WO2020237023A1 - Formulations for altering microbiome to reduce risks of hereditary and spontaneous colorectal cancers - Google Patents

Abstract

The present invention relates to methods for reducing or neutralizing microorganism produced pathogenic molecules active in the cascade of events leading to colorectal cancer. In some aspects, formulations and methods for treating, preventing, or reducing the risk of colorectal cancer, are provided.

Description

FORMULATIONS FOR ALTERING MICROBIOME TO REDUCE RISKS OF HEREDITARY AND SPONTANEOUS COLORECTAL CANCERS

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Patent Application 62/850,714, filed May 21, 2019, the entire content of which is hereby incorporated by reference in its entirety.

FIELD OF THE INVENTION

[0002] The present invention generally relates to reducing risks and manifestations of disease.

More particularly, the present invention relates to effecting alterations in signaling pathways of an intestinal microbiome as a means of reducing the risk of developing colonic adenomata and colorectal cancers.

BACKGROUND OF THE INVENTION

[0003] A growing number of diseases are associated with alterations and variations in the

composition of the communities of microorganisms residing on and within the bodies of humans and other vertebrates.

[0004] Mechanisms producing the observed associations between microbiomes and states of health are rapidly being elucidated, and it is clear that many such mechanisms involve the metabolic activity, expression of virulence factors and toxins, and mechanics of adhesion of specific strains of microorganisms in relation to an individual’s own biological and genetic characteristics, and the environment.

[0005] Known associations between alterations in the human intestinal microbiome composition and signaling mechanisms (“dysbiosis”) and disease include but are not limited to cardiometabolic disorders (the metabolic syndrome, obesity, diabetes, cardiovascular risk), cancer risk, neurodegenerative disease risk, behavioral disorders (e.g., autism, attention- deficit hyperactivity disorder (ADHD)), psychiatric disorders including mood disorders, schizophrenia, anxiety, functional gut disorders (e.g., irritable bowel syndrome (IBS), functional diarrhea), other intestinal disorders including celiac disease, non-celiac gluten sensitivity, and small intestinal bacterial overgrowth.

[0006] A need exists for methods of treating, preventing, or reducing risk of diseases associated with signaling pathways in the human intestinal microbiome. The present invention addresses and satisfies this need.

SUMMARY OF THE INVENTION

[0007] The following presents a simplified summary in order to provide a basic understanding of some aspects of the invention. This summary is not an extensive overview of the invention. It is not intended to identify key/critical elements of the invention or to delineate the scope of the invention. Its sole purpose is to present some concepts of the invention in a simplified form as a prelude to the more detailed description that is presented later.

[0008] As disclosed herein, the present invention includes methods and formulations for

identifying, preventing, treating, or reducing the risk for a colorectal cancer. In one aspect, the present invention includes a formulation for preventing, reducing the risk of, or treating, colorectal cancer in at least one subject in need thereof. In some embodiments, the formulation comprises an IgY antibody that specifically binds a Bacteroides fragilis toxin (BFT). In some embodiments, the formulation comprises an IgY antibody that specifically binds an Escherichia coli adhesin or an Escherichia coli flagellin. In some other

embodiments, formulation comprises a mixture of an IgY antibody that specifically binds a Bacteroides fragilis toxin (BFT) and an IgY antibody that specifically binds an Escherichia coli adhesin or an Escherichia coli flagellin.

[0009] In another aspect, the present invention includes a method of reducing and/or preventing DNA damage in intestinal cells in a subject, the method comprising administering to the subject an effective amount of a formulation comprising an IgY antibody that specifically binds a Bacteroides fragilis toxin (BFT) and/or a formulation comprising an IgY antibody that specifically binds an Escherichia coli adhesin or an Escherichia coli flagellin. In some embodiments, the subject is human. In some other embodiments, the subject has a hereditary condition predisposing said subject to colorectal cancer. In some embodiments, the subject has a mutation in APC, the subject has familial adenomatous polyposis (FAP), and/or the subject has a first-degree relative who has FAP or colorectal cancer.

[0010] In yet another aspect, the present invention includes a method of treating, preventing, or reducing risk of developing colorectal cancer in a subject, the method comprising

administering to the subject a therapeutically effective amount of a formulation comprising an IgY antibody that specifically binds a Bacteroides fragilis toxin (BFT) and/or a formulation comprising an IgY antibody that specifically binds an Escherichia coli adhesin or an Escherichia coli flagellin. In some embodiments, the subject is human. In some other embodiments, the subject has a hereditary condition predisposing said subject to colorectal cancer. In some embodiments, the subject has a mutation in APC, the subject has familial adenomatous polyposis (FAP), and/or the subject has a first-degree relative who has FAP or colorectal cancer.

[0011] In still another aspect, the present invention includes a method of preventing or reducing risk of colorectal cancer in a subject, the method comprising (a) determining whether the subject has an elevated risk for colorectal cancer, and (b) if the subject is determined to have an elevated risk for colorectal cancer (CRC), administering to the subject a therapeutically effective amount of a formulation comprising an IgY antibody that specifically binds a Bacteroides fragilis toxin (BFT) and/or a formulation comprising an IgY antibody that specifically binds an Escherichia coli adhesin or an Escherichia coli flagellin. In some embodiments, the subject is determined to have an elevated risk for colorectal cancer if (a) a mutation is present in an APC protein or nucleic acid in a biological sample obtained from the subject, (b) the subject has familial adenomatous polyposis (FAP), or (c) the subject has a first-degree relative who has FAP or colorectal cancer. In some embodiments, the subject is human.

[0012] In some embodiments of any one of the aspects herein, the BFT is produced by

Bacteroides fragilis strain 086-5443-2-2. In some embodiments, the IgY antibody specifically binds a BFT epitope comprising an amino acid sequence selected from SEQ ID NOS: 17-22.

[0013] In some embodiments of any one of the aspects herein, the adhesin or flagellin is

produced by Escherichia coli strain NC101 or a pks+ Escherichia coli. In some

embodiments, the IgY antibody specifically binds a Escherichia coli adhesin epitope comprising an amino acid sequence selected from: SEQ ID NOS: 1-3 or a Escherichia coli flagellin epitope comprising an amino acid sequence selected from: SEQ ID NOS: 4-16.

[0014] In some embodiments, the IgY antibody is polyclonal. In some other embodiments, the formulation further comprises a pharmaceutically acceptable carrier or excipient.

BRIEF DESCRIPTION OF THE DRAWINGS

[0015] For a more complete understanding of the nature and desired objects of the present

invention, reference is made to the following detailed description taken in conjunction with the accompanying drawing figures wherein like reference characters denote corresponding parts throughout the several views.

[0016] FIG. 1 provides a table representing each species of microorganism, and each microorganism-produced molecule, to be targeted by the formulation of IgY disclosed in this invention, along with known or theorized mechanisms of action that may promote colorectal carcinogenesis. Included in FIG. 1 are also the microorganism-produced molecules currently associated with colorectal carcinogenesis by the microorganisms involved.

[0017] FIG. 2 provides a table with an exemplary listing the various target-specific IgY molecules to be included in various embodiments of this invention. It is essential to note that IgY specific of more than one strain of each of the targeted microorganisms, including those producing microorganism-produced pathogenic molecules such as BFT and other molecules known to be involved in the microbiome event cascades leading to colorectal cancer (CRC), may be produced and included in the composition disclosed in this patent.

[0018] FIG. 3 provides a diagram showing points of interventions aimed at reducing CRC oncogenesis.

DETAILED DESCRIPTION

Definitions:

[0019] Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which this invention belongs. The following references provide one of skill with a general definition of many of the terms used in this invention: Singleton et ah, Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R. Rigger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991). As used herein, the following terms have the meanings ascribed to them below, unless specified otherwise.

[0020] The singular terms "a," "an," and "the" include plural referents unless context clearly indicates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly indicates otherwise. It is further to be understood that all base sizes or amino acid sizes, and all molecular weight or molecular mass values, given for nucleic acids or polypeptides are approximate, and are provided for description. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, suitable methods and materials are described below.

[0021] As used herein, the term“about” will be understood by persons of ordinary skill in the art and will vary to some extent on the context in which it is used. As used herein when referring to a measurable value such as an amount, a concentration, a temporal duration, and the like, the term“about” is meant to encompass variations of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and still more preferably ±0.1% from the specified value, as such variations are appropriate to perform the disclosed methods.

[0022] As used herein,“abnormal/disease reference” means a range of values associated with elevated risk established by studies of populations determined to have, or be at risk for, one or more specific diseases or disability states, a range of values associated with elevated risk established by publicly-available microbiome libraries of microbiome profiles of populations determined to have, or be at risk for, one or more specific diseases or disability states, a range of values associated with elevated risk established by private individuals, governmental, academic, nonprofit, or corporate entities that develop or maintain such libraries of microbiome profiles of populations determined to have, or be at risk for, a specific disease or disability state.

[0023] As used herein,“absolute abundance” means the measured amount, (for non-limiting example, in colony-forming units, or CFU) of a given taxonomic unit in a microbiome.

[0024] As used herein,“additive therapy” means the addition of specific microorganisms to a microbiome found to have those microorganisms present at reduced relative abundance. The most common example of additive therapy is the use of probiotics. [0025] By“ameliorate” is meant decrease, suppress, attenuate, diminish, arrest, or stabilize the development or progression of a disease.

[0026] As used herein,“animal” refers to living multi-cellular vertebrate organisms, a category that includes, for example, mammals and birds. The term“mammal” includes both human and non-human mammals.

[0027] The term“antibody,” as used herein, refers to an immunoglobulin molecule that

specifically binds with an antigen. Antibodies can be intact immunoglobulins derived from natural sources or from recombinant sources, and can be immunoreactive portions of intact immunoglobulins. Antibodies are typically tetramers of immunoglobulin molecules. The antibodies in the present invention may exist in a variety of forms including, for example, polyclonal antibodies, monoclonal antibodies, Fv, Fab and F(ab)2, as well as single chain antibodies and humanized antibodies (Harlow et al ., 1999, In: Using Antibodies: A

Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al ., 1989, In: Antibodies: A Laboratory Manual, Cold Spring Harbor, New York; Houston et al. , 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et al., 1988, Science 242:423-426).

[0028] The term“antibody fragment” refers to a portion of an intact antibody and refers to the antigenic determining variable regions of an intact antibody. Examples of antibody fragments include, but are not limited to, Fab, Fab’, F(ab’)₂, and Fv fragments, linear antibodies, scFv antibodies, single-domain antibodies such as sdAb (either VL or VH), such as camelid antibodies (Riechmann, 1999, J. Immunol. Meth. 231 :25-38), camelid VHH domains, composed of either a VL or a VH domain that exhibit sufficient affinity for the target, and multispecific antibodies formed from antibody fragments such as a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region, and an isolated complementarity-determining region (CDR) or other epitope binding fragments of an antibody. An antigen binding fragment can also be incorporated into single domain antibodies, maxibodies, minibodies, nanobodies, intrabodies, diabodies, triabodies, tetrabodies, v-NAR and bis-scFv (see, e.g ., Hollinger & Hudson, 2005, Nature Biotech.

23 : 1126-1136). Antigen binding fragments can also be grafted into scaffolds based on polypeptides such as a fibronectin type III (Fn3) (U.S. Patent No.: 6,703,199, which describes fibronectin polypeptide minibodies). The antibody fragment also includes a human antibody or a humanized antibody or a portion of a human antibody or a humanized antibody.

[0029] In one aspect of this invention, the non-human antibody is an avian antibody,

immunoglobulin Y (IgY). As will be understood by those skilled in the art, production of IgY necessarily entails changes in the host hen’s immune tissues that permit extraction of genetic material for introduction into single celled expression systems, including but not limited to yeast cells, Chinese hamster ovary (CHO) cells, and human hybridoma cells.

