WO2023141560A2 - Alkaline phosphatase for use in oncology - Google Patents

Abstract

The present disclosure relates, inter alia, to combination therapies of specific commensal gastrointestinal bacteria with therapeutic intestinal alkaline phosphatases (lAPs) for preventing, treating, and/or reducing a cancer therapy-mediated side effect, or improving efficacy of a cancer therapy relative to the treatment before or in the absence of IAP.

Description

ALKALINE PHOSPHATASE FOR USE IN ONCOLOGY

TECHNICAL FIELD

The present disclosure relates, inter alia, to combination therapies of specific gastrointestinal bacteria with therapeutic intestinal alkaline phosphatases for use in oncology.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No. 63/301 ,227, filed January 20, 2022, the entire contents of which are hereby incorporated by reference in their entirety.

DESCRIPTION OF THE TEXT FILE SUBMITTED ELECTRONICALLY

The contents of the text file submitted electronically herewith are incorporated herein by reference in their entirety: A computer readable format copy of the Sequence Listing (File name: “112492-5057-PC Sequence Listing”; Date created: January 9, 2023; File size: 24,206 bytes).

BACKGROUND

Administration of cancer therapies, such as immune checkpoint inhibitors (CPI), can be limiting due to the onset of side effect that accompany such treatment, for example gastrointestinal (Gl) distress such as diarrhea and/or colitis.

Alkaline phosphatase (“AP,” EC 3.1.3.1) is a hydrolase enzyme that can remove phosphate groups from various targets, including nucleotides and proteins. In particular, mammalian APs exert their properties by primarily targeting lipopolysaccharide (LPS; a TLR4 agonist), flagellin (a TLR5 agonist) and CpG DNA (a TLR9 agonist). APs also degrade intestine luminal NTPs (e.g., ATP, GTP, etc.), which promote the growth of good bacteria and reverse dysbiosis. Accordingly, APs may find clinical use in, for example, treating various Gl disorders.

Given the need for improved cancer therapies and mitigation of harmful side effects to allow for improved cancer patient care, there is a growing need for therapeutic compositions that prevent, treat, and/or reduce the side effects associated with cancer therapies.

SUMMARY

Accordingly, in aspects, the disclosure provides methods for preventing, treating, and/or reducing a cancer therapy-mediated side effect in a subject in need thereof, comprising administering to the subject an intestinal alkaline phosphatase (IAP).

In aspects, the disclosure provides methods for improving and/or increasing and/or enhancing efficacy of a cancer therapy relative to the treatment before or in the absence of IAP, comprising administering to the subject an IAP. In aspects, the disclosure provides methods for preventing, treating, and/or reducing a cancer therapy-mediated side effect in a subject in need thereof, comprising administering to the subject an IAP, wherein the subject is undergoing therapy with a composition comprising at least one commensal gastrointestinal bacteria.

In aspects, the disclosure provides methods for improving and/or increasing and/or enhancing efficacy of a cancer therapy relative to the treatment before or in the absence of IAP, comprising administering to the subject an IAP, wherein the subject is undergoing therapy with a composition comprising at least one commensal gastrointestinal bacteria.

In aspects, the disclosure provides methods for preventing and/or treating an immune checkpoint inhibitor (CPI)- mediated gastrointestinal (Gl) side effect in a subject in need thereof, comprising administering to the subject an IAP and an CPI selected from an agent that modulates one or more of PD-1, PD-L1 , PD-L2, ICOS, ICOSL, and CTLA-4.

In aspects, the method increases a therapeutic window of the cancer therapy relative to the treatment before or in the absence of IAP. In embodiments, the increased therapeutic window of the cancer therapy comprises one or more of increasing the subject’s likelihood of receiving maintenance therapy of the cancer therapy relative to the treatment before or in the absence of IAP; increasing the subject’s likelihood of receiving a complete regimen of the cancer therapy relative to the treatment before or in the absence of IAP; increasing the subject’s likelihood of receiving more than a complete regimen of the cancer therapy relative to the treatment before or in the absence of IAP; and increasing the dose or length of the cancer therapy relative to the treatment before or in the absence of IAP.

In aspects, the method reduces the likelihood of the subject requiring a transition to palliative care.

In aspects, the method for improving and/or increasing and/or enhancing efficacy of the cancer therapy allows for one or more of dose reduction, treatment duration reduction, and usage of an incomplete regimen of the cancer therapy, relative to the treatment before or in the absence of IAP.

In aspects, the method makes the subject suitable for treatment with a combination therapy of more than one cancer therapies.

In aspects, the cancer therapy-mediated side effect comprises a CPI-mediated gastrointestinal (Gl) side effect. In embodiments, the CPI-mediated Gl side effect comprises diarrhea and/or colitis.

In aspects, the cancer therapy comprises chemotherapy, and the cancer therapy-mediated side effect comprises a chemotherapy-mediated side effect. In embodiments, the chemotherapy-mediated side effect comprises one or more selected from alopecia, myelosuppression, renal toxicity, weight lossy pain, nausea, vomiting, diarrhea, constipation, anemia, malnutrition, hair loss, numbness, changes in tastes, loss of appetite, thinned or brittle hair, mouth sores, memory loss, hemorrhage, cardiotoxicity, hepatotoxicity, ototoxicity, and post-chemotherapy cognitive impairment. In embodiments, the chemotherapy-mediated side effect comprises one or more selected from stomatitis, esophagopharyngitis, leukopenia and/or reduced white blood count, reduced neutrophils, reduced lymphocytes, reduced platelets, increased risk of infection, vomiting, diarrhea, nausea, poor appetite, gastrointestinal ulceration and/or bleeding, thrombocytopenia, hand-foot syndrome, alopecia, mucositis, impaired hepatic function, impaired hepatic function, and hemorrhage.

In aspects, the cancer therapy comprises a radiotherapy, and the cancer therapy-mediated side effect comprises a radiation-mediated side effect. In embodiments, the radiation-mediated side effect comprises one or more selected from fatigue, nausea and vomiting, damage to the epithelial surfaces (e.g., without limitation, moist desquamation), mouth, throat and stomach sores, intestinal discomfort (e.g., without limitation, soreness, diarrhea, and nausea), swelling, infertility, fibrosis, epilation, dryness (e.g. without limitation, dry mouth (xerostomia) and dry eyes (xerophthalmia), and dryness of the armpit and vaginal mucosa), lymphedema, heart disease, cognitive decline, radiation enteropathy (e.g. without limitation, atrophy, fibrosis and vascular changes, which may produce malabsorption, diarrhea, steatorrhea and bleeding with bile acid diarrhea and vitamin B12 malabsorption commonly found due to ileal involvement), pelvic radiation disease including radiation proctitis, producing bleeding, diarrhea and urgency, and radiation cystitis.

In aspects, the present disclosure provides various combinations of one or more IAP constructs, including variants thereof, and therapeutic uses thereof. In aspects, the present disclosure provides various combinations of one or more IAP constructs, including variants thereof, and a composition comprising at least one commensal gastrointestinal bacteria (or “gastrointestinal bacteria”), including, but not limited to, Bacteroides acidifaciens, and therapeutic uses thereof. In embodiments, the IAP construct is a mammalian IAP including, but not limited to, human IAP (hlAP), calf IAP (clAP), and bovine IAP (blAP). In embodiments, the IAP is secreted from the host cell. In embodiments, the IAP is administered orally.

In aspects, the present disclosure provides an additional agent, such as, but not limited to, compositions comprising specific commensal gastrointestinal bacteria (e.g., Bacteroides acidifaciens). In embodiments, a composition comprising Bacteroides acidifaciens is administered by fecal bacteriotherapy, such as fecal microbiota transplant (FMT). In embodiments, the Bacteroides acidifaciens is administered orally.

In aspects, the present disclosure provides a co-formulation of an IAP and a composition comprising Bacteroides acidifaciens. In embodiments, the co-formulation is administered to a subject simultaneously but the release of the additional agent and the IAP from their respective dosage forms (or single unit dosage form if co-formulated) occurs sequentially.

In aspects, the present disclosure provides methods for the therapeutic use of an IAP. In embodiments, an IAP is administered to a subject undergoing therapy with a composition comprising commensal gastrointestinal bacteria, including, but not limited to, Bacteroides acidifaciens. For example, in embodiments, a subject is undergoing therapy with FMT. In other aspects, a therapeutically effective amount of a composition comprising commensal gastrointestinal bacteria, including but not limited to, Bacteroides acidifaciens, is administered to a subject undergoing therapy with IAP. In embodiments, the composition comprising Bacteroides acidifaciens is a fecal microbiota transplant. In embodiments, the composition comprising Bacteroides acidifaciens is an isolated bacterial composition. In embodiments, the present disclosure provides methods for increasing or preserving the number of commensal bacteria and/or composition of the gut microbiome of the subject. In embodiments, the present disclosure provides methods for inhibiting the growth or decreasing the number of pathogenic bacteria in the gut microbiome of the subject.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 depicts sequences pertaining to alkaline phosphatase agents present in compositions and/or formulations described herein.

DETAILED DESCRIPTION

Overview

The present disclosure is based, inter alia, on the discovery that alkaline phosphatases, such as IAP, find use in preventing, treating, reducing, and/or eliminating cancer therapy-mediated side effects. The present disclosure is also based, inter alia, on the discovery that alkaline phosphatases, such as IAP, find use in preventing, treating, reducing, and/or eliminating cancer therapy-mediated side effects, for example in subjects undergoing therapy with a composition comprising at least one commensal gastrointestinal bacteria. Such use of alkaline phosphatases (e.g. IAP) allows for more efficacious cancer treatment. In embodiments, use of alkaline phosphatases improve efficacy (e.g., anti-tumor efficacy) of cancer therapies relative to the treatment before or in the absence of IAP. In embodiments, the cancer therapy comprises immune checkpoint inhibitor (CPI) immunotherapy, chemotherapy, and/or radiotherapy.

IAP is an endogenous protein expressed by the intestinal epithelium that can be used to mitigate inflammation and maintain gut homeostasis. For example, loss of IAP expression or function is associated with increased intestinal inflammation, dysbiosis, bacterial translocation, and systemic inflammation. Its primary functions, among others, in maintaining intestinal homeostasis are generally recognized as the regulation of bicarbonate secretion and duodenal surface pH, long chain fatty acid absorption, mitigation of intestinal inflammation through detoxification of pathogen-associated molecular patterns, and regulation of the gut microbiome. Several substrates that are acted on by lAP’s phosphatase functions include lipopolysaccharide (LPS), flagellin, CpG DNA, and nucleotide di- and tri-phosphates. Specifically, IAP is a target for therapeutics due to its ability to downregulate inflammation, regulate the microbiome, tighten the gut barrier through enhanced expression of claudins and occludins, and affect metabolism of adenosine tri-phosphate and diphosphate (ATP and ADP). The present disclosure contemplates a composition comprising IAP that does not hinder cancer treatment to the subject relative to the treatment before or in the absence of IAP. In fact, according to the present disclosure, the methods described herein increase a therapeutic window of the cancer therapy relative to the treatment before or in the absence of IAP. Alkaline Phosphatases (APs)

The present disclosure is directed, in part, to pharmaceutical compositions, formulations, and uses of one or more alkaline phosphatases. Alkaline phosphatases are dimeric metalloenzymes that catalyze the hydrolysis of phosphate esters and dephosphorylate a variety of target substrates at physiological and higher pHs. Illustrative APs that may be utilized in the present disclosure include, but are not limited to, intestinal alkaline phosphatase (IAP; e.g., calf IAP or bovine IAP, chicken IAP, goat IAP), placental alkaline phosphatase (PLAP), placental-like alkaline phosphatase, germ cell alkaline phosphatase (GCAP), tissue non-specific alkaline phosphatase (TNAP; which is primarily found in the liver, kidney, and bone), bone alkaline phosphatase, liver alkaline phosphatase, kidney alkaline phosphatase, bacterial alkaline phosphatase, fungal alkaline phosphatase, shrimp alkaline phosphatase, modified IAP, recombinant IAP, and any polypeptide comprising alkaline phosphatase activity.

