodiments, the GI tract organism is a pathogen or indigenous pathobiont. In embodiments, the GI tract organism anaerobic or aerobic. In embodiments, the GI tract organism causes symptoms in a certain GI tract compartment. In embodiments, the GI tract organism is any organism known to cause symptoms in the mouth, esophagus, stomach, small intestine, large intestine, rectum, anus, or any combination thereof. In embodiments, symptoms result from infection with or overgrowth of the GI tract organism.

[0094] In embodiments, a novel antagonist of the invention reduces any one or more undesirable activity of a particular GI Tract Target known to those of skill in the art and described in the literature. In embodiments, a novel antagonist of the invention increases any activity normally decreased by ETBF infection, e.g., cell viability and/or cell growth. In embodiments, the invention includes novel antagonist methods and compositions for treating a condition in a subject infected with and/or experiencing symptoms of infection with a GI tract organism. In embodiments, treatment results in amelioration of one or more symptoms known to those of skill in the art to be characteristic of infection with the GI tract organism.

[0095] A GI tract target can include a virulence factor produced by an organism well-known to be pathogenic, e.g., bacteria for which antibiotics are typically used as first-line therapy. From among these pathogens, many antibiotic-resistant strains have arisen. In this regard the treatments including those described herein will be of utility. In embodiments, novel antagonists of the invention are useful for treating GI tract infections that are not readily or effectively treated by existing treatment modalities. In embodiments, the typical treatment modality is antibiotic treatment. In embodiments, the GI tract infection is caused by a GI tract organism that is antibiotic resistant.

[0096] The invention also includes GI tract targets produced by organisms that are constitutive in the GI tract and that contribute to disease or disorders under certain circumstances. In embodiments these organisms are commensal. In embodiments they are pathobionts. Such circumstances potentially include growth of one or more organisms beyond a tolerable threshold for an individual. This threshold can depend on an individual’s health status, age, gender, and other factors. Such growth can be defined by over-representation of the organism among the microbiota, e.g., in the microbiome profile, of a given tissue or body compartment.

Identification of constitutive GI tract organisms that can trigger or perpetuate a condition or disease provides a strategy for preventing and/or treating the condition or disease. Palm, N., et ah, 2014, “Immunoglobulin A Coating Identifies Colitogenic Bacteria in Inflammatory Bowel Disease,” Cell 158:1-11, incorporated by reference herein in its entirety, reported that commensal GI tract bacteria characterized by IgA coating preferentially drive IBD. In embodiments, the invention includes novel antagonist compositions and methods for the prevention or treatment of disease in a subject, by reducing an activity of a GI tract target produced by one or more commensal or constitutive GI tract organisms. In embodiments, the invention includes novel antagonist compositions and methods for the prevention or treatment of a condition or disease in a subject, by reducing an activity of a GI tract target produced by a GI tract organism having a high IgA coating. In embodiments, the GI tract organism is selected from Prevotellaceae , Helicobacter , and SFB (segmented filamentous bacteria), and a combination thereof. In embodiments, the GI tract organism is selected from UC Prevotellaceae , SFB, Lactobacillus , Helicobacter sp. Flexispira , and a combination thereof. In embodiments, the condition or disease is an inflammatory disease. In embodiments, the condition or disease is GI inflammation, colitis, pseudomembranous colitis, hemorrhagic colitis, peptic ulcer disease, diarrhea, hemolytic uremic syndrome, inflammatory bowel disease,

Crohn’s disease, ulcerative colitis, or cholera. In embodiments, the other condition or disease is caused by or contributed to by infection, colonization, or overgrowth of a GI tract organism. In embodiments, the condition or disease is an arthritis or inflammatory arthritis.

[0097] In embodiments, the GI tract organism is selected from the following nonlimiting list: Aeromonas, Aspergillus , Bacteroides, Bilophila , Campylobacter , Clostridioides , Coccidiosis, Crytosporidia, Enterobacter , Enterococcus , Escherichia , Firmicutes , Helicobacter , Lactobacillus , Listeria , Peptostreptococcus , Pleisiomonas , Prevotellaceae , Pseudomonas , Salmonella , Sarcina, SFB, Shigella , Staphylococcus , Streptococcus , Veillonella , Vibrio ,

Yersinia , Candida , Mycobacterium , Mycoplasma, Rotavirus, Calicivirus, Norwalk-like viruses, adenoviruses, astroviruses, sapporo-like viruses, toroviruses, coronaviruses, picomaviruses, herpes viruses, noroviruses, Proteus, Entamoeba , Giardia, and Strongyloides, single-celled parasites, multi-celled parasites, amoebae, worms, tape worms, protozoans, flukes, helminths, roundworms, pinworms, and hookworms. In embodiments, the GI tract organism is selected from the following nonlimiting list: Bacteroides fragilis , Bilophila wadsworthia, Campylobacter jejuni , Campylobacter coli , Clostridioides difficile , Clostridioides sordelli , Enterobacter cloacae , Enterococcus faecalis , Enterococcus faecium , Escherichia coli , enterotoxigenic Escherichia coli , Helicobacter pylori , Helicobacter sp. Flexispira. Listeria monocytogenes , Pleisiomonas shigelloides , UC Prevotellaceae. Pseudomonas aeruginosa , Salmonella enterica , Salmonella bongori, Salmonella enterica subsp. enterica serotype Typhimurium , Shigella dysenteriae , Staphylococcus aureus , Streptococcus anginosus, Streptococcus thermophilus , Vibrio cholera , Vibrio parahaemolyticus, Yersinia enter ocolitica, Candida albicans , Entamoeba histolytica , Giardia lamblia , and Strongyloides stercoralis. It is understood that any GI tract organism known to those of skill in the art to produce a GI tract target that causes or contributes to undesirable GI tract effects or symptoms is included.

[0098] In embodiments, the compositions and methods of the present invention are used to reduce an activity of a GI tract target produced by a GI tract microbe selected from: Clostridium difficile (e.g., TcdA, TcdB); Helicobacter pylori (e.g., VacA, CagA); Campylobacter jejuni and Campylobacter coli (e.g., Cholera toxin (CT)-like enterotoxin); Toxigenic Escherichia coli (e.g., 935 J toxin, 933 W toxin); Staphylococcus aureus (e.g., enterotoxins A, B, C, D, E, G, H, I, L, M; exfoliative toxins A, B; toxic shock syndrome toxin; SEA, SEB); Bacteroides fragilis (e.g., BFT fragilysin); and Vibrio cholera (e.g., Cholera toxin AB ).

[0099] Clostridium difficile

[0100] C. diff mitction (CDI) results from overgrowth ofC. diff. in the gastrointestinal tract, causing release of toxins that inflame the intestinal lining. C. diff. is commonly part of an individual’s normal intestinal flora but can flourish following treatment with antibiotics that kill other intestinal bacteria. The severity of gastrointestinal disease caused by CDI varies based on factors that include the health status of the infected individual, theC. diff. toxinotype, and timing of treatment. Mild CDI causes diarrhea, whereas severe CDI can cause ulcerative colitis. In very severe cases, the disease progresses to fulminant colitis and death from CDI is not rare (see, e.g., Voth and Ballard, 2005, “ Clostridium difficile Toxins: Mechanism of Action and Role in Disease,” Clinical Microbiology Reviews vol. 18 no. 2: 247-263, incorporated herein by reference). Death can result from progression of a mild case of CDI when left untreated. In the past twenty years, a hypervirulentC. diff strain that produces more toxin protein and is resistant to first-line antibiotic treatments has emerged.

[0101] Pathogenesis

[0102] C. diff. has two major toxins, or virulence factors, TcdA (308 kDa) and TcdB (270 kDa), encoded by the tcdA and tcdB gene, respectively. TcdA and TcdB are bacterial exotoxins having 47% amino acid identity. Many C. diff. toxinotypes have been identified, e.g., as described by Rupnik and Janezic, 2016, J. Clin. Microb. 54(1): 13-18. TcdA and TcdB have each been described as having four major domains, for example, as shown by Gupta et ah, 2017, J. Biol. Chem. 292(42): 17290-17301, incorporated herein by reference, which reports in Fig.

1 A therein: 1) a glucosyltransferase domain (GTD); 2) an autoprotease or autocatalytic cleavage domain (APD, also referred to as a cysteine protease domain, CPD); 3) a translocation/pore formation domain (which includes a hydrophobic region, HR); and 4) a combined repetitive oligopeptide domain (CROP domain or receptor binding domain, RBD). Structural studies reporting regions responsible for specific actions have been carried out, e.g., by Chumbler et al., 2016 Jan 11, Nature Microbiology 1:15002. doi: 10.1038/nmicrobiol.2015.2, incorporated by reference herein in its entirety.

[0103] The TcdA and TcdB RBDs each contain a highly repetitive, elongated b-solenoid structural unit containing a series of binding sites for cell surface receptor carbohydrates. Each receptor carbohydrate binding site comprises a central long repeat (LR) unit separated by several short repeat (SR) units (see, e.g., Murase et al., 2014, J. Biol. Chem. 289(4): 2331-2343, and Orth et al., J. Biol. Chem. 2014, 289(26): 18008-18021, both incorporated herein by reference). Orth et al., 2014, describes an individual TcdA or TcdB CROP unit as an LR flanked by 2-3 SRs on either side. Murase et al., 2014 (e.g., in Fig. 1 therein), reports a TcdA RBD having seven LRs, each alternating with three to five SRs. This TcdA therefore has seven CROP units. The reported TcdB RBD comprises four LRs, each alternating with two to five SRs (four CROP units). These TcdB SRs are 20-23 amino acid residues and therefore longer than those of TcdA, which are 15-21 residues (Murase et al., 2014). Fig. 1 herein shows the groupings of the SRs in the TcdA and TcdB RBD (closed black rectangles) around each LR (open white rectangles) as reported for C. diff. strain VPI 10463 by Orth et al., 2014 (see, e.g., Fig. 1 therein). The TcdB sequence of the hypervirulent strain NAP 1/B 1/027 is reportedly 70% identical and 90% homologous to that of VPI 10463 (Orth et al., 2014). The TcdB protein of this hypervirulent strain is described by, e.g., Lanis et al., 2013, PLOS Pathogens 9(8): 1-11.

[0104] In C. diff. infection , TcdA and TcdB bind to one or more target receptors on a target cell and are internalized via endocytosis. The GTD and APD are then delivered into the cytosol from endosomes. The low pH of the endosome promotes the membrane insertion of the domain comprising the GTD and APD, allowing for pore formation, and the GTD is translocated across the membrane. Release of the GTD from the rest of the toxin protein (endosome escape) is triggered when eukaryotic inositol-hexakisphosphate (InsP6) binds the APD and activates an intramolecular cleavage (autoprocessing). Once released into the host cell cytosol, the GTD inactivates small GTPases, including RhoA, Racl and Cdc42. Rac/Rho glucosylation can trigger actin depolymerization, cell rounding, and eventually cell death (cytopathic effect). At high concentrations, TcdB has been reported to trigger necrosis, causing colonic tissue damage independent of the toxin’s glucosylation activity.

[0105] Novel Antagonist Targeted to C. difficile Toxins

[0106] In embodiments, a novel antagonist of the invention reduces an activity of a GI tract target, wherein the GI tract target is a C. diff toxin protein. In embodiments, the novel antagonist is a novel binding protein that binds to a target site of a C. diff toxin protein, or a novel endopeptidase that cleaves a target site of a C. diff. toxin protein. In embodiments, the toxin protein is a Ted A or a TcdB of any known C. diff. strain or toxinotype known to those of skill in the art, e.g., a toxinotype as described by Rupnik and Janezic, 2016, J. Clin. Microb. 54(1): 13-18. In embodiments, the TcdA or TcdB is from the prototypical 630 strain. In embodiments, the TcdA or TcdB is from the hypervirulent R20291 strain. In embodiments, the novel antagonist reduces an activity of TcdB of a strain that does not produce TcdA. In embodiments, the novel antagonist reduces an activity of TcdB of a strain that produces a TcdA having a truncation or deletion of the CROP region encoded by the 3’ end of tcdA.

[0107] In embodiments, an activity of a C. diff toxin protein is reduced by cleaving a target cleavage site of a C. diff toxin protein with a novel endopeptidase of the invention, or by binding of a novel binding protein of the invention to a target binding site of a C. diff toxin protein. In embodiments, the cytopathicity of the C. diff toxin protein is thereby reduced. In embodiments, the binding of a C. diff toxin protein activity to a GI tract binding partner is thereby reduced. In embodiments, C. diff infection in a subject is treated by cleaving a target cleavage site of the C. diff toxin protein with the novel endopeptidase, or by binding of a novel binding protein of the invention to a target binding site of a C. diff toxin protein.

[0108] In embodiments, a novel endopeptidase cleaves TcdA, TcdB, or both TcdA and TcdB, or a novel binding protein binds to TcdA, TcdB, or both TcdA and TcdB. A toxin protein of any known C. diff toxinotype or strain can be cleaved by the novel endopeptidase or bound by the novel binding protein. In embodiments, the TcdA and/or TcdB is from a prototypical 630 strain. In embodiments, the TcdA and/or TcdB is from hypervirulent strain R20291. In embodiments, a novel endopeptidase of the present invention cleaves TcdA and/or TcdB at a target site, also referred to herein as a target cleavage site. A target cleavage site refers to the location of the break in the toxin amino acid sequence resulting from cleavage. In embodiments, a novel binding protein of the present invention binds to TcdA and/or TcdB at a target site of the novel binding protein, also referred to herein as a target binding site. A target binding site refers to, e.g., the amino acid sequence or motif with which the novel binding protein interacts. In embodiments, the novel binding protein interacts with the target binding site covalently or noncovalently. In embodiments, the target binding or cleavage site is located anywhere in the glucosyltransferase domain (GTD), the autoprotease domain (APD), the translocation/pore formation domain, or in the receptor binding domain (RBD). In embodiments, a novel antagonist TcdA and/or TcdB target binding or cleavage site is in one or more of the following locations: within the glucosyl transferase domain; at the junction of the GTD and the APD; within the APD; at the junction of the APD and the translocation/pore formation domain; within the translocation/pore formation domain; at the junction of the translocation/pore formation domain and the RBD, within the CROPS, and; within the RBD. The target cleavage site can be referred to by the amino acids flanking the cleavage site, or by its location relative to a toxin domain. The target binding site can be referred to by the amino acids involved in the binding, or by its location relative to a toxin domain. It will be understood by those of skill in the art that among different toxinotypes, the features and junctions of the toxin domains, though recognizable based on conserved sequences and published reports, may have slightly different amino acid numbering.

[0109] In embodiments, a novel endopeptidase of the present invention disrupts and/or cleaves a TcdA or TcdB protein, or both, in the glucosyltransferase domain, the autoprotease or autocatalytic cleavage domain (cysteine protease domain), the translocation/pore formation domain (within or outside of the hydrophobic region, and/or within or outside of the pore forming region), or the combined repetitive oligopeptide domain. In embodiments, a novel endopeptidase of the present invention disrupts and/or cleaves a TcdA or TcdB protein, or both, at the junction of the glucosyltransferase domain and the autoprotease or autocatalytic cleavage domain (cysteine protease domain), at the junction of the autoprotease or autocatalytic cleavage domain (cysteine protease domain) and the translocation/pore formation domain (within or outside of the hydrophobic region, and/or within or outside of the pore-forming region), or at the junction of the translocation/pore formation domain (within or outside of the hydrophobic region, and/or within or outside of the pore-forming region) and the combined repetitive oligopeptide domain. The targeted TcdA and/or TcdB protein can be one from any C. diff. strain, variant, or any toxinotype known in the art, including but not limited to VPI 10463, 630, R20291, and NAPl/B 1/027. The amino acids corresponding to the TcdA and TcdB domains of any known strain to be targeted by a novel antagonist as described herein will be recognizable to those of skill in the art.

[0110] In embodiments, a novel binding protein of the present invention disrupts and/or binds to a TcdA or TcdB protein, or both, in the glucosyltransferase domain, the autoprotease or autocatalytic cleavage domain (cysteine protease domain), the translocation/pore formation domain (within or outside of the hydrophobic region, and/or within or outside of the pore forming region), or the combined repetitive oligopeptide domain. In embodiments, a novel binding protein of the present invention disrupts and/or cleaves a TcdA or TcdB protein, or both, at the junction of the glucosyltransferase domain and the autoprotease or autocatalytic cleavage domain (cysteine protease domain), at the junction of the autoprotease or autocatalytic cleavage domain (cysteine protease domain) and the translocation/pore formation domain (within or outside of the hydrophobic region, and/or within or outside of the pore-forming region), or at the junction of the translocation/pore formation domain (within or outside of the hydrophobic region, and/or within or outside of the pore-forming region) and the combined repetitive oligopeptide domain. The targeted TcdA and/or TcdB protein can be one from any C. diff. strain, variant, or any toxinotype known in the art, including but not limited to VPI 10463, 630, R20291, and NAP1/B 1/027. The amino acids corresponding to the TcdA and TcdB domains of any known strain to be targeted by a novel antagonist as described herein will be recognizable to those of skill in the art. The genome sequence of VPI 10493 and its TcdA and TcdB protein sequences are described at, e.g., GenBank: MUJV01000001.1 (genome sequence), incorporated herein by reference. See, e.g., SEQ ID NOS: 1 and 2 herein.

[0111] Method for Reducing a C. diff. Toxin Protein Cytopathicity

[0112] The present invention includes novel antagonist compositions and methods for reducing the cytopathicity of a GI tract target.

[0113] In embodiments, the toxin protein is a C. diff toxin protein. In embodiments, a novel antagonist, e.g., a novel endopeptidase or novel binding protein, reduces the cytopathic effect of a C. diff. TcdA or TcdB on a host cell. The cytopathicity is the toxin’s capacity to induce structural changes in target cells (the cytopathic or cytopathogenic effect, or CPE), of the toxin on a target cell.

[0114] In embodiments, the CPE is represented by and can be evaluated based on: cell rounding; total destruction of a host cell monolayer; subtotal destruction of a host cell monolayer; focal degeneration (a localized attack of a host cell monolayer); swelling and clumping; foamy degeneration (vacuolization); syncytium formation; formation of inclusion bodies, and any combination thereof.

[0115] In embodiments, a novel antagonist reduces host cell death caused by C. diff TcdA or TcdB. Cytopathicity can be measured by any method known in the art or described herein. For example, cytopathicity can be evaluated using a neutralization assay, e.g., as described by Orth et ah, 2014, a cell rounding and competition assay as described by, e.g., Chung et ah, 2018, Frontiers in Microbiology Vol. 9, Art 2314: 1-11, incorporated herein by reference, a cytotoxicity neutralization assay as described by, e.g., Chen et ak, 2008, Gastroenterology 135:1984-1992, or a hemagglutination assay as described by, e.g., Dingo et ak, 2008, Glycobiology.

[0116] In embodiments, the cytopathicity is reduced by novel endopeptidase cleavage at a target cleavage site of a TcdA and/or a TcdB, and/or by binding of a novel binding protein to a target binding site of a TcdA and/or a TcdB. In embodiments, a subject is treated for CDI by reducing the cytopathicity of a toxin protein produced by the C. diff. in the subject by cleavage of or binding to TcdA and/or TcdB by a novel antagonist of the invention.