Antibodies produced in such systems are known as“engineered antibodies” or“antibody fragments” or“nanobodies.” It is therefore possible to screen native polyclonal IgY for antibodies with the desired effects on a microorganism-produced molecule, identify those with the strongest desirable characteristics, and proceed to produce engineered antibodies with identical or superior characteristics to those in the native polyclonal mixture produced by the hen.

[0030] In some other aspects, the non-human antibody is a mammalian antibody, of the

immunoglobulin class G (IgG), A (IgA and secretory IgA), or M (IgM). In some

embodiments, the antibody or fragment thereof is a monomeric IgA, such as the IgA described in Virdi et al. Nat. Biotechnol. 2019 May;37(5):527-530. In some embodiments, the antibody or fragment thereof is a VHH, such as for example, llama-derived single chain antibody fragments (VHH) as described in Garaicoechea et al., 2015 PloS ONE

10(8):e0133665.

[0031] The term“antigen” or“Ag” as used herein is defined as a molecule that provokes an immune response. This immune response may involve either antibody production, or the activation of specific immunologically-competent cells, or both. The skilled artisan will understand that any macromolecule, including virtually all proteins or peptides, can serve as an antigen. Furthermore, antigens can be derived from recombinant or genomic DNA. A skilled artisan will understand that any DNA, which comprises a nucleotide sequence or a partial nucleotide sequence encoding a protein that elicits an immune response therefore encodes an“antigen” as that term is used herein. Furthermore, one skilled in the art will understand that an antigen need not be encoded solely by a full-length nucleotide sequence of a gene. It is readily apparent that the present invention includes, but is not limited to, the use of partial nucleotide sequences of more than one gene and that these nucleotide sequences are arranged in various combinations to elicit the desired immune response. Moreover, a skilled artisan will understand that an antigen need not be encoded by a“gene” at all. It is readily apparent that an antigen can be generated synthesized or can be derived from a biological sample. Such a biological sample can include, but is not limited to a tissue sample, a tumor sample, a cell or a biological fluid.

[0032] As used herein,“Bacteroidetes” refers to the bacterial phylum Bacteroidetes, or to any microorganism classified as a member of that phylum.

[0033] As used herein,“immunoglobulin” refers to a polypeptide ligand comprising at least a light chain or heavy chain immunoglobulin variable region which specifically binds an epitope of a protein or a fragment of a protein. Immunoglobulins can include a heavy chain and a light chain, each of which has a variable region, termed the variable heavy (VH) region and the variable light (VL) region. Together, the VH region and the VL region are responsible for binding the antigen recognized by the immunoglobulin. This includes intact immunoglobulins and the variants and portions of them well known in the art, such as Fab' fragments, F(ab)'2 fragments, single chain Fv proteins ("scFv"), and disulfide stabilized Fv proteins ("dsFv"). A scFv protein is a fusion protein in which a light chain variable region of an immunoglobulin and a heavy chain variable region of an immunoglobulin are bound by a linker, while in dsFvs, the chains have been mutated to introduce a disulfide bond to stabilize the association of the chains. The term also includes recombinant forms such as chimeric immunoglobulins (for example, humanized murine immunoglobulins), heteroconjugate immunoglobulins (such as, bispecific immunoglobulins), and immunoglobulins produced by genetically-modified bacteria or yeast under defined conditions. See also, Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co., Rockford, IL); Kuby, Immunology, 3rd Ed., W.H. Freeman & Co., New York, 1997.

[0034] As used herein,“biofilm” refers to any group of microorganisms in which cells stick to each other and also to a surface.

[0035] As used herein,“biomarker” refers to any measurable analyte used in the diagnosis or assessment of a disease, disease process, or disease risk; nonlimiting examples include relative abundance and diversity as characteristics of a microbiome, fecal calprotectin as an indicator of intestinal inflammation, or serum cholesterol as an indicator of cardiovascular disease risk. [0036] As used herein, the terms“comprising,”“including,”“containing” and“characterized by” are exchangeable, inclusive, open-ended and do not exclude additional, unrecited elements or method steps. Any recitation herein of the term“comprising,” particularly in a description of components of a composition or in a description of elements of a device, is understood to encompass those compositions and methods consisting essentially of and consisting of the recited components or elements.

[0037] As used herein, the term“consisting of’ excludes any element, step, or ingredient not specified in the claim element.

[0038] The term“colonic” refers herein to any attribute or structure of or pertaining to that

portion of the vertebrate intestinal tract called the colon, or large intestine.

[0039] As used herein,“colonic adenoma” (plural,“adenomata”) means a benign tumor of

colonic epithelial tissue with glandular origin, glandular characteristics, or both, in which the glandular structures of the colonic wall are involved.

[0040] As used herein,“colonic mucosa” means the innermost layer of epithelial cells lining the colon and surrounding the lumen of the colon.

[0041] As used herein,“colorectal cancers” refers to a cancer of the colon or rectum of the large intestine.

[0042] As used herein,“customized” means developed in response to one or more measured characteristic of an individual or population, said characteristic being relevant to a

microbiome profile. For example, a mixture of IgYs is“customized” to the needs of a subject or group of subjects when it is formulated by reference to a microbiome profile of said subject or group of subjects. When“customized” is used in reference to an individual subject, or a formulation for use in a specific individual subject, the term includes the sense of “personalized.”

[0043] As used herein,“defined population” means a distinct population of individuals having in common at least one common characteristic relevant to disease or disease risk. Nonlimiting examples of defined populations include all those living in a defined geographical or climatic area, those relying on a common water source, those with serum cholesterol levels within a specified range, those with defining symptoms of a disease (e.g., irritable bowel syndrome), those at elevated risk for cancer, those at elevated risk for neurodegenerative diseases, and those with psychiatric, behavioral, or mood disorders. [0044] By“disease” is meant any condition or disorder that damages or interferes with the normal function of a cell, tissue, or organ, system or entire organism.

[0045] As used herein,“diversity” means a measure of the number of distinct genera, species, and other taxa or operational taxonomic units present within a defined microbiome from a body site. Common indices of diversity include, but are not limited to, the Shannon index and the Simpson index.

[0046] As used herein,“dysbiosis” means an imbalance, maladaptation, or disruption of a

normal microbiome.

[0047] The terms“effective amount” or“therapeutically effective amount” are used

interchangeably herein, and refer to an amount of an agent (e.g., an antibody, such as an IgY antibody) formulation, material, or composition, as described herein effective to achieve a particular biological result. Such results may include, but are not limited to, the treatment of a disease or condition as determined by any means suitable in the art.

[0048] As used herein,“epitope” means a molecular structure found on a microorganism, or represented by a functional microorganism-produced molecule, that is recognized by the immune system and leads to the production of specific immunoglobulins directed against the epitope.

[0049] As used herein,“familial adenomatous polyposis,” or FAP, refers to an autosomal

dominant inherited condition in which numerous adenomatous polyps form mainly in the epithelium of the large intestine; such polyps start out benign, but undergo malignant transformation into colon cancer when they are left untreated.

[0050] As used herein,“Firmicutes” or“Firmicute” refers to the bacterial phylum Firmicutes, or to any microorganism classified as a member of that phylum.

[0051] As used herein,“formulation” means a mixture of at least one IgY specific of at least one microbiome constituent (bacteria, virus, protozoan, fungus, or protist), or of at least one microorganism-produced molecule, or of IL-17.

[0052] As used herein,“genotoxin” refers to a chemical agent that damages genetic information within a cell, causing mutations, which may lead to cancer.

[0053] As used herein,“Interleukin- 17,” or“IL-17,” refers to a pro-inflammatory signaling

molecule, or cytokine, associated with various inflammatory and autoimmune disorders. [0054] As used herein,“lumen” means the inside space of a tubular structure, specifically the colon.

[0055] As used herein,“normal reference” and“healthy reference” mean a reference range or typical microbiome composition established by studies of microbiome compositions of populations determined to be in states of good health, a reference range established by publicly-available microbiome libraries of microbiome profiles of populations in states of good health, and a reference range established by private individuals, governmental, academic, nonprofit, or corporate entities that develop or maintain such libraries of microbiome profiles of populations in states of good health.

[0056] As used herein,“oncotoxin” refers to chemical compounds capable of triggering

cancerous changes within cells.

[0057] As used herein,“immunoglobulin Y” (“IgY”) is a type of immunoglobulin which is the major immunoglobulin in bird, reptile, and lungfish blood. It is also found in high concentrations in chicken egg yolk. As with the other immunoglobulins, IgY is a class of proteins which are formed by the immune system in reaction to certain foreign substances, and specifically recognize them. IgY is composed of two light and two heavy chains.

Structurally, these two types of immunoglobulin differ primarily in the heavy chains, which in IgY have a molecular mass of about 65,100 atomic mass units (amu). The light chains in IgY have a molar mass of about 18,700 amu. The molar mass of IgY thus amounts to about 167,000 amu.

[0058] As used herein,“IgY specific of one or more microorganisms” means IgY molecules produced by immunizing a hen with one or more specific antigens derived from one or more specific microorganisms, such that the IgY produced is capable of binding to said antigens when encountered by the IgY molecule.

[0059] As used herein,“IgY specific of one or more microorganism-produced molecule” means IgY molecules produced by immunizing a hen with one or more specific antigens derived from one or more specific microorganism-produced molecules, such that the IgY produced is capable of binding to said antigens when encountered by the IgY molecule. In some embodiments, the microorganism-produced molecule is an E. coli adhesin. In some embodiments, the microorganism-produced molecule is an E. coli flagellin. In some other embodiments, the microorganism-produced molecule is B. fragilis toxin (BFT). [0060]“Immunoassay” refers to a biochemical test that measures the presence or concentration of a substance in a sample, such as a biological sample, using the reaction of an

immunoglobulin to its cognate antigen, for example the specific binding of an

immunoglobulin to a protein. Both the presence of antigen or the amount of antigen present can be measured. For measuring proteins, for each the antigen and the presence and amount (abundance) of the protein can be determined or measured. Measuring the quantity of antigen can be achieved by a variety of methods. One of the most common is to label either the antigen or immunoglobulin with a detectable label.

[0061] An“individual”,“patient” or“subject”, as these terms are used interchangeably herein, includes a member of any animal species including, but are not limited to, birds, humans and other primates, and other mammals including commercially relevant mammals such as cattle, pigs, horses, sheep, cats, and dogs. Preferably, the subject is a human.

[0062] As used herein,“ in vivo” refers to processes that take place within a living organism, herein most commonly, a laying hen.

[0063] As used herein,“manifestation of a disease” means any sign, symptom, diagnostic test result, severity score, quality-of-life measure, or measure of disability that is associated with a specific disease, disease process, or disease risk.

[0064] As used herein,“metagenomic analysis” means an analysis, by culture-independent and sequencing-based studies, of the collective set of genomes of mixed microorganism communities (metagenomes), with the aim of exploring their compositional and functional characteristics.

[0065] As used herein,“microbiome” or“microbiota” refers to the ecological community of commensal, symbiotic and pathogenic microorganisms that are normally found dwelling in specific sites of an animal or human body. The term is used to emphasize the importance of microorganisms inhabiting the human body in health and disease. By the original definitions the terms“microbiome” and“microbiota” are largely synonymous, and are used

synonymously herein. Nonlimiting examples of individual microbiomes include the intestinal (“gut”) microbiome, the oropharyngeal microbiome, the skin microbiome, and the vaginal microbiome.

[0066] As used herein,“microbiome composition” means the kind, amount, and diversity of detectable microorganisms in a microbiome.“Microbiome composition” in this sense is the biomarker that indicates the state of an individual microbiome or that of microbiomes typical to a group of subjects with or at risk for a disease or condition.

[0067] As used herein,“microbiome event cascade” means a verified sequence of molecular events initiated by microorganism members of a microbiome and having deleterious events on other microorganisms or on human tissue; microbiome event cascades may include intact mi coorganisms, fragments of microorganisms, and signaling molecules, virulence factors, and toxins produced by a microbial member of a microbiome.