In embodiments, the present disclosure contemplates the use of mammalian alkaline phosphatases including, but not limited to, intestinal alkaline phosphatase (IAP), placental alkaline phosphatase (PLAP), germ cell alkaline phosphatase (GCAP), and tissue non-specific alkaline phosphatase (TNAP).

Intestinal Alkaline Phosphatase (IAP)

In embodiments, the alkaline phosphatase is IAP IAP is produced in the proximal small intestine and is bound to the enterocytes via a glycosyl phosphatidylinositol (GPI) anchor. Some IAP is released into the intestinal lumen in conjunction with vesicles shed by the cells and as soluble protein stripped from the cells via phospholipases. The enzyme then traverses the small and large intestine such that some active enzyme can be detected in the feces. In embodiments, the IAP is human IAP (hlAP). In embodiments, the IAP is calf IAP (clAP), also known as bovine IAP (blAP). There are multiple isozymes of blAP, for example, with blAP II and IV having higher specific activity than blAP I. In embodiments, the IAP is any one of the clAP or blAP isozymes (e.g., blAP I, II, and IV). In embodiments, the IAP is blAP II. In embodiments, the IAP is blAP IV.

In embodiments, the IAP of the present disclosure has greater specific enzymatic activity than commercially- available APs, e.g., calf IAP (clAP).

IAP variants

Also included within the definition of lAPs are IAP variants. An IAP variant has at least one or more amino acid modifications, generally amino acid substitutions, as compared to the parental wild-type sequence. In embodiments, an IAP of the disclosure comprises an amino sequence having at least about 60% (e.g. about 60%, or about 61%, or about 62%, or about 63%, or about 64%, or about 65%, or about 66%, or about 67%, or about 68%, or about 69%, or about 70%, or about 71 %, or about 72%, or about 73%, or about 74%, or about 75%, or about 76%, or about 77%, or about 78%, or about 79%, or about 80%, or about 81 %, or about 82%, or about 83%, or about 84%, or about 85%, or about 86%, or about 87%, or about 88%, or about 89%, or about 90%, or about 91%, or about 92%, or about 93%, or about 94%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99%) sequence identity with any of the sequences disclosed herein. In addition, IAP variants retain most or all of their biochemical activity, measured as described herein.

In embodiments, an IAP of the disclosure comprises an amino sequence having at least about 60% (e.g. about 60%, or about 61%, or about 62%, or about 63%, or about 64%, or about 65%, or about 66%, or about 67%, or about 68%, or about 69%, or about 70%, or about 71 %, or about 72%, or about 73%, or about 74%, or about 75%, or about 76%, or about 77%, or about 78%, or about 79%, or about 80%, or about 81 %, or about 82%, or about 83%, or about 84%, or about 85%, or about 86%, or about 87%, or about 88%, or about 89%, or about 90%, or about 91%, or about 92%, or about 93%, or about 94%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99%) sequence identity with any one of SEQ ID NOs: 1-6 and 10-14.

In embodiments, an IAP of the disclosure comprises an amino sequence having at least about 60% (e.g. about 60%, or about 61%, or about 62%, or about 63%, or about 64%, or about 65%, or about 66%, or about 67%, or about 68%, or about 69%, or about 70%, or about 71 %, or about 72%, or about 73%, or about 74%, or about 75%, or about 76%, or about 77%, or about 78%, or about 79%, or about 80%, or about 81 %, or about 82%, or about 83%, or about 84%, or about 85%, or about 86%, or about 87%, or about 88%, or about 89%, or about 90%, or about 91 %, or about 92%, or about 93%, or about 94%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99%) sequence identity with any one of SEQ ID NOs: 5, 6, and 10-14.

In embodiments, an IAP of the disclosure comprises an amino sequence having at least about 60% (e.g. about 60%, or about 61%, or about 62%, or about 63%, or about 64%, or about 65%, or about 66%, or about 67%, or about 68%, or about 69%, or about 70%, or about 71 %, or about 72%, or about 73%, or about 74%, or about 75%, or about 76%, or about 77%, or about 78%, or about 79%, or about 80%, or about 81 %, or about 82%, or about 83%, or about 84%, or about 85%, or about 86%, or about 87%, or about 88%, or about 89%, or about 90%, or about 91 %, or about 92%, or about 93%, or about 94%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99%) sequence identity with SEQ ID NO: 11. In embodiments, the IAP of the disclosure comprises, consists of, and/or consists essentially of SEQ ID NO: 11 .

GPI anchored proteins

Mammalian alkaline phosphatases are GPI anchored proteins. They have signal peptides and are translated into the secretory pathway. Once in the endoplasmic reticulum (ER), the proteins are glycosylated and folded. There are two disulfide bonds as well as a single free cysteine that is apparently not accessible on the surface. In the late ER, the carboxy terminus is removed and the GPI anchor is appended. GPI anchoring is therefore a process that occurs at the carboxy terminus of the alkaline phosphatase. The inclusion of stop codons at the anchor site enables secretion of biologically active protein (presumably the homodimer). While there is no consensus sequence, the carboxy terminus includes three amino acids, termed omega, omega +1, and omega +2 which are followed by a short stretch of hydrophilic amino acids and then a stretch of hydrophobic amino acids. Without wishing to be bound by theory, it is believed that the hydrophobicity is critical for embedding the carboxy terminus in the ER membrane. There, an enzymatic reaction replaces the carboxy terminus with the GPI anchor. In embodiments, the IAP of the disclosure is a secreted protein; that is, in embodiments, the IAP is not GPI anchored, leading to secretion rather than intracellular retention. This can be accomplished in several ways. In embodiments, the IAP may lack the GPI anchor site (e.g., have the DAAH site removed, leading to secretion). In embodiments, the IAP comprises a stop codon that is inserted immediately before the GPI anchor site. In embodiments, the IAP comprises a stop codon after the aspartate in the DAAH consensus site (e.g., at amino acid 503 of hlAP and blAP IV or amino acid 506 of blAP II). Figure 1 depicts HIAP with a stop codon (SEQ ID NO: 3) and blAP II with a stop codon (SEQ ID NO: 4).

Human IAP

In embodiments, the IAP is human IAP (hlAP). In embodiments, the IAP is hlAP comprising the amino acid sequence of SEQ ID NO: 1 as depicted in Figure 1 or a variant as described herein, as long as the hlAP variant retains at least 75, 80, 85, 90, 95, 96, 97, 98, 99 or 100% of the phosphatase activity as compared to the wild type enzyme using an assay as outlined herein and/or known in the art.

Included within the definition of hlAP are amino acid modifications, with amino acid substitutions finding particular use in the present disclosure. For example, without wishing to be bound by theory, it is believed that a cysteine at the carboxy terminus of the IAP (e.g., at position 500 of SEQ ID NO: 1) may interfere with protein folding. Accordingly, in embodiments, the IAP includes a mutation of the cysteine (e.g., at position 500 of SEQ ID NO: 1). In embodiments, the cysteine is replaced with any amino acid, although glycine finds particular use in embodiments. Furthermore, the C-terminal cysteine can also be deleted.

As will be appreciated by those in the art, additional amino acid modifications can be made in hlAP as discussed herein. For example, in embodiments, a stop codon may be inserted after the aspartate in the DAAH consensus site (e.g., at amino acid 503 of hlAP). Figure 1 depicts hlAP with an inserted stop codon (SEQ ID NO: 3).

Fusion Proteins

In embodiments, the present disclosure provides for chimeric proteins. In embodiments, the present disclosure provides for chimeric fusion proteins. For example, In embodiments, the present disclosure provides an isolated or recombinant alkaline phosphatase comprising a crown domain and a catalytic domain, wherein said crown domain and said catalytic domain are obtained from different alkaline phosphatases (e.g., human and bovine alkaline phosphatases). In embodiments, the alkaline phosphatases are both human APs. In embodiments, the present disclosure provides for recombinant fusion proteins comprising human IAP and a domains of human placental alkaline phosphatases. In embodiments, the present disclosure provides for chimeric hlAP-placenta fusion proteins.

In embodiments, the IAP of the disclosure is a fusion protein. In embodiments, the IAP comprises an alkaline phosphatase fused to a protein domain that replaces the GPI anchor sequence. In embodiments, the alkaline phosphatase is fused to a protein domain that promotes protein folding and/or protein purification and/or protein dimerization and/or protein stability. In embodiments, the IAP fusion protein has an extended serum half-life. In embodiments, the alkaline phosphatase is fused to an immunoglobulin Fc domain and/or hinge region. In embodiments, the immunoglobulin Fc domain and/or hinge region is derived from the Fc domain and/or hinge region of an antibody (e.g., of IgG, IgA, IgD, and IgE, inclusive of subclasses (e.g. lgG1 , lgG2, lgG3, and lgG4, and lgA1 and lgA2)). In embodiments, the IAP of the disclosure comprises an alkaline phosphatase fused to the hinge region and/or Fc domain of IgG.

In embodiments, the IAP of the disclosure is a pro-enzyme. In embodiments, the activity of the proenzyme is suppressed by a carboxy terminus. In embodiments, protease removal of the carboxy terminus reactivates the enzymatic activity of the alkaline phosphatase. In embodiments, the pro-enzyme is more efficiently secreted than the enzyme without the carboxy terminus.

In embodiments, for generation of the pro-enzyme, the native carboxy terminus of the alkaline phosphatase is replaced with the analogous sequence from hPLAP. In embodiments, a mutation is made in the hydrophobic carboxy tail to promote protein secretion without cleavage of the carboxy terminus. In an illustrative embodiment, a single point mutation such as a substitution of leucine with e.g., arginine is generated in the hydrophobic carboxy terminus (e.g. allpllagtl is changed to e.g., allplragtl) to result in secretion of the enzyme without removal of the carboxy terminus.

Bovine lAPs

In embodiments, the IAP is bovine IAP (blAP). blAP II

In embodiments, the IAP is bovine IAP II (blAP II) or a variant as described herein, as long as the blAP variant retains at least 75, 80, 85, 90, 95, 96, 97, 98, 99 or 100% of the phosphatase activity using an assay as outlined herein. In embodiments, the blAP II comprises the signal peptide and carboxy terminus of blAP I. In embodiments, the blAP II comprises an aspartate at position 248 (similar to blAP IV). In embodiments, the blAP II comprises the amino acid sequence of SEQ ID NO: 2. Figure 1 depicts BlAP II with 248D assignment in which the signal peptide and sequence past 480 are derived from blAP I (SEQ ID NO: 2).

In embodiments, the blAP II comprise amino acid variants as described herein. For example, in embodiments, a stop codon may be inserted after the aspartate in the DAAH consensus site (e.g., at amino acid 506 of blAP II). Figure 1 depicts blAP II with an inserted stop codon (SEQ ID NO: 4).

In embodiments, the blAP II comprises, consists of, or consists essentially of the amino acid sequence of SEQ ID NO: 11.