[0117] In embodiments, the reduction in cytopathicity of a C. diff. toxin by the novel antagonist results from, e.g., a reduction in target cell receptor binding, a reduction in membrane translocation, a reduction in endosome escape/autoprocessing, a reduction in glucosyltransferase activity, or any combination thereof. Reduction in cytopathicity of a C. diff toxin resulting from target site cleavage by a novel endopeptidase can occur by disruption of a key GI tract target locus. Reduction in cytopathicity of a C. diff toxin resulting from target site binding by a novel binding protein can occur by disruption of a key GI tract target locus. Key GI tract target loci may include, e.g, a binding site or other feature directly or indirectly responsible for binding of the GI tract target to a target cell receptor, membrane translocation, endosome escape/autoprocessing, or glucosyltransferase activity. In embodiments, target site cleavage by a novel endopeptidase can disrupt a receptor binding site of the C. diff toxin (a direct effect) or it can interfere allosterically with binding by changing the conformation of the receptor binding site (an indirect effect). In embodiments, target site binding by a novel endopeptidase can disrupt a receptor binding site of the C. diff toxin (a direct effect) or it can interfere allosterically with binding by changing the conformation of the receptor binding site (an indirect effect). In embodiments, the reduction in any GI tract target activity is determined by comparison with a control. In some embodiments, the control is a non-optimized enzyme or binding protein. In some embodiments, the control is any other drug or treatment for CDI known in the art. In some embodiments, the control is the absence of enzyme. In some embodiments, the control includes the use of a nonspecific target, e.g., a different cleavage or binding substrate.

[0118] In some embodiments, the cytopathicity includes induction of necrosis. In some embodiments, the cytopathicity does not include induction of necrosis.

[0119] C. diff. Toxin Receptor Binding

[0120] TcdA and TcdB target a broad range of cell types, notably, colonic epithelial cells. Vero cells are commonly used for toxin binding and neutralization assays. Certain receptors identified for TcdA have been reported to comprise the disaccharide Gal b I -4GlcNac (Voth and Ballard, 2005). Receptors identified for TcdB include poliovirus receptor like 3 (PVRL3, or NECTIN3), chondroitin sulfate proteoglycan 4 (CSPG4), and members of the Frizzled protein family (FZD1, FZD2, and FZD7) (Gupta et ak, 2017). [0121] A multiple receptor model for host cell entry has been proposed, wherein either TcdA or TcdB docks onto the cell surface by binding to a low affinity low affinity receptor/oligosaccharide via its RBD (CROP-dependent binding), followed by binding to a high affinity CROP-independent receptor (Gupta et al., 2017). Manse et al., 2015, FEBS Letters 589: 3945-3951, incorporated herein by reference) report that three independent regions (TcdB 1372- 1493, 1493-1848, and CROPs) contribute to binding and entry of TcdB into mammalian cells, and that residues 1372-1493 are sufficient for interaction with PVRL3. In embodiments, a novel endopeptidase of the invention cleaves TcdB, or a novel binding protein binds TcdB, at a position within or overlapping amino acids 1372-1493 or 1493-1848.

[0122] Exemplary amino acid numbering for the regions of TcdB is provided by Manse et al., 2015, reporting the GTD at amino acids 1-543, the CPD (APD) at 544-807, the translocation domain at about 810-1350, including the pore-forming region at 830-990 and the HR at 956- 1128, a secondary receptor binding domain at 1349-1811, and the RBD at 1851-2366 (see, e.g., Fig. 1 therein). In embodiments, a novel endopeptidase of the invention cleaves TcdB, or a novel binding protein binds to TcdB, at a position within or overlapping amino acids 1-543, 544- 807, 819-1350, 830-990, 956-1128, 1349-1811, or 1851-2366. Exemplary TcdA sequences are provided at, e.g., GenBank: AGG91568.1 (strain VPI 10463), SwissProt P16154 (TcdA003), and SwissProt C9YJ37 (TcdA027). Exemplary TcdB sequences are provided at, e.g., GenBank CAA63562.1 (strain VPI 10463), SwissProt P18177 (TcdB003/CTD003), SwissProt C9YJ35 (TcdB027/CTD027). In embodiments, a novel endopeptidase of the invention cleaves TcdA or TcdB, or a novel binding protein binds to TcdA or TcdB, at a position within or overlapping the glucosyltransferase domain. In embodiments, a novel endopeptidase of the invention cleaves TcdA or TcdB, or a novel binding protein binds to TcdA or TcdB, at a position within or overlapping the autoprotease or autocatalytic cleavage domain (cysteine protease domain). In embodiments, a novel endopeptidase of the invention cleaves TcdA or TcdB, or a novel binding protein binds to TcdA or TcdB, at a position within or overlapping the translocation/pore formation domain. In embodiments, a novel endopeptidase of the invention cleaves TcdA or TcdB, or a novel binding protein binds to TcdA or TcdB, at a position within or overlapping the hydrophobic region (HR) of the translocation/pore formation domain. In embodiments, a novel endopeptidase of the invention cleaves TcdA or TcdB, or a novel binding protein binds to TcdA or TcdB, at a position within or overlapping the combined repetitive oligopeptide domain (CROP domain or receptor binding domain).

[0123] More recently, as many as three regions of TcdA and TcdB have been implicated in receptor binding: one in the C-terminal binding domain (CROP domain), and two in the intermediate delivery domain, referred to herein above as the translocation/pore formation domain (Chung et al., 2018).

[0124] Method for Reducing Receptor Binding by a C. diff. Toxin Protein

[0125] In embodiments, cleavage of a C. diff. toxin protein at a target site by a novel antagonist of the invention reduces the binding of the C. diff toxin protein to a cell receptor for the C. diff toxin protein. In embodiments, the target binding or cleavage site is within the RBD. In embodiments, the target binding or cleavage site is outside of the RBD. In embodiments, cleavage at the target site partially removes, fully removes and/or inactivates at least one CROP unit receptor carbohydrate binding site in the RBD. In embodiments, binding of a novel binding protein to the target site partially blocks, fully blocks and/or inactivates at least one CROP unit receptor carbohydrate binding site in the RBD. In embodiments, partial blocking or removal, complete blocking or removal, and/or inactivation of the at least one CROP unit eliminates or reduces the receptor binding capacity of the at least one CROP unit. In embodiments, a novel endopeptidase of the present invention cleaves TcdA or TcdB at the junction of the translocation domain and the RBD, thereby removing all CROPs in the toxin. In embodiments, a novel binding protein of the present invention blocks, comprises an epitope that binds to, or targets, TcdA or TcdB anywhere within the CROPs. It is understood that the number of CROPs in a toxin RBD may differ depend on the C. diff strain/toxinotype, and that this number will be readily determined by one of skill in the art.

[0126] In embodiments, cleavage of TcdA by a novel endopeptidase inactivates 1 CROP unit to all CROP units in the RBD. In embodiments, binding of TcdA by a novel binding protein inactivates 1 CROP unit to all CROP units in the RBD. In embodiments, 1 to 7 CROP units are inactivated. In embodiments, binding of a TcdA by the novel binding protein inactivates 1 CROP unit to 2 CROP units, 1 CROP unit to 3 CROP units, 1 CROP unit to 4 CROP units, 1 CROP unit to 5 CROP units, 1 CROP unit to 6 CROP units, 1 CROP unit to 7 CROP units, 2 CROP units to 3 CROP units, 2 CROP units to 4 CROP units, 2 CROP units to 5 CROP units, 2

CROP units to 6 CROP units, 2 CROP units to 7 CROP units, 3 CROP units to 4 CROP units, 3

CROP units to 5 CROP units, 3 CROP units to 6 CROP units, 3 CROP units to 7 CROP units, 4

CROP units to 5 CROP units, 4 CROP units to 6 CROP units, 4 CROP units to 7 CROP units, 5

CROP units to 6 CROP units, 5 CROP units to 7 CROP units, or 6 CROP units to 7 CROP units. In embodiments, bindingof TcdA by the novel binding protein inactivates 1 CROP unit, 2 CROP units, 3 CROP units, 4 CROP units, 5 CROP units, 6 CROP units, or 7 CROP units. In embodiments, binding of TcdA by the novel binding protein inactivates at least 1 CROP unit, at least 2 CROP units, at least 3 CROP units, at least 4 CROP units, at least 5 CROP units, or at least 6 CROP units. In embodiments, binding of TcdA by the novel binding protein inactivates at most 2 CROP units, at most 3 CROP units, at most 4 CROP units, at most 5 CROP units, at most 6 CROP units, or at most 7 CROP units.

[0127] In embodiments, binding of TcdB by the novel binding protein inactivates 1 CROP unit to all CROP units in the RBD. In embodiments, 1 to 4 CROP units are inactivated. In embodiments, binding of TcdB by the novel binding protein inactivates 1 CROP unit to 2 CROP units, 1 CROP unit to 3 CROP units, 1 CROP unit to 4 CROP units, 2 CROP units to 3 CROP units, 2 CROP units to 4 CROP units, or 3 CROP units to 4 CROP units. In embodiments, binding of TcdB by the novel endopeptidase inactivates 1 CROP unit, 2 CROP units, 3 CROP units, or 4 CROP units. In embodiments, binding of TcdB by the novel endopeptidase inactivates at least 1 CROP unit, at least 2 CROP units, or at least 3 CROP units. In embodiments, binding of TcdB by the novel endopeptidase inactivates at most 2 CROP units, at most 3 CROP units, or at most 4 CROP units.

[0128] In embodiments, partial blocking, complete blocking and/or inactivation of the at least one CROP unit, or inactivation of all CROPS, results in a reduction in binding of the TcdA or TcdB protein to at least one cell receptor for that protein.

[0129] As reported by Gupta, 2017, TcdA and TcdB may each bind to two different receptors on a target cell, one with low affinity and CROP-dependent, and a second with high affinity and CROP -independent. In embodiments, a novel antagonist acts on a TcdA or TcdB target cleavage or binding site to reduce binding of the toxin to at least two receptors on a target cell.

In embodiments, one of the at least two receptors is: CROP-dependent; CROP -independent; low affinity and CROP-dependent; high affinity and CROP -independent, or any combination thereof.

[0130] In embodiments, any method as described herein uses, or any composition described herein comprises, two or more different novel antagonists, wherein each novel antagonist cleaves or binds to a target cleavage or binding site of TcdA and/or TcdB at a different location. In embodiments, the two or more different novel antagonists cleave or bind to the same toxin protein, at different locations. In embodiments, a novel binding protein and novel endopeptidase are used together to target different target binding or cleavage sites of the TcdA and/or TcdB. In embodiments, the two or more different novel antagonists each cleave or bind to both TcdA and TcdB. In embodiments, at least one of the two or more different novel antagonists cleave or bind to TcdA and at least one cleaves TcdB. In embodiments, at least one of the two or more different novel antagonists cleaves or binds to both toxins and at least one cleaves or binds to only one toxin. In embodiments, two or more different novel antagonists are used to cleave or bind to TcdA and/or TcdB at a combination of the following locations: within the glucosyl transferase domain; at the junction of the glucosyl transferase and the translocation domain; within the translocation domain; at the junction of the translocation domain and the RBD, within the CROPS, and; within the RBD.

[0131] In some embodiments of the present invention, a novel antagonist of the invention cleaves a target cleavage site or binds to a target binding site of a C. diff. toxin protein to reduce CROP-dependent toxin binding. In embodiments, cleavage of a TcdA or TcdB target cleavage site by a novel endopeptidase of the invention or binding to a TcdA or TcdB target binding site by a novel binding protein of the invention reduces CROP-dependent toxin binding by removing, disrupting all or part of the RBD or the translocation/pore formation domain. In embodiments, cleavage of a TcdA or TcdB target cleavage site by a novel endopeptidase of the invention or binding to a TcdA or TcdB target binding site by a novel binding protein of the invention reduces CROP-dependent toxin binding by cleavage or blocking at any point N- terminal to the RBD. In some embodiments, cleavage of a TcdA or TcdB target cleavage site by a novel endopeptidase of the invention or binding to a TcdA or TcdB target binding site by a novel binding protein of the invention reduces CROP-independent toxin binding. In embodiments, cleavage of a TcdA or TcdB target cleavage site by a novel endopeptidase of the invention or binding to a TcdA or TcdB target binding site by a novel binding protein of the invention reduces CROP-independent toxin binding by disrupting TcdB amino acids 1372- 1493, 1493-1848, or both. In embodiments, cleavage of a TcdA or TcdB target cleavage site by a novel endopeptidase of the invention or binding to a TcdA or TcdB target binding site by a novel binding protein of the invention reduces binding of the toxin to a GI tract binding partner by disrupting a secondary binding domain in the toxin. In embodiments, the secondary binding domain is disrupted by cleavage at any point N-terminal to the secondary binding domain. In embodiments, a TcdB secondary binding domain is disrupted by cleavage of or binding by a novel antagonist between amino acids 1348 and 1349, to between amino acids 1811 and 1312.

In embodiments, two or more binding domains are disrupted by the cleavage or binding.

[0132] C. diff. Toxin Receptor Binding Assays

[0133] TcdA and TcdB protein useful in any assay described herein can be prepared recombinantly and purified according to known methods, or obtained commercially, e.g., from The Native Antigen Company (Oxfordshire, UK), and Abeam (Boston, MA).

[0134] Any suitable assay for evaluating ligand binding known in the art can be used to compare receptor binding of a cleaved C. diff. toxin to receptor binding of an uncleaved C. diff toxin (or another appropriate control), or to compare receptor binding in the presence and absence of a novel binding protein. In embodiments, an assay used is: a labeled ligand-binding assay, e.g., a fluorescent ligand binding assay, a radioligand binding assays, or a bioluminescent binding assay using nanoluciferase; a label-free ligand binding assay, e.g., surface plasmon resonance (SPR), plasmon-waveguide resonance (PWR), SPR imaging for affinity-based biosensors, nanofluidic fluorescence microscopy (NFM), whispering gallery microresonator (WGM), resonant waveguide grating (RWG), or biolayer interferometry biosensor (BIB); a structure- based ligand binding assay, e.g., nuclear magnetic resonance (NMR) or X-ray crystallography; a thermodynamic binding assay, e.g., thermal denaturation (TDA) or isothermal titration calorimetry (ITC); or a whole cell ligand-binding assay, e.g., urface acoustic wave (SAW) biosensor or RWG biosensor. Ligand binding assays are described in the literature, e.g., by Yakimchuk, 2011, Mater. Methods 1 : 199, incorporated herein by reference in its entirety.

[0135] In embodiments, a receptor pull-down assay is used to evaluate toxin-receptor binding, e.g., as described by Chung et ah, 2018. In embodiments, membrane-bound toxin is measured following incubation of target cells with the cleaved or uncleaved toxin, or in the presence of a novel binding protein, e.g., as described by Orth et ah, 2014. In embodiments, a cell binding assay as described by Kroh et ah, 2017, J. Biol. Chem. 292(35): 14401-14412 is used to evaluate binding of the C. diff. toxin to a target receptor. Each of these references is incorporated herein.

[0136] In embodiments, the reduction in receptor binding resulting from disruption of a target site of TcdA or TcdB by the novel antagonist is about 75 % to about 100 %. In some embodiments, the reduction in receptor binding is about 75 % to about 100 %. In some embodiments, the reduction in receptor binding is about 75 % to about 80 %, about 75 % to about 82 %, about 75 % to about 84 %, about 75 % to about 86 %, about 75 % to about 88 %, about 75 % to about 90 %, about 75 % to about 92 %, about 75 % to about 94 %, about 75 % to about 96 %, about 75 % to about 98 %, about 75 % to about 100 %, about 80 % to about 82 %, about 80 % to about 84 %, about 80 % to about 86 %, about 80 % to about 88 %, about 80 % to about 90 %, about 80 % to about 92 %, about 80 % to about 94 %, about 80 % to about 96 %, about 80 % to about 98 %, about 80 % to about 100 %, about 82 % to about 84 %, about 82 % to about 86 %, about 82 % to about 88 %, about 82 % to about 90 %, about 82 % to about 92 %, about 82 % to about 94 %, about 82 % to about 96 %, about 82 % to about 98 %, about 82 % to about 100 %, about 84 % to about 86 %, about 84 % to about 88 %, about 84 % to about 90 %, about 84 % to about 92 %, about 84 % to about 94 %, about 84 % to about 96 %, about 84 % to about 98 %, about 84 % to about 100 %, about 86 % to about 88 %, about 86 % to about 90 %, about 86 % to about 92 %, about 86 % to about 94 %, about 86 % to about 96 %, about 86 % to about 98 %, about 86 % to about 100 %, about 88 % to about 90 %, about 88 % to about 92 %, about 88 % to about 94 %, about 88 % to about 96 %, about 88 % to about 98 %, about 88 % to about 100 %, about 90 % to about 92 %, about 90 % to about 94 %, about 90 % to about 96 %, about 90 % to about 98 %, about 90 % to about 100 %, about 92 % to about 94 %, about 92 % to about 96 %, about 92 % to about 98 %, about 92 % to about 100 %, about 94 % to about 96 %, about 94 % to about 98 %, about 94 % to about 100 %, about 96 % to about 98 %, about 96 % to about 100 %, or about 98 % to about 100 %. In some embodiments, the reduction in receptor binding is about 75 %, about 80 %, about 82 %, about 84 %, about 86 %, about 88 %, about 90 %, about 92 %, about 94 %, about 96 %, about 98 %, or about 100 %. In some embodiments, the reduction in receptor binding is at least about 75 %, about 80 %, about 82 %, about 84 %, about 86 %, about 88 %, about 90 %, about 92 %, about 94 %, about 96 %, or about 98 %. In some embodiments, the reduction in receptor binding is at most about 80 %, about 82 %, about 84 %, about 86 %, about 88 %, about 90 %, about 92 %, about 94 %, about 96 %, about 98 %, or about 100 %.

[0137] Reduction of C. diff. Toxin Membrane Translocation

[0138] In some embodiments of the present invention, disruption of a target site of a C. diff. toxin protein by a novel antagonist of the invention reduces membrane translocation. In embodiments, cleavage at a TcdA or TcdB target cleavage site by a novel endopeptidase or binding at a TcdA or TcdB target binding site reduces membrane translocation by disrupting the translocation domain. In embodiments, membrane translocation is reduced by cleavage of or binding to TcdB at a sequence within amino acids 810-1350. In embodiments, membrane translocation is reduced by cleavage of or binding to TcdB at a sequence within the pore forming region at amino acids 830-990 or within the HR at amino acids 956-1128. In embodiments, novel endopeptidase cleavage of TcdA or TcdB reduces membrane translocation by cleavage at any point N-terminal to the translocation domain, e.g., TcdB amino acid 956. In embodiments, a novel binding protein to a TcdA or TcdB reduces membrane translocation by binding to a target binding site at any point N-terminal to the translocation domain, e.g., TcdB amino acid 956. In embodiments, the resulting reduction in membrane translocation is about 75% to about 100%.

[0139] Reduction of C. diff. Toxin Endosome Escape/ Autocatalytic Cleavage

[0140] In some embodiments of the present invention, cleavage of a C. diff. toxin protein by a novel antagonist of the invention reduces endosome escape by reducing autocatalytic cleavage (autoprocessing). Autocatalytic cleavage can be evaluated by any method described in the literature. In embodiments, the novel endopeptidase reduces TcdA or TcdB endosome escape/autocatalytic cleavage by disrupting the APD. In embodiments, endosome escape/autocatalytic cleavage is reduced by disruption of TcdB at a sequence within amino acids 544-807. In embodiments, TcdA or TcdB endosome escape/autocatalytic cleavage is reduced by disruption at any point N-terminal to the APD, e.g., TcdB amino acid 544. In embodiments, the reduction in endosome escape/autocatalytic cleavage resulting from toxin disruption by the novel antagonist is about 75% to about 100%.

[0141] Reduction of C. diff. Toxin Glucosyltransferase Activity

[0142] In some embodiments of the present invention, disruption of a C. diff. toxin protein by a novel antagonist of the invention reduces glucosyltransferase activity. In embodiments, TcdA or TcdB glucosyltransferase activity is reduced by disruption of the GTD. In embodiments, glucosyltransferase activity is reduced by disruption, e.g., cleavage by a novel endopeptidase or binding by a novel binding protein, of TcdB at a sequence within amino acids 1-543. In embodiments, the reduction in glucosyltransferase activity resulting from disruption by the novel endopeptidase is about 75% to about 100%.