[0068] As used herein, a“microbiome profile” is a quantitative report revealing the kind and amount of selected microorganisms (“microbiome composition”) present in a sample of a microbiome. In this sense the“microbiome profile” is the laboratory report that provides information about the biomarker,“microbiome composition.” Components of the report include but are not limited to diversity, relative abundance of specific strains, species, genera, and other taxonomic units, and the Firmicutes/Bacteroidetes (F/B) ratio. A microbiome profile may be generated by at least one of a multiplicity of techniques known to those in the art, for example, multiplex real-time polymerase chain reaction (PCR), pyrosequencing, whole genome sequencing, shotgun analysis, and others.

[0069] As used herein,“microorganism” refers to any microscopic life form capable of self- replication. Nonlimiting examples include bacteria, viruses, protozoans, prions, fungi, and yeasts.

[0070] A“mutation” as used herein is a change in a sequence (e.g., an amino acid sequence of a protein, a nucleic acid sequence encoding a protein, or a nucleic acid sequence in a non coding region) resulting in an alteration from a given reference sequence (which may be, for example, a wildtype amino acid sequence of a protein, or a wildtype DNA sequence or RNA sequence). In some embodiments, the mutation is a deletion and/or insertion and/or duplication and/or substitution of at least one amino acid. In some embodiments, the mutation produces a change in at least one function and/or activity of a protein. In some embodiments, the mutation is in a adenomatous polyposis coli (APC) gene. In some embodiments, the mutation in APC results in loss of APC function. In some embodiments, the mutation in the APC gene results in a truncated APC protein.

[0071] As used herein,“naturally-occurring pathogenic molecules” refers to molecules that

occur in nature in foods or in the environment, and that are known to play an active role in disease production. A common example of a naturally-occurring pathogenic molecule is the wheat protein, gliadin, which becomes pathogenic if it passes from the intestinal lumen into the bloodstream, where it can trigger an autoimmune reaction in subjects having the necessary genetic pre-load.

[0072] As used herein,“non-pathogen” refers to a microorganism that does not fulfill Koch’s Postulates establishing a causative relationship between a microorganism and a disease, but rather that is a natural or normal member of a microbiome.

[0073] By“defined microbial consortia” is meant a purified and/or isolated population of known microbes.

[0074] By“undesirable gut microbiome” is meant a community of microbes comprising a

pathogen or having a biological activity associated with a pathogenic process.

[0075] By“normal gut flora” is meant a population of microbes that is substantially similar to the population of microbes present in the gut of a healthy control subject or a group of subjects determined to be in good health and free of the disease of interest.

[0076] As used herein, a“normal microbiome reference level” means the approximate relative abundance of at least one microorganism comprising a microbiome of a subject in good health, or the range of relative abundance of said at least one microorganism comprising a representative microbiome of a group or population of subjects determined to be in good health, or to have been demonstrated not to have or be at risk for the microbiome-related disease or condition of immediate interest.

[0077] As used herein,“nucleotide” means the organic molecules that serve as subunits of

nucleic acids such as DNA and RNA. The terms“nucleotide sequence,”“DNA sequence,” and“RNA sequence” refer to covalently-bonded linear sequences of nucleotides, DNA, or RNA that can be detected using automated biochemical methods know to those skilled in the art. Living organisms contain many unique nucleotide sequences, and the detection of such sequences forms the basis for identification of specific organisms present.

[0078] As used herein,“operational taxonomic unit” means a group of individual

microorganisms closely related on the basis of close similarity of DNA or RNA sequences.

[0079] As used herein,“taxon” and“taxa” refer to the singular and plural, respectively, of one or more groups of one or more populations of an organism or organisms seen by taxonomists to form a unit in the hierarchy of the organization of life (taxonomy). Common examples of taxa include, but are not limited to, phylum (plural phyla), class, order, family, genus, species, subspecies and strains.

[0080] As used herein,“pathobiont” means any potentially pathological (disease-causing)

organism which, under normal circumstances, lives as a symbiont, or member of a normal microbiome.

[0081] As used herein,“pathogen” means a microorganism that does fulfill Koch’s Postulates establishing a causative relationship between a microorganism and a disease, that is, the pathogen always produces the infectious disease, and in the absence of the pathogen, the disease never occurs.

[0082] As used herein,“functional microorganism-produced molecule” means a molecule, produced by at least one microorganism taxon, that produces biological effects in a host of a microbiome. Such molecules may facilitate communications between microorganisms in a microbiome, between microorganisms and the host (signaling molecules), or may be used by one taxon of microorganisms to suppress growth of other taxa (e.g., bacteriocins). Functional microorganism-produced molecules may also directly produce disease (e.g., bacterial exotoxins or endotoxins), or may contribute to disease or disease risk by means of their interactions with host tissues (e.g., virulence factors). In some embodiments, the functional microorganism-produced molecule is an E. coli adhesin , an E. coli flagellin, or a B. fragilis toxin (BFT).

[0083] As used herein,“pharmaceutically acceptable protective agent” means a coating,

encapsulation, covalent bond, physical structure, or other means of protecting the therapeutic mixture of IgY or probiotics against degradation in the stomach or upper gastrointestinal tract. Examples of such protective agents include, but are not limited to, acid-resistant capsules, liposomes, nanospheres, and other methodologies known to those practiced in the art.

[0084] As used herein, the terms“peptide,”“polypeptide,” and“protein” are used

interchangeably, and refer to a molecule comprised of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and no limitation is placed on the maximum number of amino acids that can comprise a protein’s or peptide’s sequence. Polypeptides include any peptide or protein comprising two or more amino acids joined to each other by peptide bonds. As used herein, the term refers to both short chains, which also commonly are referred to in the art as peptides, oligopeptides and oligomers, for example, and to longer chains, which generally are referred to in the art as proteins, of which there are many types. “Polypeptides” include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others. The polypeptides include natural peptides, recombinant peptides, synthetic peptides, or a combination thereof.

[0085] As used herein,“prebiotic” means a non-living organic compound known to foster the growth or colonization of a probiotic microorganism.

[0086] As used herein,“probiotic” or“live biotherapeutic” means ingested microorganisms associated with benefits for humans and animals. The terms are used interchangeably herein. A probiotic formulation may contain multiple individual organisms from a single species or other taxonomic unit, or may be a mixture of multiple taxonomic units.

[0087] As used herein,“reference value” for a microbiome profile reporting a microbiome

composition means at least one of the following: the relative abundance of at least one microorganism in a microbiome profile generally recognized as representative of a group of subjects in good general health, the relative abundance of at least one microorganism in a microbiome profile generally recognized as representative of a group of subjects having in common the absence of a specific disease or disease risk, or the relative abundance of at least one microorganism in a microbiome profile generally recognized as representative of a group of subjects having in common a disease or a disease risk. It is understood that the expression “higher than a reference value” means that the relative abundance of at least one

microorganism on an individual subject’s microbiome profile, or the microbiome profile of a group of subjects, is greater than that of the reference value to a degree known to be associated with a disease or a disease risk. It is further understood that the expression“lower than a reference value” means that the relative abundance of at least one microorganism on an individual subject’s microbiome profile, or the microbiome profile of a group of subjects, is lower than that of the reference value to a degree known to be associated with a disease or a disease risk.

[0088] As used herein,“relative abundance” refers to the proportional contribution of a

taxonomic unit to the overall composition of a microbiome. For example, in a theoretical, simplified microbiome containing five total taxonomic units of organisms, all found in identical proportions, each taxonomic unit would have a relative abundance of 20%, while in another, two taxonomic units might be present at 35% each, and the remaining three would then be present at 10% each.

[0089] As used herein,“replacement therapy” refers to a complete, or near-complete,

replacement of an entire microbiome with microorganisms from another source. The most relevant and common example of replacement therapy for an intestinal microbiome is a fecal microbial transplant (FMT), in which microorganisms from fecal matter of a healthy subject are transferred into the colon of a subject in need of modulation of the intestinal microbiome, with the result that the microorganisms establish and maintain a new microbiome in the intestine of the recipient.

[0090] As used herein,“sample” or“biological sample” refers to anything, which may contain an analyte (e.g., polypeptide, polynucleotide, or fragment thereof) for which an analyte assay is desired. The sample may be a biological sample, such as a biological fluid or a biological tissue. In one embodiment, a biological sample is a salivary sample. Such a sample may include diverse cells, proteins, and genetic material. Examples of biological tissues also include organs, tumors, lymph nodes, arteries and individual cell(s). Examples of biological fluids include urine, blood, plasma, serum, saliva, semen, stool, sputum, cerebral spinal fluid, tears, mucus, amniotic fluid or the like.

[0091] By the term“specifically binds,” as used herein with respect to an antibody, e.g., an IgY antibody, is meant an antibody which recognizes a specific antigen, but does not substantially recognize or bind other molecules in a sample. For example, an antibody that specifically binds to an antigen from one species may also bind to that antigen from one or more species. But, such cross-species reactivity does not itself alter the classification of an antibody as specific. In another example, an antibody that specifically binds to an antigen may also bind to different allelic forms of the antigen. However, such cross reactivity does not itself alter the classification of an antibody as specific. In some instances, the terms“specific binding” or“specifically binding,” can be used in reference to the interaction of an antibody, a protein, or a peptide with a second chemical species, to mean that the interaction is dependent upon the presence of a particular structure (e.g., an antigenic determinant or epitope) on the chemical species; for example, an antibody recognizes and binds to a specific protein structure rather than to proteins generally. If an antibody is specific for epitope“A”, the presence of a molecule containing epitope A (or free, unlabeled A), in a reaction containing labeled“A” and the antibody, will reduce the amount of labeled A bound to the antibody. In some embodiments, the antibody is an IgY. In some embodiments, the antibody, e.g., IgY antibody, specifically binds a microorganism-produced molecule. In some embodiments, the antibody (e.g., IgY antibody) specifically binds an E. cob adhesin. In some embodiments, the antibody (e.g., IgY antibody) specifically binds an E. cob flagellin. In some other embodiments, the antibody (e.g., IgY antibody) specifically binds B. fragibs toxin (BFT).

[0092] As used herein,“subtractive therapy” means the deliberate reduction in absolute or

relative abundance of at least one microorganism in a microbiome, with the intent of modulating a microbiome to more closely resemble one associated with health. Examples of subtractive therapy include the use of targeted antibiotics, and the use of the still more highly-targeted IgY.

[0093] As used herein,“therapeutic agent” refers to a substance that demonstrates some

therapeutic effect by restoring or maintaining health, such as by alleviating the signs, symptoms, or abnormal diagnostic test results associated with a disease or physiological disorder, or delaying (including preventing) progression or onset of a disease. In some instances, the therapeutic agent is a chemical, biological, or pharmaceutical agent, or a prodrug. A therapeutic agent may be an agent which prevents or inhibits one or more signs or symptoms, or laboratory findings associated with disease or disease risk. In one some embodiments, the therapeutic agent is an antibody, e.g., a IgY antibody, that specifically binds a microorganism-produced molecule, such as an E. cob adhesin, an E. cob flagellin, or a B. fragibs toxin (BFT).

[0094] A "therapeutically effective amount" or "effective amount" or "therapeutically effective dose" is that amount or dose sufficient to inhibit or prevent onset or advancement, to treat outward symptoms, or to cause regression, of a disease. The therapeutically effective amount or dose also can be considered as that amount or dose capable of relieving symptoms caused by the disease. Thus, a therapeutically effective amount or dose of an antibody (e.g., IgY antibody) is that amount or dose sufficient to achieve a stated therapeutic effect. The therapeutically effective amount may vary depending the subject and disease condition being treated, e.g., the weight and age of the subject, the severity of the disease condition, the manner of administration and the like, which can readily be determined by one of ordinary skill in the art.