BlAP II with stop codon and no leader sequence (SYN-020) (SEQ ID NO: 11):

LIPAEEENPAFWNRQAAQALDVAKKLQPIQTAAKNVILFLGDGMGVPTVTATRILKGQMNGKL GPETPLAMDQFPYVALSKTYNVDRQVPDSAGTATAYLCGVKGNYRTIGVSAAARYNQCNTTR GNEVTSVINRAKKAGKAVGWTTTRVQHASPAGAYAHTVNRNWYSDADLPADAQKNGCQDI AAQLVYNMDIDVILGGGRMYMFPEGTPDPEYPDDASVNGVRKDKQNLVQEWQAKHQGAQY VWNRTALLQAADDSSVTHLMGLFEPADMKYNVQQDHTKDPTLAEMTEAALQVLSRNPRGFY LFVEGGRIDHGHHDGKAYMALTEAIMFDNAIAKANELTSELDTLILVTADHSHVFSFGGYTLRG TSIFGLAPGKALDSKSYTSILYGNGPGYALGGGSRPDVNGSTSEEPSYRQQAAVPLASETHG GEDVAVFARGPQAHLVHGVQEETFVAHIMAFAGCVEPYTDCNLPAPATATSIPD.

Expression Variants

In embodiments, the IAP of the disclosure is efficiently expressed and secreted from a host cell. In embodiments, the IAP of the disclosure is efficiently transcribed in a host cell. In embodiments, the IAP exhibits enhanced RNA stability and/or transport in a host cell. In embodiments, the IAP is efficiently translated in a host cell. In embodiments, the IAP exhibits enhanced protein stability.

In embodiments, the lAPs are efficiently expressed in a host cell. In embodiments, the Kozak sequence of the DNA construct encoding the IAP is optimized. The Kozak sequence is the nucleotide sequence flanking the ATG start codon that instructs the ribosome to start translation. There is flexibility in the design of a Kozak sequence, but one canonical sequence is GCCGCCACCATGG (SEQ ID NO: 15). The purine in the -3 position and the G in the +4 position are the most important bases for translation initiation. For hlAP, blAP II, and blAP IV, the second amino acid, that is, the one after the initiator methionine, is glutamine. Codons for glutamine all have a C in the first position. Thus, their Kozak sequences all have an ATGC sequence. Accordingly, In embodiments, the ATGC sequence is changed to ATGG. This can be achieved by changing the second amino acid to a glycine, alanine, valine, aspartate, or glutamic acid, all of whose codons have a G in the first position. These amino acids may be compatible with signal peptide function. In alternative embodiments, the entire signal peptide is substituted for peptide having a canonical Kozak sequence and is derived from a highly expressed protein such as an immunoglobulin.

In embodiments, the signal peptide of the IAP may be deleted and/or substituted. For example, the signal peptide may be deleted, mutated, and/or substituted (e.g., with another signal peptide) to ensure optimal protein expression.

In embodiments, the DNA construct encoding the IAP of the disclosure comprises untranslated DNA sequences. Such sequences include an intron, which may be heterologous to the IAP protein or native to the IAP protein including the native first and/or second intron and/or a native 3’ UTR. Without wishing to be bound by theory, it is believed that inclusion of these sequences enhances protein expression by stabilizing the mRNA. Accordingly, in embodiments, the DNA construct encoding the IAP of the disclosure comprises the 5’UTR and/or the 3’UTR. Provided in Figure 1 are illustrative IAP DNA sequences with a first intron and a 3’UTR, including hlAP with native first intron (shown as bolded and underlined) (SEQ ID NO: 7); and hlAP with native 3’ UTR (shown as bolded and underlined) (SEQ ID NO: 8).

In embodiments, the IAP of the disclosure comprises a nucleotide sequence having at least about 60% (e.g. about 60%, or about 61 %, or about 62%, or about 63%, or about 64%, or about 65%, or about 66%, or about 67%, or about 68%, or about 69%, or about 70%, or about 71 %, or about 72%, or about 73%, or about 74%, or about 75%, or about 76%, or about 77%, or about 78%, or about 79%, or about 80%, or about 81 %, or about 82%, or about 83%, or about 84%, or about 85%, or about 86%, or about 87%, or about 88%, or about 89%, or about 90%, or about 91 %, or about 92%, or about 93%, or about 94%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99%) sequence identity with any of the sequences disclosed herein.

In embodiments, the IAP of the disclosure may comprise an amino acid sequence having one or more amino acid mutations relative to any of the protein sequences described herein. In embodiments, the one or more amino acid mutations may be independently selected from substitutions, insertions, deletions, and truncations.

In embodiments, the substitutions may also include non-classical amino acids (e.g. selenocysteine, pyrrolysine, N-formylmethionine [3-alanine, GABA and 6-Aminolevulinic acid, 4-aminobenzoic acid (PABA), D-isomers of the common amino acids, 2,4-diaminobutyric acid, a-amino isobutyric acid, 4-aminobutyric acid, Abu, 2-amino butyric acid, y-Abu, s-Ahx, 6-amino hexanoic acid, Aib, 2-amino isobutyric acid, 3-amino propionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosme, citrulline, homocitrulline, cysteic acid, t-butylglycine, t- butylalanine, phenylglycine, cyclohexylalanine, [3-alanine, fluoro-amino acids, designer amino acids such as - methyl amino acids, C a-methyl amino acids, N a-methyl amino acids, and amino acid analogs in general).

Mutations may be made to the IAP of the disclosure to select for agents with desired characteristics. For example, mutations may be made to generate lAPs with enhanced catalytic activity or protein stability. In embodiments, directed evolution may be utilized to generate lAPs of the disclosure. For example, error-prone PCR and DNA shuffling may be used to identify mutations in the bacterial alkaline phosphatases that confer enhanced activity.

Commensal Gastrointestinal bacteria

The present disclosure provides, in part, pharmaceutical compositions, formulations, and uses of commensal gastrointestinal bacteria. High densities of non-pathogenic bacteria, otherwise known as commensal bacteria, inhabit the gastrointestinal system, specifically the lower small intestine and colon. Distinct differences exist between the mucosal immune response to pathogens and commensals. In fact, certain commensal gastrointestinal bacteria have been shown to influence and promote immunological tolerance and gut homeostasis. Specific commensals, such as, Bacteroides acidifaciens, have been shown to prevent obesity and improve insulin sensitivity and overall metabolic syndrome in animal models.

In aspects, the present disclosure contemplates pharmaceutical compositions, formulations, and uses of commensal gastrointestinal bacteria. Such bacteria can include, but are not limited to, Bacteroides acidifaciens. Other commensal bacteria of the present disclosure can include, but are not limited to, bacterial members of the genus Bacteroides, anaerobic gram-positive cocci, such as Peptostreptococcus sp., Clostridiales sp., Eubacterium sp., Lactobacillus sp., Clostridium sp, members of phila Firmicutes, and members of phila Proteobacteria. In embodiments, the present disclosure contemplates pharmaceutical compositions, formulations, and uses of compositions comprising a variety of bacterial strains. In aspects, the present disclosure contemplates pharmaceutical compositions, formulations, and uses of the commensal bacteria Bacteroides acidifaciens. For example, in embodiments, a subject is undergoing therapy with a composition comprising Bacteroides acidifaciens. In embodiments, the composition comprising Bacteroides acidifaciens is co-administered with IAP. In embodiments, the composition comprising Bacteroides acidifaciens is co-formulated with IAP.

Fecal Microbiota Transplant (FMT)

In embodiments, the present disclosure provides for administration of fecal microbiota transplant (FMT), and in embodiments, the FMT occurs in conjunction with administration of IAP to a subject in need thereof. In embodiments, the FMT comprises at least one commensal gastrointestinal bacterial strain. For example, in embodiments, the FMT comprises Bacteroides acidifaciens. In embodiments, the composition comprising Bacteroides acidifaciens is a FMT. Without wishing to be bound by theory, it is posited that FMT can repopulate the microbiome of a subject in need thereof with diverse microorganisms by taking material containing microorganisms from a donor and transplanting those materials to said subject. In embodiments, the FMT comprises human stool or derivatives thereof. In such embodiments, the human stool or derivates are obtained from a human donor. In embodiments, the FMT comprises a synthetic material containing bacteria that are isolated from a human donor’s gastrointestinal tract or other environments and grown in pure or mixed cultures. In embodiments, the FMT is formulated as a pill or tablet.

The fecal transplant can be administered in a variety of ways, including, but not limited to, oral administration, colonoscopy, sigmoidoscopy, enema, naso-gastric intubation, naso-duodenal intubation, and naso-jejunal intubation.

Isolated Bacterial Compositions

In embodiments, the composition comprising at least one commensal gastrointestinal bacteria, including but not limited to Bacteroides acidifaciens, is an isolated bacterial composition. Specifically, the bacteria may have been isolated from human stool, the human Gl tract, or other environments and grown in pure or mixed cultures. In embodiments, the bacteria are isolated from a human donor. For example, bacteria may be isolated from a first subject (e.g., a first human subject), and formulated as described herein, and administered to a second subject (e.g., a second human subject) as described herein. Following isolation (techniques for which are apparent to one skilled in the art), the isolated bacteria can be formulated according to a desired administration route. Exemplary formulations of isolated bacterial compositions can include, but are not limited to, tablets, pills, powders, capsules, lyophilized compositions, and aqueous formulations. In embodiments, the isolated bacterial composition is formulated in a capsule comprising Bacteroides acidifaciens in an aqueous phase.

In embodiments, the present disclosure provides for a composition comprising a consortia of bacteria (e.g., a multitude of bacterial strains). In embodiments, the composition comprises a defined amount of bacterial strains, including but not limited to, the variety of commensal gastrointestinal bacteria previously mentioned. Accordingly, the composition can comprise from about 1-10, from about 1-20, from about 1-30, from about 1-40, from about 1-50, from about 1-60, from about 1-70, from about 1-80, from about 1-90, or from about 1-100 commensal gastrointestinal bacterial strains.

In embodiments, the bacteria are viable. The term “viable,” as used herein, generally refers to the ability of an organism, such as bacteria, to survive under particular conditions. In embodiments, the term “viable” relates to the ability of an organism, such as bacteria, to survive upon administration and/or delivery and to retain metabolic activity once released into the target region.

Cancer Therapy

In aspects, the present disclosure contemplates methods for improving and/or increasing and/or enhancing efficacy of a cancer therapy relative to the treatment before or in the absence of IAP. In aspects, the present disclosure contemplates methods for improving and/or increasing and/or enhancing efficacy of a cancer therapy relative to the treatment before or in the absence of IAP, for example in a subject undergoing therapy with a composition comprising at least one commensal gastrointestinal bacteria. Non-limiting examples of cancer therapy include an immune checkpoint inhibitor immunotherapy (e.g., used in tumor treatment), chemotherapy, and radiotherapy. In embodiments, methods of the present disclosure improve efficacy of the cancer therapy by increasing the therapeutic window of the cancer therapy relative to the treatment before or in the absence of IAP. Non-limiting examples of increased therapeutic window include increasing the subject’s likelihood of receiving maintenance therapy of the cancer therapy relative to the treatment before or in the absence of IAP; increasing the subject’s likelihood of receiving a complete regimen of the cancer therapy relative to the treatment before or in the absence of IAP; increasing the subject’s likelihood of receiving more than a complete regimen of the cancer therapy relative to the treatment before or in the absence of IAP; and increasing the dose or length of the cancer therapy relative to the treatment before or in the absence of IAP. In embodiments, the method reduces the likelihood of the subject requiring a transition to palliative care relative to the treatment before or in the absence of IAP. In embodiments, the method improved and/or increased and/or enhanced efficacy of the cancer therapy allows for one or more of dose reduction, treatment duration reduction, and usage of an incomplete regimen of the cancer therapy relative to the treatment before or in the absence of IAP. In embodiments, the method makes the subject suitable for treatment with a combination therapy of more than one cancer therapies. In embodiments, the efficacy of a cancer therapy is not hindered by administration of an IAP construct and/or a composition comprising at least one commensal gastrointestinal bacteria. In embodiments, administration of an IAP construct and/or a composition comprising at least one commensal gastrointestinal bacteria does not decrease efficacy of a cancer therapy. In further embodiments, administration of an IAP construct and/or a composition comprising at least one commensal gastrointestinal bacteria improves anti-tumor efficacy associated with a cancer therapy relative to the treatment before or in the absence of IAP. In embodiments of the present disclosure, the efficacy of cancer therapy is enhanced, for example, as measured by the cancer therapy’s increased antitumor effects relative to the treatment before or in the absence of IAP. In aspects, the present disclosure contemplates methods for preventing, treating, and/or reducing a cancer therapy-mediated side effect in a subject in need thereof.