[0143] Glucosyltransferase activity can be evaluated by Western Blot or other detection analysis, as described in the art. Enzymatic activity can be assayed, e.g., using glucosylhydrolase/glucosylation assay methods described in the art, for example in U.S. Pat. No. 7,226,597, “Mutants of Clostridium difficile toxin B and methods of use,” incorporated herein by reference in its entirety. Specifically, glucosylation reactions can be carried out in a reaction mix containing 50 mM n-2hydroxyethylpiperazine-n'-2-ethane sulfonic acid, 100 mM KC1, 1 mM MnCh, 1 mM MgCh, 100 pgram/ml BSA, 0.2 mM GDP, 40 pM[¹⁴C]UDP-glucose (303 Ci/mol; ICN Pharmaceuticals), 100 mM UDP-glucose and 3 pmol of TcdB or 10 pmol of each fusion protein. The assay is allowed to incubate overnight at 37° C and the cleaved glucose is separated using AG1-X2 anion exchange resin and counted in a liquid scintillation counter.

[0144] Helicobacter pylori

[0145] Helicobacter pylori is a gram-negative bacterium commonly found in the stomach and discovered in the 1980’s to colonize the gastric mucosa. Infection with H. pylori can cause chronic gastritis, peptic ulcer diseases, gastric cancer, and gastric mucosa-associated lymphoid tissue (MALT) lymphoma. When indicated, infection is typically treated with proton pump inhibitors and/or antibiotics, however antibiotic resistance of infecting strains is increasing.

[0146] H. pylori Pathogenesis

[0147] H. pylori thrives in the highly acidic stomach environment, in part by producing factors including urease, which promotes ammonia production, that reduce stomach acidity, and flagellin. Urease and its catalytic products also play a role in pathogenicity, e.g., by disrupting tight junctions. Sequences within that flagellin DO domain help the bacterium evade recognition by the toll-like receptor (TLR5).

[0148] The interaction with host cell receptors is mediated by bacterial outer membrane proteins including blood group antigen-binding adhesin Bab A, Sab A, outer inflammatory protein OipA, and outer membrane protein HopQ. BabA interacts with di-fucosylated glycan found on Leb and mono-fucosylated glycan found on HI -antigen, A-antigen, and B-antigen of blood groups O, A, and B respectively. H. pylori factors and its pathogenesis are described in the literature, e.g., by Ansari, S., and Yamaoka, Y., et al., 2019, “ Helicobacter pylori Virulence Factors Exploiting Gastric Colonization and its Pathogenicity,” Toxins 11: 677, Chmiela, M., and Kupcinskas, T, 2019, “Review: Pathogenesis of Helicobacter pylori infection,” Helicobacter 24 (Suppl. I):el2638, Yahiro, K., et al., 2016, Toxins 8:152, each incorporated by reference herein.

[0149] Treatment of H. Pylori Infection with Novel Antagonists

[0150] In embodiments, the invention includes compositions comprising a novel antagonist that reduces an activity of an H. pylori GI tract target. In embodiments, a novel antagonist of the invention reduces any undesirable activity of an H. pylori GI tract target known to those of skill in the art and described in the literature, including, but not limited to, receptor binding, translocation, cell entry, and intracellular effects, e.g., VacA-induced apoptosis or necrosis, g- glutamyl transpeptidase-induced apoptosis, CagA-dependent downregulation of cathepsin C (CtsC) via Src/ERK and Janus kinase (JAK), OipA-mediated apoptosis or mucosal damage, BabA- or SapA-mediated cell attachment and colonization, and CagA-dependent activation of transcription 3 (STAT 3) pathways. In embodiments, the novel antagonist is active at a stomach pH. In embodiments, a novel antagonist of the invention increases any activity normally decreased by an H. pylori GI tract target, e.g., cell viability and/or cell growth. In embodiments, the invention includes novel antagonist methods and compositions for treating a condition in a subject infected with and/or suspected to be infected with H. pylori. The invention includes the use of a novel antagonist and methods of using the novel antagonist to cleave a target cleavage site or bind to a target binding site of a GI tract target, wherein the GI tract target is a virulence factor, epithelial cell colonizing factor, or epithelial cell pathogenicity factor, of H. pylori.

[0151] In embodiments, the GI tract target is an epithelial cell colonizing factor selected from H. pylori VacA (vacuolating cytotoxin), CagA (cytotoxin associated protein), HtrA (protease), and g-glutamyl transpeptidase. In embodiments, the GI tract target is an epithelial cell colonizing factor selected from H. pylori BabA (binds with the epithelial cell receptor Leb), SabA (binds with sialyl-Lex antigen), OipA (bacterial adherence to the gastric epithelium), and HopQ outer membrane adhesin (bacterial adherence to the gastric epithelium). In embodiments, the GI tract target is an epithelial cell pathogenicity factor selected from CagT (helps in the translocation of CagA), CagY (binds integrin), CagL (helps in the translocation of CagA and binds integrin) In embodiments, the GI tract target is IceA (induced by contact with epithelium). In embodiments, the GI tract target binding partner is the transferrin receptor (TFRC), toll-like receptor 4 (TLR4), epithelial cell receptor Leb, sialyl Lex antigen, the gastric epithelium, integrin, or a certain carcinoembryonic antigen-related cell adhesion molecule (CEACAM). In embodiments, the GI tract target is HopQ and the Gl-tract binding partner is a CEACAM. In embodiments, the novel antagonist prevents the translocation of CagA. In embodiments, the GI tract target is CagA, wherein cleavage of a target cleavage site or binding to a target binding site of CagA by the novel antagonist prevents CagA translocation. In embodiments, the GI tract target is VacA and the GI tract target binding partner is receptor-like protein tyrosine phosphatase (RPTP) a, RPTPP, low-density lipoprotein receptor-related protein- 1 (LRP1), CD 18, fibronectin, or sphingomyelin. In embodiments, the GI tract target is VacA and the target cleavage site or the target binding site is located in the p55 region (C -terminal region of about 55 kDa) of VacA. In embodiments, the GI tract target is BabA and the GI tract target binding partner is epithelial cell receptor Leb.

[0152] Campylobacter

[0153] Campylobacter are gram-negative bacteria that are the most common bacterial cause of diarrheal illness in the United States, with most cases caused by Campylobacter jejuni and Campylobacter coli. About 20 cases of infection are diagnosed each year for every 100,000 people, with many more cases undiagnosed or unreported. The CDC estimates that Campylobacter infection affects 1.5 million U.S. residents every year. Campylobacter infection is acquired from food, particularly raw or undercooked poultry, by untreated drinking water, and through contact with infected animals.

[0154] The acidic stomach environment provides a natural defense against Campylobacter, but when successful infection leads to injury of GI compartments including the jejunum, ileum, and colon. Campylobacter infection typically causes bloody diarrhea, fever, stomach cramps, nausea and vomiting. Symptoms usually start two to five days after infection and last about one week, however some complications including irritable bowel syndrome, temporary paralysis, and arthritis can result. In people with weakened immune systems, Campylobacter can spread to the bloodstream and causes a life-threatening infection. Antibiotic treatment is available, however antibiotic-resistant strains Campylobacter continue to arise.

[0155] Campylobacter Pathogenesis [0156] Most Campylobacter strains, as well as many other gram-negati ve Proteobacteria including Shigella dysenteriae , Haemophilus ducreyi , and E. coli , produce cytolethal distending toxins (CDTs). CDTs, which cause apoptosis, are 3-subunit AB toxins, with an active ("A") subunit that directly damages DNA and a binding ("B") subunit that helps the toxin attach to the target cells. CdtB is the active subunit and a homolog to mammalian DNase I, and CdtA and CdtC make up the binding subunit. CDTs are reviewed by, e.g., Guerra, L. et al., 2011, “The Biology of the Cytolethal Distending Toxins,” Toxins 3:172-190, incorporated herein by reference.

[0157] Campylobacter jejuni also produces an enterotoxin resembling cholera toxin (CT) and the E. coli heat-labile enterotoxin (LT), that has been referred to as cholera toxin-like toxin (CTLT) (e.g., Albert, M. J. et al., 2007, “Identification of a Campylobacter jejuni Protein That Cross-Reacts with Cholera Toxin,” Inf. and Imm. 75(6): 3070-3073, incorporated herein by reference.)

[0158] Treatment of Campylobacter Infection with Novel Antagonists

[0159] In embodiments, the invention includes novel antagonist methods and compositions for treating a condition in a subject infected with and/or suspected to be infected with Campylobacter. In embodiments, the invention includes compositions comprising a novel antagonist that reduces an activity of an Campylobacter GI tract target. The invention includes the use of a novel antagonist and methods of using the novel antagonist to cleave a target cleavage site or bind to a target binding site of a Campylobacter GI tract target. In embodiments, the novel antagonist is active at a pH characteristic of the small intestine and/or large intestine. In embodiments, the novel antagonist is active at a pH characteristic of the jejunum, ileum, and/or colon.

[0160] In embodiments, the GI tract target is a cytolethal distending toxin or a CTLT. In embodiments, the cytolethal distending toxin is from a Proteobacterium. In embodiments, the cytolethal distending toxin is from a Campylobacter , a Shigella , a Haemophilus, or an Escherichia. In embodiments, the cytolethal distending toxin is from Campylobacter jejuni or Campylobacter coli. In embodiments, the target cleavage site or target binding site is within the CdtA or CdtC subunit of a cytolethal distending toxin. In embodiments, the GI tract binding partner is on the plasma membrane. In embodiments, the GI tract binding partner is an N-linked glycoprotein. In embodiments, the GI tract binding partner is a receptor for advanced glycation end products (RAGE). In embodiments, the GI tract target is a cytolethal distending toxin and the GI tract binding partner is an N-linked glycoprotein or a receptor for advanced glycation end products (RAGE). In embodiments, the target cleavage site or target binding site is a nuclear localization signal (NLS) of the GI tract target. In embodiments, the GI tract target is CdtB and the target cleavage site or target binding site is a nuclear localization signal. In embodiments, the GI tract target is CdtB and the target cleavage site or target binding site is NLS1 and/or NLS2. In embodiments, novel antagonist cleavage at a target cleavage site or binding at a target binding site that is NLS1, NLS2, or both, results in a reduction in cell cycle arrest and nuclear localization of one or more CDT compononents, e.g, the holotoxin.

[0161] In embodiments, the invention includes methods of reducing an activity of a Campylobacter GI Tract Target. In embodiments, a novel antagonist of the invention reduces any undesirable activity of a Campylobacter GI Tract Target known to those of skill in the art and described in the literature, including, but not limited to, plasma membrane binding, cell entry, translocation to the nucleus, and intranuclear effects, e.g., cell cycle arrest or DNase activity (and thereby a reduction in DNA damage). In embodiments, a novel a novel antagonist of the invention increases any activity normally decreased by Campylobacter infection, e.g., cell viability and/or cell growth. In embodiments, the invention includes novel antagonist methods and compositions for treating a condition in a subject infected with and/or experiencing symptoms of infection with Campylobacter.

[0162] Toxigenic Escherichia coli

[0163] Toxigenic, or pathogenic Escherichia coli include: Shiga toxin-producing E. coli (STEC), or Verocytotoxin-producing E. coli (VTEC) or enterohemorrhagic E. coli (EHEC); enterotoxigenic E. coli (ETEC); enteropathogenic E. coli (EPEC); enteroaggregative E. coli (EAEC); enteroinvasive E. coli (EIEC); and diffusely adherent A. coli (DAEC). An estimated 265,000 STEC infections occur each year in the United States. According to the CDC, STEC 0157 causes about 36% of these infections, and non-0157 STEC cause the rest. STEC include E. coli 0157:H7 (“0157”), commonly responsible for foodbome disease outbreaks, and non- 0157 STECs, e.g., 0145, 026, 0111, and 0103. Symptoms of infection with STEC often begin slowly with mild belly pain or non-bloody diarrhea that worsens over several days. Hemolytic uremic syndrome (HUS) may develop on an average 7 days after the first symptoms, when the diarrhea is improving.

[0164] Toxigenic A. coli Pathogenesis

[0165] E. coli 0157:H7 produces Shiga toxin (Stx), disrupts protein synthesis in the epithelial cells lining intestinal mucosa, leading to cell death, sloughing of the mucosa, and bloody diarrhea. The Shiga toxin has systemic effects on vascular endothelial cells, resulting in a vasculitis, and manifests in hemolytic uremic syndrome, abdominal pain, and rarely, thrombotic thrombocytopenic purpura. E. coli 0157:H7 Shiga toxin initiates the inflammatory cascade that causes leukocyte aggregation, apoptosis of the affected cells, platelet aggregation, microthrombi formation, hemolysis, and renal dysfunction. E. coli 0157:H7 Shiga toxin damages the kidney and can result in vasculitic injury that affects multiple organ systems and multiple organ failures. Variants of Stxl and Stx2 include Stxlc, Stx Id, Stx2c, Stx2d, Stx2d-activatable (Stx2-act),

Stx2e, and Stx2f.

[0166] Shiga toxins are ABs holotoxins. The active domain (A), comprises an N-glycosidase that depurinates the 28S rRNA of the 60S ribosomal subunit, which stops proteins synthesis and leads to cell death. The A subunit is about 32 kDa and is proteolytically cleaved by trypsin or furin into an Ai subunit of about 28 kDa, and an A2 peptide of about 5 kDa which are connected through a single disulfide bond. The Ai subunit contains the active domain. The A2 peptide non-covalently tethers the active domain to the binding domain. Binding domain B consists of five identical monomers of about 7.7 kDa that form a pentamer through which the C-terminus of the A2 peptide traverses. Each of the B subunit monomers has two cysteine residues that form a disulfide bond within each monomer. The B pentamer binds the eukaryotic receptor globotriaosyl ceramide (Gb3) (Gb4 for Stx2e). Shiga toxin has one moiety that binds to the cell surface, and another, enzymatically active moiety, that enters the cytosol and inhibits protein synthesis by inactivating ribosomes. The toxin travels from the cell surface to endosomes, the Golgi apparatus and the ER before the ribosome-inactivating moiety enters the cytosol. Shiga toxin binds to the neutral glycosphingolipid Gb3 at the cell surface and is therefore dependent on this lipid for transport into the cells. Certain E. coli 0157:H7 strains have been reported to harbor toxin-converting phages designated 935 J and 933 W. The “Shiga-like” produced toxins have homology with S. dysenteriae type 1 and Shigella flexneri. Shiga and Shiga-like toxins are described in the literature, e.g., in U.S. Pat. App. No. 2009/0258010, “Methods, compositions, and kits for treating shiga toxin associated conditions,” and Strockbine, N. A., et ah, 1986, “Two Toxin-Converting Phages from Escherichia coli 0157:H7 Strain 933 Encode Antigenically Distinct Toxins with Similar Biologic Activities,” Inf. And Imm. 53(1)”135-140, both incorporated by reference herein.

[0167] Treatment of Toxigenic Escherichia coli Infection with Novel Antagonists

[0168] In embodiments, the invention includes novel antagonist methods and compositions for treating a condition in a subject infected with and/or suspected to be infected with a toxigenic E. coli. In embodiments, the invention includes compositions comprising a novel antagonist that reduces an activity of a toxigenic E. coli GI tract target. The invention includes the use of a novel antagonist and methods of using the novel antagonist to cleave a target cleavage site or bind to a target binding site of a toxigenic E. coli GI tract target. In embodiments, the invention includes methods of reducing an activity of a toxigenic E. coli GI Tract Target. In embodiments, a novel antagonist of the invention reduces any undesirable activity of a toxigenic E. coli GI Tract Target known to those of skill in the art and described in the literature, including, but not limited to, receptor binding, transport into cells, leukocyte aggregation, apoptosis of the affected cells, platelet aggregation, microthrombi formation, hemolysis, and renal dysfunction. In embodiments, a novel antagonist of the invention increases any activity normally decreased by toxigenic if coli infection, e.g., cell viability and/or cell growth.

[0169] In embodiments, the GI tract target is a Shiga toxin or Shiga-like toxin. In embodiments, the GI tract binding partner is Gb3 or Gb4. In embodiments, the GI tract target is a Shiga toxin and the GI tract binding partner is Gb3. In embodiments, the GI tract target is Stx2e and the GI tract binding partner is Gb4. In embodiments, the target cleavage site or target binding site is in the B pentamer. In embodiments, target site cleavage or target binding site in the B pentamer disrupts binding of a Shiga or Shiga-like toxin to the Gb3 or Gb4 receptor. In embodiments, the target cleavage site or target binding site is in the Ai domain. In embodiments, target site cleavage or target binding site in the Ai domain reduces an activity of the Shiga or Shiga-like toxin.

[0170] Staphylococcus aureus

[0171] Staphylococcus aureus is a Gram-positive bacterium that is commonly present on the skin and upper respiratory tract. It is carried by about one third of the general population, often with no adverse effects, but can cause infection when over-represented. S. aureus is responsible for common and serious diseases, including skin infections, food poisoning, and toxic shock syndrome.

[0172] S. aureus Pathogenesis

[0173] More than 20 S. aureus enterotoxins have been identified, with varying numbers produced by different strains. Staphylococcal enterotoxins bind to T cell receptors, stimulating T cells and causing cytokine production. GI inflammatory injury is mediated through the SE superantigenic effect on MHC class II expressing mucosal professional APCs (macrophages and dendritic cells), nonprofessional APCs (such as myofibroblasts) and TCR expressing CD4+ T cells. SE can cross the intestinal epithelial barrier in and bind to class II MHC molecules expressed on subepithelial myofibroblasts. This results in production of proinflammatory cytokines and chemokines, including IL-6, IL-8 and MCP-1. MCP-1 may lead to increased chemotaxis of professional immune cells (CD4+ T cells, macrophages, DC) from gut associated lymphoid tissue (GALT) to the site of SE associated inflammation in GI mucosa. Those MHC class II:SEs:TCR interactions may in turn result in hyperactivation of APCs and T cells, leading to T cell proliferation and high levels of proinflammatory cytokines and chemokines. This can result in acute inflammation and shock. Staphylococcal enterotoxins (SEs) include, e.g., SEA, SEB, SEC, SED, SEE, SEF, SEG, SHE, SEI, and so forth, through SEV, Toxic Shock Syndrome Toxin-1 (TSST-1). Crystallographic studies show that SEs are elliptical in shape and have two major unequal domains composed mostly of b-strands and a few a-helices. The two domains are separated by a shallow cavity. The larger of the two domains contains both the amino and carboxyl termini. Mutational analysis of both SEA and SEB implicates this cavity in the binding to T cell receptors (TcR). Tyrosine 66 of SEA was identified by mutational analysis to interact with the TcR V b 7 and 8.1. Amino acids 45-58 of SEB are reportedly involved in the binding to class II major histocompatibility complex (MHC) molecules that are expressed by antigen presenting cells (APC). Several SEs have a Zn-binding site that contributes to their interaction with class II MHC molecules. Amino acids 118-175 are similar to the COOH-terminal end of the human and mouse CD74 protein (invariant chain) that binds class II MHC molecules early during their synthesis in the endoplasmic reticulum and serves as a scaffold for their assembly. Class II MHC molecule HLA-DRl has two chains, a and b, that Staphylococcal enterotoxins reportedly bind, either to both chains (SEA and SED), or one chain. SEA has been shown to bind to other class II MHC isoforms, including HLA-DP and HLA-DQ. SEB and SEC were shown to interact with HLA-DQ.

[0174] The Staphylococcal enterotoxins and their interactions with binding partners have been described at length in the literature, e.g., as reviewed by Pinchuk et ah, 2010, “Staphylococcal Enterotoxins” Toxins 2: 2177-2197, incorporated herein by reference.

[0175] Treatment of Staphylococcus aureus Infection with Novel Antagonists

[0176] In embodiments, the invention includes novel antagonist methods and compositions for treating a condition in a subject infected with and/or suspected to be infected with S. aureus. In embodiments, the invention includes compositions comprising a novel antagonist that reduce an activity of an S. aureus GI tract target. The invention includes the use of a novel antagonist and methods of using the novel antagonist to cleave a target cleavage site or bind to a target binding site of a S. aureus GI tract target. In embodiments, a novel antagonist of the invention reduces any undesirable activity of S. aureus GI tract target known to those of skill in the art and described in the literature, including, but not limited to, cell surface protein or receptor binding, superantigen effect, and upregulation of cytokine production. In embodiments, a novel antagonist of the invention increases any activity normally decreased by S. aureus infection, e.g., cell viability and/or cell growth. In embodiments, the invention includes novel antagonist methods and compositions for treating a condition in a subject infected with and/or experiencing symptoms of infection with S. aureus.