[0095] As used herein,“topical administration” means application or administration of a

therapeutically effective amount of a substance (here, IgY or probiotic microorganisms) to the skin or mucous membrane surfaces of a living vertebrate.

[0096] As used herein, the terms "treatment" and "treating" refer to an approach for obtaining beneficial or desired results including, but not limited to, therapeutic benefit and/or a prophylactic benefit. By therapeutic benefit is meant eradication or amelioration of the underlying disorder being treated. Also, a therapeutic benefit is achieved with the eradication or amelioration of one or more of the physiological symptoms associated with the underlying disorder such that an improvement is observed in the patient, notwithstanding that the patient may still be afflicted with the underlying disorder. For prophylactic benefit, the compositions may be administered to a patient at risk of developing a particular disease, or to a patient reporting one or more of the physiological symptoms of a disease, even though a diagnosis of this disease may not have been made.

[0097] Throughout this disclosure, various aspects of the invention may be presented in a range format. It should be understood that the description in range format is merely for

convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range and, when appropriate, partial integers of the numerical values within ranges. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

Description:

[0098] As research into the structure and function of individual microorganism species in

microbiomes has expanded, mechanistic pathways have emerged that demonstrate how specific virulence factors and signaling molecules directly contribute to the risk for and manifestations of certain conditions. Recent studies have established increasingly close associations between composition of a microbiome with respect to species and strains of bacteria, presence of specific microorganism-produced pathogenic molecules such as signaling molecules, virulence factors, and toxins, and the risks for a number of intra- and extra-intestinal diseases.

[0099] Specifically, studies of subjects with colorectal cancer (CRC) have revealed the presence of biofilms enriched with certain bacterial species in affected subjects, especially in the right sided (or proximal) portions of the colon. These species represent two members of the normal gastrointestinal flora, Escherichia coli (E. coli, Phylum Proteobacteria, Class Gammaproteobacteria) and Bacteroides fragilis (B. fragilis, Phylum Bacteroidetes, Class Bacteroidia), as well as three bacteria most commonly found as pathogens in the oral microbiome, Fusobacterium nucleatum (F. nucleatum, Phylum fusobacteria, Order Fusobacteriales), Parvimonas micra (P. micra, Phylum Firmicutes, Class Tissierella), and Peptostreptococcus stomatis (P. stomatis, Phylum Firmicutes, Class Clostridia). Both B. fragilis and E. coli are known to elaborate tumor-promoting toxins, respectively fragilysin (EC 3.4.24.74, or Bacteroides fragilis (entero)toxin, BFT, which thins the natural protective mucin layer in the colon), and colibactin (which alkylates DNA with an unusual electrophilic cyclopropane, and which is produced by E. coli strains carrying the polyketide synthetase (pks) genomic structure).

[00100] Furthermore, B. fragilis toxin is known to trigger a multi-step inflammatory cascade requiring IL-17 as part of the oncogenic process.

[00101] Studies of subjects with familial adenomatous polyposis (FAP) have also identified the presence of patchy biofilms composed predominantly of E. coli and B. fragilis, and genomic methods have demonstrated enrichment in these populations of genes encoding BFT and colibactin.

[00102] Animal studies show that mice colonized with cocolonized with colibactin-expressing E. coli and enterotoxigenic B. fragilis have increased IL-17 in the colon, DNA damage in the colonic epithelium, and faster tumor onset and greater mortality, compared to mice with either bacterial strain alone.

[00103] Other studies have shown that pks+ E. coli strains can themselves directly induce DNA alkylation of the pattern identified in adults with CRC; such strains appear to require adhesion to colonocyte surfaces in order to express the colibactin toxin. [00104] All of these microorganisms and protein molecules are therefore appealing targets for interventions aimed at reducing CRC oncogenesis (FIG. 3). To date no comprehensive and specifically targeted means for selectively and precisely reducing the abundance of any of these pathobionts, or of their microorganism-produced pathogenic molecules, or of IL-17 in situ in the intestinal tract has been disclosed.

[00105] This invention discloses methods for reducing the risk of, or treating, colorectal cancers in subjects at risk for those malignancies, and for detecting the presence of microorganisms, microorganism-produced molecules, and IL-17, in fecal, colonic brushings/washings, and biopsy specimens, in subjects at risk for those malignancies. Subjects at risk for CRC include those with FAP, who currently face total colectomy at an early age as the only effective preventive measure.

[00106] In one aspect of this invention, a mixture of IgY is devised, such that the IgY

immunoglobulins target signaling molecules, virulence factors, or toxins produced by E. coli and B. fragilis. In another aspect, the mixture of IgY will include IgY specific of F.

nucleatum, P. micra, and P. stomatis. In a further aspect, the IgY mixture contains IgY immunoglobulins that target BFT or flagellins or adhesins produced by pathogenic strains of E. coli. In still a further aspect, the IgY mixture contains IgY immunoglobulins that target IL-17.

[00107] In another aspect, the produced target-specific IgY molecules will be used in

immunoglobulin-based assays, for non-limiting example, ELISA, aimed at the quantitative detection and measurement of the microorganisms associated with CRC and FAP, their microbiome-produced molecules, and IL-17 in clinical specimens from subjects with or at risk for FAP and CRC.

[00108] This invention solves several problems that are associated with the current state of the art of modifying the risk of CRC, both in subjects with FAP, and in those with sporadic polyposis or CRC.

[00109] First, the use of IgY permits targeting of individual species or strains of microorganisms with much greater specificity than can be accomplished with antibiotic use, which inevitably destroys microorganisms other than those targeted for intervention. That degree of specificity is further enhanced in this invention because the IgY can be developed to target not intact microorganisms, but rather microorganism-produced molecules including signaling molecules, virulence factors, and toxins involved in microbiome event cascades leading to CRC. The mixture of IgY is formulated in response to known pathobionts associated with elevation of CRC risk, rather than applying a“one-size-fits-all” approach, as is the case when antibiotics are used.

[00110] Further advantages of the invention include the ability to continuously reformulate the IgY mixture in response to results of diagnostic or detection methods obtained after a period of treatment, to permit increased or decreased dosing of the specific IgY in the mixture. In addition, there is no evidence to date that microorganisms targeted by IgY develop resistance to IgY, as opposed to the high risk of microorganisms developing resistance to antibiotics, particularly if repeatedly or continuously administered. Should resistance to a specific IgY emerge, new IgY can be produced in response to epitopes specific of the new microorganism.

[00111] An additional advantage of this invention is that the formulations described produce interventions within the colon itself and are not absorbed systemically. This feature greatly limits the risk of systemic adverse events. A related advantage of using IgY instead of mammalian-derived antibodies (e.g., IgG) is that IgY does not bind to mammalian Fc receptors, nor does it fix mammalian complement. As a result, the risk of inflammatory reactions to the administered antibodies is greatly reduced.

[00112] An additional advantage of this invention is the large amounts of IgY that can be

produced by laying hens and transferred to their eggs; published figures suggest an average of 100 mg/IgY per egg yolk; a simple calculation shows that a“small” commercial flock of 10,000 hens could produce 325 kg of IgY/year.

[00113] The present invention also provides a novel approach to research on microorganisms and microorganism-produced molecules associated with FAP and CRC in living animals or in model microbiome systems, by allowing researchers to selectively and specifically alter the abundance of individual strains, species, or other taxa, or of the concentrations of signaling molecules, virulence factors, and toxins, and observing the impact on microbiome

composition, function, and host phenotype. This is a desirable new capability because current science does not permit such selective modifications, limiting the study of the contribution of any individual microorganism or groups of microorganisms to host biological function.

[00114] A variety of methods known in the art for administering the IgY composition of the

invention are contemplated by the present invention. The composition described herein may be administered to a patient orally, transarterially, subcutaneously, intradermally, intratumorally, intranodally, intramedullary, intramuscularly, by intravenous (z.v.) injection, intraperitoneally, topically, intraorally, intravaginally, or by ingestion. In other instances, the IgY(s) of the invention are injected directly into a site of inflammation in the subject, a local disease site in the subject, a lymph node, an organ, a tumor, and the like. In other instances, the IgY(s) of the invention are infused directly into the colon of a subject with FAP or at risk for CRC, at the time of colonoscopy. In such instances, additional infusion of

microorganisms beneficial to colonic health is contemplated; such organisms are often obligate anaerobes, and the pure nitrogen atmosphere of colonoscopy presents an attractive opportunity to instill these organisms.

[00115] In the following description, examples and figures, the methods of the invention should not be construed as being limited solely to the microbiome of an individual subject but rather applicable as well to the microbiome of a population. It is understood that the processes described apply both to the modification of an individual subject’s microbiome composition and function, and to that of a microbiome composition and function representative of a population of subjects having in common a disease or disease risk influenced by microbiome composition. In the case of populations of subjects, the microbiome profile is one

representative of the population of interest, derived from population-level studies. For instance, if the population of interest represents patients with irritable bowel syndrome (IBS), then the microbiome profile is understood to mean an aggregate microbiome profile representative of a known population of IBS patients.

[00116] FIG. 1 provides a table representing each species of microorganism to be targeted by the formulation of IgY disclosed in this invention, along with known or theorized mechanisms of action that may promote colorectal carcinogenesis. Included in FIG. 1 are also the

microorganism-produced molecules currently associated with colorectal carcinogenesis by the microorganisms involved.

[00117] In at least one embodiment of the invention, at least one IgY specific of E. coli or B. fragilis, or of a microorganism-produced molecule such as a signaling molecule, virulence factor, or toxin, is included in the formulation. In at least one other embodiment, the formulation includes at least one IgY specific of F. nucleatum, P. micra, or P. stomatis. In at least one other embodiment, the formulation includes at least one IgY specific of colibactin or BFT. In at least one other embodiment, the formulation includes at least one IgY specific of IL-17.

[00118] In at least one embodiment, the IgY in the formulation will be generated against specific strains of at least one of the five microorganisms disclosed in this patent, such strains, for non-limiting example, consisting of those known to be high producers of BFT or colibactin.

[00119] In at least one embodiment, the IgY in the formulation will be generated against at least one microorganism-produced signaling molecule, virulence factor, or toxin known to be involved in a microbiome event cascade leading to CRC.

[00120] FIG. 2 provides a table listing the various target-specific IgY molecules to be included in various embodiments of this invention. It is essential to note that IgY specific of more than one strain of each of the targeted microorganisms, including those producing microorganism- produced pathogenic molecules such as BFT and colibactin, and IgY specific of a

microorganism-produced pathogenic molecule known to be involved in a microbiome event cascade leading to CRC, may be produced and included in the composition disclosed in this patent.

[00121] It is contemplated that ongoing studies will provide: 1/ a Table showing the

concentrations of target-specific IgY in eggs from specifically-immunized hens, compared with those from unimmunized, control hens; 2/ a series of graphs showing the binding characteristics of experimentally-produced IgY against the target microorganisms and microorganism-produced molecules and IL-17, indicating the degree to which each target- specific IgY binds to its cognate microorganism or molecule; 3/ a Table showing the growth inhibition of target microorganisms by their cognate target-specific IgY, under conditions in which the target microorganisms will be grown alone, with IgY from unimmunized eggs, and with target-specific IgY against each microorganism; 4/ a series of graphs showing growth inhibition of the target microorganisms; 5/ a series of graphs showing inhibition of the action of BFT on mammalian cells in culture, and of DNA damage caused by pks+ E. coli on mammalian cells in culture, and 6/ a drawing of an example rapid detection kit capable of identifying and semi-quantifying the presence of each of the target microorganisms and of bft, colibactin, and IL-17 in a specimen of feces, colonic washings/brushings, or biopsy.

Production of IgY for Colorectal Cancer Preventive Therapy:

[00122] This invention discloses the use of specially-prepared mixtures containing at least one IgY specific of at least one: A) Microorganism found to be associated by its excess with FAP and CRC, or B) Microorganism-produced pathogenic molecule known to be involved in a microbiome event cascade leading to CRC, or C) human IL-17.