In embodiments, the cancer therapy-mediated side effect comprises one or more of stomatitis, esophagopharyngitis, leukopenia and/or reduced white blood count, risk of infection, vomiting, diarrhea, nausea, poor appetite, damage to the Gl tract, including gastrointestinal ulceration and/or bleeding, thrombocytopenia, hand-foot syndrome, alopecia, mucositis, impaired hepatic function, and hemorrhage.

In embodiments, the cancer therapy-mediated side effect comprises one or more of diarrhea, dehydration, suppression of the immune system, hematological damage, lowered white blood cell counts, neutropenia, cholinergic syndrome, blood clots, infection, and abdominal pain.

In embodiments, the cancer therapy-mediated side effect comprises hematological damage. In embodiments, the hematological damage is one or more of leukopenia and/or reduced white blood count and reduction of other types of blood cells, including neutrophils, lymphocytes and platelets. In embodiments, hematological damage is assessed by a blood test.

In embodiments, the cancer therapy-mediated side effect comprises impaired hepatic function. In embodiments, impaired hepatic function is assessed by testing for levels of various liver enzymes. Exemplary indicia of impaired hepatic function include, by way of non-limiting example, elevated aminotransferase levels, elevated plasma ammonia levels, changes in one or more of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase, 5' nucleotidase, a gamma-glutamyl transpeptidase (GGT), and bilirubin. In embodiments, impaired hepatic function is assessed by a blood test and/or biopsy that involves a screen for markers of liver function (e.g. liver enzymes) in embodiments, hepatic imaging may be used to test for impaired hepatic function. In embodiments, the hepatic imaging may be one or more of ultrasounds, CT, MR, and PET, PET/CT and may involve various imaging agents (e.g. gadolinium chelates). Techniques for imaging may be found in Ros and Mortele, Hepatic Imaging. An overview Clin Liver Dis. 2002 Feb;6(1):1-16, the contents of which are hereby incorporated by reference in their entirety.

In embodiments, the cancer therapy-mediated side effect comprises damage to the Gl tract. In embodiments, the damage to the Gl tract is assessed by measurement one or more of (a) the height and width of villi of the small intestines and/or number of surface enterocytes and goblet cells in the small and large intestines; (b) status of crypts (one or more of depth, size and shape, presence of apoptotic bodies, number of and granules in Paneth’s cells, luminal migration of epithelial nuclei, loss of goblet cells, presence of atrophy and distortion); and (c) state of the lamina propria (one or more of presence of transitory cells, lymphoid accumulations, edema, blood vessel congestion and hemorrhage) in, for example, a biopsy.

In embodiments, the cancer therapy-mediated side effect comprises diarrhea and/or vomiting. In embodiments, the cancer therapy-mediated side effect is measured by monitoring a subject’s temperature. For example, temperature in excess of about 37.5°C (99.5°F) or about 38°C (100.4°F) and beyond indicates cancer therapy-mediated side effects.

In embodiments, the subject is a cancer patient. In embodiments, the cancer is selected from basal cell carcinoma, biliary tract cancer; bladder cancer; bone cancer; brain and central nervous system cancer; breast cancer; cancer of the peritoneum; cervical cancer; choriocarcinoma; colon and rectum cancer; connective tissue cancer; cancer of the digestive system; endometrial cancer; esophageal cancer; eye cancer; cancer of the head and neck; gastric cancer (including Gl cancer); glioblastoma; hepatic carcinoma; hepatoma; intra-epithelial neoplasm; kidney or renal cancer; larynx cancer; leukemia; liver cancer; lung cancer (e.g., small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous carcinoma of the lung); melanoma; myeloma; neuroblastoma; oral cavity cancer (lip, tongue, mouth, and pharynx); ovarian cancer; pancreatic cancer; prostate cancer; retinoblastoma; rhabdomyosarcoma; rectal cancer; cancer of the respiratory system; salivary gland carcinoma; sarcoma; skin cancer; squamous cell cancer; stomach cancer; testicular cancer; thyroid cancer; uterine or endometrial cancer; cancer of the urinary system; vulval cancer; lymphoma including Hodgkin's and non-Hodgkin's lymphoma, as well as B-cell lymphoma (including low grade/follicular nonHodgkin's lymphoma (NHL); small lymphocytic (SL) NHL; intermediate grade/follicular NHL; intermediate grade diffuse NHL; high grade immunoblastic NHL; high grade lymphoblastic NHL; high grade small non-cleaved cell NHL; bulky disease NHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom's Macroglobulinemia; chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); Hairy cell leukemia; chronic myeloblastic leukemia; as well as other carcinomas and sarcomas; and post-transplant lymphoproliferative disorder (PTLD), as well as abnormal vascular proliferation associated with phakomatoses, edema (such as that associated with brain tumors), and Meigs’ syndrome.

Immune Checkpoint Inhibitor (CPI) Immunotherapy

The present disclosure provides, in part, pharmaceutical compositions, formulations, and uses of immune checkpoint inhibitor immunotherapies. In embodiments, the present disclosure provides for the treatment of and/or administration to a subject who suffers from a side effect of immune checkpoint inhibitor immunotherapies. In embodiments, the subject is undergoing therapy with a composition comprising at least one commensal gastrointestinal bacteria.

Cancer immunotherapy involves the utilization of naturally derived or synthetically generated components to stimulate or enhance the immune system to fight cancer. Immune checkpoint inhibitor immunotherapies are effective in fighting cancer due to the priming and activation of the immune system in order to produce antitumor effects, often involving highly specific targeting. Along with the promise of cancer immunotherapy, there is the need to maintain the immune system’s complex counterbalance between identification and eradication of foreign antigens and the processes necessary for suppressing an uncontrolled immune response. Despite important clinical benefits, checkpoint inhibition is associated with a unique spectrum of side effects, or immune-related adverse events, including, but not limited to, dermatologic, Gl, hepatic, endocrine, and other less common inflammatory events. In various embodiments of the present disclosure, the CPI-mediated Gl side effect is diarrhea and/or colitis. Generally, treatment of these moderate or severe immune checkpoint inhibitor immunotherapy-mediated side effects can require interruption of the checkpoint inhibitor immunotherapy and use of corticosteroid immunosuppression

In aspects, the present disclosure contemplates methods for improving and/or increasing and/or enhancing efficacy of an immune checkpoint inhibitor immunotherapy (e.g., used in tumor treatment) relative to the treatment before or in the absence of IAP. For example, in embodiments, the methods of the present disclosure improve efficacy of the CPI by increasing the therapeutic window of the CPI relative to the treatment before or in the absence of IAP. In embodiments, the efficacy of a CPI therapy is not hindered by administration of an IAP construct and/or a composition comprising at least one commensal gastrointestinal bacteria. In embodiments, administration of an IAP and/or a composition comprising at least one commensal gastrointestinal bacteria construct does not decrease efficacy of a CPI therapy. In further embodiments, administration of an IAP construct and/or a composition comprising at least one commensal gastrointestinal bacteria improves anti-tumor efficacy associated with a CPI therapy relative to the treatment before or in the absence of IAP. In embodiments of the present disclosure, the efficacy of an immune checkpoint inhibitor immunotherapy is enhanced by administration of an IAP and/or a composition comprising at least one commensal gastrointestinal bacteria, for example, as measured by the immunotherapies’ increased antitumor effects relative to the treatment before or in the absence of IAP.

In aspects, the present disclosure provides pharmaceutical compositions, formulations, and uses of CPI immunotherapies. Such CPIs can include, but are not limited to, one or more agents that modulate one or more of programmed cell death protein-1 (PD-1), programmed death-ligand 1 (PD-L1), programmed death-ligand 2 (PD-L2), inducible T-cell costimulator (ICOS), inducible T-cell costimulator ligand (ICOSL), and cytotoxic T- lymphocyte-associated protein 4 (CTLA-4). In embodiments, the subject is undergoing therapy with an immune checkpoint inhibitor immunotherapy selected from an agent that modulates one or more of programmed cell death protein-1 (PD-1), programmed death-ligand 1 (PD-L1), programmed death-ligand 2 (PD-L2), inducible T- cell costimulator (ICOS), inducible T-cell costimulator ligand (ICOSL), and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4).

In other aspects, the present disclosure contemplates methods for preventing and/or treating CPI-mediated Gl side effects by administering a combination of an IAP and an CPI selected from an agent that modulates one or more of PD-1 , PD-L1, PD-L2, ICOS, ICOSL, and CTLA-4.

In embodiments, the agent that modulates one or more of PD-1, PD-L1 , PD-L2, ICOS, ICOSL, and CTLA-4 is an antibody or antibody format specific for one or more of PD-1, PD-L1, PD-L2, ICOS, ICOSL, and CTLA-4. In embodiments, the antibody or antibody format specific for one or more of PD-1, PD-L1 , PD-L2, ICOS, ICOSL, and CTLA-4 is selected from one or more of a monoclonal antibody, polyclonal antibody, antibody fragment, Fab, Fab', Fab'-SH, F(ab')2, Fv, single chain Fv, diabody, linear antibody, bispecific antibody, multispecific antibody, chimeric antibody, humanized antibody, human antibody, and fusion protein comprising the antigen-binding portion of an antibody.

For example, in embodiments, the present disclosure provides for a CPI that is an agent that modulates PD-1 , wherein the agent is an antibody or antibody format specific for PD-1. In embodiments, the antibody or antibody format specific for PD-1 is selected from nivolumab, pembrolizumab, and pidilizumab. In other embodiments, the present disclosure provides for a CPI that is an agent that modulates PD-L1 , wherein the agent is an antibody or antibody format specific for PD-L1. In embodiments, the antibody or antibody format specific for PD-L1 is selected from BMS-936559, atezolizumab, avelumab and durvalumab. In embodiments, the present disclosure provides for a CPI that is an agent that modulates PD-L2, wherein the agent is an antibody or antibody format specific for PD-L2. In other emobiments, the present disclosure provides for a CPI that is an agent that modulates ICOS, wherein the agent is an antibody or antibody format specific for ICOS. In embodiments, the antibody or antibody format specific for ICOS comprises JTX-2011. In other emobiments, the present disclosure provides for an CPI that is an agent that modulates ICOSL, wherein the agent is an antibody or antibody format specific for ICOSL. In other emobiments, the present disclosure provides for an CPI that is an agent that modulates CTLA-4, wherein the agent is an antibody or antibody format specific for CTLA-4. In embodiments, the antibody or antibody format specific for CTLA-4 is selected from tremelimumab and Ipilimumab.