[0177] In embodiments, the S. aureus GI tract target is selected from a Staphylococcal enterotoxin, an Exfoliative Toxin, a cytotoxin, and an enzyme. In embodiments, the S. aureus GI tract target is selected from: SEA, SEB, SEC, SED, SEE, SEF, SEG, SHE, SEI, SEJ, SEK, SEL, SEM, SEN, SEO, SEP, SEQ, SER, SES, SET, SEU, SEV, Exfoliative Toxin A,

Exfoliative Toxin B, and TSST-1. In embodiments, the GI tract binding partner is a cell surface protein. In embodiments, the cell surface protein is an MHC Class II molecule. In embodiments, the MHC Class II molecule selected from HLA-DRl, HLA-DP and HLA-DQ. In embodiments, the GI tract binding partner is the HLA-DRl a chain or b chain. In embodiments, the GI tract target is SEA and the GI tract binding partner is HLA-DRl, HLA-DP or HLA-DQ. In embodiments, the GI tract target is SEB or SEC and the GI tract binding partner is HLA-DQ. In embodiments, the GI tract target is SEA and the GI tract target cleavage site or GI tract target binding site is Tyrosine 66. In further such embodiments, the GI tract binding partner is TcR V b 7 or 8.1. In embodiments, the GI tract target is SEB and the GI tract target cleavage site or GI tract target binding site is at or within amino acids 45-58. In embodiments, the GI tract target cleavage site or GI tract target binding site is a Zn binding site.

[0178] Enterotoxigenic Bacteroides fragilis

[0179] Bacteroides fragilis is a Gram-negative bacterium that is normally present in the human colon. Many species of B. fragilis are commensal, but enterotoxigenic B. fragilis (ETBF) is associated with colonic disease, and as an opportunistic pathogen B. fragilis can cause extraintestinal infections upon entry of the bloodstream or surrounding tissue. Extraintestinal infections include abdominal abscesses and bloodstream infections. ETBF associated with diarrheal disease can affect humans and many animals. Infection is treated by antibiotics, but strains resistant to ampicillin, metronidazole, clindamycin, or tetracycline are common.

[0180] . fragilis Pathogenesi s

[0181] Enterotoxigenic/?. Fragilis produce enterotoxins including BFT-1, BFT-2, and BFT-3. ETBF and its toxins are described in detail in the literature, e.g., by Allen, J. et al., 2019, “Epigenetic Changes Induced by Bacteroides fragilis Toxin,” Inf. and Imm. 87(6): 1-12, Sears, C. L., 2009, “Enterotoxigenic Bacteroides fragilis: a Rogue among Symbiotes,” Clin. Microb. Rev. 22(2): 349-369, and in W02020/010254, “Compositions and methods for treating inflammatory bowel disease,” each incorporated by reference herein in their entirety. Each BFT consists of three protein domains - the signal peptide, proprotein, and mature toxin, which contains a zinc-dependent metalloprotease motif. The proprotein is cleaved from the holotoxin A Cl 1 family cysteine protease, fragipain (Fpn) at a cleavage site to release the mature BFT. Sears 2009 reports the cleavage site as Arg_2ii-Arg_2i2. It has been proposed that ETBF colonizes the colon, where BFT is released and attaches to a specific colonic epithelial cell (CEC) receptor, triggering CEC signal transduction. This results in the cleavage of E-cadherin and CEC protein synthesis, and increased expression of c-Myc, cyclooxygenase-2 (COX-2), and chemokines/cytokines, including IL-8 and TGF-b. The cleavage initiates decreased barrier function of the colonic mucosa, potentially increasing exposure of the mucosal immune system to ETBF antigens and mucosal inflammation. c-Myc expression stimulates CEC proliferation, and the release of chemokines and cytokines by CECs into the submucosa enhance mucosal inflammation.

[0182] Treatment of Enterotoxigenic Bacteroides fragilis Infection with Novel Antagonists

[0183] In embodiments, the invention includes novel antagonist methods and compositions for treating a condition in a subject infected with and/or suspected to be infected with ETBF. In embodiments, the invention includes compositions comprising a novel antagonist that reduces an activity of an ETBF GI tract target. The invention includes the use of a novel antagonist and methods of using the novel antagonist to cleave a target cleavage site or bind to a target binding site of an ETBF GI tract target. In embodiments, the novel antagonist is active at a pH characteristic of the small intestine and/or large intestine.

[0184] In embodiments, the GI tract target is a BFT selected from BFT-1, BFT-2, BFT-3, and any combination thereof. In embodiments, the GI tract binding partner is colonic epithelial cell (CEC) receptor. In embodiments, the GI tract target is a BFT and the GI tract binding partner is a CEC receptor. In embodiments, the GI tract target is a BFT and the target cleavage site or target binding site is within the mature BFT protein. In embodiments, the GI tract target is a BFT and the target cleavage site or target binding site is the zinc-dependent metalloprotease motif. In embodiments, the GI tract target is a BFT and the target cleavage site or target binding site is the fragipain cleavage site of the holotoxin. In embodiments, the GI tract target is a BFT and the target cleavage site or target binding site is at Arg_2ii-Arg_2i2 of the holotoxin. In embodiments, the GI tract target is a BFT and the target cleavage site or target binding site is at Arg_2ii-Arg_2i2 of the holotoxin, or cleavage at the target cleavage site or binding at the target binding site disrupts at Arg_2ii-Arg_2i2 of the holotoxin.

[0185] In embodiments, the invention includes methods of reducing an activity of an ETBF GI Tract Target. In embodiments, a novel antagonist of the invention reduces any undesirable activity of an ETBF GI Tract Target known to those of skill in the art and described in the literature, including, but not limited to, receptor binding, epigenetic changes, epithelial cell rounding and detachment, neutrophilic inflammation, necrosis, hemorrhage, and combinations thereof. In embodiments, a novel antagonist of the invention increases any activity normally decreased by ETBF infection, e.g., cell viability and/or cell growth. In embodiments, the invention includes novel antagonist methods and compositions for treating a condition in a subject infected with and/or experiencing symptoms of infection with ETBF.

[0186] Vibrio cholerae

[0187] Vibrio cholerae is a Gram-negative bacterium that causes cholera, which can be contracted by ingesting contaminated food or water. About 5 to 10 percent of infected individuals develop severe cholera. Cholera symptoms include profuse watery diarrhea and vomiting leading to dehydration. Severe cholera can cause acute renal failure, severe electrolyte imbalances, coma, shock and death. Large amounts of infectious Vibrio cholerae bacteria are contained in the diarrhea produced by cholera patients and can readily spread disease.

[0188] V. cholerae Pathogenesis

[0189] V cholerae colonize the human small intestine and secrete the major virulence factor, cholera toxin (CT). CT belongs to the protein family of AB toxins, which comprise one catalytically active A-subunit and a homopentamer of non-toxic B-subunits. The CT B- pentamer (CTB) facilitates binding to epithelial cells in the small intestine and cellular uptake of the holotoxin. Once in the cytosol, the A-subunit uses the signalling pathways of the cell, causing the secretion of chloride ions into the intestinal lumen. The resulting osmotic gradient causes severe watery diarrhea. Cellular uptake involves movement from the plasma membrane of host cells to the endoplasmic reticulum by binding to the lipid raft ganglioside GM1. CT primary binding sites on its B-pentamer recognize GM1 ganglioside, and secondary binding sites on the B-pentamer interact with fucosylated histo-blood group antigens (HBGAs). GM1 binding is described by, e.g., Wolf A., et ak, 2008, “Attenuated Endocytosis and Toxicity of a Mutant Cholera Toxin with Decreased Ability To Cluster Ganglioside GM1 Molecules,” Inf. and Imm. 76(4): 1476-1484, and HBGA binding is described in detail by Heim et ak, 2019, “Crystal structures of cholera toxin in complex with fucosylated receptors point to importance of secondary binding site” Scientific Reports, Nature Research 9:12243, each incorporated herein by reference. Key primary binding site residues include Trp88 and Gly33, and key HBGA binding site residues include His 18 and His94. The 5-HT₃ receptors also have been shown to be important in CT pathogenesis (see, e.g., Mourad, F. H., et ak, 1995, “Role of 5- hydroxytrptamine type receptors in rat intestinal fluid and electrolyte secretion induced by cholera and Escherichia coli enterotoxins,” Gut 37:340-345, incorporated herein by reference.

[0190] Treatment of V cholerae Infection with Novel Antagonists

[0191] In embodiments, the invention includes novel antagonist methods and compositions for treating a condition in a subject infected with and/or suspected to be infected with Vibrio cholerae. In embodiments, the invention includes compositions comprising a novel antagonist that reduces an activity of a V cholerae GI tract target. The invention includes the use of a novel antagonist and methods of using the novel antagonist to cleave a target cleavage site or bind to a target binding site of a V cholerae GI tract target. In embodiments, the novel antagonist is active at a pH characteristic of the small intestine.

[0192] In embodiments, the GI tract target is CT. In embodiments, the GI tract binding partner is a GM1 and/or HBGA. In embodiments, the GI tract target is CT and the target cleavage site or target binding site is the within CT B-pentamer. In embodiments, the GI tract target is a CT and the target cleavage site or target binding site is a GM1 binding site in the CT B-pentamer.

In embodiments, the GI tract target is CT and cleavage at the target cleavage site or binding at the target binding site disrupts 1, 2, 3, 4, or 5 binding sites of the B-pentamer. In embodiments, the GI tract target is a CT, and cleavage at the target cleavage site or binding at the target binding site disrupts Trp88 or Gly33. In embodiments, the GI tract target is a CT, and the target cleavage site or target binding site is at Trp88 or Gly33. In embodiments, the GI tract target is a CT, and cleavage at the target cleavage site or binding at the target binding site disrupts Trp88 or Gly33.

[0193] In embodiments, the GI tract target is CT, the GI tract binding partner is GM1, and the target cleavage site or target binding site is the within CT B-pentamer. In embodiments, the GI tract target is a CT, the GI tract binding partner is GM1, and the target cleavage site or target binding site is a GM1 binding site in the CT B-pentamer. In embodiments, the GI tract target is CT, the GI tract binding partner is GM1, and cleavage at the target cleavage site or binding at the target binding site disrupts 1, 2, 3, 4, or 5 binding sites of the B-pentamer. In embodiments, the GI tract target is a CT, the GI tract binding partner is GM1, and the target cleavage site or target binding site is at Trp88 or Gly33. In embodiments, the GI tract target is a CT, the GI tract binding partner is GM1, and cleavage at the target cleavage site or binding at the target binding site disrupts Trp88 or Gly33.

[0194] In embodiments, the GI tract target is CT and the target cleavage site or target binding site is an HBGA binding site. In embodiments, the GI tract target is CT and the target cleavage site or target binding site is Hisl8 and/or His94. In embodiments, the GI tract target is CT and cleavage at the target cleavage site or binding at the target binding site disrupts Hisl8 and/or His94. In embodiments, the GI tract target is CT, the GI tract binding partner is HBGA, and the target cleavage site or target binding site is Hisl8 and/or His94. In embodiments, the GI tract target is CT, the GI tract binding partner is HBGA, and cleavage at the target cleavage site or binding at the target binding site disrupts Hisl8 and/or His94.

[0195] In embodiments, the invention includes methods of reducing an activity of a Vibrio cholerae GI Tract Target. In embodiments, a novel antagonist of the invention reduces any undesirable activity of a V cholerae GI Tract Target known to those of skill in the art and described in the literature, including, but not limited to, epithelial cell binding, cellular uptake, toxicity, and a combination thereof. In embodiments, a novel antagonist of the invention increases any activity normally decreased by ETBF infection, e.g., cell viability and/or cell growth. In embodiments, the invention includes novel antagonist methods and compositions for treating a condition in a subject infected with and/or experiencing symptoms of infection with V cholerae.

[0196] Commensal and Indigenous Pathobiont-induced Disorders

[0197] As described above, the invention includes GI tract targets produced by organisms that are constitutive in the GI tract and that contribute to disease or disorders under certain circumstances. Identification of constitutive GI tract organisms that trigger or perpetuate a condition provides a strategy for preventing and/or treating disease. It was reported by Palm, N., et ah, 2014, “Immunoglobulin A Coating Identifies Colitogenic Bacteria in Inflammatory Bowel Disease,” Cell 158:1-11, and U.S. Pat. Nos. 9,758,838 and 10,428,392, both titled “Compositions and methods for identifying secretory antibody -bound microbes,” each incorporated by reference herein in its entirety, that commensal GI tract bacteria characterized by IgA coating preferentially drive IBD. Methods for identifying such microorganisms are described therein. The potential role of secretory-antibody bound bacteria in inflammatory disease is also discussed in WO 2016/033439, “Compositions and methods for the treating an inflammatory disease or disorder,” incorporated herein by reference.

[0198] In embodiments, the invention includes novel antagonist compositions and methods for the prevention or treatment of disease in a subject, that reduce an activity of a GI tract target produced by one or more commensal or constitutive GI tract organisms. In embodiments, the invention includes novel antagonist compositions and methods for the prevention or treatment of a condition or disease in a subject, by reducing an activity of a GI tract target produced by a GI tract organism having a high secretory antibody coating. In embodiments, the GI tract organism having a high secretory antibody coating is a commensal bacteria or an indigenous pathobiont.

In embodiments, the secretory antibody is selected from IgAl, IgA2, and IgM. In embodiments, the GI tract organism is a secretory antibody-bound bacteria selected from the group consisting of Segmented Filamentous Bacteria (SFB), Heliobacter flexispira , Lactobacillus , Helicobacter , S24-7, Erysipelotrichaceae , Prevotellaceae , Eubacterium , Eubacterium biforme , Eubacterium dolichum , Ruminococcus gnavus, Acidaminococcus, Actinomyces , Allobaculum , Anaerostipes, Bacteroides, Bacteroides fragilis , Bifidobacterium , Bifidobacterium adolescentis , Blautia , Blautia obeum , Blautia producta , Clostridium , Clostridium perfringens , Collinsella aerofaciens , Coprococcus , Coprococcus catus, Dialister , Eggerthella lenta , Erysipelotrichaceae , Faecalibacterium prausnitzii , Haemophilus parainfluenzae , Lachnospiraceae , Lactobacillus , Lactobacillus mucosae , Lactobacillus reuteri , Lactobacillus zeae , Oscillospira , Rikenellaceae , Roseburia, Roseburia faecis , Ruminococcaceae , Ruminococcus , Ruminococcus bromii , SMB53, Streptococcus , Streptococcus luteciae , Sutterella , Turicibacter , UC Clostridiales , UC Erysipelotrichaceae , UC Ruminococcaceae , Veillonella , Veillonella dispar , and Weissella, and any combination thereof. In embodiments, the GI tract organism is selected from Prevotellaceae, Helicobacter, and SFB (segmented filamentous bacteria), and a combination thereof. In embodiments, the GI tract organism is selected from UC Prevotellaceae, SFB, Lactobacillus, Helicobacter sp. Flexispira, and a combination thereof.

[0199] In embodiments, the GI tract organism is selected from Acidaminococcus spp., Actinomyces spp. , Akkermansia muciniphila, Allobaculum spp., Anaerococcus spp., Anaerostipes spp. , Bacteroides spp. , Bacteroides Other, Bacteroides acidifaciens, Bacteroides coprophilus, Bacteroides fragilis, Bacteroides ovatus, Bacteroides uniformis, Barnesiellaceae spp., Bifidobacterium adolescentis, Bifidobacterium Other, Bifidobacterium spp., Bilophila spp., Blautia obeum, Blautia producta, Blautia Other, Blautia spp., Bulleidia spp., Catenibacterium spp., Citrobacter spp., Clostridiaceae spp., Clostridiales Other, Clostridiales spp., Clostridium perfringens, Clostridium spp., Clostridium Other, Collinsella aerofaciens, Collinsella spp., Collinsella stercoris, Coprococcus catus, Coprococcus spp., Coriobacteriaceae spp., Desulfovibrionaceae spp., Dialister spp., Dorea formicigenerans, Dorea spp., Dorea Other, Eggerthella lenta, Enterobacteriaceae Other, Enter obacteriaceae spp., Enterococcus spp., Erysipelotrichaceae spp. , Eubacterium biforme, Eubacterium biforme, Eubacterium dolichum, Eubacterium spp., Faecalibacterium prausnitzii, Fusobacterium spp., Gemellaceae spp., Haemophilus parainfluenzae, Haemophilus Other, Helicobacter spp., Helicobacter Lachnospiraceae Other, Lachnospiraceae spp., Lactobacillus reuteri, Lactobacillus mucosae, Lactobacillus zeae, Lactobacillus spp., Lactobacillaceae spp., Lactococcus spp.,

Leuconostocaceae spp., Megamonas spp. , Megasphaera spp.,Methanobrevibacter spp., Mitsuokella multacida , Mitsuokella spp. , Mucispirillum schaedleri , Odoribacter spp. , Oscillospira spp., Parabacteroides distasonis , Parabacteroides spp., Paraprevotella spp., Paraprevotellaceae spp., Parvimonas spp., Pediococcus spp., Pediococcus Other, Peptococcus spp., Peptoniphilus spp., Peptostreptococcus anaerobius , Peptostreptococcus Other, Phascolarctobacterium spp. , Prevotella copri, Prevotella spp. , Prevotella stercorea, Prevotellaceae, Proteus spp., Rikenellaceae spp., Roseburia faecis, Roseburia spp., Ruminococcaceae Other, Ruminococcaceae spp., Ruminococcus bromii, Ruminococcus gnavus, Ruminococcus spp., Ruminococcus Other, Ruminococcus torques, Slackia spp., S24-7 spp., SMB53 spp., Streptococcus anginosus, Streptococcus luteciae, Streptococcus spp.,

Streptococcus Other, Sutter ella spp., Turicibacter spp., UC Bulleidia, UC Enter obacteriaceae, UC Faecalibacterium, UC Parabacteroides, UC Pediococcus, Varibaculum spp., Veillonella spp., Sutterella, Turicibacter, UC Clostridiales, UC Erysipelotrichaceae, UC Ruminococcaceae, Veillonella parvula, Veillonella spp., Veillonella dispar , and Weissella.

[0200] In embodiments, the GI tract organism is a secretory antibody-bound bacteria and the condition or disease is an inflammatory disease. In embodiments, the condition or disease is GI inflammation, colitis, pseudomembranous colitis, hemorrhagic colitis, peptic ulcer disease, diarrhea, hemolytic uremic syndrome, inflammatory bowel disease, Crohn’s disease, ulcerative colitis, or cholera. In embodiments, the condition or disease is caused by or contributed to by infection, colonization, or overgrowth of a GI tract organism. In embodiments, the condition or disease is an arthritis or inflammatory arthritis.

[0201] In embodiments, the invention includes methods of reducing an activity of a secretory antibody-bound bacteria. In embodiments, a novel antagonist of the invention reduces any undesirable activity of secretory antibody-bound bacteria selected from a group including, but not limited to, receptor binding, cell-surface binding, epithelial colonization, transport into cells, nuclear transport, and cell damage. In embodiments, a novel antagonist of the invention increases any activity normally decreased by secretory antibody -bound bacteria infection, e.g., cell viability and/or cell growth. In embodiments, the invention includes novel antagonist methods and compositions for treating a condition in a subject infected with and/or experiencing symptoms of infection with one or more secretory antibody -bound bacteria.