[00123] The production of IgY from domestic chicken eggs is known to those skilled in the art.

In its essence, it involves selection of a healthy laying hen, and selectively injecting that hen, subcutaneously or intramuscularly, with an antigen of interest. An antigen of interest may be an intact, inactivated microorganism, a complete biomolecule known to be involved in a microbiome event cascade leading to CRC, or at least one specific epitope of said

biomolecule. It may be advantageous to use multiple individual epitopes representing receptor binding domains or other specific domains of a microorganism produced molecule as the antigenic material. As is known to those skilled in the art, such epitopes may be chemically conjugated to an additional chemical moiety intended to increase the antigenicity of the epitope when injected into a laying hen. Over a several -week period, the hen’s immune system responds to the antigen by producing large amounts of IgY, which become concentrated in her eggs. A booster injection is commonly given to enhance immunoglobulin production, and full immunoglobulin production is reached at around week 10-12.

[00124] Alternatively, to enhance IgY production and reduce costs associated with immunizing large groups of hens, a viral vector, for non-limiting example, a recombinant vesicular stomatitis virus (rVSV) may be injected or otherwise administered to a flock of hens, such that the virus establishes an asymptomatic infection in the hen, continuously producing the epitope or antigen of interest. Use of rVSV against several viral epitopes has been reported, and has been shown to raise IgY production roughly three-fold compared with standard serial injections, and to result in lifelong production of the desired IgY without need for further injections.

[00125] A laying hen produces on the order of 350 eggs/year, each egg containing roughly 100 mg IgY. Production rates vary by breed or genetic lines, as well as on a septadian biorhythm. Eggs are collected, yolks are separated from whites, and pooled from multiple hens having received the same antigen injection, and then, as is known by those skilled in the art, undergo a small number of physical and chemical steps for purification and extraction of the IgY proteins. Because yolks of immunized hens also contain naturally-occurring IgY, the extracted IgY proteins may be further affinity-purified, to selectively harvest only the specific IgY desired for therapeutic use. Alternatively, the IgY proteins may be administered without affinity-purification, so long as sufficient amounts of the active IgY are found in the product to achieve the goals of subtractive therapy.

[00126] Specific details, including preparation and optimization of antigen doses, choice of adjuvant (immune-boosting material), immunization interval, and techniques for isolating and purifying IgY, are known by those skilled in the art and are available in the scientific literature.

[00127] Once a sufficient quantity of IgY has been prepared against the target microorganisms, their pathogenic molecules, and IL-17, quantities of IgY will then be prepared for oral administration to a subject at risk for FAP or CRC. The administered IgY has been shown to survive passage through the upper gastrointestinal tract in quantities sufficient to bind to microorganisms in the colon. Should greater quantities of IgY be required to achieve the desired subtractive effect, techniques for preventing destruction of ingested proteins such as IgY (e.g., encapsulation, liposomes, nanospheres, and others), which are known to those skilled in the art, may be used in formulation to protect the active IgY.

Specific Embodiments of the Invention:

[00128] Multiple embodiments of the present invention are possible.

[00129] In one embodiment, the formulation to be administered to a subject is comprised of lyophilized or spray-dried egg yolks from eggs of chickens immunized against whole-cell preparations of at least one of the target microorganisms (E. coli, B. fragilis, F. nucleatum, P. micra, and P. stomatis), without specific extraction or purification of the target-specific IgY.

[00130] In another embodiment, the target-specific IgY is generated by immunizing laying hens with at least one strain or subspecies of the aforementioned bacteria known to be a producer of (or hold the genetic code for) at least one microorganism-produced pathogenic molecule, or at least one epitope specific to a microorganism-produced molecule known to be involved in a microbiome event cascade leading to CRC.

[00131] In another embodiment, the formulation is comprised of lyophilized or spray-dried egg yolks from the eggs of chickens immunized against at least one of the microorganism- produced pathogenic molecules.

[00132] In another embodiment, the formulation includes lyophilized or spray-dried egg yolks from the eggs of chickens immunized against human IL-17. [00133] In another embodiment, the target-specific IgY to be administered to a subject is extracted and purified, using techniques known those skilled in the art, from yolks of eggs from chickens immunized against at least one of the target microorganisms, or at least one of the target microorganism-produced molecules (for non-limiting example, BFT, E. coli adhesins, E. coli flagellins), or against human IL-17.

[00134] In another embodiment, the target-specific IgY is produced by immunizing laying

chickens with at least one purified cellular component of the target microorganisms, as a means of increasing target-specificity and enhancing binding characteristics of the antibodies. For non-limiting example, hens may be immunized with a cell-surface polysaccharide, a lipopolysaccharide, a flagellin, a porin, or an adhesion molecule, and the resulting IgY used in crude form from lyophilized or spray-dried yolks from specifically- immunized hens’ eggs.

[00135] In another embodiment, the aforementioned purified cellular component of target

microorganisms, or at least one peptide epitope thereof, is chemically conjugated to another molecule known to enhance the avian immune response, as a means of increasing the amount and specificity of the resulting IgY. In a similar embodiment, the at least one microorganism- produced pathogenic molecule is similarly complexed to an immune response-stimulating molecule (adjuvant).

[00136] In one embodiment, the formulation administered to the at least one subject is composed of at least one IgY targeting at least one of the aforementioned bacteria, or at least one of the aforementioned microorganism-produced pathogenic molecules, or human IL-17. It will be understood that such formulations may include at least one, several, or all of the target- specific IgY described in this disclosure.

[00137] In another embodiment, the selected formulation is packaged as dried egg powder to be ingested by the at least one subject by mixing with an aqueous solution such as water, juice, or milk. In another embodiment, the selected formulation is packaged as dried egg powder in capsules resistant to digestion in the upper portions of the gastrointestinal (GI) tract, for release in the distal ileum or the proximal colon.

[00138] In another embodiment, the selected formulation is comprised of IgY purified from the crude yolk extract. In this embodiment, the IgY composition is packaged into digestion- resistant capsules or tablets. [00139] In another embodiment, the selected formulation of dried egg powder, or alternatively of purified IgY, is dissolved in a physiologically-inert aqueous solution (e.g., phosphate- buffered saline (PBS), or 0.9% saline), and is infused into the colon during or at the end of a colonoscopy procedure. This embodiment may be particularly advantageous in subjects with FAP, who undergo frequent colonoscopy procedures.

[00140] In yet another embodiment, at least one of the aforementioned target-specific IgY serves as the indicator molecule in an immunodetection or immunodiagnostic kit or device. In this embodiment, one enabling example consists of target-specific IgY mounted on a substrate, and used in a simple, or in a sandwich ELISA using techniques known to those skilled in the art. When the IgY encounters a specific target, it binds to the target, and the addition of a reporter molecule (e.g., a dye, a fluorescent molecule) after washing the substrate reveals, semi-quantitatively, the presence of the target microorganism in the specimen.

[00141] In one embodiment, the target-specific IgY is bound to a test strip and sold as a kit

including the reagents necessary for activation and detection of the reporter molecule. In a related embodiment, the kit is used in an automated reading device capable of measuring the intensity of the signal produced by IgY-target binding, and producing a semi quantitative indication to a user. Such a system is advantageous because it delivers rapid results and will not require extraction of DNA or RNA as is done in molecular detection systems. Clinicians or end-users could then determine the amount of target microorganisms of interest in an individual specimen at baseline, and could follow the abundance of said microorganisms during a course of treatment aimed at reducing or eliminating abundance of microorganisms.

[00142] In another embodiment, the target-specific IgY is bound to the surface of microbeads of latex or another inert substance, and used in an immuno-agglutination assay, in which a positive and semi quantitative signal is derived from the degree to which the beads agglutinate in the presence of the target microorganism or microorganism-produced molecule. This embodiment also lends itself to production as a kit capable of simultaneous detection and quantification of at least one of the microorganisms or microorganism- produced molecules described as targets of this invention.

Advantages of the invention include:

[00143] Use of IgY for targeted therapy (reducing abundance of specific microorganisms in a microbiome, or reducing activity of microorganism-produced pathogenic molecules) is microorganism-specific and event-cascade-specific, allowing only selected species or other taxonomic units to be reduced in quantity, or allowing only the signaling molecules, virulence factors, or toxins involved in a specific microbiome event cascade to be reduced. This is an advantage over all other known subtractive therapies, which can only subtract or delete larger, and non-taxonomically-defmed, classes of microorganisms in a broad fashion. For example, antibiotics are not species- specific, and can only be targeted at larger classes of microorganisms (for example, gram-negative, gram-positive bacteria), and have no direct impact on microorganism-produced pathogenic molecules. IgY molecules are capable of targeting individual offending species and strains, while leaving intact other members of a microbiome community that are not directly contributory to an observed state of pathology.

[00144] Use of IgY as described in this invention permits a colon cancer risk-reducing therapy to be applied directly within the colon, without systemic absorption. This sharply reduces the risk of systemic adverse events. The risk of local adverse events such as undesired inflammatory responses is reduced because IgY antibodies do not bind to or activate mammalian Fc receptors, nor do they fix mammalian complement. Furthermore, exposure of intraluminal IgY to the host immune system, while non-zero, is considerably limited by comparison with immune system exposure to therapeutic molecules introduced into host tissue or circulation.

[00145] Microbiome-focused therapy with IgY will also allow calibrated, or titrated removal of microorganisms and/or their signaling molecules, virulence factors, and toxins, recalling that none of the microorganisms that contribute to imbalance in the microbiome are explicitly pathogens, so that complete removal of such microorganisms is distinctly undesirable. Other subtractive therapies are“all-or-nothing” in their targeting, reducing populations of targeted microorganisms to zero or near-zero states.

[00146] While antibiotic resistance is a known and widespread threat to public health as well as health of the individual, it is not expected, and has to date not been shown, that use of IgY against bacterial targets, or their signaling molecules, virulence factors, or toxins, produces resistance, and, in fact, the mechanisms by which IgY operate to destroy or remove selected species depend only on evolutionarily-determined surface markers, not on any adaptable physiology of the target microorganism. Moreover, should bacteria in fact be naturally selected to reduce or eliminate display of target molecular markers (including signaling molecules, virulence factors, and toxins released by bacteria) in response to persistent IgY therapy (at present an entirely theoretical risk), the nature of IgY production makes it possible to choose from any other identifying molecular feature of the microorganism, thereby renewing the ability to identify and subtract it from the population.

[00147] No genetic modification of any living microorganism is required for IgY production or administration in microbiome modification, unlike recently-disclosed approaches that rely on engineered microbes to control, amplify, or subtract from the healthy microbiome. Such engineered approaches carry with them the real threat of environmental release of

microorganisms with synthetic properties and unknown consequences. Similarly, IgY therapy obviates the need for bacteriophage viruses, another means of attempting selective reduction therapy. Bacteriophages carry the small but nonzero risks of untoward viral events, and are incapable of addressing pathologies induced by non-bacterial microbiome

constituents or microorganism-produced molecules.

[00148] The features of IgY related in the preceding paragraph also represent an advantageous factor in regulatory considerations. Polyclonal antibodies in general are well-understood by regulatory agencies, unlike many other emerging microbiome-targeted therapeutics such as live biotherapeutics and phage viruses. This can hasten regulatory approval, permitting useful therapeutics to become available in a much shorter time-frame.

[00149] IgY has now been developed to target not only microorganisms, but specific functional molecules. Unlike other subtractive therapies to date, this may allow IgY to be used against bacterial toxins, lipopolysaccharides, and other microbe-generated molecules that cause damage to host tissues, or facilitate interactions with other microorganisms that are deleterious to the host.

[00150] Production of IgY is relatively simple and non-invasive, compared to current methods of producing immunoglobulins for human experimental or therapeutic purposes. IgY production may require as few as two injections per hen. Hens may be housed humanely under cage-free conditions, and would be expected to have lifespans comparable with those of other domestic fowl.