Chemotherapy

The present disclosure provides, in part, pharmaceutical compositions, formulations, and uses of chemotherapy. In embodiments, the present disclosure provides for the treatment of and/or administration to a subject who suffers from a side effect of chemotherapy. In embodiments, the subject is undergoing therapy with a composition comprising at least one commensal gastrointestinal bacteria.

In embodiments, the chemotherapy-mediated side effect comprises one or more selected from alopecia, myelosuppression, renal toxicity, weight loss, pain, nausea, vomiting, diarrhea, constipation, anemia, malnutrition, hair loss, numbness, changes in tastes, loss of appetite, thinned or brittle hair, mouth sores, memory loss, hemorrhage, cardiotoxicity, hepatotoxicity, ototoxicity, and post-chemotherapy cognitive impairment.

In embodiments, the chemotherapy-mediated side effect comprises one or more selected from stomatitis, esophagopharyngitis, leukopenia and/or reduced white blood count, reduced neutrophils, reduced lymphocytes, reduced platelets, increased risk of infection, vomiting, diarrhea, nausea, poor appetite, gastrointestinal ulceration and/or bleeding, thrombocytopenia, hand-foot syndrome, alopecia, mucositis, impaired hepatic function, impaired hepatic function, and hemorrhage.

In aspects, the present disclosure contemplates methods for improving and/or increasing and/or enhancing efficacy of chemotherapy relative to the treatment before or in the absence of IAP. For example, in embodiments, the methods of the present disclosure improve efficacy of the chemotherapy by increasing the therapeutic window of the chemotherapy relative to the treatment before or in the absence of IAP. In embodiments, the efficacy of chemotherapy is not hindered by administration of an IAP construct and/or a composition comprising at least one commensal gastrointestinal bacteria. In embodiments, administration of an IAP and/or a composition comprising at least one commensal gastrointestinal bacteria construct does not decrease efficacy of chemotherapy. In further embodiments, administration of an IAP construct and/or a composition comprising at least one commensal gastrointestinal bacteria improves anti-tumor efficacy associated with chemotherapy relative to the treatment before or in the absence of IAP. In embodiments of the present disclosure, the efficacy of chemotherapy is enhanced by administration of an IAP and/or a composition comprising at least one commensal gastrointestinal bacteria, for example, as measured by the chemotherapy’s increased antitumor effects relative to the treatment before or in the absence of IAP.

In aspects, the disclosure provides a method of treating a cancer by improving the effectiveness of chemotherapy in a subject in need thereof relative to the treatment before or in the absence of IAP comprising, administering to the subject an IAP. In aspects, the disclosure provides a method of treating a cancer by improving the effectiveness of chemotherapy in a subject in need thereof relative to the treatment before or in the absence of IAP comprising, administering to the subject an IAP, wherein the subject is undergoing therapy with a composition comprising at least one commensal gastrointestinal bacteria. In embodiments, the IAP described herein acts as an adjuvant to a chemotherapeutic treatment described herein. In embodiments, the IAP described herein improves the anti-cancer effect and/or increases the therapeutic window of any of the chemotherapeutic treatments described herein relative to the treatment before or in the absence of IAP. In embodiments, administering to the subject undergoing therapy with a composition comprising at least one commensal gastrointestinal bacteria an IAP described herein does not interfere with treatment of cancer.

In embodiments, the chemotherapy comprises one or more of alkylating agents such as thiotepa and CYTOXAN cydosphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethiylenethiophosphoramide and trimethylolomelamine; acetogenins (e.g., bullatacin and bullatacinone); a camptothecin (including the synthetic analogue topotecan); bryostatin; cally statin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); cryptophycins (e.g., cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189 and CB 1-TM1); eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics such as the enediyne antibiotics (e.g., calicheamicin, especially calicheamicin gammall and calicheamicin omegall (see, e.g., Agnew, Chem. Inti. Ed. Engl., 33: 183-186 (1994)); dynemicin, including dynemicin A; bisphosphonates, such as clodronate; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores), aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN doxorubicin (including morpholinodoxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxy doxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals such as minoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; demecolcine; diaziquone; elformithine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK polysaccharide complex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin; sizofuran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2"-trichlorotriethylamine; trichothecenes (e.g., T-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxoids, e.g., TAXOL paclitaxel (Bristol-Myers Squibb Oncology, Princeton, N.J.), ABRAXANE Cremophor-free, albumin-engineered nanoparticle formulation of paclitaxel (American Pharmaceutical Partners, Schaumberg, 111.), and TAXOTERE doxetaxel (Rhone-Poulenc Rorer, Antony, France); chloranbucil; GEMZAR gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin, oxaliplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; NAVELBINE, vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; irinotecan (Camptosar, CPT-11) (including the treatment regimen of irinotecan with 5-FU and leucovorin); topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid; capecitabine; combretastatin; leucovorin (LV); oxaliplatin, including the oxaliplatin treatment regimen (FOLFOX); lapatinib (Tykerb); inhibitors of PKC-a, Raf, H-Ras, EGFR (e.g., erlotinib (Tarceva)) and VEGF-A that reduce cell proliferation; and pharmaceutically acceptable salts, acids or derivatives of any of the above.

Radiotherapy

The present disclosure provides, in part, pharmaceutical compositions, formulations, and uses of radiotherapy. In embodiments, the present disclosure provides for the treatment of and/or administration to a subject who suffers from a side effect of radiotherapy. In embodiments, the subject is undergoing therapy with a composition comprising at least one commensal gastrointestinal bacteria. In embodiments, the present methods reduce or eliminate a side effect of radiotherapy, including acute side effects, long-term side effects), or cumulative side effects. In embodiments, the present methods reduce or eliminate a local or systemic side effect of radiotherapy. In embodiments, the radiation-mediated side effect comprises one or more selected from fatigue, nausea and vomiting, damage to the epithelial surfaces (e.g., without limitation, moist desquamation), mouth, throat and stomach sores, intestinal discomfort (e.g., without limitation, soreness, diarrhea, and nausea), swelling, infertility, fibrosis, epilation, dryness (e.g. without limitation, dry mouth (xerostomia) and dry eyes (xerophthalmia), and dryness of the armpit and vaginal mucosa), lymphedema, heart disease, cognitive decline, radiation enteropathy (e.g. without limitation, atrophy, fibrosis and vascular changes, which may produce malabsorption, diarrhea, steatorrhea and bleeding with bile acid diarrhea and vitamin B12 malabsorption commonly found due to ileal involvement), pelvic radiation disease including radiation proctitis, producing bleeding, diarrhea and urgency, and radiation cystitis.

In embodiments, the radiotherapy is pelvic radiotherapy. In such embodiments, the IAP described herein, optionally IAP, which may be administered orally, reduces or eliminates Gl-related side effects as described herein. In such embodiments, the IAP described herein, optionally IAP, which may be administered orally, reduces or eliminates lower body-related side effects as described herein.

In embodiments, the radiotherapy is pelvic radiotherapy the IAP described herein, optionally IAP, which may be administered orally, reduces or eliminates one or more of radiation enteropathy, atrophy, fibrosis and vascular changes, malabsorption, diarrhea, steatorrhea, bleeding with bile acid diarrhea, malabsorption (e.g. vitamin malabsorption, e.g. vitamin B12 malabsorption). In embodiments, the radiotherapy is pelvic radiotherapy the IAP described herein, optionally IAP, which may be administered orally, reduces or eliminates radiation proctitis, producing bleeding, diarrhea and urgency, and radiation cystitis.

In embodiments, the radiotherapy is delivered as one or more of external-beam radiation therapy, brachytherapy, and systemic radiation therapy.

In embodiments, the radiotherapy is an external-beam radiation therapy, selected from 3-dimensional conformal radiation therapy (3D-CRT), intensity-modulated radiation therapy (IMRT, e.g. RAPIDARC), image-guided radiation therapy (IGRT), electromagnetic-guided radiation therapy (e.g. CALYPSO) tomotherapy, stereotactic radiosurgery (SRS), stereotactic body radiation therapy (SBRT, e.g. CYBERKNIFE, GAMMAKNIFE, X-KNIFE, CLINAC), Intraoperative radiation therapy (IORT), and proton therapy.

In embodiments, the radiotherapy is a brachytherapy, selected from interstitial brachytherapy, intracavitary brachytherapy, and episcleral brachytherapy.

In embodiments, the radiotherapy is a systemic radiation therapy, selected from a radioactive iodine and a radioactive biologic. For example, the radiotherapy may be radioactive iodine (¹³¹l), ibritumomab tiuxetan (ZEVALIN), tositumomab and iodine I 131 tositumomab (BEXXAR), samarium-153-lexidronam (QUADRAMET), and strontium-89 chloride (METASTRON). In embodiments, the radiotherapy comprises a dose of about 20 Gy, or about 30 Gy, or about 40 Gy, or about 50 Gy, or about 60 Gy, or about 70 Gy, or about 80 Gy, or about 90 Gy, or about 100 Gy, optionally fractionated.

In aspects, the present disclosure contemplates methods for improving and/or increasing and/or enhancing efficacy of radiotherapy relative to the treatment before or in the absence of IAP. For example, in embodiments, the methods of the present disclosure improve efficacy of the radiotherapy by increasing the therapeutic window of the radiotherapy relative to the treatment before or in the absence of IAP. In embodiments, the efficacy of radiotherapy is not hindered by administration of an IAP construct and/or a composition comprising at least one commensal gastrointestinal bacteria. In embodiments, administration of an IAP and/or a composition comprising at least one commensal gastrointestinal bacteria construct does not decrease efficacy of radiotherapy. In further embodiments, administration of an IAP construct and/or a composition comprising at least one commensal gastrointestinal bacteria improves anti-tumor efficacy associated with radiotherapy relative to the treatment before or in the absence of IAP. In embodiments of the present disclosure, the efficacy of radiotherapy is enhanced by administration of an IAP and/or a composition comprising at least one commensal gastrointestinal bacteria, for example, as measured by the radiotherapy’s increased antitumor effects relative to the treatment before or in the absence of IAP.

The present disclosure provides, in part, methods for preventing, treating, and/or reducing cancer therapy- mediated Gl side effects by further administering to a subject a corticosteroid and/or an agent targeting tumor necrosis factor alpha (TNF-a) in order to provide temporary immunosuppression. In embodiments, the subject is undergoing therapy with one or more corticosteroids. In embodiments, the subject is undergoing therapy with one or more agents targeting TNF-a. In embodiments, the one or more agents targeting TNF-a is an antibody or a fusion protein. In embodiments, the one or more agents targeting TNF-a is selected from infliximab (Remicade), infliximab-dyyb (Inflectra), infliximab-abda (Renflexis) and Flixabi. In embodiments, administering a corticosteroid and/or an agent targeting TNF-a to a subject undergoing cancer therapy, for example CPI, interrupts the cancer therapy.