[0202] Treatment of a Secretory Antibody-bound Bacterial Condition

[0203] The present invention includes a method for preventing and/or treating a condition or disease in a subject infected with and/or experiencing symptoms of infection with one or more secretory antibody-bound bacteria. In embodiments, the condition or disease is GI inflammation, colitis, pseudomembranous colitis, hemorrhagic colitis, peptic ulcer disease, diarrhea, hemolytic uremic syndrome, inflammatory bowel disease, Crohn’s disease, ulcerative colitis, or cholera. In embodiments, the other condition or disease is caused by or contributed to by infection, colonization, or overgrowth of a GI tract organism. In embodiments, the condition or disease is an arthritis. In embodiments, the condition or disease is selected from: a GI tract ulcer, gastritis, a GI tract cancer, an inflammatory or autoimmune condition, C. diff. infection,

H. pylori infection, Campylobacter jejuni infection, Campylobacter coli infection, Toxigenic Escherichia coli infection, Stapvhylococcus aureus infection, and Bacteroides fragilis infection.

[0204] In embodiments, the inflammatory or autoimmune condition or disease, is selected from, e.g., inflammatory bowel disease (IBD), Crohn’s disease, ulcerative colitis (UC), multiple sclerosis (MS), Type 1 diabetes mellitus, chronic biliary systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), psoriatic arthritis, autoimmune hepatitis, pemphigus, psoriasis, ankylosing spondylitis, Hashimoto’s thyroiditis, vasculitis, gout, scleroderma, and Graves disease. Additional autoimmune diseases are recognized in the art and described in the literature, e.g., in U.S. Pat. No. 10,485,882, “Nanoparticle compositions for sustained therapy,” incorporated herein by reference in its entirety.

[0205] Subjects

[0206] In embodiments, the subject is a mammal. In embodiments, the subject is a primate or ape. In embodiments, the subject is a human. In embodiments, the subject is any subject susceptible to or having the condition or disease being treated, as known to those of skill in the art. In embodiments, the subject is of any age. In embodiments, the subject is bovine, ovine, equine, porcine, avian, canine, or feline. In embodiments, the subject is a food animal, e.g., a cow, a pig, a sheep, or a turkey. In embodiments, the subject is a companion animal, e.g., a dog or cat.

[0207] In embodiments wherein the condition or disease is CDI, the subject has CDI. In embodiments, the subject is an asymptomatic carrier of C. diff. The treated subject can be any subject susceptible to infection with C. diff , including, but not limited to, a human (of any age, e.g., an adult, teen, or child), a cow, a horse, a pig, a dog, a cat, an ape, a monkey, or a bird.

[0208] Treatment Evaluation

[0209] In embodiments, the novel antagonist is evaluated based on its effect on an activity of the GI tract target, including, but not limited to, a reduction in GI tract target binding to a receptor; reduction in adhesion of the GI tract target to a cell or tissue; reduction in harm to a cell or tissue resulting from infection by a pathogen comprising the GI tract target; a reduction of or mitigation of a disease symptom resulting from infection by a pathogen comprising the GI tract target, e.g., diarrhea, vomiting, or abdominal pain; a reduction of or modulation of any other symptom or effect of infection with the GI tract pathogen corresponding to the GI tract target, including but not limited to an inflammatory condition or disease; an increase in a function of the subject, wherein the function is decreased by infection with the GI tract pathogen corresponding to the GI tract target; or any combination thereof.

[0210] In embodiments, administration of a novel antagonist of the invention to a subject with a condition or disease caused by a GI tract organism results in decreased disease symptoms. In embodiments, the disease symptoms are any symptoms known in the art to be characteristic of the GI tract infection. In embodiments, the disease symptoms are any known to be caused by an activity of the GI tract target. In embodiments, disease activity, disease symptoms, or any other characteristics of a disease or condition are measured using any appropriate method known in the art. In embodiments, disease activity in the subject is measured before, during, or after treatment. In embodiments, the disease activity measured following treatment is substantially reduced compared to the disease activity measured prior to treatment.

[0211] The effectiveness of treating a condition or disease in a subject using the inventive methods can be evaluated based on any appropriate end point known to those skilled in the art and/or described in the literature. In embodiments, the effect of treatment is evaluated based on resolution of any known symptoms of the disease by any known method for symptom evaluation. For example, resolution of diarrhea may be assessed at the end of a period of therapy (e.g., as defined by three or fewer unformed bowel movements over two consecutive days following treatment). In embodiments, e.g., for CDI infection, the period of therapy can be from about 5 days to about 25 days. In embodiments, evaluation is based on clinical recurrence of infection or disease symptoms, e.g., CDI, within a follow-up period (e.g., as defined by more than three unformed bowel movements in a 24-hour period, positive stool testing for C. diff. toxin A and/or B, and the need for retreatment for CDI). In embodiments, evaluation is based on clinical cure without relapse at the end of a follow-up period (e.g., defining relapse as diarrhea with a positive stool test for C. difficile toxin). In embodiments, the follow-up period is from about 4 to about 16 weeks. End points for clinical trials of antibiotic treatments for GI tract infections are known in the art. For example, endpoints for CDI are described in the literature, e.g., by Lewis and Anderson, 2013, “Treatment of Clostridium difficile infection: recent trial results,” Clin. Investig. (Lond) 3(9): 875-886, incorporated herein by reference. In an asymptomatic carrier, the effectiveness of treatment may be evaluated based on positive stool testing for C. diff toxin A and/or B following the period of therapy, or at the end of the defined follow-up period. Useful assays for toxin detection are commercially available and described by, e.g., Eastwood et al., 2009, J. Clin. Micro. 47(10): 3211-3217, incorporated by reference herein. Treatment modalities are also described by, e.g., Lowy et al., 2010, NEJM 362(3): 197- 205, and Wilcox et al., 2017, NEJM 376(4): 305-317, incorporated by reference herein.

[0212] In embodiments, the novel antagonist has optimal activity in the colon, e.g., due to resistance to colonic proteases, including, but not limited to reducing enzymes (e.g., nitroreductase, azoreductase, N-oxide reductase, sulfoxide reductase, and hydrogenase) and hydrolytic enzymes (e.g., esterases, amidases, glycosidases, glucuronidase, and sulfatase), as described in the literature (see, e.g., Jayaprakash and Mathew, 2012, “Colon Specific Drug Delivery Systems: A Review on Various Pharmaceutical Approaches,” J. App. Pharm. Sci.02(01): 163-169, incorporated herein by reference).

[0213] Novel Antagonist Compositions

[0214] In some embodiments, provided herein, is a novel antagonist, novel binding protein or endopeptidase, of any embodiment as described herein. In some embodiments, provided herein, is a pharmaceutical composition comprising the composition of any novel antagonist, the novel binding protein, or the novel endopeptidase and a pharmaceutically acceptable excipient. Pharmaceutical compositions can be formulated in a conventional manner using one or more pharmaceutically acceptable inactive ingredients that facilitate processing of the active compounds into preparations that can be used pharmaceutically. A proper formulation is dependent upon the route of administration chosen and a summary of pharmaceutical compositions can be found, for example, in Remington: The Science and Practice of Pharmacy (Gennaro, 21st Ed. Mack Pub. Co., Easton, PA (2005); Hoover, John E., Remington’s Pharmaceutical Sciences, Mack Publishing Co., Easton, Pennsylvania 1975; Liberman, H.A. and Lachman, L., Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, N.Y., 1980; and Pharmaceutical Dosage Forms and Drug Delivery Systems, Seventh Ed. (Lippincott Williams & Wilkins 1999), herein incorporated by reference. In some embodiments, the pharmaceutical composition facilitates administration of the compound to an organism.

[0215] In some embodiments, provided herein, is the novel antagonist, the novel binding protein, or the novel endopeptidase, described herein for use a medicament. In some embodiments, provided herein, is a method of treating a disease or a condition in a subject in need thereof, comprising administering to the subject the pharmaceutical composition described herein. In some embodiments, provided herein, is the pharmaceutical composition, the novel antagonist, the novel binding protein, or the novel endopeptidase described herein for use in a method of treating a disease or a condition in a subject in need thereof. In some embodiments, provided herein, is the use of the pharmaceutical composition, the novel antagonist, the novel binding protein, or the novel endopeptidase described herein for the manufacture of a medicament for treating a disease or a condition in a subject in need thereof. In some embodiments, the disease or the condition results from the activity of a GI tract target produced by a microbial organism as described herein. In some embodiments, the disease or the condition results from the activity of a GI tract target produced by one or more commensal or constitutive GI tract organisms as described herein. In some embodiments, the disease or the condition is GI inflammation, colitis, pseudomembranous colitis, hemorrhagic colitis, peptic ulcer disease, diarrhea, hemolytic uremic syndrome, inflammatory bowel disease, Crohn’s disease, ulcerative colitis, or cholera. In some embodiments, the condition or disease is caused by or contributed to by infection, colonization, or overgrowth of a GI tract organism. In some embodiments, the condition or disease is an arthritis or inflammatory arthritis.

[0216] A novel antagonist composition, e.g., a pharmaceutical composition, of the present invention can be administered by any suitable method known in the art for delivering a composition to the gastrointestinal tract, including, e.g., the colon (large intestine) and small intestine. In embodiments, a composition of the present invention is formulated for delivery to the colon. In embodiments, a composition of the present invention is formulated for administration by any route as appropriate, e.g., orally, rectally, or intragastrically. In embodiments, the novel antagonist composition described herein is combined with one or more pharmaceutically suitable or acceptable carrier (also referred to herein as a pharmaceutically suitable (or acceptable) excipient, physiologically suitable (or acceptable) excipient, or physiologically suitable (or acceptable) carrier selected on the basis of a chosen route of administration and standard pharmaceutical practice as described, e.g., in Remington (2005). [0217] An oral formulation for colonic delivery is designed to withstand the lower pH of the stomach and the proximal part of the small intestine, and preferably disintegrate at the neutral or slightly alkaline pH of the terminal ileum or the ileocecal junction, or at the colon pH (about 5.5 to about 7.5). In embodiments, a novel antagonist composition of the present invention is administered orally to a subject for delivery to the colon as a pill, capsule, tablet, or the like. In embodiments, the oral formulation has an enteric coating. In embodiments, a colon selective delivery system is used. As nonlimiting examples, the composition can be formulated using a pH sensitive coating, a delayed time-controlled release system, a bioadhesive system, polysaccharide-based delivery, microbially triggered drug delivery, pressure controlled drug delivery, Colon Targeted Delivery System (CODESTM), Osmotic Controlled Drug Delivery (ORDS-CT, e.g., OROS, Alza Corporation), or a suitable combination thereof, e.g., a timed- release, pH sensitive system. Considerations and options for colon-targeted oral delivery formulations are described at length in the literature, e.g., by Archana and Divya, 2016, Int. J. of Pharm. Science and Research 1(5): 40-47, Amidon et al., 2015, AAPS PharmSciTech 16(4)

DOI: 10.1208/sl2249-015-0350-9, and Philip and Philip, 2010, Oman Med. J. 25(2): 70-78, each incorporated herein by reference in its entirety.

[0218] In embodiments, a composition for intrarectal administration comprises a rectally suitable carrier, and is supplied in any appropriate formulation known to those of skill in the art, including, but not limited to, a solution, foam, gel, enema, or suppository. In embodiments, for intrarectal administration, a device, e.g., a syringe, bag, or a pressurized container for localized delivery within the rectum or colon is further provided. In embodiments, a composition for intragastric administration comprises an intragastric suitable carrier and is provided, e.g., through a nasogastric or nasoduodenal tube.

[0219] In embodiments, the novel antagonist composition is formulated in combination with one or more other active agent, e.g., a probiotic, a prebiotic, a nutritional supplement, a vitamin, or the like. In embodiments, the one or more other active agent is an antibiotic or a non antimicrobial therapy. In embodiments, the one or more other active agent is an antibiotic selected from: a macrocyclic antibiotic, e.g., fidaxomicin; metronidazole, vancomycin; a quinolonyl-oxazolidinone antibiotic, e.g., Act-0179811 (cadazolid); athiopeptide antibiotic, e.g., LFF571; a lipoglycodepsipeptide antibiotic, e.g., ramoplanin; a lipopeptide antibiotic, e.g., CB-183,315 (surotomycin). In embodiments, the one or more other active agent is a non antimicrobial therapy selected from: tolevamer; an anti-TcdA antibody, e.g., actoxumab; an anti- TcdB antibody, e.g., bezlotoxumab; a therapeutic vaccine; a probiotic, e.g., Saccharomyces boulardii , or a Lactobacillus spp; fecal microbiota transplantation; stool replacement therapy; and a synthetic stool transplant. In embodiments, the novel antagonist composition is formulated and provided as a medical food according to the Medical Foods Guidance Documents & Regulatory Information promulgated by the United States Food and Drug Administration, publicly available on the FDA website and incorporated herein by reference.

[0220] Dosage

[0221] A therapeutically effective amount of the novel antagonist composition or formulation as used herein refers to an amount of the enzyme or formulation that is effective for treating a disease or condition. Dosage regimens can be adjusted to provide the optimum desired response, as determined by those of skill in the art. A suitable dosage range may be 0.1 ug/kg- 100 mg/kg body weight; alternatively, it may be 0.5 ug/kg to 50 mg/kg; 1 ug/kg to 25 mg/kg, or 5 ug/kg to 10 mg/kg body weight. The antagonist can be delivered as a single dose, or may be administered multiple times, e.g., 2 to ten or more times and at desired intervals as desired or needed. [0222] While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

[0223] Exemplary Embodiments of the Invention - Novel Endopeptidase [0224] In certain embodiments, the invention includes:

1. A method for cleaving at least one C. difficile toxin protein, comprising: contacting the at least one C. difficile toxin protein with a novel endopeptidase that cleaves the at least one C. difficile toxin protein at a target site, under conditions suitable for the target site cleavage of the at least one C. difficile toxin protein by the novel endopeptidase to occur; wherein the at least one C. difficile toxin protein is a TcdA protein, a TcdB protein, or both; and wherein the target site cleavage results in a reduction in cytopathicity of the C. difficile toxin protein in comparison with the same at least one C. difficile toxin protein that has not been cleaved.

2. The method of embodiment 1, wherein the at least one C. difficile toxin protein is a TcdA protein.

3. The method of embodiment 1 or 2, wherein the target site cleavage reduces binding of the TcdA protein to the C. difficile toxin protein receptor, thereby reducing the cytopathicity.

4. The method of embodiment 2 or 3, wherein the target site cleavage of the TcdA protein removes a part of or the full receptor binding domain (RBD) of the TcdA protein.

5. The method of any of embodiments 2-4, wherein the target site cleavage removes at least one C-terminal combined repetitive oligopeptide (CROP) in the RBD.