[00151] IgY can be produced in polyvalent mixtures from a single hen, to boost productivity. In this fashion, a hen may be immunized to a large number of antigens (up to 20), resulting in eggs enriched in not one, but several or many IgY molecules, each with its own target. [00152] IgY production is relatively inexpensive, compared to most other technology endeavors; it involves standard animal husbandry, including immunization of individual hens. Egg collection, separation, and processing can also be done using existing food-service technologies.

[00153] Furthermore, the rapid pace with which new IgY molecules can be produced is expected to improve efficiency and shorten discovery periods for therapeutic mixtures. Should an initial formulation prove less effective than hoped, it can be rapidly re-formulated with new IgY targeting different antigens, epitopes, microorganisms, or microorganism-produced molecules, and then rapidly subjected to renewed testing.

[00154] Subtractive therapy of any kind, including IgY, is advantageous over existing means of manipulating the microbiome, which to date are limited largely to dietary changes, probiotics, and prebiotics, none of which appear, alone, to produce lasting changes in the microbiome. The addition of IgY subtractive therapy to additive therapy such as pre- and probiotic therapies may result in improved persistence of the added microorganisms, and may contribute favorably to increased microbiome diversity, a desirable change.

[00155] Regarding methods of detection and surveillance for those with or at risk for colorectal cancer and FAP, advantages of this invention include rapid result production, including the potential for point-of-care utilization. For non-limiting example, fluid from a routine colonoscopy may be applied to a detection method as disclosed in this invention, that method producing a signal that indicates at least presence or absence of any of the microorganisms or their microorganism-produced molecules in real time, permitting stratification of risk to a higher degree of resolution than in presently possible.

[00156] A related advantage of rapid detection methodologies relates to ongoing monitoring or surveillance of subjects with FAP or family history of CRC, in that rising levels of at least one of the targeted bacteria or microorganism-produced molecules on regular testing would alert the subject and care providers to a potentially increased risk for emergence of malignancy.

Unique elements of the invention include:

• Application of IgY technology to regulation of the microbiome (with the advantages enumerated above).

• Provision of a means of“fine tuning” the microbiome by titrating or calibrating doses to observed effects.

• Low cost.

• Rapid response times to observed microbiome imbalances.

• Rapid revision of prototype formulations to achieve optimal results prior to marketing or human testing.

• Rapid revision of therapeutic formulations in response to changes in individual

microbiome dynamics, or to changes in population-level dynamics (e.g., new pathways, new molecular targets as new scientific results arise).

• The potential for a personalized approach that matches each individual’s microbiome profile, across dimensions of time and space, allowing continuous revision and modification of individual therapy.

• An approach that is not theoretically limited to specific classes of microorganisms (e.g., non-beta-lactamase producers, as in certain antibiotics), but rather can be applied to all known existing microorganisms and their signaling molecules, virulence factors, or toxins, as well as those discovered in the future.

• Rapid point-of-care quantitative or semiquantitative detection of targeted microorganisms and their microorganism-produced molecules implicated in FAP and CRC, permitting ongoing monitoring and surveillance.

• An experimental tool (target-specific IgY) that permits selective removal or reduction in abundance of targeted strains, species, or other taxonomic units, or of microorganism- produced pathogenic molecules, as part of studies of microbiome composition-function studies.

Kits:

[00157] The invention includes a kit comprising at least an antibody (e.g., IgY antibody) of the invention, an applicator, and an instructional material for use thereof. The instructional material included in the kit comprises instructions for preventing or treating a disease, e.g., colorectal cancer, associated with characteristics of a microbiome in a subject. The instructional material recites the amount of, and frequency with which, the at least one antibody of the invention should be administered to the subject. In other embodiments, the kit further comprises at least one additional antitumor agent. In some other embodiments, the kit further comprises reagents for detection of a mutation (e.g., a mutation in an APC gene or an APC protein) in a sample obtained from a subject.

Administration/Dosage/Formulations:

[00158] The regimen of administration may affect what constitutes an effective amount. The therapeutic formulations may be administered to the subject either prior to or after the onset of a disease or disorder contemplated in the invention. Further, several divided dosages, as well as staggered dosages may be administered daily or sequentially, or the dose may be continuously infused, or may be a bolus injection. Further, the dosages of the therapeutic formulations may be proportionally increased or decreased as indicated by the exigencies of the therapeutic or prophylactic situation.

[00159] Administration of the compositions of the present invention to a patient, preferably a mammal, more preferably a human, may be carried out using known procedures, at dosages and for periods of time effective to treat a disease or disorder contemplated in the invention. An effective amount of the therapeutic agent necessary to achieve a therapeutic effect may vary according to factors such as the state of the disease or disorder in the patient; the age, sex, and weight of the patient; and the ability of the therapeutic agent to treat a disease or disorder contemplated in the invention. Dosage regimens may be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation. A non-limiting example of an effective dose range for a therapeutic agent of the invention is from about 1 and 5,000 mg/kg of body weight/per day. One of ordinary skill in the art would be able to study the relevant factors and make the

determination regarding the effective amount of the therapeutic agent without undue experimentation.

[00160] Actual dosage levels of the active ingredients in the pharmaceutical compositions of this invention may be varied so as to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.

[00161] The therapeutically effective amount or dose of an agent (e.g., IgY antibody) of the present invention depends on the age, sex and weight of the patient, the current medical condition of the patient and the progression of a disease or disorder contemplated in the invention. [00162] A medical doctor, e.g, physician or veterinarian, having ordinary skill in the art may readily determine and prescribe the effective amount of the pharmaceutical composition required. For example, the physician or veterinarian could start doses of the agents of the invention employed in the pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.

[00163] Suitable doses of an agent (e.g., IgY antibody) of the present invention may vary across a wide range of values, depending on the degree of decrease or increase in the relative abundance of targeted microorganisms desired. The dose may be administered in a single dosage or in multiple dosages, for example from 1 to 4 or more times per day. When multiple dosages are used, the amount of each dosage may be the same or different. For example, a dose of 1 g per day may be administered as two 0.5 g doses, with about a 12-hour interval between doses.

[00164] In one embodiment, the compositions of the invention are administered to the patient in dosages that range from one to five times per day or more. In another embodiment, the compositions of the invention are administered to the patient in range of dosages that include, but are not limited to, once every day, every two, days, every three days to once a week, and once every two weeks. It is readily apparent to one skilled in the art that the frequency of administration of the various combination compositions of the invention varies from individual to individual depending on many factors including, but not limited to, age, disease or disorder to be treated, gender, overall health, and other factors. Thus, the invention should not be construed to be limited to any particular dosage regime and the precise dosage and composition to be administered to any patient is determined by the attending physical taking all other factors about the patient into account.

[00165] It is understood that the amount of an agent (e.g., IgY antibody) dosed per day may be administered, in non-limiting examples, every day, every other day, every 2 days, every 3 days, every 4 days, or every 5 days. For example, with every other day administration, a 5 mg per day dose may be initiated on Monday with a first subsequent 5 mg per day dose administered on Wednesday, a second subsequent 5 mg per day dose administered on Friday, and so on.

[00166] In the case wherein the patient’s status does improve, upon the doctor’s discretion the administration of theagent (e.g., IgY antibody) of the invention is optionally given continuously; alternatively, the dose of drug being administered is temporarily reduced or temporarily suspended for a certain length of time (i.e., a“drug holiday”). The length of the drug holiday optionally varies between 2 days and 1 year, including by way of example only, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 12 days, 15 days, 20 days, 28 days, 35 days, 50 days, 70 days, 100 days, 120 days, 150 days, 180 days, 200 days, 250 days, 280 days, 300 days, 320 days, 350 days, or 365 days. The dose reduction during a drug holiday includes from 10%-100%, including, by way of example only, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%.

[00167] Once improvement of the patient’s conditions has occurred, a maintenance dose is

administered if necessary. Subsequently, the dosage or the frequency of administration, or both, is reduced, as a function of the disease or disorder, to a level at which the improved disease is retained. In one embodiment, patients require intermittent treatment on a long term basis upon any recurrence of symptoms.

[00168] The agent (e.g., IgY antibody) for use in the method of the invention may be formulated in unit dosage form. The term“unit dosage form” refers to physically discrete units suitable as unitary dosage for patients undergoing treatment, with each unit containing a

predetermined quantity of active material calculated to produce the desired therapeutic effect, optionally in association with a suitable pharmaceutical carrier. The unit dosage form may be for a single daily dose or one of multiple daily doses (e.g., about 1 to 4 or more times per day). When multiple daily doses are used, the unit dosage form may be the same or different for each dose.

[00169] Toxicity and therapeutic efficacy of such therapeutic regimens are optionally determined in cell cultures or experimental animals, including, but not limited to, the determination of the LD_5O (the dose lethal to 50% of the population) and the ED₅₀ (the dose therapeutically effective in 50% of the population). The dose ratio between the toxic and therapeutic effects is the therapeutic index, which is expressed as the ratio between LD₅₀ and ED_50. The data obtained from cell culture assays and animal studies are optionally used in formulating a range of dosage for use in human. The dosage of such agents lies preferably within a range of circulating concentrations that include the ED₅₀ with minimal toxicity. The dosage optionally varies within this range depending upon the dosage form employed and the route of administration utilized.

[00170] In one embodiment, the compositions of the invention are formulated using one or more pharmaceutically acceptable excipients or carriers. In one embodiment, the pharmaceutical compositions of the invention comprise a therapeutically effective amount of an agent (e.g., IgY antibody) of the invention and a pharmaceutically acceptable carrier.

[00171] The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity may be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms may be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal and the like. In many cases, it is preferable to include isotonic agents, for example, sugars, sodium chloride, or polyalcohols such as mannitol and sorbitol, in the composition.

[00172] In one embodiment, the present invention is directed to a packaged pharmaceutical composition comprising a container holding a therapeutically effective amount of an agent (e.g., IgY antibody) of the invention, alone or in combination with a second pharmaceutical agent; and instructions for using the agent to treat, prevent, or reduce one or more symptoms of a disease or disorder contemplated in the invention.

[00173] Formulations may be employed in admixtures with conventional excipients, i.e.,

pharmaceutically acceptable organic or inorganic carrier substances suitable for any suitable mode of administration, known to the art. The pharmaceutical preparations may be sterilized and if desired mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure buffers, coloring, flavoring and/or aromatic substances and the like. They may also be combined where desired with other active agents, e.g, analgesic agents.

[00174] Suitable compositions and dosage forms include, for example, dispersions, suspensions, solutions, syrups, granules, beads, powders, pellets, liquid sprays for nasal or oral administration, dry powder or aerosolized formulations for inhalation, and the like. It should be understood that the formulations and compositions that would be useful in the present invention are not limited to the particular formulations and compositions that are described herein.

Oral Administration:

[00175] For oral application, particularly suitable are tablets, dragees, liquids, drops,

suppositories, or capsules, caplets and gelcaps. The compositions intended for oral use may be prepared according to any method known in the art and such compositions may contain one or more agents selected from the group consisting of inert, non-toxic pharmaceutically excipients which are suitable for the manufacture of tablets. Such excipients include, for example an inert diluent such as lactose; granulating and disintegrating agents such as cornstarch; binding agents such as starch; and lubricating agents such as magnesium stearate. The tablets may be uncoated or they may be coated by known techniques for elegance or to delay the release of the active ingredients. Formulations for oral use may also be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert diluent.