In embodiments, the present disclosure provides for methods of preventing, treating, and/or reducing a cancer therapy-mediated Gl side effect by administering IAP and/or a composition comprising commensal gastrointestinal bacteria, including but not limited to Bacteroides acidifaciens, to a subject that is further undergoing treatment with a corticosteroid and/or an agent targeting TNF-a. In embodiments, the administration of IAP and/or a composition comprising commensal gastrointestinal bacteria, including but not limited to Bacteroides acidifaciens, reduces the dose or frequency of corticosteroid administration needed for immunotherapy-mediated Gl side effect treatment. In embodiments, the administration of IAP and/or a composition comprising commensal gastrointestinal bacteria, including but not limited to Bacteroides acidifaciens, obviates corticosteroid administration for immunotherapy-mediated Gl side effect treatment. In embodiments, the administration of IAP and/or a composition comprising commensal gastrointestinal bacteria, including but not limited to Bacteroides acidifaciens, reduces the dose or frequency of administration of one or more agents targeting TNF-a needed for cancer therapy-mediated Gl side effect treatment. In embodiments, the administration of IAP and/or a composition comprising commensal gastrointestinal bacteria, including but not limited to Bacteroides acidifaciens, obviates administration of one or more agents targeting TNF-a for cancer therapy-mediated Gl side effect treatment. In embodiments, the cancer-therapy mediated side effect comprises one or more immunotherapy-mediated Gl side effects.

Methods of Making IAP of the Disclosure

The lAPs of the disclosure are made using standard molecular biology techniques. For example, nucleic acid compositions encoding the lAPs of the disclosure are also provided, as well as expression vectors containing the nucleic acids and host cells transformed with the nucleic acid and/or expression vector compositions. As will be appreciated by those in the art, the protein sequences depicted herein can be encoded by any number of possible nucleic acid sequences, due to the degeneracy of the genetic code.

As is known in the art, the nucleic acids encoding the components of the disclosure can be incorporated into expression vectors as is known in the art, and depending on the host cells, used to produce the IAP compositions of the disclosure. Generally, the nucleic acids are operably linked to any number of regulatory elements (promoters, origin of replication, selectable markers, ribosomal binding sites, inducers, etc.). The expression vectors can be extra-chromosomal or integrating vectors.

The nucleic acids and/or expression vectors of the disclosure are then transformed into any number of different types of host cells as is well known in the art, including mammalian, bacterial, yeast, insect and/or fungal cells, with mammalian cells (e.g. CHO cells), finding use in many embodiments.

The lAPs of the disclosure are made by culturing host cells comprising the expression vector(s) as is well known in the art. Once produced, traditional purification steps are done.

Formulations

The present disclosure provides the described IAP and/or composition comprising at least one commensal gastrointestinal bacteria (e.g. Bacteroides acidifaciens) (and/or additional agents) in various formulations. Any IAP and/or or composition comprising Bacteroides acidifaciens (and/or additional agents) described herein can take the form of tablets, pills, pellets, capsules, capsules containing liquids, capsules containing multiparticulates, powders, solutions, emulsion, drops, suppositories, emulsions, aerosols, sprays, suspensions, delayed-release formulations, sustained-release formulations, controlled-release formulations, or any other form suitable for use.

The formulations comprising the IAP and/or composition comprising at least one commensal gastrointestinal bacteria (e.g. Bacteroides acidifaciens) (and/or additional agents) may conveniently be presented in unit dosage forms. For example, the dosage forms may be prepared by methods which include the step of bringing the therapeutic agents into association with a carrier, which constitutes one or more accessory ingredients. For example, the formulations are prepared by uniformly and intimately bringing the therapeutic agent into association with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, shaping the product into dosage forms of the desired formulation (e.g., wet or dry granulation, powder blends, etc., followed by press tableting).

In embodiments, the IAP and/or composition comprising at least one commensal gastrointestinal bacteria (e.g. Bacteroides acidifaciens) (and/or additional agents) described herein are formulated as compositions adapted for a mode of administration described herein.

In embodiments, the IAP and the composition comprising at least one commensal gastrointestinal bacteria (e.g. Bacteroides acidifaciens) are co-formulated.

In embodiments, the formulation comprising IAP is resistant to compression and therefore suitable for tableting. In embodiments, the formulation comprising at least one commensal gastrointestinal bacteria (e.g. Bacteroides acidifaciens) is resistant to compression and therefore suitable for tableting. The IAP can be provided in a powder form that is then tableted, e.g., by physical compression of dried materials.

In embodiments, the IAP and/or composition comprising at least one commensal gastrointestinal bacteria (e.g. Bacteroides acidifaciens) of the disclosure is stable and/or active in the Gl tract, e.g. in one or more of the mouth, esophagus, stomach, duodenum, small intestine, duodenum, jejunum, ileum, large intestine, colon transversum, colon descendens, colon ascendens, colon sigmoidenum, cecum, and rectum. In embodiments, the IAP and/or composition comprising Bacteroides acidifaciens is stable in the large intestine, optionally selected from one or more of colon transversum, colon descendens, colon ascendens, colon sigmoidenum and cecum. In embodiments, the IAP and/or composition comprising Bacteroides acidifaciens is stable in the small intestine, optionally selected from one or more of duodenum, jejunum, and ileum. In embodiments, the IAP and/or composition comprising at least one commensal gastrointestinal bacteria (e.g. Bacteroides acidifaciens) is resistant to proteases in the Gl tract, including for example, the small intestine. In embodiments, the IAP and/or composition comprising at least one commensal gastrointestinal bacteria (e.g. Bacteroides acidifaciens) is substantially active at a pH of about 5.0 or above. For example, the IAP and/or composition comprising at least one commensal gastrointestinal bacteria (e.g. Bacteroides acidifaciens) may be substantially active at a pH of about 6.0 to about 12, e.g. about 6.0, or about 6.1, or about 6.2, or about 6.3, or about 6.4, or about 6.5, or about 6.6, or about 6.7, or about 6.8, or about 6.9, or about 7.0, or about 7.1 , or about 7.2, or about 7.3, or about 7.4, or about 7.5, or about 8.0, or about 8.5, or about 9.0, or about 9.5, or about 10.0, or about 10.5, or about 11.0, or about 11.5, or about 12.0 (including, for example, via formulation, as described herein). In embodiments, stable refers to an enzyme that has a long enough half-life and maintains sufficient activity for therapeutic effectiveness.

In embodiments, the IAP and/or composition comprising at least one commensal gastrointestinal bacteria (e.g. Bacteroides acidifaciens) of the disclosure is stable in chyme. In order to assess stability of IAP and/or the composition comprising at least one commensal gastrointestinal bacteria (e.g. Bacteroides acidifaciens) in chyme, samples of lAPs and/or compositions comprising at least one commensal gastrointestinal bacteria (e.g. Bacteroides acidifaciens) are incubated in human chyme at 37°C. Stability is then evaluated by assessing aliquots withdrawn from the incubated samples at 0, 0.5, 1, 2, 3, 4, 5, and 6 hours for AP activity using a para- nitrophenyl phosphate (pNPP) AP substrate. Different chyme specimens can be used for evaluation of stability, including mixed chyme samples. Chyme samples are characterized for pH, liquid content, and protease activity.

In embodiments, the IAP described herein includes derivatives that are modified, i.e., by the covalent attachment of any type of molecule to the alkaline phosphatase such that covalent attachment does not prevent the activity of the enzyme. For example, but not by way of limitation, derivatives include alkaline phosphatases that have been modified by, inter alia, glycosylation, lipidation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein, etc. Any of numerous chemical modifications can be carried out, including, but not limited to specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc. Additionally, the derivative can contain one or more non-classical amino acids. In embodiments, the IAP is glycosylated to ensure proper protein folding.

Pharmaceutically acceptable salts

The IAP and/or composition comprising at least one commensal gastrointestinal bacteria (e.g. Bacteroides acidifaciens) described herein can possess a sufficiently basic functional group, which can react with an inorganic or organic acid, or a carboxyl group, which can react with an inorganic or organic base, to form a pharmaceutically acceptable salt. A pharmaceutically acceptable acid addition salt is formed from a pharmaceutically acceptable acid, as is well known in the art. Such salts include the pharmaceutically acceptable salts listed in, for example, Journal of Pharmaceutical Science, 66, 2-19 (1977) and The Handbook of Pharmaceutical Salts; Properties, Selection, and Use. P. H. Stahl and C. G. Wermuth (eds.), Verlag, Zurich (Switzerland) 2002, which are hereby incorporated by reference in their entirety.

The term “pharmaceutically acceptable salt” also refers to a salt of the alkaline phosphatases having an acidic functional group, such as a carboxylic acid functional group, and a base. Suitable bases include, but are not limited to, hydroxides of alkali metals such as sodium, potassium, and lithium; hydroxides of alkaline earth metal such as calcium and magnesium; hydroxides of other metals, such as aluminum and zinc; ammonia, and organic amines, such as unsubstituted or hydroxy-substituted mono-, di-, or tri-alkylamines, dicydohexylamine; tributyl amine; pyridine; N-methyl, N-ethylamine; diethylamine; triethylamine; mono-, bis-, or tris-(2-OH-lower alkylamines), such as mono-; bis-, or tris-(2-hydroxyethyl)amine, 2-hydroxy-tert-butylamine, or tris- (hydroxymethyl)methylamine, N,N-di-lower alkyl-N-(hydroxyl-lower alkyl)-amines, such as N,N-dimethyl-N-(2- hydroxyethyl)amine or tri-(2-hydroxyethyl)amine; N-methyl-D-glucamine; and amino acids such as arginine, lysine, and the like.

In embodiments, the compositions described herein are in the form of pharmaceutically acceptable salts. In embodiments, the formulation comprises 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, about 40%, about 41 %, about 42%, about 43%, about 43%, about 44%, about 45%, about 46%, about 47%, about 48%, about 49%, or about 50% by weight pharmaceutically acceptable salts. Pharmaceutical excipients

Further, any IAP and/or composition comprising at least one commensal gastrointestinal bacteria (e.g. Bacteroides acidifaciens) described herein can be administered to a subject as a component of a composition that comprises a pharmaceutically acceptable carrier or vehicle. Such compositions can optionally comprise a suitable amount of a pharmaceutically acceptable excipient so as to provide the form for proper administration.

Pharmaceutical excipients can be liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. The pharmaceutical excipients can be, for example, saline, gum acacia, gelatin, starch paste, talc, keratin, colloidal silica, urea and the like. In addition, auxiliary, stabilizing, thickening, lubricating, and coloring agents can be used. In embodiments, the pharmaceutically acceptable excipients are sterile when administered to a subject. Water is a useful excipient when any agent described herein is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid excipients, specifically for injectable solutions. Suitable pharmaceutical excipients also include starch, glucose, cellulose, hypromellose, lactose, sucrose, trehalose, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, povidone, crosspovidone, water, ethanol and the like. Any agent described herein, if desired, can also comprise minor amounts of wetting or emulsifying agents, or pH buffering agents. Other examples of suitable pharmaceutical excipients are described in Remington’s Pharmaceutical Sciences 1447-1676 (Alfonso R. Gennaro eds., 19th ed. 1995), incorporated herein by reference.

A suitable pharmaceutical excipient for the purposes of tableting can be Ludipress (Lactose, povidone, crospovidone; CAS-No.: 5989-81-1 + 9003-39-8).

Where necessary, the IAP and/or composition comprising Bacteroides acidifaciens and/or pharmaceutical compositions (and/or additional agents) can include a solubilizing agent. Also, the agents can be delivered with a suitable vehicle or delivery device. Combination therapies outlined herein can be co-delivered in a single delivery vehicle or delivery device.