6. The method of any of embodiments 2-5, wherein the target site cleavage removes all CROPs in the RBD. The method of embodiment 2, wherein the target site cleavage reduces membrane translocation of the TcdA protein into a target cell, thereby reducing the cytopathicity. The method of embodiment 1 or 7, wherein the target site cleavage reduces autocatalytic cleavage of the TcdA protein, thereby reducing membrane translocation of the TcdB protein into the target cell. The method of embodiment 1 or 2, wherein the target site cleavage reduces endosome escape of the TcdA protein, thereby reducing the cytopathicity. The method of embodiment 1 or 2, wherein the target site cleavage reduces glucosyltransferase activity of the TcdA protein, thereby reducing the cytopathicity. The method of embodiment 1, wherein the at least one C. difficile toxin protein is a TcdB protein. The method of embodiment 1 or 11, wherein the target site cleavage reduces binding of the cleaved C. difficile TcdB protein to the C. difficile toxin protein receptor, thereby reducing the cytopathicity. The method of embodiment 11 or 12, wherein the target site cleavage of the TcdB protein removes a part of or the full RBD of the TcdB protein. The method of embodiment 13, wherein the target site cleavage removes at least one CROP in the RBD. The method of embodiment 13 or 14, wherein the target site cleavage removes all CROPs in the RBD. The method of embodiment 11, wherein the target site cleavage reduces membrane translocation of the TcdB protein into a target cell, thereby reducing the cytopathicity. The method of embodiment 16, wherein the target site cleavage reduces autocatalytic cleavage of the TcdB protein, thereby reducing membrane translocation of the TcdB protein into the target cell. The method of embodiment 11, wherein the target site cleavage reduces endosome escape of the TcdB protein, thereby reducing the cytopathicity. The method of embodiment 11, wherein the target site cleavage reduces glucosyltransferase activity of the TcdB protein, thereby reducing the cytopathicity. The method of any of embodiments 3-6 and 12-15, wherein: the reduction in binding of the at least one C. difficile toxin protein to the cell receptor is represented by a reduction in binding affinity; the novel endopeptidase cleaves the at least one toxin protein with a high specificity; the novel endopeptidase cleaves the at least one toxin protein with a high activity; or a combination thereof. The method of any of embodiments 3-6 and 12-15, wherein the reduction in binding of the at least one C. difficile toxin protein to the cell receptor is about 80% to about 100%, in comparison with the at least one C. difficile toxin protein that has not been cleaved. The method of any of embodiments 7, 8, 16 and 17, wherein the reduction in membrane translocation is about 80% to about 100%, in comparison with the at least one C. difficile toxin protein that has not been cleaved. The method of embodiment 9 or 18, wherein the reduction in endosome escape is about 80% to about 100%, in comparison with the at least one C. difficile toxin protein that has not been cleaved. The method of embodiment 10 or 19, wherein the reduction in glucosyltransferase activity is about 80% to about 100%, in comparison with the at least one C. difficile toxin protein that has not been cleaved. The method of any of embodiments 1-24, wherein the reduction in cytopathicity is about 80% to about 100%. A method for reducing the cytopathicity of at least one C. difficile toxin protein comprising: contacting the at least one C. difficile toxin protein with a novel endopeptidase that cleaves the at least one C. difficile toxin protein at a target site, under conditions suitable for the target site cleavage of the at least one C. difficile toxin protein by the novel endopeptidase to occur; wherein the at least one C. difficile toxin protein is a TcdA protein, a TcdB protein, or both; and wherein the cytopathicity of the cleaved C. difficile toxin protein is reduced in comparison with the same at least one C. difficile toxin protein that has not been cleaved. The method of embodiment 25, wherein the C. difficile toxin protein is a TcdA protein. The method of embodiment 26, wherein the target site cleavage of the TcdA protein reduces binding of the cleaved TcdA protein to the C. difficile toxin protein receptor, thereby reducing the cytopathicity. The method of any one of embodiments 26-28, wherein the target site cleavage removes a part of or the full RBD of the at least one TcdA protein. The method of embodiment 28, wherein the target site cleavage removes at least one CROP in the RBD. The method of embodiment 29, wherein the target site cleavage removes all CROPs in the RBD. The method of embodiment 26, wherein the target site cleavage reduces membrane translocation of the TcdA protein into a target cell, thereby reducing the cytopathicity. The method of embodiment 32, wherein the target site cleavage reduces autocatalytic cleavage of the TcdA protein, thereby reducing the membrane translocation. The method of embodiment 26, wherein the target site cleavage reduces endosome escape of the TcdA protein, thereby reducing the cytopathicity. The method of embodiment 26, wherein the target site cleavage reduces glucosyltransferase activity of the TcdA protein, thereby reducing the cytopathicity. The method of embodiment 26, wherein the C. difficile toxin protein is a TcdB protein. The method of embodiment 36, wherein the target site cleavage of the TcdB protein reduces binding of the TcdB protein to the C. difficile toxin protein receptor, thereby reducing the cytopathicity. The method of any one of embodiments 26, 36, and 37 wherein the target site cleavage removes a part of or the full RBD of the TcdB protein. The method of embodiment 38, wherein the target site cleavage removes at least one CROP in the RBD. The method of embodiment 39, wherein the target site cleavage removes all CROPs in the RBD. The method of embodiment 36, wherein the target site cleavage reduces membrane translocation of the TcdB protein into a target cell, thereby reducing the cytopathicity. The method of embodiment 36 or 41, wherein the target site cleavage reduces autocatalytic cleavage of the TcdB protein, thereby reducing membrane translocation of the TcdB protein into the target cell. The method of embodiment 36, wherein the target site cleavage reduces endosome escape of the TcdB protein, thereby reducing the cytopathicity. The method of any of embodiments 28-31 and 37-40, wherein: the reduction in binding of the at least one C. difficile toxin protein to the cell receptor is represented by a reduction in binding affinity; the novel endopeptidase cleaves the at least one toxin protein with a high specificity, the novel endopeptidase cleaves the at least one toxin protein with a high activity; or a combination thereof. The method of any of embodiments 28-31 and 37-40, wherein the reduction in binding of the at least one C. difficile toxin protein to the cell receptor is about 80% to about 100%, in comparison with the at least one C. difficile toxin protein that has not been cleaved. The method of any of embodiments 32, 33, 41 and 42, wherein the reduction in membrane translocation is about 80% to about 100%, in comparison with the at least one C. difficile toxin protein that has not been cleaved. The method of embodiment 34 or 43, wherein the reduction in endosome escape is about 80% to about 100%, in comparison with the at least one C. difficile toxin protein that has not been cleaved. The method of embodiment 35 or 125, wherein the reduction in glucosyltransferase activity is about 80% to about 100%, in comparison with the at least one C. difficile toxin protein that has not been cleaved. The method of any of embodiments 26-48, wherein the reduction in cytopathicity is about 80% to about 100%. A method for treating C. difficile infection in a subject, comprising: administering to the subject a therapeutically effective amount of a composition comprising a novel endopeptidase that cleaves at least one C. difficile toxin protein at a target site; wherein the at least one C. difficile toxin protein is a TcdA protein, a TcdB protein, or both; and wherein the cytopathicity of the at least one cleaved C. difficile toxin protein is reduced in comparison with the same at least one C. difficile toxin protein that has not been cleaved. The method of embodiment 50, wherein the C. difficile toxin protein is a TcdA protein. The method of embodiment 51, wherein the target site cleavage of the TcdA protein reduces binding of the cleaved C. difficile TcdA protein to the C. difficile toxin protein receptor, thereby reducing the cytopathicity. The method of embodiment 51 or 52, wherein the target site cleavage removes a part of or the full RBD of the TcdA protein. The method of embodiment 53, wherein the target site cleavage removes at least one CROP in the RBD. The method of embodiment 54, wherein the target site cleavage removes all CROPs in the RBD. The method of embodiment 51, wherein the target site cleavage reduces membrane translocation of the TcdA protein into a target cell. The method of embodiment 56, wherein the target site cleavage reduces autocatalytic cleavage of the TcdA protein, thereby reducing membrane translocation of the TcdA protein into the target cell. The method of embodiment 51, wherein the target site cleavage reduces endosome escape of the TcdA protein. The method of embodiment 51, wherein the target site cleavage reduces glucosyltransferase activity of the TcdA protein. The method of embodiment 50, wherein the C. difficile toxin protein is a TcdB protein. The method of embodiment 60, wherein the target site cleavage of the TcdB protein reduces binding of the TcdB protein to the C. difficile toxin protein receptor, thereby reducing the cytopathicity. The method of embodiment 50 or 60, wherein the target site cleavage removes a part of or the full RBD of the TcdB protein. The method of embodiment 62, wherein the target site cleavage removes at least one CROP in the RBD. The method of embodiment 63, wherein the target site cleavage removes all CROPs in the RBD. The method of embodiment 60, wherein the target site cleavage reduces membrane translocation of the TcdB protein into a target cell. The method of embodiment 65, wherein the target site cleavage reduces autocatalytic cleavage of the TcdB protein, thereby reducing membrane translocation of the TcdA protein into the target cell. The method of embodiment 60, wherein the target site cleavage reduces endosome escape of the TcdB protein. The method of embodiment 60, wherein the target site cleavage reduces glucosyltransferase activity of the TcdB protein. The method of any of embodiments 52-55 and 61-64, wherein: the reduction in binding of the at least one C. difficile toxin protein to the cell receptor is represented by a reduction in binding affinity; the novel endopeptidase cleaves the at least one toxin protein with a high specificity, the novel endopeptidase cleaves the at least one toxin protein with a high activity; or a combination thereof. The method of any of embodiments 52-55 and 61-64, wherein the reduction in binding of the at least one C. difficile toxin protein to the cell receptor is about 80% to about 100%, in comparison with the at least one C. difficile toxin protein that has not been cleaved. The method of any of embodiments 56, 57, 65 and 66, wherein the reduction in membrane translocation is about 80% to about 100%, in comparison with the at least one C. difficile toxin protein that has not been cleaved. The method of embodiment 58 or 67, wherein the reduction in endosome escape is about 80% to about 100%, in comparison with the at least one C. difficile toxin protein that has not been cleaved. The method of embodiment 59 or 68, wherein the reduction in glucosyltransferase activity is about 80% to about 100%, in comparison with the at least one C. difficile toxin protein that has not been cleaved. The method of any of embodiments 50-73, wherein the reduction in cytopathicity is about 80% to about 100%. The method of any of embodiments 50-73, wherein the composition is formulated for colonic delivery. The method of embodiment 75, wherein the composition is formulated for oral or rectal administration. The method of embodiment 76, wherein the composition is formulated for oral administration and comprises an enteric coating. The method of any of embodiments 50-78, wherein the composition comprises at least one other active agent, is administered in combination with at least one other active agent, or both. The method of embodiment 78, wherein the at least one other active agent is a prebiotic or probiotic agent. A novel endopeptidase capable of a target site cleavage of at least one C. difficile toxin protein selected from a TcdA protein and a TcdB protein, wherein the target site cleavage results in reduced cytopathicity of the at least one C. difficile toxin protein. The novel endopeptidase of embodiment 80, wherein the C. difficile toxin protein is a TcdA protein. The novel endopeptidase of embodiment 81, wherein the target site cleavage of the Ted A protein reduces binding of the Ted A protein to the C. difficile toxin protein receptor, thereby reducing the cytopathicity. The novel endopeptidase of embodiment 81 or 82, wherein the target site cleavage removes a part of or the full RBD of the TcdA protein, thereby reducing binding of the Ted A protein to a C. difficile toxin protein receptor. The novel endopeptidase of embodiment 83, wherein at least one CROP in the TcdA protein RBD is removed by the target site cleavage. The novel endopeptidase of embodiment 84, wherein all CROPs in the RBD are removed by the target site cleavage. The novel endopeptidase of embodiment 81, wherein the target site cleavage reduces membrane translocation of the TcdA protein into a target cell. The novel endopeptidase of embodiment 86, wherein the target site cleavage reduces autocatalytic cleavage of the TcdA protein, thereby reducing membrane translocation of the TcdA protein into a target cell. The novel endopeptidase of embodiment 81, wherein the target site cleavage reduces endosome escape of the TcdA protein. The novel endopeptidase of embodiment 81, wherein the target site cleavage reduces glucosyltransferase activity of the TcdA protein. The novel endopeptidase of embodiment 80, wherein the C. difficile toxin protein is TcdB. The novel endopeptidase of embodiment 90, wherein the target site cleavage of the TcdB protein reduces binding of the TcdB protein to the C. difficile toxin protein receptor, thereby reducing the cytopathicity. The novel endopeptidase of embodiment 90 or 91, wherein the target site cleavage removes a part of or the full RBD of the TcdB protein, thereby reducing binding of the TcdB protein to a C. difficile toxin protein receptor. The novel endopeptidase of embodiment 92, wherein at least one CROP in the RBD is removed by the target site cleavage. The novel endopeptidase of embodiment 93, wherein all CROPs in the RBD are removed by the target site cleavage. The novel endopeptidase of embodiment 90, wherein the target site cleavage reduces membrane translocation of the TcdB protein into a target cell. The novel endopeptidase of embodiment 95, wherein the target site cleavage reduces autocatalytic cleavage of the TcdB protein, thereby reducing membrane translocation of the TcdB protein into a target cell. The novel endopeptidase of embodiment 90, wherein the target site cleavage reduces endosome escape of the TcdB protein. The novel endopeptidase of embodiment 90, wherein the target site cleavage reduces glucosyltransferase activity of the TcdB protein. The novel endopeptidase of any of embodiments 82-85 and 91-94, wherein: the reduction in binding of the at least one C. difficile toxin protein to the cell receptor is represented by a reduction in binding affinity; the novel endopeptidase cleaves the at least one toxin protein with a high specificity; the novel endopeptidase cleaves the at least one toxin protein with a high activity; or a combination thereof. . The novel endopeptidase of any of embodiments 82-85 and 91-94, wherein the reduction in binding of the at least one C. difficile toxin protein to the cell receptor is about 80% to about 100%, in comparison with the at least one C. difficile toxin protein that has not been cleaved. . The novel endopeptidase of any of embodiments 86, 87, 95, and 96, wherein the reduction in membrane translocation is about 80% to about 100%, in comparison with the at least one C. difficile toxin protein that has not been cleaved. . The novel endopeptidase of embodiment 88 or 97, wherein the reduction in endosome escape is about 80% to about 100%, in comparison with the at least one C. difficile toxin protein that has not been cleaved. . The novel endopeptidase of embodiment 89 or 98, wherein the reduction in glucosyltransferase activity is about 80% to about 100%, in comparison with the at least one C. difficile toxin protein that has not been cleaved. . The novel endopeptidase of any of embodiments 80-103, wherein the reduction in cytopathicity is about 80% to about 100%. . A composition comprising the novel endopeptidase of any of embodiments 80-104. . The composition of embodiment 105, formulated for colonic delivery. . The composition of embodiment 105 or 106, formulated for oral or rectal administration. . The composition of embodiment 107, wherein the composition is formulated for oral administration and comprises an enteric coating. . The composition of any of embodiments 105-108, wherein the composition comprises at least one other active agent, is administered in combination with at least one other active agent, or both. . The composition of embodiment 109, wherein the at least one other active agent is a prebiotic or probiotic agent. . A method for treating C. difficile infection in a subject in need thereof, comprising administering to the subject the composition any of embodiments 105-110. . A method for reducing the binding of at least one C. difficile toxin protein to a cell receptor specific for the at least one C. difficile toxin protein, comprising: contacting the at least one C. difficile toxin protein with a novel endopeptidase that cleaves the at least one C. difficile toxin protein at a target site, under conditions suitable for the target site cleavage of the at least one C. difficile toxin protein by the novel endopeptidase to occur; wherein the at least one C. difficile toxin protein is a TcdA protein, a TcdB protein, or both; and wherein the binding of the cleaved C. difficile toxin protein to a C. difficile toxin protein receptor is reduced in comparison with the same at least one C. difficile toxin protein that has not been cleaved. . The method of embodiment 112, wherein the at least one C. difficile toxin protein is a TcdA protein. . The method of embodiment 113, wherein the target site cleavage reduces binding of the TcdA protein to the C. difficile toxin protein receptor. . The method of embodiment 113 or 114, wherein the target site cleavage of the TcdA protein removes a part of or the full RBD of the TcdA protein. . The method of any of embodiments 113-115, wherein the target site cleavage removes at least one CROP in the RBD. . The method of any of embodiments 113-116, wherein the target site cleavage removes all CROPs in the RBD. . The method of embodiment 112, wherein the at least one C. difficile toxin protein is a TcdB protein. . The method of embodiment 118, wherein the target site cleavage reduces binding of the cleaved C. difficile TcdB protein to the C. difficile toxin protein receptor. . The method of any of embodiments 118 or 119, wherein the target site cleavage of the TcdB protein removes a part of or the full RBD of the TcdB protein. . The method of embodiment 120, wherein the target site cleavage removes at least one CROP in the RBD. . The method of embodiment 120 or 121, wherein the target site cleavage removes all CROPs in the RBD. . The method of any of embodiments 113-122, wherein: the reduction in binding of the at least one C. difficile toxin protein to the cell receptor is represented by a reduction in binding affinity; the novel endopeptidase cleaves the at least one toxin protein with a high specificity; the novel endopeptidase cleaves the at least one toxin protein with a high activity; or a combination thereof. 124. The method of any of embodiments 113-122, wherein the reduction in binding of the at least one C. difficile toxin protein to the cell receptor is about 80% to about 100%, in comparison with the at least one C. difficile toxin protein that has not been cleaved.

125. The method of embodiment 36, wherein the target site cleavage reduces glucosyltransferase activity of the TcdB protein, thereby reducing the cytopathicity.

[0225] Exemplary Embodiments of the Invention - Novel Binding Protein [0226] In certain other embodiments, the invention includes:

1. A method for reducing cytopathicity of at least one C. difficile toxin protein, comprising: contacting the at least one C. difficile toxin protein with a novel binding protein that binds the at least one C. difficile toxin protein at a target site, under conditions suitable for the binding of the at least one C. difficile toxin protein by the novel binding protein to occur; wherein the at least one C. difficile toxin protein is a TcdA protein, a TcdB protein, or both; and wherein the target site binding results in a reduction in cytopathicity of the C. difficile toxin protein.

3. The method of embodiment 1 or 2, wherein the target site binding reduces binding of the TcdA protein to the C. difficile toxin protein receptor, thereby reducing the cytopathicity.

4. The method of embodiment 2 or 3, wherein the target site binding of the TcdA protein disrupts a part of or the full receptor binding domain (RBD) of the TcdA protein.

5. The method of any of embodiments 2-4, wherein the target site binding disrupts at least one C-terminal combined repetitive oligopeptide (CROP) in the RBD.