[00176] For oral administration, the agents (e.g., IgY antibodies) may be in the form of tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g., polyvinylpyrrolidone, hydroxypropylcellulose or

hydroxypropylmethylcellulose); fillers (e.g, cornstarch, lactose, microcrystalline cellulose or calcium phosphate); lubricants (e.g, magnesium stearate, talc, or silica); disintegrates (e.g, sodium starch gly collate); or wetting agents (e.g, sodium lauryl sulphate). If desired, the tablets may be coated using suitable methods and coating materials such as OP ADR Y™ film coating systems available from Colorcon, West Point, Pa. (e.g, OP ADR Y™ OY Type, OYC Type, Organic Enteric OY-P Type, Aqueous Enteric OY-A Type, OY-PM Type and

OP ADR Y™ White, 32K18400). Liquid preparation for oral administration may be in the form of solutions, syrups or suspensions. The liquid preparations may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g, sorbitol syrup, methyl cellulose or hydrogenated edible fats); emulsifying agent (e.g, lecithin or acacia); non-aqueous vehicles (e.g, almond oil, oily esters or ethyl alcohol); and preservatives (e.g, methyl or propyl p-hydroxy benzoates or sorbic acid).

[00177] Granulating techniques are well known in the pharmaceutical art for modifying starting powders or other particulate materials of an active ingredient. The powders are typically mixed with a binder material into larger permanent free-flowing agglomerates or granules referred to as a“granulation.” For example, solvent-using“wet” granulation processes are generally characterized in that the powders are combined with a binder material and moistened with water or an organic solvent under conditions resulting in the formation of a wet granulated mass from which the solvent must then be evaporated.

[00178] Melt granulation generally consists in the use of materials that are solid or semi-solid at room temperature {i.e. having a relatively low softening or melting point range) to promote granulation of powdered or other materials, essentially in the absence of added water or other liquid solvents. The low melting solids, when heated to a temperature in the melting point range, liquefy to act as a binder or granulating medium. The liquefied solid spreads itself over the surface of powdered materials with which it is contacted, and on cooling, forms a solid granulated mass in which the initial materials are bound together. The resulting melt granulation may then be provided to a tablet press or be encapsulated for preparing the oral dosage form. Melt granulation improves the dissolution rate and bioavailability of an active (i.e., drug) by forming a solid dispersion or solid solution.

[00179] U.S. Patent No. 5,169,645 discloses directly compressible wax-containing granules having improved flow properties. The granules are obtained when waxes are admixed in the melt with certain flow improving additives, followed by cooling and granulation of the admixture. In certain embodiments, only the wax itself melts in the melt combination of the wax(es) and additives(s), and in other cases both the wax(es) and the additives(s) will melt.

[00180] The present invention also includes a multi-layer tablet comprising a layer providing for the delayed release of one or more agents of the invention, and a further layer providing for the immediate release of a medication for treatment of a disease or disorder. Using a wax/pH-sensitive polymer mix, a gastric insoluble composition may be obtained in which the active ingredient is entrapped, ensuring its delayed release.

Parenteral Administration:

[00181] For parenteral administration, the agents (e.g., IgY antibodies) may be formulated for injection or infusion, for example, intravenous, intramuscular or subcutaneous injection or infusion, or for administration in a bolus dose and/or continuous infusion. Solutions, suspensions or emulsions in an oily or aqueous vehicle, optionally containing other formulatory agents such as suspending, stabilizing and/or dispersing agents may be used. Additional Administration Forms: [00182] Additional dosage forms of this invention include dosage forms as described in U.S. Patents Nos. 6,340,475, 6,488,962, 6,451,808, 5,972,389, 5,582,837, and 5,007,790.

Additional dosage forms of this invention also include dosage forms as described in U.S. Patent Applications Nos. 2003/0147952, 2003/0104062, 2003/0104053, 2003/0044466, 2003/0039688, and 2002/0051820. Additional dosage forms of this invention also include dosage forms as described in PCT Applications Nos. WO 03/35041, WO 03/35040, WO 03/35029, WO 03/35177, WO 03/35039, WO 02/96404, WO 02/32416, WO 01/97783, WO 01/56544, WO 01/32217, WO 98/55107, WO 98/11879, WO 97/47285, WO 93/18755, and WO 90/11757.

Controlled Release Formulations and Drug Delivery Systems:

[00183] In certain embodiments, the formulations of the present invention may be, but are not limited to, short-term, rapid-offset, as well as controlled, for example, sustained release, delayed release and pulsatile release formulations.

[00184] The term sustained release is used in its conventional sense to refer to a drug

formulation that provides for gradual release of a drug over an extended period of time, and that may, although not necessarily, result in substantially constant blood levels of a drug over an extended time period. The period of time may be as long as a month or more and should be a release which is longer that the same amount of agent administered in bolus form.

[00185] For sustained release, the agents (e.g., IgY antibody) may be formulated with a suitable polymer or hydrophobic material which provides sustained release properties to the agents. As such, the agents for use the method of the invention may be administered in the form of microparticles, for example, by injection or in the form of wafers or discs by implantation.

[00186] In certain embodiments, the agents (e.g., IgY antibodies) of the invention are

administered to a patient, alone or in combination with another pharmaceutical agent, using a sustained release formulation.

[00187] The term delayed release is used herein in its conventional sense to refer to a drug

formulation that provides for an initial release of the drug after some delay following drug administration and that mat, although not necessarily, includes a delay of from about 10 min up to about 12 hours.

[00188] The term pulsatile release is used herein in its conventional sense to refer to a drug

formulation that provides release of the drug in such a way as to produce pulsed plasma profiles of the drug after drug administration.

[00189] The term immediate release is used in its conventional sense to refer to a drug

formulation that provides for release of the drug immediately after drug administration.

[00190] As used herein, short-term refers to any period of time up to and including about 8

hours, about 7 hours, about 6 hours, about 5 hours, about 4 hours, about 3 hours, about 2 hours, about 1 hour, about 40 min, about 20 min, or about 10 min and any or all whole or partial increments thereof after drug administration after drug administration.

[00191] As used herein, rapid-offset refers to any period of time up to and including about 8 hours, about 7 hours, about 6 hours, about 5 hours, about 4 hours, about 3 hours, about 2 hours, about 1 hour, about 40 min, about 20 min, or about 10 min, and any and all whole or partial increments thereof after drug administration.

[00192] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific procedures, embodiments, claims, and examples described herein. Such equivalents were considered to be within the scope of this invention and covered by the claims appended hereto. For example, it should be understood, that modifications in reaction conditions, including but not limited to reaction times, reaction size/volume, and experimental reagents, such as solvents, catalysts, pressures, atmospheric conditions, e.g ., nitrogen atmosphere, and reducing/oxidizing agents, with art-recognized alternatives and using no more than routine experimentation, are within the scope of the present application.

[00193] It is to be understood that wherever values and ranges are provided herein, all values and ranges encompassed by these values and ranges, are meant to be encompassed within the scope of the present invention. Moreover, all values that fall within these ranges, as well as the upper or lower limits of a range of values, are also contemplated by the present application.

[00194] The following examples further illustrate aspects of the present invention. However, they are in no way a limitation of the teachings or disclosure of the present invention as set forth herein.

EXAMPLES

[00195] EXAMPLE 1: Producing type-specific IgY against microorganisms implicated in familial adenomatous polyposis (FAP) and colorectal cancer (CRC), and determining target-specificity, physiological effects on mammalian cells, and growth inhibition of the target bacteria.

[00196] Materials and Methods: Peptide epitopes determined by computer-assisted epitope mapping of BFT and E. coli adhesins and flagellins synthesized (Novopep Limited,

Shanghai, China) conjugated to keyhole limpet hemocyanin (KLH), and used as antigens for generation of target-specific IgY. Epitopes for BFT and for E. coli adhesins/flagellins are listed in FIG. 1. All epitopes for each target molecule mixed together as antigens and injected as a single injection.

[00197] Hen immunizations prepared to a volume of 0.5 mL, with 30% of the volume consisting of the lyophilized epitope peptides solubilized in water or 4M urea (50 meg epitope/dose), and the remaining 70% consisting of the commercial avian adjuvant Montanide ISA 70 VG (Seppic, NJ, USA). Specific microorganisms represented will be Escherichia coli strain NC101, an adherent and invasive strain, and Bacteroides fragilis strain 086-5443-2-2. These epitopes, listed in FIG. 1, are understood to be representative, and by no means exclusive epitopes for these antigens.

[00198] Immunization of laying hens will begin as follows.

[00199] Two female Red Sexlink (Barred Rock/Rhode Island Red crossbred) chickens

approximately 21 weeks of age will be acquired, acclimatized, and housed for each IgY to be produced. Animals will be housed on an existing poultry farm, leg-banded to permit positive identification of each pair, and kept pairwise in segregated housing with internal laying boxes, and fenced external runs to preclude intermingling of hens and eggs. Animal care will be provided by the poultry farmer, who will routinely examine all birds for good health status. All birds will be kept in the segregated housing with food (custom-mixed balanced ration for laying hens containing com, beans, and poultry pre mix (minerals, vitamins, trace elements)) and water ad libitum for two weeks for acclimatization and health monitoring.

Egg collection will begin as soon as hens began laying in their individual nesting boxes. All collected eggs will be labeled to correspond to the pair of hens that produced them, and cross-checked against a master list. A sample of eggs from each pair of hens obtained prior to immunization will be stored for baseline IgY determination.

[00200] After the two-week acclimatization period, each pair of hens will be immunized with lmL/hen of the assigned bacterial antigen mixed with adjuvant, by injecting 0.5 mL of the mixture into each breast muscle. Booster immunizations will be given at appropriate intervals (for example, 2, 4, 6, and 8 weeks) after the initial immunization. Two additional hens will remain unimmunized, but housed, segregated, in the same facility as immunized hens. Eggs from these hens will be used to extract non-specific IgY for determination of baseline level of IgYs specific of any of the target antigens, in later steps.

[00201] Beginning at 3 weeks after initial immunization, eggs will be collected daily from all pairs of laying hens, labeled indelibly to correspond with the antigen type used, and then stored at 4°C until purification of IgY and further analysis.

[00202] All of the following steps will take place for eggs from each pair of immunized hens and for eggs from the unimmunized hens. For purification of egg yolk IgY antibodies, a water dilution method as previously described will be used. Intact egg yolks will be physically separated from whites and rolled on paper towels to remove adherent whites. The yolk membrane will be punctured, and yolk allowed to flow into a graduated cylinder. Yolks from multiple eggs from each pair of similarly-immunized hens will be pooled before further use. The pooled yolks at Vi will be mixed gently with 2 volumes of a 5.25% (w/v) polyethylene glycol (PEG) 6000 in phosphate-buffered saline (PBS) to produce a final volume of yolk/PEG containing 3.5% PEG 6000. This mixture will be centrifuged at 13,000 x g for 20 minutes, the lipid-rich layer discarded, and the aqueous layer retained. To this aqueous material will be added PEG 6000 at 8% (w/v), the mixture vortexed and placed on a roller table for 10 minutes, after which the mixture will be centrifuged again at 13,000 x g for 20 minutes. The supernatant will be discarded and the pellet resuspended in PBS. To this mixture will be added PEG 6000 at 12% w/v, vortexed, roller mixed, and again centrifuged at 13,000 x g for 20 minutes. The supernatant will be discarded, and the remaining pellet resuspended in PBS. This final aqueous material will be dialyzed against a solution of 0.1% sodium chloride overnight, then dialyzed against a solution of PBS for three hours to rid the material of residual PEG. At this point, the mixture will be frozen, labeled, at -20C until further use in analyses.

[00203] IgY quality will be assessed by analysis on SDS-PAGE, stained with Coomassie

brilliant blue R-250, to assure that bands representing the heavy and light chains of IgY were detected. [00204] To evaluate immunogenicity of the antigens and target-specificity of the resulting antibodies, enzyme-linked immunosorbent assays (ELISA) will be carried out as follows.