In embodiments, the IAP and/or composition comprising at least one commensal gastrointestinal bacteria (e.g. Bacteroides acidifaciens) (and/or additional agents) described herein are formulated as compositions adapted for oral administration. Compositions for oral delivery can be in the form of tablets, lozenges, aqueous or oily suspensions, granules, powders, sprinkles, emulsions, capsules, syrups, or elixirs, for example. Orally administered compositions can comprise one or more agents, for example, sweetening agents such as fructose, aspartame or saccharin; flavoring agents such as peppermint, oil of Wintergreen, or cherry; coloring agents; and preserving agents, to provide a pharmaceutically palatable preparation. Moreover, where in tablet or pill form, the compositions can be coated to delay disintegration to provide a sustained action over an extended period of time. Selectively permeable membranes surrounding an osmotically active agent driving any IAP (and/or additional agents) described herein are also suitable for orally administered compositions. In these latter platforms, fluid from the environment surrounding the capsule is imbibed by the driving compound, which swells to displace the agent or agent composition through an aperture. These delivery platforms can provide an essentially zero order delivery profile as opposed to the spiked profiles of immediate release formulations. A time-delay material such as glycerol monostearate or glycerol stearate can also be useful. Oral compositions can include excipients such as mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, ethacrylic acid and derivative polymers thereof, and magnesium carbonate. In embodiments, the excipients are of pharmaceutical grade. Suspensions, in addition to the active compounds, may contain suspending agents such as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar, tragacanth, etc., and mixtures thereof.

In embodiments, the IAP and/or composition comprising at least one commensal gastrointestinal bacteria (e.g. Bacteroides acidifaciens) (and/or additional agent) are formulated as solid dosage forms such as tablets, dispersible powders, granules, and capsules. In embodiments, the IAP and/or composition comprising Bacteroides acidifaciens (and/or additional agent) are formulated as a capsule. In embodiments, the IAP and/or composition comprising at least one commensal gastrointestinal bacteria (e.g. Bacteroides acidifaciens) (and/or additional agent) are formulated as a tablet. In yet embodiments, the IAP and/or composition comprising at least one commensal gastrointestinal bacteria (e.g. Bacteroides acidifaciens) (and/or additional agent) are formulated as a soft-gel capsule. In embodiments, the IAP and/or composition comprising Bacteroides acidifaciens (and/or additional agent) are formulated as a gelatin capsule.

In embodiments, the formulations of the IAP and/or composition comprising at least one commensal gastrointestinal bacteria (e.g. Bacteroides acidifaciens) may additionally comprise a pharmaceutically acceptable carrier or excipient. As one skilled in the art will recognize, the formulations can be in any suitable form appropriate for the desired use and route of administration.

In some dosage forms, the agents described herein are mixed with at least one inert, pharmaceutically acceptable excipient or carrier such as sodium citrate, dicalcium phosphate, etc., and/or a) fillers or extenders such as starches, lactose, sucrose, glucose, mannitol, silicic acid, microcrystalline cellulose, and Bakers Special Sugar, etc., b) binders such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose, acacia, polyvinyl alcohol, polyvinylpyrrolidone, methylcellulose, hydroxypropyl cellulose (HPC), and hydroxymethyl cellulose etc., c) humectants such as glycerol, etc., d) disintegrating agents such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, sodium carbonate, cross-linked polymers such as crospovidone (cross-linked polyvinylpyrrolidone), croscarmellose sodium (cross-linked sodium carboxymethylcellulose), sodium starch glycolate, etc., e) solution retarding agents such as paraffin, etc., f) absorption accelerators such as quaternary ammonium compounds, etc., g) wetting agents such as, for example, cetyl alcohol and glycerol monostearate, etc., h) absorbents such as kaolin and bentonite clay, etc., and i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, glyceryl behenate, etc., and mixtures of such excipients. One of skill in the art will recognize that particular excipients may have two or more functions in the oral dosage form. In the case of an oral dosage form, for example, a capsule or a tablet, the dosage form may also comprise buffering agents.

Surface active agents

The formulation can additionally include a surface active agent. Surface active agents suitable for use in the present disclosure include, but are not limited to, any pharmaceutically acceptable, non-toxic surfactant. Classes of surfactants suitable for use in the compositions of the disclosure include, but are not limited to polyethoxylated fatty acids, PEG-fatty acid diesters, PEG-fatty acid mono- and di-ester mixtures, polyethylene glycol glycerol fatty acid esters, alcohol-oil transesterification products, polyglycerized fatty acids, propylene glycol fatty acid esters, mixtures of propylene glycol esters-glycerol esters, mono- and diglycerides, sterol and sterol derivatives, polyethylene glycol sorbitan fatty acid esters, polyethylene glycol alkyl ethers, sugar esters, polyethylene glycol alkyl phenols, polyoxyethylene-olyoxypropylene block copolymers, sorbitan fatty acid esters, lower alcohol fatty acid esters, ionic surfactants, and mixtures thereof. In embodiments, compositions of the disclosure may comprise one or more surfactants including, but not limited to, sodium lauryl sulfate, polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80, and triethyl citrate.

The formulation can also contain pharmaceutically acceptable plasticizers to obtain the desired mechanical properties such as flexibility and hardness. Such plasticizers include, but are not limited to, triacetin, citric acid esters, triethyl citrate, phthalic acid esters, dibutyl sebacate, cetyl alcohol, polyethylene glycols, polysorbates and other plasticizers.

The formulation can also include one or more application solvents. Some of the more common solvents that can be used include, for example, a delayed-release coating composition include isopropyl alcohol, acetone, methylene chloride and the like.

The formulation can also include one or more alkaline materials. Alkaline material suitable for use in compositions of the disclosure include, but are not limited to, sodium, potassium, calcium, magnesium and aluminum salts of acids such as phosphoric acid, carbonic acid, citric acid and other aluminum/magnesium compounds. In addition, the alkaline material may be selected from antacid materials such as aluminum hydroxides, calcium hydroxides, magnesium hydroxides and magnesium oxide.

In embodiments, the formulation can additionally include magnesium and/or zinc. Without wishing to be bound by theory, the inclusion of magnesium and/or zinc in the formulation promotes protein folding (e.g., dimer formation) and bioactivity of the IAP. In embodiments, the formulation can include magnesium at a concentration of from about 1 piM to greater than 5 mM (e.g., from about 1 piM to more than 5 mM), inclusive of all ranges and values therebetween. In embodiments, the magnesium is present in the formulation at 1.0 mM. In embodiments, the formulation can include zinc at a concentration of about 1 piM to greater than 1 mM (e.g., from about 1 piM to more than 1 mM), inclusive of all ranges and values therebetween. In embodiments, the zinc is present in the formulation at 0.1 mM. In embodiments, the formulation of the present disclosure is substantially free of metal chelators. In embodiments, the pH of the formulation ensures that the IAP is properly folded (e.g., dimer formation) and is bioactive. In embodiments, the formulation is maintained at a pH such that the amino acids which coordinate the binding of magnesium and/or zinc within the IAP are not protonated. Protonation of such coordinating amino acids may lead to loss of metal ions and bioactivity and dimer disassociation. In embodiments, the pH of the formulation is greater than about 6, about 6.5, about 7, about 7.5, about 8, about 8.5, about 9, about 9.5, about 10, about 10.5, about 11 , about 11 .5, or about 12.

Besides inert diluents, the oral compositions can also include adjuvants such as sweetening, flavoring, and perfuming agents.

Delivery

Various methods may be used to formulate and/or deliver the agents described herein to a location of interest. For example, the IAP and/or composition comprising at least one commensal gastrointestinal bacteria (e.g. Bacteroides acidifaciens) (and/or additional agents) described herein may be formulated for delivery to the Gl tract. The Gl tract includes organs of the digestive system such as mouth, esophagus, stomach, duodenum, small intestine, large intestine and rectum and includes all subsections thereof (e.g. the small intestine may include the duodenum, jejunum and ileum; the large intestine may include the colon transversum, colon descendens, colon ascendens, colon sigmoidenum and cecum). For example, the IAP and/or composition comprising Bacteroides acidifaciens (and/or additional agents) described herein may be formulated for delivery to one or more of the stomach, small intestine, large intestine and rectum and includes all subsections thereof (e.g. duodenum, jejunum and ileum, colon transversum, colon descendens, colon ascendens, colon sigmoidenum and cecum). In embodiments, the compositions described herein may be formulated to deliver to the gut. In embodiments, the compositions described herein may be formulated to deliver to the upper or lower Gl tract. In embodiments, the IAP and/or composition comprising Bacteroides acidifaciens (and/or additional agents) may be administered to a subject, by, for example, directly or indirectly contacting the mucosal tissues of the Gl tract.

In embodiments, the administration of the IAP and/or composition comprising at least one commensal gastrointestinal bacteria (e.g. Bacteroides acidifaciens) (and/or additional agents) is into the Gl tract via, for example, oral delivery, nasogastral tube, intestinal intubation (e.g. an enteral tube or feeding tube such as, for example, a jejunal tube or gastro-jejunal tube, etc.), direct infusion (e.g., duodenal infusion), endoscopy, colonoscopy, sigmoidoscopy or enema.

For example, in embodiments, the present disclosure provides modified release formulations comprising at least one IAP (and/or additional agents), wherein the formulation releases a substantial amount of the IAP (and/or additional agents) into one or more regions of the Gl tract. For example, the formulation may release at least about 60% of the IAP after the stomach and into one or more regions of the Gl tract.

In embodiments, the modified-release formulation of the present disclosure releases at least 60% of the IAP (or additional agents) after the stomach into one or more regions of the intestine. For example, the modified-release formulation releases at least 60%, at least 61 %, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least

74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least

82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least

90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least

98%, at least 99%, or 100% of the IAP (or additional agents) in the intestines.

In embodiments, the modified-release formulation of the present disclosure releases at least 60% of the IAP (or additional agents) in the small intestine. For example, the modified-release formulation releases at least 60%, at least 61 %, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the IAP (or additional agents) in the small intestine (e.g., one or more of duodenum, jejunum, ileum, and ileocecal junction).

In embodiments, the modified-release formulation of the present disclosure releases at least 60% of the IAP (or additional agents) in the large intestine. For example, the modified-release formulation releases at least 60%, at least 61 %, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the IAP (or additional agents) in the large intestine (e.g., one or more of cecum, ascending, transverse, descending or sigmoid portions of the colon, and rectum).

In embodiments, the modified-release formulation does not substantially release the IAP (or additional agents) in the stomach.

In embodiments, the modified-release formulation releases the IAP and/or composition comprising Bacteroides acidifaciens (or additional agents) above a specific pH. For example, in embodiments, the modified-release formulation is substantially stable in an acidic environment and substantially unstable (e.g., dissolves rapidly or is physically unstable) in a near neutral to alkaline environment. In embodiments, stability is indicative of not substantially releasing while instability is indicative of substantially releasing. For example, in embodiments, the modified-release formulation is substantially stable at a pH of about 7.0 or less, or about 6.5 or less, or about 6.0 or less, or about 5.5 or less, or about 5.0 or less, or about 4.5 or less, or about 4.0 or less, or about 3.5 or less, or about 3.0 or less, or about 2.5 or less, or about 2.0 or less, or about 1.5 or less, or about 1.0 or less. In embodiments, the present formulations are stable in lower pH areas and therefore do not substantially release in, for example, the stomach. In embodiments, the modified-release formulation is substantially stable at a pH of about 1 to about 5 or lower and substantially unstable at pH values that are greater. In these embodiments, the modified-release formulation does not substantially release in the stomach. In these embodiments, the modified- release formulation substantially releases in the small intestine (e.g. one or more of the duodenum, jejunum, and ileum) and/or large intestine (e.g. one or more of the cecum, ascending colon, transverse colon, descending colon, and sigmoid colon). In embodiments, modified-release formulation is substantially stable at a pH of about 4 to about 7 or lower and consequentially is substantially unstable at pH values that are greater and therefore is not substantially released in the stomach and/or proximal small intestine (e.g. one or more of the duodenum, jejunum). In these embodiments, the modified-release formulation substantially releases in the distal small intestine or large intestine (e.g. one or more of the cecum, ascending colon, transverse colon, descending colon, and sigmoid colon). In embodiments, the pH values recited herein may be adjusted as known in the art to account for the state of the subject, e.g. whether in a fasting or postprandial state.