6. The method of any of embodiments 2-5, wherein the target site binding disrupts all CROPs in the RBD.

7. The method of embodiment 2, wherein the target site binding reduces membrane translocation of the TcdA protein into a target cell, thereby reducing the cytopathicity. The method of embodiment 1 or 7, wherein the target site binding reduces autocatalytic cleavage of the TcdA protein, thereby reducing membrane translocation of the TcdB protein into the target cell. The method of embodiment 2, wherein the target site binding reduces endosome escape of the TcdA protein, thereby reducing the cytopathicity. The method of embodiment 2, wherein the target site binding reduces glucosyltransferase activity of the TcdA protein, thereby reducing the cytopathicity. The method of embodiment 1, wherein the at least one C. difficile toxin protein is a TcdB protein. The method of embodiment 1 or 11, wherein the target site binding reduces binding of the C. difficile TcdB protein to the C. difficile toxin protein receptor, thereby reducing the cytopathicity. The method of any of embodiment 11 or 12, wherein the target site binding of the TcdB protein disrupts a part of or the full RBD of the TcdB protein. The method of embodiment 13, wherein the target site binding disrupts at least one CROP in the RBD. The method of embodiment 14, wherein the target site binding disrupts all CROPs in the RBD. The method of embodiment 11, wherein the target site binding reduces membrane translocation of the TcdB protein into a target cell, thereby reducing the cytopathicity. The method of embodiment 17, wherein the target site binding reduces autocatalytic cleavage of the TcdB protein, thereby reducing membrane translocation of the TcdB protein into the target cell. The method of embodiment 11, wherein the target site binding reduces endosome escape of the TcdB protein, thereby reducing the cytopathicity. The method of embodiment 11, wherein the target site binding reduces glucosyltransferase activity of the TcdB protein, thereby reducing the cytopathicity. The method of any of embodiments 3-6 and 12-15, wherein: the reduction in binding of the at least one C. difficile toxin protein to the cell receptor is represented by a reduction in binding affinity; the novel binding protein disrupts the at least one toxin protein with a high specificity; the novel binding protein disrupts the at least one toxin protein with a high activity; or a combination thereof. The method of any of embodiments 3-6 and 12-15, wherein the reduction in binding of the at least one C. difficile toxin protein to the cell receptor is about 80% to about 100%. The method of any of embodiments 7, 8, 16 and 17, wherein the reduction in membrane translocation is about 80% to about 100%. The method of embodiment 9 or 18, wherein the reduction in endosome escape is about 80% to about 100%. The method of embodiment 10 or 19, wherein the reduction in glucosyltransferase activity is about 80% to about 100%. The method of any of embodiments 1-24, wherein the reduction in cytopathicity is about 80% to about 100%. A method for reducing the cytopathicity of at least one C. difficile toxin protein comprising: contacting the at least one C. difficile toxin protein with a novel binding protein that disrupts the at least one C. difficile toxin protein at a target site, under conditions suitable for the target site binding of the at least one C. difficile toxin protein by the novel binding protein to occur; wherein the at least one C. difficile toxin protein is a TcdA protein, a TcdB protein, or both; and wherein the cytopathicity of the C. difficile toxin protein is reduced. The method of embodiment 25, wherein the C. difficile toxin protein is a TcdA protein. The method of embodiment 26, wherein the target site binding of the TcdA protein reduces binding of the TcdA protein to the C. difficile toxin protein receptor, thereby reducing the cytopathicity. The method of any one of embodiments 26-28, wherein the target site binding disrupts a part of or the full RBD of the at least one TcdA protein. The method of embodiment 28, wherein the target site binding disrupts at least one CROP in the RBD. The method of embodiment 29, wherein the target site binding disrupts all CROPs in the RBD. The method of embodiment 26, wherein the target site binding reduces membrane translocation of the TcdA protein into a target cell, thereby reducing the cytopathicity. The method of any one of embodiment 32, wherein the target site binding reduces autocatalytic cleavage of the TcdA protein, thereby reducing the membrane translocation. The method of embodiment 26, wherein the target site binding reduces endosome escape of the TcdA protein, thereby reducing the cytopathicity. The method of embodiment 26, wherein the target site binding reduces glucosyltransferase activity of the TcdA protein, thereby reducing the cytopathicity. The method of embodiment 26, wherein the C. difficile toxin protein is a TcdB protein. The method of embodiment 36, wherein the target site binding of the TcdB protein reduces binding of the C. difficile TcdB protein to the C. difficile toxin protein receptor, thereby reducing the cytopathicity. The method of any one of embodiments 26, 36, and 37, wherein the target site binding disrupts a part of or the full RBD of the TcdB protein. The method of embodiment 38, wherein the target site binding disrupts at least one CROP in the RBD. The method of embodiment 38 or 39, wherein the target site binding disrupts all CROPs in the RBD. The method of embodiment 36, wherein the target site binding reduces membrane translocation of the TcdB protein into a target cell, thereby reducing the cytopathicity. The method of embodiment 36 or 41, wherein the target site binding reduces autocatalytic cleavage of the TcdB protein, thereby reducing membrane translocation of the TcdB protein into the target cell. The method of embodiment 36, wherein the target site binding reduces endosome escape of the TcdB protein, thereby reducing the cytopathicity. The method of any of embodiments 28-31 and 37-40, wherein: the reduction in binding of the at least one C. difficile toxin protein to the cell receptor is represented by a reduction in binding affinity; the novel binding protein disrupts the at least one toxin protein with a high specificity, the novel binding protein disrupts the at least one toxin protein with a high activity; or a combination thereof. The method of any of embodiments 28-31 and 37-40, wherein the reduction in binding of the at least one C. difficile toxin protein to the cell receptor is about 80% to about 100%. The method of any of embodiments 32, 33, 41 and 42, wherein the reduction in membrane translocation is about 80% to about 100%. The method of embodiment 34 or 43, wherein the reduction in endosome escape is about 80% to about 100%. The method of embodiment 35 or 125, wherein the reduction in glucosyltransferase activity is about 80% to about 100%. The method of any of embodiments 26-48, wherein the reduction in cytopathicity is about 80% to about 100%. A method for treating C. difficile infection in a subject, comprising: administering to the subject a therapeutically effective amount of a composition comprising a novel binding protein that binds at least one C. difficile toxin protein at a target site; wherein the at least one C. difficile toxin protein is a TcdA protein, a TcdB protein, or both; and wherein the cytopathicity of the at least one C. difficile toxin protein is reduced. The method of embodiment 50, wherein the C. difficile toxin protein is a TcdA protein. The method of embodiment 51, wherein the target site binding of the TcdA protein reduces binding of the C. difficile TcdA protein to the C. difficile toxin protein receptor, thereby reducing the cytopathicity. The method of embodiment 51 or 52, wherein the target site binding disrupts a part of or the full RBD of the TcdA protein. The method of embodiment 53, wherein the target site binding disrupts at least one CROP in the RBD. The method of embodiment 54, wherein the target site binding disrupts all CROPs in the RBD. The method of embodiment 51, wherein the target site binding reduces membrane translocation of the TcdA protein into a target cell. The method of embodiment 56, wherein the target site binding reduces autocatalytic cleavage of the TcdA protein, thereby reducing membrane translocation of the TcdA protein into the target cell. The method of embodiment 51, wherein the target site binding reduces endosome escape of the TcdA protein. The method of embodiment 51, wherein the target site binding reduces glucosyltransferase activity of the TcdA protein. The method of embodiment 50, wherein the C. difficile toxin protein is a TcdB protein. The method of embodiment 60, wherein the target site binding of the TcdB protein reduces binding of the C. difficile TcdB protein to the C. difficile toxin protein receptor, thereby reducing the cytopathicity. The method of embodiment 50 or 60, wherein the target site binding disrupts a part of or the full RBD of the TcdB protein. The method of embodiment 62, wherein the target site binding disrupts at least one CROP in the RBD. The method of embodiment 63, wherein the target site binding disrupts all CROPs in the RBD. The method of embodiment 60, wherein the target site binding reduces membrane translocation of the TcdB protein into a target cell. The method of embodiment 65, wherein the target site binding reduces autocatalytic cleavage of the TcdB protein, thereby reducing membrane translocation of the TcdA protein into the target cell. The method of embodiment 60, wherein the target site binding reduces endosome escape of the TcdB protein. The method of embodiment 60, wherein the target site binding reduces glucosyltransferase activity of the TcdB protein. The method of any of embodiments 52-55 and 61-64, wherein: the reduction in binding of the at least one C. difficile toxin protein to the cell receptor is represented by a reduction in binding affinity; the novel binding protein disrupts the at least one toxin protein with a high specificity, the novel binding protein disrupts the at least one toxin protein with a high activity; or a combination thereof. The method of any of embodiments 52-55 and 61-64, wherein the reduction in binding of the at least one C. difficile toxin protein to the cell receptor is about 80% to about 100%. The method of any of embodiments 56, 57, 65 and 66, wherein the reduction in membrane translocation is about 80% to about 100%. The method of embodiment 58 or 67, wherein the reduction in endosome escape is about 80% to about 100%. The method of embodiment 59 or 68, wherein the reduction in glucosyltransferase activity is about 80% to about 100%. The method of any of embodiments 50-73, wherein the reduction in cytopathicity is about 80% to about 100%. The method of any of embodiments 50-73, wherein the composition is formulated for colonic delivery. The method of embodiment 75, wherein the composition is formulated for oral or rectal administration. The method of embodiment 76, wherein the composition is formulated for oral administration and comprises an enteric coating. The method of any of embodiments 50-78, wherein the composition comprises at least one other active agent, is administered in combination with at least one other active agent, or both. The method of embodiment 78, wherein the at least one other active agent is a prebiotic or probiotic agent. A novel binding protein capable of a target site binding of at least one C. difficile toxin protein selected from a TcdA protein and a TcdB protein, wherein the target site binding results in reduced cytopathicity of the at least one C. difficile toxin protein. The novel binding protein of embodiment 80, wherein the C. difficile toxin protein is a TcdA protein. The novel binding protein of embodiment 81, wherein the target site binding of the TcdA protein reduces binding of the TcdA protein to the C. difficile toxin protein receptor, thereby reducing the cytopathicity. The novel binding protein of embodiment 81 or 82, wherein the target site binding disrupts a part of or the full RBD of the TcdA protein, thereby reducing binding of the TcdA protein to a C. difficile toxin protein receptor. The novel binding protein of embodiment 83, wherein at least one CROP in the TcdA protein RBD is disrupted by the target site binding. The novel binding protein of embodiment 84, wherein all CROPs in the RBD are disrupted by the target site binding. The novel binding protein of embodiment 81, wherein the target site binding reduces membrane translocation of the TcdA protein into a target cell. The novel binding protein of embodiment 86, wherein the target site binding reduces autocatalytic cleavage of the TcdA protein, thereby reducing membrane translocation of the TcdA protein into a target cell. The novel binding protein of embodiment 81, wherein the target site binding reduces endosome escape of the TcdA protein. The novel binding protein of embodiment 81, wherein the target site binding reduces glucosyltransferase activity of the TcdA protein. The novel binding protein of embodiment 80, wherein the C. difficile toxin protein is TcdB. The novel binding protein of embodiment 90, wherein the target site binding of the TcdB protein reduces binding of the TcdB protein to the C. difficile toxin protein receptor, thereby reducing the cytopathicity. The novel binding protein of embodiment 90 or 91, wherein the target site binding disrupts a part of or the full RBD of the TcdB protein, thereby reducing binding of the TcdB protein to a C. difficile toxin protein receptor. The novel binding protein of embodiment 92, wherein at least one CROP in the RBD is removed by the target site binding. The novel binding protein of embodiment 93, wherein all CROPs in the RBD are removed by the target site binding. The novel binding protein of embodiment 90, wherein the target site binding reduces membrane translocation of the TcdB protein into a target cell. The novel binding protein of embodiment 95, wherein the target site binding reduces autocatalytic cleavage of the TcdB protein, thereby reducing membrane translocation of the TcdB protein into a target cell. The novel binding protein of embodiment 90, wherein the target site binding reduces endosome escape of the TcdB protein. The novel binding protein of embodiment 90, wherein the target site binding reduces glucosyltransferase activity of the TcdB protein. The novel binding protein of any of embodiments 82-85 and 91-94, wherein: the reduction in binding of the at least one C. difficile toxin protein to the cell receptor is represented by a reduction in binding affinity; the novel binding protein disrupts the at least one toxin protein with a high specificity; the novel binding protein disrupts the at least one toxin protein with a high activity; or a combination thereof. . The novel binding protein of any of embodiments 82-85 and 91-94, wherein the reduction in binding of the at least one C. difficile toxin protein to the cell receptor is about 80% to about 100% . The novel binding protein of any of embodiments 86, 87, 95, and 96, wherein the reduction in membrane translocation is about 80% to about 100%. . The novel binding protein of embodiment 88 or 97, wherein the reduction in endosome escape is about 80% to about 100% . The novel binding protein of embodiment 89 or 98, wherein the reduction in glucosyltransferase activity is about 80% to about 100% . The novel binding protein of any of embodiments 80-103, wherein the reduction in cytopathicity is about 80% to about 100%. . A composition comprising the novel binding protein of any of embodiments 80- 104. . The composition of embodiment 105, formulated for colonic delivery. . The composition of embodiment 105 or 106, formulated for oral or rectal administration. . The composition of embodiment 107, wherein the composition is formulated for oral administration and comprises an enteric coating. . The composition of any of embodiments 105-108, wherein the composition comprises at least one other active agent, is administered in combination with at least one other active agent, or both. . The composition of embodiment 109, wherein the at least one other active agent is a prebiotic or probiotic agent. . A method for treating C. difficile infection in a subject in need thereof, comprising administering to the subject the composition any of embodiments 105-110. . A method for reducing the binding of at least one C. difficile toxin protein to a cell receptor specific for the at least one C. difficile toxin protein, comprising: contacting the at least one C. difficile toxin protein with a novel binding protein that disrupts the at least one C. difficile toxin protein at a target site, under conditions suitable for the target site binding of the at least one C. difficile toxin protein by the novel binding protein to occur; wherein the at least one C. difficile toxin protein is a TcdA protein, a TcdB protein, or both; and wherein the binding of the C. difficile toxin protein to a C. difficile toxin protein receptor is reduced. . The method of embodiment 112, wherein the at least one C. difficile toxin protein is a TcdA protein. . The method of embodiment 113, wherein the target site binding reduces binding of the TcdA protein to the C. difficile toxin protein receptor. . The method of embodiment 113 or 114, wherein the target site binding of the TcdA protein disrupts a part of or the full RBD of the TcdA protein. . The method of any of embodiments 113-115, wherein the target site binding disrupts at least one CROP in the RBD. . The method of any of embodiments 113-116, wherein the target site binding disrupts all CROPs in the RBD. . The method of embodiment 112, wherein the at least one C. difficile toxin protein is a TcdB protein. 119. The method of embodiment 118, wherein the target site binding reduces binding of the C. difficile TcdB protein to the C. difficile toxin protein receptor.

120. The method of any of embodiments 118 or 119, wherein the target site binding of the TcdB protein disrupts a part of or the full RBD of the TcdB protein.

121. The method of embodiment 120, wherein the target site binding disrupts at least one CROP in the RBD.

122. The method of embodiment 120 or 121, wherein the target site binding disrupts all CROPs in the RBD.

123. The method of any of embodiments 113-122, wherein: the reduction in binding of the at least one C. difficile toxin protein to the cell receptor is represented by a reduction in binding affinity; the novel binding protein disrupts the at least one toxin protein with a high specificity; the novel binding protein disrupts the at least one toxin protein with a high activity; or a combination thereof.

124. The method of any of embodiments 113-122, wherein the reduction in binding of the at least one C. difficile toxin protein to the cell receptor is about 80% to about 100%.

[0227] Additional Exemplary Embodiments of the Invention [0228] In certain additional embodiments, the invention includes:

1. A novel antagonist that reduces an activity of a gastrointestinal (GI) tract target.

2. The novel antagonist of additional embodiment 1, wherein the novel antagonist is a novel binding protein comprising at least one binding epitope that specifically binds to a target binding site of a GI tract target that is involved in specific binding of the GI tract target to a GI tract target binding partner, wherein binding of the novel binding protein to the target binding site results in reduction of an activity of the GI tract target.

3. The novel antagonist of additional embodiment 2, wherein the novel antagonist is a novel binding protein comprising a non-antibody binding protein. The novel antagonist of additional embodiment 2, wherein the novel antagonist is a novel binding protein comprising an antibody fragment. The novel antagonist of any one of additional embodiments 2-4, wherein the novel antagonist is a novel binding protein, and wherein the target binding site on the GI tract target is located at a binding interface of the GI tract target and the GI tract target binding partner. The novel antagonist of any one of additional embodiments 2-4, wherein the novel antagonist is a novel binding protein, and wherein the target binding site of the GI tract target is not located at a binding interface of the GI tract target and the GI tract target binding partner. The novel antagonist of any one of additional embodiments 2-6, wherein the novel antagonist is a novel binding protein, and wherein when bound to the target binding site of the GI tract target, the novel binding protein blocks binding of the GI tract target to the GI tract target binding partner. The novel antagonist of any one of additional embodiments 2-6, wherein the novel antagonist is a novel binding protein, and wherein when bound to the target binding site of the GI tract target, the novel binding protein prevents binding of the GI tract target to the GI tract target binding partner by inducing conformational change of the GI tract target. The novel antagonist of additional embodiment 1, wherein the novel antagonist is a novel endopeptidase that cleaves a target cleavage site of the GI tract target, wherein the cleavage results in a reduced activity of the GI tract target. The novel antagonist of any one of additional embodiments 1-9, wherein the activity of the GI tract target is selected from: receptor binding, enzyme activity, membrane translocation, cytopathicity, and any combination thereof. The novel antagonist of any one of additional embodiments 1-10, wherein the GI tract target is from a microbe, wherein the microbe is a commensal microbe, an exogenous pathogen, or an indigenous pathobiont. The novel antagonist of additional embodiment 11, wherein the microbe is a bacterium, virus, fungus, archaeon, or protozoan. The novel antagonist of additional embodiment 11 or 12, wherein the microbe is selected from: Aeromonas; Aspergillus ; Bacteroides; Bilophila ; Campylobacter ; Clostridioides ; Coccidiosis ; Crytosporidia; Enter obacter, Enterococcus ; Escherichia ; Firmicutes ; Helicobacter ; Lactobacillus ; Listeria ; Peptostreptococcus, Pleisiomonas ; Prevotellaceae, Pseudomonas, Salmonella ; Sarcina; SFB; Shigella, Staphylococcus ; Streptococcus, Veillonell , Vibrio, Yersinia, a secretory antibody-coated bacterium; Candida ; Mycobacterium, Mycoplasma; Rotavirus; Calicivirus; Norwalk-like viruses; adenoviruses; astroviruses; sapporo-like viruses; toroviruses; coronaviruses; picornaviruses; herpes viruses; noroviruses; Proteus; Entamoeba ; Giardia, and Strongyloides, single-celled parasites; multi-celled parasites; and amoebae. The novel antagonist of any one of additional embodiments 11-13, wherein the microbe is drug-resistant or hypervirulent. The novel antagonist of any one of additional embodiments 11-14, wherein the microbe is selected from: Bacteroides fragilis, Bilophila wadsworthia, Campylobacter jejuni, Campylobacter coli, Clostridioides difficile, Clostridioides sordelli, Enterobacter cloacae, Enterococcus faecalis, Enterococcus faecium, Escherichia coli, enterotoxigenic Escherichia coli, Helicobacter pylori, Helicobacter sp. Flexispira, Listeria monocytogenes, Pleisiomonas shigelloides, UC Prevotellaceae, Pseudomonas aeruginosa, Salmonella enterica, Salmonella bongori, Salmonella enterica subsp. enterica serotype Typhimurium, Shigella dysenteriae, Staphylococcus aureus, Streptococcus anginosus, Streptococcus thermophilus, Vibrio cholera, Vibrio parahaemolyticus, Yersinia enterocolitica, Candida albicans, Entamoeba histolytica, Giardia lamblia, and Strongyloides stercoralis. The novel antagonist of any one of additional embodiments 1-15, wherein the GI tract target is selected from: a microbial toxin protein, a microbial virulence factor, and a microbial adhesin protein. The novel antagonist of any one of additional embodiments 13-16, wherein the microbe is Clostridioides difficile, wherein the GI tract target is a microbial toxin protein, and wherein the microbial toxin protein is TcdA or TcdB. The novel antagonist of additional embodiment 17, wherein the activity of the GI tract target is selected from: receptor binding, glucosyl transferase activity, membrane translocation, cytopathicity, and any combination thereof. The novel antagonist of additional embodiment 17 or 18, wherein the novel antagonist is a novel binding protein, and wherein the specific epitope binding to the TcdA or TcdB protein reduces the activity of the respective TcdA or TcdB. The novel antagonist of additional embodiment 17 or 18, wherein the novel antagonist is a novel endopeptidase, and wherein cleavage of the target cleavage site of the TcdA or TcdB protein reduces the activity of the respective TcdA or TcdB. The novel antagonist of any one of additional embodiments 16-19, wherein the novel antagonist is a novel binding protein that specifically binds to the C-terminal combined repetitive oligopeptide (CROP) region in the receptor binding domain (RBD) of the TcdA or TcdB. The novel antagonist of any one of additional embodiments 16-18 and 20, wherein the novel antagonist is a novel endopeptidase, wherein the cleavage of the target cleavage site occurs in the C-terminal combined repetitive oligopeptide (CROP) region in the receptor binding domain (RBD) of the respective TcdA or TcdB. The novel antagonist of any one of additional embodiments 2-8, 10-19, and 21, wherein the novel antagonist is a novel binding protein and wherein the binding epitope binds to the GI tract target with high specificity, high affinity, or both. The novel antagonist of any one of additional embodiments 1, 9-20, and 22, wherein the novel antagonist is a novel endopeptidase that cleaves the GI tract target with high specificity, high activity, or both. The novel antagonist of any one of additional embodiments 1-24, wherein the novel antagonist reduces the activity of the GI tract target by about 80% to about 100%, in comparison with a control. The novel antagonist of any one of additional embodiments 1-25, wherein the novel antagonist is not native to a known biological species. The novel antagonist of any one of additional embodiments 2-8, 10-19, 21, 23, 25 and 26, wherein the novel antagonist is a novel binding protein, and wherein the at least one binding epitope comprises a binding domain of a receptor specific for the GI tract target. A method for reducing an activity of a GI tract target, comprising: i. contacting the GI tract target with a novel antagonist of any one of additional embodiments 1-27 under conditions that allow interaction between the novel antagonist and the GI tract target; ii. wherein the interaction between the novel antagonist and the GI tract target results in reduction of an activity of the GI tract target. The method of additional embodiment 28, wherein the activity of the GI tract target is selected from: receptor binding, enzyme activity, membrane translocation, cytopathicity, and any combination thereof. The method of additional embodiment 29, wherein the reduction in the activity of the GI tract target contacted with the novel antagonist is about 80% to about 100%, in comparison with a control. The method of any one of additional embodiments 28-30, wherein the novel antagonist is a novel binding protein comprising at least one binding epitope that specifically binds to a GI tract target at a binding site on the GI tract target that is involved in specific binding of the GI tract target to a GI tract target binding partner, wherein binding of the novel binding protein to the target binding site results in reduction of an activity of the GI tract target. The method of additional embodiment 31, wherein the novel antagonist is a novel binding protein, and wherein the GI tract target is Clostridioides difficile TcdA or TcdB, and wherein the specific epitope binding to the TcdA or TcdB protein reduces an activity of the respective TcdA or TcdB. The method of any one of additional embodiments 28-30, wherein the novel antagonist is a novel endopeptidase capable of a cleaving a target cleavage site of the GI tract target, wherein the cleavage results in a reduced activity of the GI tract target. The method of additional embodiment 33, wherein the novel antagonist is a novel endopeptidase, and wherein the GI tract target is Clostridioides difficile TcdA or TcdB, and wherein the cleavage reduces an activity of the respective TcdA or TcdB. A composition comprising the novel antagonist of any one of additional embodiments 1- 27. The composition of additional embodiment 35, formulated for oral or rectal administration. The composition of additional embodiment 35 or 36, formulated for delivery to a site in the GI tract. The composition of additional embodiment 37, wherein the site in the GI tract is located in the esophagus, the stomach, the small intestine, the large intestine, and the rectum, or any combination thereof.

-HO- The composition of additional embodiment 38, wherein the site in the GI tract is located in the duodenum, the jejunum, the ileum, the caecum, the ascending colon, the transverse colon, the descending colon, the sigmoid colon, the rectum, or any combination thereof. The composition of any one of additional embodiments 35-39, wherein the composition is formulated for oral administration and comprises an enteric coating. The composition of any one of additional embodiments 35-40, wherein the composition comprises at least one other active agent, is administered in combination with at least one other active agent, or both. The composition of additional embodiment 41, wherein the at least one other active agent is a prebiotic or probiotic agent. A method for treating a condition in a subject in need thereof, comprising administering to the subject the composition of any one of additional embodiments 35-42. The method of additional embodiment 43, wherein the subject has a condition resulting from production of a GI tract target. The method of additional embodiment 44, wherein the GI tract target is from a microbe that is a bacterium, virus, fungus, or protozoan. The method of additional embodiment 45, wherein the microbe is a bacterium selected from: Aeromonas; Aspergillus ; Bacteroides, Bilophila ; Campylobacter ; Clostridioides ; Coccidiosis ; Crytosporidia; Enter obacter, Enterococcus ; Escherichia ; Firmicutes ; Helicobacter ; Lactobacillus ; Listeria ; Peptostreptococcus, Pleisiomonas ;

Prevotellaceae, Pseudomonas, Salmonella ; Sarcina; SFB; Shigella, Staphylococcus ; Streptococcus, Veillonell , Vibrio, Yersinia, a secretory antibody-coated bacterium; Candida ; Mycobacterium, Mycoplasma; Rotavirus; Calicivirus; Norwalk-like viruses; adenoviruses; astroviruses; sapporo-like viruses; toroviruses; coronaviruses; picornaviruses; herpes viruses; noroviruses; Proteus; Entamoeba ; Giardia; and Strongyloides; single-celled parasites; multi-celled parasites; and amoebae. The method of additional embodiment 45 or 46, wherein the microbe is drug-resistant or hypervirulent. The method of any one of additional embodiments 45-47, wherein the microbe is selected from: Bacteroides fragilis, Bilophila wadsw or thia, Campylobacter jejuni ; Campylobacter coir, Clostridioides difficile ; Clostridioides sordellr, Enterobacter cloacae ; Enterococcus faecalis, Enterococcus faecium, Escherichia coir, enterotoxigenic Escherichia coir, Helicobacter pylori ; Helicobacter sp. Flexispira ; Listeria monocytogenes ; Pleisiomonas shigelloides ; UC Prevotellaceae Pseudomonas aeruginosa ; Salmonella enterica ; Salmonella bongori; Salmonella enterica subsp. enterica serotype Typhimurium Shigella dysenteriae Staphylococcus aureus ; Streptococcus anginosus; Streptococcus thermophilus ; Vibrio cholera ; Vibrio parahaemolyticus; Yersinia enterocolitica ; Candida albicans ; Entamoeba histolytica ; Giardia lamblia ; and Strongyloides stercoralis. The method of any one of additional embodiments 45-48, wherein the microbe is Clostridioides difficile , and wherein the GI tract target is TcdA, TcdB, or both. The method of any one of additional embodiments 43-49, wherein the condition is selected from: a GI tract ulcer; gastritis; a GI tract cancer; an inflammatory or autoimmune condition; C. diff. infection; secretory antibody-coated bacteria infection; H. pylori infection; Campylobacter jejuni infection; Campylobacter coli infection;

Toxigenic Escherichia coli infection; Staphylococcus aureus infection; Bacteroides fragilis infection; Vibrio cholera infection; and a combination thereof. The method of any one of additional embodiments 43-50, further comprising measuring disease activity in the subject, before, during, or any time after treatment. The method of additional embodiment 51, wherein the disease activity measured following treatment is substantially reduced compared to the disease activity measured prior to treatment. A composition of any one of additional embodiments 35-42 for use in the manufacture of a medicament for treating a disease or a condition in a subject in need thereof.