[00205] Wells in a 96-well plate will be coated with 100 microliters (mcl) of a solution of each epitope against BFT and each epitope of the E. coli adhesins/flagellins, and incubated overnight. Extracted IgY from each pair of immunized hens (1 pair BFT epitopes, 1 pair E. coli epitopes) will be added in serial 1 : 10 dilutions and incubated in coated microplate wells in PBS-Tween with 2% nonfat milk at 37C for 1 hour. Microplate wells will be washed three times with lx PBS and 0.05% Tween-20. Next 100 mcl of goat-anti-chicken IgY conjugated to horseradish peroxidase (HRP) will be added and washed three times with lx PBS and 0.05% Tween-20. Finally, an HRP substrate will be added, and plates will be scanned using spectrophotometry at 652 nm to detect target-specific IgY. Total protein concentration will be determined by the Bradford method.

[00206] During IgY production, eggs from each pair of immunized hens will be collected

weekly. Eggs will be separated and the yolks processed as described above to extract the IgY, and stored, labeled, at -20C until further testing and use.

[00207] Samples from each batch of target-specific IgY, and 1 batch of non-target-specific IgY extracted from the eggs of unimmunized hens will be used in the following steps for analysis.

[00208] For functional analysis of anti-BFT IgY, a culture of Bacteroides fragilis strain 086- 5443-2-2 will be grown anaerobically under conditions conducive of BFT expression. After growth has reached the exponential stage, determined by attaining an optical density of 0.6- 0.8, the culture will be centrifuged and the supernatant, containing BFT, will be collected, filtered through a 0.2 micron filter to eliminate bacterial organisms, and then incubated with a culture of Vero cells. A second sample of the same material will be incubated with anti- BFT IgY solution at serial dilutions, and then incubated with Vero cells in culture. The Vero cells will be examined microscopically to detect cytopathic effects, primarily a visible “rounding” of the cells. The number of rounded cells in the IgY-treated cultures will be divided by the number of rounded cells in the BFT-only cultures to express a percentage reduction in cytopathic effects. Any reduction in cytopathic effects of 50% or greater compared with the control condition will be considered a positive result.

[00209] For functional analysis of anti-E.coli adhesin/flagellin IgY, a culture of E. coli NC101 will be grown aerobically to an optical density of 0.6 to 0.8. In a control condition, a diluted specimen of E. coli will be co-incubated with Vero cells in culture, while in the active condition the E. coli will first be incubated with anti-adhesin/flagellin IgY, and then co incubated with Vero cells in culture. Vero cell cultures will then be washed, and analyzed for DNA damage characteristic of colibactin intoxication of Vero cells, using an ELISA assay to determine the percentage of g-H2AC, a sensitive marker for DNA double-strand breaks. Any reduction in DNA double-strand breaks of 50% or greater compared with the control condition will be considered a positive result.

[00210] Growth inhibition assays will be conducted to detect the degree of inhibition induced by each IgY at various concentrations. Isolates of Bacteroides fragilis strain 086-5443-2-2 will be grown anaerobically in 5 mL thioglycolate broth, and NC101 E. coli will be grown aerobically in 5 mL TS broth, and their turbidity adjusted to 0.05-0.2 optical density (OD) units on a spectrophotometer, consistent with bacterial concentrations of 10^L6 to 10^L8 CFU/mL. IgY in PBS will be prepared and filter-sterilized through a 0.22-micron filter. 100 microliters of prepared IgY will be added to 100 microliters of the appropriate aerobic or anaerobic culture medium, and incubated at 37 °C for up to 3 days, during which time growth will be determined by periodic optical density measurements until the end of the experiment.

[00211] In summary, this example will demonstrate a) that IgY production against target

epitopes is practical and successful, b) that each target-specific IgY inhibits the pathological effects of each target (cytopathic effects and DNA damage by anti-BFT IgY, and DNA damage by anti-E. coli adhesin/flagellin IgY), and, c) any inhibition of growth of the target microorganisms in culture. This information will then be used for formulation of test products for use in mammalian subjects, specifically mice co-col onized with toxin-producing strains of E. coli and B. fragilis, and for inclusion in a rapid-detection kit using

immunological techniques to identify presence and amounts of any of the 5 bacterial and 3 molecular targets described herein.

[00212] EXAMPLE 2: Demonstration of Impact of Target-Specific IgY Administration on Bacterial and Molecular Targets in a Living Animal Model

[00213] Materials and Methods: Appropriate quantities of the target-specific IgY prepared in Example 1 will be mixed and shipped to a participating laboratory for administration to experimental cancer-prone mice. As a control solution, an equivalent concentration of IgY extracted from eggs of unimmunized hens will be shipped for administration to control mice.

[00214] Animal experimental materials and methods will reflect those used by Dejea, et al

(Dejea, C. M., P. Fathi, et al. (2018). "Patients with familial adenomatous polyposis harbor colonic biofilms containing tumorigenic bacteria." Science 359(6375): 592-597). This protocol used tumor-prone Apc^+mi6 heterozygous multiple intestinal neoplasia (Min) mice and specific pathogen free (SPF) C57BL/6J mice treated with azoxymethane (AOM) (10 mg/kg weekly for 6 weeks).

[00215] As described in the technique provided by Dejea et al, prior to inoculation of bacterial strains in either mouse model, 6-week-old mice will be given water containing 500 mg/L cefoxitin for 48 hours. Cefoxitin treatment results in an absence of detectable bacteria by culture or 16S rRNA qPCR by 24 hours. After removal of antibiotic water for 24 hrs, mice will be inoculated by oral gavage with 10 colony-forming units (cfu) ETBF (piglet 86-5443-2-2), 1 Ox cfu pks+E. coli (murine NCI 01, expressing a fluorescent ampicillin resistance plasmid sfGFP- g

pBAD) or a mixture containing 10 cfu of each strain.

[00216] After cocolonization with both target microorganisms has been established, groups of 12 mice will each be assigned to receive a) an oral formulation of the selected IgY described above, b) an oral formulation of IgY from non-immunized hens’ eggs, or c) no treatment for 6 weeks.

[00217] Fecal specimens will be obtained for culture and 16-S PCR analysis to determine the bacterial composition of the animals’ microbiomes and specifically the abundance of the target microorganisms at baseline and at 4 and 7 days, and 4 and 15 weeks, following the beginning of the intervention, and animals will undergo daily biometric determinations. Animals will be monitored for the emergence of tumors, and will be sacrificed at the end of the study for quantitative determination of tumor counts, weights, and volumes in each group. Assays for colibactin, BFT and IL-17 will also be performed.

[00218] EXPECTED RESULTS: It is anticipated that mice treated with target-specific IgY

formulations will express fewer, smaller, and lighter tumors than those in either control group. It is further expected that histopathological analysis of specimens from the animals colonic mucosa will illustrate reductions in biofilm formation, invasion, and tissue damage. It is further expected that animals treated with the experimental formulation will survive significantly longer and in greater numbers than those in either control group.

Claims

CLAIMS:

1. A formulation for treating and/or preventing colorectal cancer in a subject, the formulation comprising an IgY antibody that specifically binds a Bacteroides fragilis toxin (BFT).

2. A formulation for treating and/or preventing colorectal cancer in a subject, the formulation comprising an IgY antibody that specifically binds an Escherichia coli adhesin or an Escherichia coli flagellin.

3. A formulation for treating and/or preventing colorectal cancer in a subject, the formulation comprising a mixture of an IgY antibody that specifically binds a Bacteroides fragilis toxin (BFT) and an IgY antibody that specifically binds an Escherichia coli adhesin or an Escherichia coli flagellin.

4. The formulation of claim 1 or 3, wherein the BFT is produced by Bacteroides fragilis strain 086-5443-2-2.

5. The formulation of any one claims 1 and 3-4, wherein the IgY antibody specifically binds a BFT epitope comprising an amino acid sequence selected from SEQ ID NOS: 17-22.

6. The formulation of any one of claims 2-5, wherein the adhesin or flagellin is produced by Escherichia coli strain NC101 or a pks+ Escherichia coli.

7. The formulation of any one of claims 2-7, wherein the IgY antibody specifically binds a Escherichia coli adhesin epitope comprising an amino acid sequence selected from: SEQ ID NOS: 1-3 or & Escherichia coli flagellin epitope comprising an amino acid sequence selected from: SEQ ID NOS: 4-16.

8 The formulation of any one of claims 1-7, wherein the IgY antibody is polyclonal.

9. The formulation of any one of claims 1-8, wherein the formulation further comprises a pharmaceutically acceptable carrier or excipient.

10. A method of reducing and/or preventing DNA damage in intestinal cells in a subject, the method comprising administering to the subject an effective amount of a formulation comprising an IgY antibody that specifically binds a Bacteroides fragilis toxin (BFT) and/or a formulation comprising an IgY antibody that specifically binds an Escherichia coli adhesin or an Escherichia coli flagellin.

11. A method of treating, preventing, or reducing risk of developing colorectal cancer in a subject, the method comprising administering to the subject a therapeutically effective amount of a formulation comprising an IgY antibody that specifically binds a Bacteroides fragilis toxin (BFT) and/or a formulation comprising an IgY antibody that specifically binds an Escherichia coli adhesin or an Escherichia coli flagellin.

12. The method of claim 10 or 11, wherein the BFT is produced by Bacteroides fragilis strain 086-5443-2-2.

13. The method of any one of claims 10-12, wherein the adhesin or flagellin is produced by Escherichia coli strain NC101 or a pks+ Escherichia coli.

14. The method of any one of claims 10-13, wherein the IgY antibody specifically binds a BFT epitope comprising an amino acid sequence selected from SEQ ID NOS: 17-22, a

Escherichia coli adhesin epitope comprising an amino acid sequence selected from: SEQ ID NOS: 1-3, or & Escherichia coli flagellin epitope comprising an amino acid sequence selected from: SEQ ID NOS: 4-16.

15. The method of any one of claims 10-14, wherein the IgY antibody is polyclonal.

16. The method of any one of claims 10-15, wherein the formulation further comprises a pharmaceutically acceptable carrier or excipient.

17. The method of any one of claims 10-16, wherein the subject is human.

18. The method of any one of claims 10-17, wherein the subject has a hereditary condition predisposing said subject to colorectal cancer.

19. The method of any one of claims 10-18, wherein the subject has a mutation in APC, the subject has familial adenomatous polyposis (FAP), and/or the subject has a first-degree relative who has FAP or colorectal cancer.

20. A method of preventing or reducing risk of colorectal cancer in a subject, the method comprising

(a) determining whether the subject has an elevated risk for colorectal cancer, and

(b) if the subject is determined to have an elevated risk for colorectal cancer (CRC) , administering to the subject a therapeutically effective amount of a formulation comprising an IgY antibody that specifically binds a Bacteroides fragilis toxin (BFT) and/or a formulation comprising an IgY antibody that specifically binds an Escherichia coli adhesin or an Escherichia coli flagellin.

21. The method of claim 20, wherein the subject is determined to have an elevated risk for colorectal cancer if (a) a mutation is present in an APC protein or nucleic acid in a biological sample obtained from the subject, (b) the subject has familial adenomatous polyposis (FAP), or

(c) the subject has a first-degree relative who has FAP or colorectal cancer.

22. The method of claim 20 or 21, wherein the BFT is produced by Bacteroides fragilis strain 086-5443-2-2.

23. The method of any one of claims 20-22, wherein the adhesin or flagellin is produced by Escherichia coli strain NC101 or a pks+ Escherichia coli.

24. The method of any one of claims 20-23, wherein the IgY antibody specifically binds a BFT epitope comprising an amino acid sequence selected from SEQ ID NOS: 17-22, a Escherichia coli adhesin epitope comprising an amino acid sequence selected from: SEQ ID NOS: 1-3, or & Escherichia coli flagellin epitope comprising an amino acid sequence selected from: SEQ ID NOS: 4-16.

25. The method of any one of claims 20-24, wherein the IgY antibody is polyclonal.

26. The method of any one of claims 20-25, wherein the formulation further comprises a pharmaceutically acceptable carrier or excipient.

27. The method of any one of claims 20-26, wherein the subject is human.