EXAMPLES

[0229] The following examples are given for the purpose of illustrating various embodiments of the disclosure and are not meant to limit the present disclosure in any fashion. The present examples, along with the methods described herein are presently representative embodiments, are exemplary, and are not intended as limitations on the scope. Changes therein and other uses which are encompassed within the spirit of the disclosure as defined by the scope of the claims will occur to those skilled in the art.

[0230] In Examples 1-7, TcdA and TcdB are commercially obtained, or prepared by methods described in the literature. For example, native TcdB can be produced by culturing C. difficile (VPI 1046) and isolating supernatants as described by Qa’Dan, et al., 2000, Infect. Immun. 68:2470-2474. Native TcdA is removed by a thyroglobulin affinity chromatography protocol as described by Krivan and Wilkins, 1987, Infect. Immun. 55:1873-1877 (31, 32). After TcdA is removed, TcdB is purified by anion-exchange chromatography (Q-Sepharose HP; GE Healthcare) in 20 mM Tris-HCl (pH 8.0) and 20 mM CaCh. The purity of the native TcdB obtained is demonstrated by a single 270-kDa band obtained by SDS-PAGE analysis with Coomassie staining.

Example 1: Design of a Novel Endopeptidase that Selectively Cleaves a C. Diff. Toxin B Protein (TcdB)

[0231] An appropriate protease is selected as the starting enzyme for optimization. The starting protease is active at colonic pH (about 5.5 to about 7.0) and physiological temperature (about 37 degrees C), is easily produced in substantial amounts in E. coli , and can be purified quickly and at low cost.

[0232] The endopeptidase is optimized to cleave TcdB at an amino acid sequence N-terminal to the secondary binding domain using the Rosetta Molecular Modeling Suite to model the binding pocket of the endopeptidase. Theoretical mutations are generated using Rosetta, and selected based on a reduction in the overall energy of the new endopeptidase-target sequence substrate complex relative to the endopeptidase’ s native substrate, or based on an increased of not more than 5 Rosetta energy units. Candidate optimized endopeptidases are then constructed and evaluated for enzymatic activity. The most active candidate is selected for further characterization.

[0233] The optimized endopeptidase cleaves TcdB at an amino acid sequence N-terminal to the secondary binding domain to yield fragments of 1347 and 1019 amino acids. Toxin cleavage is evaluated by incubating the optimized endopeptidase with TcdB, and separating and quantitating the resulting peptide species by SDS-CGE. At least 95% of the 2366 amino acid TcdB protein is cleaved into the respective 1347 and 1019 amino acid fragments. Further analysis of the fragments confirming the expected cleavage point is carried out using mass spectrometry.

[0234] Specificity of the target site cleavage of TcdB by the optimized endopeptidases is demonstrated in a competition assay using substrate analogues. The catalytic efficiency of the optimized endopeptidases is compared with control proteases, including the unoptimized, starting enzyme, and rejected candidate optimized endopeptidases.

Example 2: Design and Evaluation of a Novel Endopeptidase that Selectively Cleaves a C. Diff. Toxin A Protein

[0235] An optimized endopeptidase that selectively cleaves a C. diff. TcdA protein is designed and evaluated in a manner similar to that described for TcdB in Example 1.

Example 3: Reduction of Receptor Binding In Vitro by Cleavage of C. Diff. Toxin Protein

[0236] Binding of TcdA and/or TcdB to a target receptor following incubation with an optimized endopeptidase of Example 1 or 2 is compared with binding of the uncleaved toxin to the target receptor. Binding is evaluated by a receptor pull-down assay as described by Chung et al., 2018.

[0237] Binding of the cleaved TcdA and/or TcdB to the target receptor is also evaluated by measurement of membrane-bound TcdA and/or TcdB following incubation of cells with the cleaved or uncleaved toxin as described by Orth et al., 2014.

Example 4: Reduction of Cvtopathicitv by Cleavage of C. Diff. Toxin Protein

[0238] The cytotoxicity of TcdB following incubation with the optimized endopeptidase designed in Example 1 is compared with that of uncleaved TcdB in a neutralization assay. Vero cells are used to test for neutralization of the cytotoxic effects of TcdB due to their high sensitivity to this toxin. Vero cells are seeded at 2000 cells/well in 96-well dishes and incubated overnight. Following demonstration of the toxin’s ability to sufficiently reduce (e.g., 95% or greater) Vero cell viability, purified TcdB toxin from strain VPI 10463 (commercially obtained) is diluted in culture medium to a final concentration of 10 pg/ml in the presence of the optimized endopeptidase, a control with no endopeptidase, and a vehicle control with no endopeptidase or TcdB. The samples are incubated at 37 °C for 2 h, aliquots removed for separate evaluation of toxin cleavage, and then added to cells. After 24 h at 37 °C, the medium is aspirated, plates are washed two times with phosphate-buffered saline, 200 microliters/well of complete medium is added, and plates are incubated at 37 °C for 48 h. Medium is removed, and 100 microliters /well of 10% cold TCA is added, followed by incubation for 60 min at 4 °C to fix the cells. TCA is removed, wells are washed four times with distilled water, and 100 microliters/well of 100 micrograms/ml sulforhodamine B (Sigma) in 10% acetic acid is added. Plates are incubated for 15 min at room temperature and then washed four times with 10% acetic acid and air-dried. 150 microliters/ well of 10 mM Tris is added, and plates are incubated with shaking at room temperature for an additional 10 min. Plates are read in a SpectraMax plate reader (Molecular Biosystems) at an absorbance wavelength of 570 nm. The percentage of cell survival is calculated by defining A570 values in empty wells as representing 0% cell survival and wells containing cells incubated in the absence of toxin representing 100% cell survival.

[0239] Cytotoxicity is further evaluated in a neutralization assay described by Chen et al., 2008. 3T3 fibroblasts in monolayer culture are exposed to TcdB following incubation with the optimized endopeptidase, or uncleaved TcdB. Plates are incubated for 24 hours at 37°C, and the cytopathic effect under each condition is analyzed by evaluating cell rounding by microscopic examination at 200x magnification.

Example 5: Treatment of C. Diff. Infection in a Subject with a Novel Endopeptidase

Composition

[0240] The effect of treatment on subjects having CDI with the optimized endopeptidase of Example 1 or 2 in comparison with a control untreated subject is evaluated by monitoring subjects for recurrence of infection. Assays described by Eastwood et al., 2009 are used for toxin detection. The effect of treatment is evaluated based on resolution of diarrhea as defined by three or fewer unformed bowel movements over two consecutive days following treatment and positive stool testing for C. diff. toxin A or B.

[0241] Treatment is also evaluated for up to 25 days. Evaluation is based on clinical recurrence of CDI within the follow-up period, with recurrence defined by more than three unformed bowel movements in a 24-hour period.

Example 6: Design and Evaluation of a Novel Binding Protein that Selectively Binds to a C.

Diff. Toxin A or Toxin B Protein

[0242] A binding protein that binds to Ted A and/or TcdB in the secondary binding domain is designed using the Rosetta Molecular Modeling Suite to model the binding site of the novel binding protein using methods described herein. The binding protein is optimized by selecting the most active candidate(s) and performing additional optimization by Rosetta and/or site saturation mutagenesis at selected residues for increased performance. The resulting novel binding protein is assayed for selectivity of binding to TcdA and/or TcdB. Example 7: Treatment of C Diff. Infection in a Subject with a Novel Binding Protein

Composition

[0243] A novel binding protein of Example 6 is used for treatment of subjects having CDI as described in Example 5, and similarly evaluated for effect on disease symptoms.

Table 7: Table of Sequences Listed

Claims

CLAIMS What Is Claimed Is:

2. The novel antagonist of claim 1, wherein the novel antagonist is a novel binding protein comprising at least one binding epitope that specifically binds to a target binding site of a GI tract target that is involved in specific binding of the GI tract target to a GI tract target binding partner, wherein binding of the novel binding protein to the target binding site results in reduction of an activity of the GI tract target.

3. The novel antagonist of claim 2, wherein the novel antagonist is a novel binding protein comprising a non-antibody binding protein.

4. The novel antagonist of claim 2 or 3, wherein the novel antagonist is a novel binding protein, and wherein the target binding site on the GI tract target is located at a binding interface of the GI tract target and the GI tract target binding partner.

5. The novel antagonist of any one of claims 2-4, wherein the novel antagonist is a novel binding protein, and wherein when bound to the target binding site of the GI tract target, the novel binding protein blocks binding of the GI tract target to the GI tract target binding partner.

6. The novel antagonist of claim 1, wherein the novel antagonist is a novel endopeptidase that cleaves a target cleavage site of the GI tract target, wherein the cleavage results in a reduced activity of the GI tract target.

7. The novel antagonist of any one of claims 1-6, wherein the activity of the GI tract target is selected from: receptor binding, enzyme activity, membrane translocation, cytopathicity, and any combination thereof.

8. The novel antagonist of any one of claims 1-7, wherein the GI tract target is from a microbe, wherein the microbe is a commensal microbe, an exogenous pathogen, or an indigenous pathobiont.

9. The novel antagonist of claim 8, wherein the microbe is a bacterium, virus, fungus, archaeon, or protozoan.

10. The novel antagonist of claim 8 or 9, wherein the microbe is selected from: Aeromonas, Aspergillus , Bacteroides, Bilophila , Campylobacter , Clostridioides , Coccidiosis, Crytosporidia, Enterobacter , Enterococcus , Escherichia , Firmicutes , Helicobacter , Lactobacillus , Listeria , Peptostreptococcus , Pleisiomonas , Prevote llaceae, Pseudomonas , Salmonella , Sarcina, SFB, Shigella , Staphylococcus , Streptococcus , Veillonella , Vibrio ,

Yersinia , a secretory antibody-coated bacterium, Candida , Mycobacterium , Mycoplasma, Rotavirus, Calicivirus, Norwalk-like viruses, adenoviruses, astroviruses, sapporo-like viruses, toroviruses, coronaviruses, picornaviruses, herpes viruses, noroviruses, Proteus, Entamoeba , Giardia , and Strongy loides, single-celled parasites, multi-celled parasites, and amoebae.

11. The novel antagonist of any one of claims 8-10, wherein the microbe is selected from: Bacteroides fragilis, Bilophila wadsworthia, Campylobacter jejuni, Campylobacter coli, Clostridioides difficile, Clostridioides sordelli, Enterobacter cloacae, Enterococcus faecalis, Enterococcus faecium, Escherichia coli, enterotoxigenic Escherichia coli, Helicobacter pylori, Helicobacter sp. Flexispira, Listeria monocytogenes, Pleisiomonas shigelloides, UC Prevotellaceae, Pseudomonas aeruginosa, Salmonella enterica, Salmonella bongori, Salmonella enterica subsp. enterica serotype Typhimurium, Shigella dysenteriae, Staphylococcus aureus, Streptococcus anginosus, Streptococcus thermophilus, Vibrio cholera, Vibrio parahaemolyticus, Yersinia enterocolitica, Candida albicans, Entamoeba histolytica, Giardia lamblia, and Strongyloides stercoralis.

12. The novel antagonist of any one of claims 8-11, wherein the microbe is Clostridioides difficile, wherein the GI tract target is a microbial toxin protein, and wherein the microbial toxin protein is TcdA or TcdB.

13. The novel antagonist of claim 12, wherein the activity of the GI tract target is selected from: receptor binding, glucosyl transferase activity, membrane translocation, cytopathicity, and any combination thereof.

14. The novel antagonist of claim 12 or 13, wherein the novel antagonist is a novel binding protein, and wherein the specific epitope binding to the TcdA or TcdB protein reduces the activity of the respective TcdA or TcdB.

15. The novel antagonist of claim 12 or 13, wherein the novel antagonist is a novel endopeptidase, and wherein cleavage of the target cleavage site of the TcdA or TcdB protein reduces the activity of the respective TcdA or TcdB.

16. The novel antagonist of any one of claims 12-14, wherein the novel antagonist is a novel binding protein that specifically binds to the C-terminal combined repetitive oligopeptide (CROP) region in the receptor binding domain (RBD) of the TcdA or TcdB.

17. The novel antagonist of any one of claims 12, 13, and 15, wherein the novel antagonist is a novel endopeptidase, wherein the cleavage of the target cleavage site occurs in the C-terminal combined repetitive oligopeptide (CROP) region in the receptor binding domain (RBD) of the respective TcdA or TcdB.

18. The novel antagonist of any one of claims 1-17, wherein the novel antagonist reduces the activity of the GI tract target by about 80% to about 100%, in comparison with a control.

19. A method for reducing an activity of a GI tract target, comprising: contacting the GI tract target with a novel antagonist of any one of claims 1-27 under conditions that allow interaction between the novel antagonist and the GI tract target; wherein the interaction between the novel antagonist and the GI tract target results in reduction of an activity of the GI tract target.

20. The method of claim 19, wherein the activity of the GI tract target is selected from: receptor binding, enzyme activity, membrane translocation, cytopathicity, and any combination thereof.

21. The method of claim 20, wherein the reduction in the activity of the GI tract target contacted with the novel antagonist is about 80% to about 100%, in comparison with a control.

22. The method of any one of claims 19-21, wherein the novel antagonist is a novel binding protein comprising at least one binding epitope that specifically binds to a GI tract target at a binding site on the GI tract target that is involved in specific binding of the GI tract target to a GI tract target binding partner, wherein binding of the novel binding protein to the target binding site results in reduction of an activity of the GI tract target.

23. The method of claim 22, wherein the novel antagonist is a novel binding protein, and wherein the GI tract target is Clostridioides difficile TcdA or TcdB, and wherein the specific epitope binding to the TcdA or TcdB protein reduces an activity of the respective TcdA or TcdB.

24. The method of any one of claims 19-21, wherein the novel antagonist is a novel endopeptidase capable of a cleaving a target cleavage site of the GI tract target, wherein the cleavage results in a reduced activity of the GI tract target.

25. The method of claim 23, wherein the novel antagonist is a novel endopeptidase, and wherein the GI tract target is Clostridioides difficile TcdA or TcdB, and wherein the cleavage reduces an activity of the respective TcdA or TcdB.

26. A composition comprising the novel antagonist of any one of claims 1-25.

27. The composition of claim 26, formulated for oral or rectal administration.

28. The composition of claim 26 or 27, formulated for delivery to a site in the GI tract.

29. The composition of claim 28, wherein the site in the GI tract is located in the esophagus, the stomach, the small intestine, the large intestine, and the rectum, or any combination thereof.

30. The composition of claim 28 or 29, wherein the site in the GI tract is located in the duodenum, the jejunum, the ileum, the caecum, the ascending colon, the transverse colon, the descending colon, the sigmoid colon, the rectum, or any combination thereof.

31. The composition of any one of claims 26-30, wherein the composition is formulated for oral administration and comprises an enteric coating.

32. The composition of any one of claims 26-31, wherein the composition comprises at least one other active agent, is administered in combination with at least one other active agent, or both.

33. A method for treating a condition in a subject in need thereof, comprising administering to the subject the composition of any one of claims 26-32.

34. The method of claim 33, wherein the subject has a condition resulting from production of a GI tract target, wherein the GI tract target is from a microbe that is a bacterium, virus, fungus, or protozoan.

35. The method of claim 34, wherein the microbe is a bacterium selected from: Aeromonas; Aspergillus ; Bacteroides, Bilophila ; Campylobacter ; Clostridioides ; Coccidiosis ; Crytosporidia; Enterobacter, Enterococcus ; Escherichia ; Firmicutes ; Helicobacter ; Lactobacillus ; Listeria ; Peptostreptococcus, Pleisiomonas ; Prevotellaceae; Pseudomonas, Salmonella ; Sarcina; SFB; Shigella, Staphylococcus ; Streptococcus, Veillonella, Vibrio, Yersinia, a secretory antibody-coated bacterium; Candida ; Mycobacterium, Mycoplasma; Rotavirus; Calicivirus; Norwalk-like viruses; adenoviruses; astroviruses; sapporo-like viruses; toroviruses; coronaviruses; picornaviruses; herpes viruses; noroviruses; Proteus; Entamoeba ; Giardia; and Strongy hides, single-celled parasites; multi-celled parasites; and amoebae.

36. The method of claim 34 or 35, wherein the microbe is selected from: Bacteroides fragilis, Bilophila wadsworthia, Campylobacter jejuni ; Campylobacter coir, Clostridioides difficile ; Clostridioides sordellr, Enterobacter cloacae ; Enterococcus faecalis, Enterococcus faecium, Escherichia coir, enterotoxigenic Escherichia coir, Helicobacter pylori, Helicobacter sp. Flexispira ; Listeria monocytogenes ; Pleisiomonas shigelloides ; UC Prevotellaceae Pseudomonas aeruginosa ; Salmonella enterica ; Salmonella bongori; Salmonella enterica subsp. enterica serotype Typhimurium Shigella dysenteriae Staphylococcus aureus ; Streptococcus anginosus; Streptococcus Ihermophihts Vibrio cholera ; Vibrio parahaemolyticus; Yersinia enterocolitica Candida albicans ; Entamoeba histolytica ; Giardia lamblia ; and Strongyloides stercoralis.

37. The method of claim 34, wherein the microbe is Clostridioides difficile , and wherein the GI tract target is TcdA, TcdB, or both.

38. The method of any one of claims 34-37, wherein the condition is selected from: a GI tract ulcer; gastritis; a GI tract cancer; an inflammatory or autoimmune condition; C. diff. infection; secretory antibody-coated bacteria infection; H. pylori infection; Campylobacter jejuni infection; Campylobacter coli infection; Toxigenic Escherichia coli infection; Staphylococcus aureus infection; Bacteroides fragilis infection; Vibrio cholera infection; and a combination thereof.

39. The method of any one of claims 33-38, further comprising measuring disease activity in the subject, before, during, or any time after treatment.

40. The method of claim 39, wherein the disease activity measured following treatment is substantially reduced compared to the disease activity measured prior to treatment.

41. The method of any one of claims 38-40, wherein the inflammatory or autoimmune condition is selected from: GI inflammation; inflammatory bowel disease (IBD); Crohn’s disease; ulcerative colitis (UC); multiple sclerosis (MS); Type 1 diabetes mellitus; chronic biliary systemic lupus erythematosus (SLE); rheumatoid arthritis (RA); psoriatic arthritis; autoimmune hepatitis; pemphigus; psoriasis; ankylosing spondylitis; Hashimoto’s thyroiditis; vasculitis; gout; scleroderma; and Graves disease; and a combination thereof.

42. A composition of any one of claims 26-32 for use in the manufacture of a medicament for treating a disease or a condition in a subject in need thereof.