In embodiments, the modified-release formulation is substantially stable in gastric fluid and substantially unstable in intestinal fluid and, accordingly, is substantially released in the small intestine (e.g. one or more of the duodenum, jejunum, and ileum) and/or large intestine (e.g. one or more of the cecum, ascending colon, transverse colon, descending colon, and sigmoid colon).

In embodiments, the modified-release formulation is stable in gastric fluid or stable in acidic environments. These modified-release formulations release about 30% or less by weight of the alkaline phosphatase and/or additional agent in the modified-release formulation in gastric fluid with a pH of about 4 to about 5 or less, or simulated gastric fluid with a pH of about 4 to about 5 or less, in about 15, or about 30, or about 45, or about 60, or about 90 minutes. Modified-release formulations of the of the disclosure may release from about 0% to about 30%, from about 0% to about 25%, from about 0% to about 20%, from about 0% to about 15%, from about 0% to about 10%, about 5% to about 30%, from about 5% to about 25%, from about 5% to about 20%, from about 5% to about 15%, from about 5% to about 10% by weight of the alkaline phosphatase and/or additional agent in the modified-release formulation in gastric fluid with a pH of 4-5, or less or simulated gastric fluid with a pH of 4-5 or less, in about 15, or about 30, or about 45, or about 60, or about 90 minutes. Modified-release formulations of the disclosure may release about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, or about 10% by weight of the total alkaline phosphatase and/or additional agent in the modified- release formulation in gastric fluid with a pH of 5 or less, or simulated gastric fluid with a pH of 5 or less, in about 15, or about 30, or about 45, or about 60, or about 90 minutes.

In embodiments, the modified-release formulation is unstable in intestinal fluid. These modified-release formulations release about 70% or more by weight of the alkaline phosphatase and/or additional agent in the modified-release formulation in intestinal fluid or simulated intestinal fluid in about 15, or about 30, or about 45, or about 60, or about 90 minutes. In embodiments, the modified-release formulation is unstable in near neutral to alkaline environments. These modified-release formulations release about 70% or more by weight of the alkaline phosphatase and/or additional agent in the modified-release formulation in intestinal fluid with a pH of about 4-5 or greater, or simulated intestinal fluid with a pH of about 4-5 or greater, in about 15, or about 30, or about 45, or about 60, or about 90 minutes. A modified-release formulation that is unstable in near neutral or alkaline environments may release 70% or more by weight of alkaline phosphatase and/or additional agent in the modified-release formulation in a fluid having a pH greater than about 5 (e.g., a fluid having a pH of from about 5 to about 14, from about 6 to about 14, from about 7 to about 14, from about 8 to about 14, from about 9 to about 14, from about 10 to about 14, or from about 11 to about 14) in from about 5 minutes to about 90 minutes, or from about 10 minutes to about 90 minutes, or from about 15 minutes to about 90 minutes, or from about 20 minutes to about 90 minutes, or from about 25 minutes to about 90 minutes, or from about 30 minutes to about 90 minutes, or from about 5 minutes to about 60 minutes, or from about 10 minutes to about 60 minutes, or from about 15 minutes to about 60 minutes, or from about 20 minutes to about 60 minutes, or from about 25 minutes to about 90 minutes, or from about 30 minutes to about 60 minutes.

Examples of simulated gastric fluid and simulated intestinal fluid include, but are not limited to, those disclosed in the 2005 Pharmacopeia 23NF/28USP in Test Solutions at page 2858 and/or other simulated gastric fluids and simulated intestinal fluids known to those of skill in the art, for example, simulated gastric fluid and/or intestinal fluid prepared without enzymes.

In embodiments, the modified-release formulation of the disclosure is substantially stable in chyme. For example, there is, in embodiments, a loss of less than about 50% or about 40%, or about 30%, or about 20%, or about 10% of IAP activity in about 10, or 9, or 8, or 7, or 6, or 5, or 4, or 3, or 2, or 1 hour from administration.

In embodiments, the modified-release formulations of the present disclosure are designed for immediate release (e.g. upon ingestion). In embodiments, the modified-release formulations may have sustained-release profiles, i.e. slow release of the active ingredient(s) in the body (e.g., Gl tract) over an extended period of time. In embodiments, the modified-release formulations may have a delayed-release profile, i.e. not immediately release the active ingredient(s) upon ingestion; rather, postponement of the release of the active ingredient(s) until the composition is lower in the Gl tract; for example, for release in the small intestine (e.g., one or more of duodenum, jejunum, ileum) or the large intestine (e.g., one or more of cecum, ascending, transverse, descending or sigmoid portions of the colon, and rectum). For example, a composition can be enteric-coated to delay release of the active ingredient(s) until it reaches the small intestine or large intestine.

Enteric coating

In embodiments, formulations of the present disclosure (e.g. IAP as a powder or tablet) are coated to provide protection of the active agent in the Gl tract, including the stomach. For example, in embodiments, the present formulations can be encapsulated in an enterically-coated capsule. Additionally, in embodiments, the formulations (e.g. IAP as a powder or tablet) itself is coated with one or more coatings, e.g. one or more modified-release coatings as described herein (e.g. after a step of granulating the powder). Further, in embodiments, the present powder formulations (e.g. IAP as a powder) can be compressed into a tablet that is enterically coated. In embodiments, modified-release formulations of the present disclosure may utilize one or more modified- release coatings such as delayed-release coatings to provide for effective, delayed yet substantial delivery of the alkaline phosphatase to the Gl tract together with, optionally, additional agents.

In embodiments, modified-release formulations of the present disclosure may utilize one or more modified- release coatings such as delayed-release coatings to provide for effective, delayed yet substantial delivery of the IAP to the intestines together with, optionally, other additional agents.

In embodiments, the delayed-release coating includes an enteric agent that is substantially stable in acidic environments and substantially unstable in near neutral to alkaline environments. In embodiments, the delayed- release coating contains an enteric agent that is substantially stable in gastric fluid. The enteric agent can be selected from, for example, solutions or dispersions of methacrylic acid copolymers, cellulose acetate phthalate, hydroxypropylmethyl cellulose phthalate, polyvinyl acetate phthalate, carboxymethylethylcellulose, and EUDRAGIT®-type polymer (poly(methacrylic acid, methylmethacrylate), hydroxypropyl methylcellulose acetate succinate, cellulose acetate trimellitate, shellac or other suitable enteric coating polymers. The polymers are described in international pharmacopeias such as Ph.Eur., USP/NF, DMF, and JPE. The EUDRAGIT®-type polymers include, for example, EUDRAGIT® FS 30D, L 30 D-55, L 100-55, L 100, L 12,5, L 12,5 P, RL 30 D, RL PO, RL 100, RL 12,5, RS 30 D, RS PO, RS 100, RS 12,5, NE 30 D, NE 40 D, NM 30 D, S 100, S 12,5, and S 12,5 P. Similar polymers include Kollicoat® MAE 30 DP and Kollicoat® MAE 100 P. In embodiments, one or more of EUDRAGIT® FS 30D, L 30 D-55, L 100-55, L 100, L 12,5, L 12,5 P RL 30 D, RL PO, RL 100, RL 12,5, RS 30 D, RS PO, RS 100, RS 12,5, NE 30 D, NE 40 D, NM 30 D, S 100, S 12,5 S 12,5 P, Kollicoat® MAE 30 DP and Kollicoat® MAE 100 P is used. In embodiments, the enteric agent may be a combination of the foregoing solutions or dispersions. In embodiments, the delayed-release coating includes the enteric agent EUDRAGIT® L 100.

By way of non-limiting example, there are various EUDRAGIT formulations that dissolve at rising pH, with formulations that dissolve at pH >5.5 (EUDRAGIT L30 D-550), pH >6.0 (EUDRAGIT L12, 5), and pH >7.0 (EUDRAGIT FS 30D). Since the ileum has the highest pH in the small intestine, ranging from 7.3 to 7.8, the use of EUDRAGIT FS 30D as an enteric agent, may delay dissolution until the ileum thereby localizing the release of the IAP to the ileum. However, the jejunum has a pH that can range from 6.6 to 7.4, therefore, various EUDRAGIT formulations can be used to target release to this segment of the intestine. The different types of EUDRAGIT can be combined with each other, or multiple different types of EUDRAGIT coatings can be combined to fine tune the dissolution profile to achieve targeted delivery to achieve optimal function. For example, EUDRAGIT L100, EUDRAGIT S100, and triethyl citrate may be mixed together at a ratio of, for example, about 72.7/18.2/9.1 , to form a coating that substantially releases at a pH of greater than about 6.2. In another example, EUDRAGIT L100, EUDRAGIT S100, and triethyl citrate may be mixed together at a ratio of, for example, about 30/60.9/9, to form a coating that substantially releases at a pH of greater than about 6.7. In a further example, DuoCoat™ (Kuecept, Ltd.) may be used that uses two coatings of enteric polymers (like EUDRAGIT), an outer layer, and an inner layer of partially neutralized enteric polymer and a buffer agent. The DuoCoat™ technology allows more rapid release of the therapeutic agent initiated at the targeted pH compared to a single coating of the enteric polymer (Liu et al., 2010, European J. Pharmaceutics and Biopharmaceuticals 47:311 , the entire contents of all of which are incorporated herein by reference). Release was demonstrated to be targeted to the ileum and/or ileoceacal junction in 10 healthy volunteers (Varum et al., 2013, European J. Pharmaceutics and Biopharmaceuticals 84:573, the entire contents of all of which are incorporated herein by reference).

In embodiments, one or more coating system additives are used with the enteric agent. For example, one or more PlasACRYLTM additives may be used as an anti-tacking agent coating additive. Illustrative PlasACRYLTM additives include, but are not limited to PlasACRYLTM HTP20 and PlasACRYLTM T20.

In embodiments, the delayed-release coating may degrade as a function of time when in aqueous solution without regard to the pH and/or presence of enzymes in the solution. Such a coating may comprise a water insoluble polymer. Its solubility in aqueous solution is therefore independent of the pH. The term “pH independent’ as used herein means that the water permeability of the polymer and its ability to release pharmaceutical ingredients is not a function of pH and/or is only very slightly dependent on pH. Such coatings may be used to prepare, for example, sustained release formulations. Suitable water insoluble polymers include pharmaceutically acceptable non-toxic polymers that are substantially insoluble in aqueous media, e.g., water, independent of the pH of the solution. Suitable polymers include, but are not limited to, cellulose ethers, cellulose esters, or cellulose ether-esters, i.e., a cellulose derivative in which some of the hydroxy groups on the cellulose skeleton are substituted with alkyl groups and some are modified with alkanoyl groups. Examples include ethyl cellulose, acetyl cellulose, nitrocellulose, and the like. Other examples of insoluble polymers include, but are not limited to, lacquer, and acrylic and/or methacrylic ester polymers, polymers or copolymers of acrylate or methacrylate having a low quaternary ammonium content, or mixture thereof and the like. Other examples of insoluble polymers include EUDRAGIT RS®, EUDRAGIT RL®, and EUDRAGIT NE®. Insoluble polymers useful in the present disclosure include polyvinyl esters, polyvinyl acetals, polyacrylic acid esters, butadiene styrene copolymers, and the like. In embodiments, colonic delivery is achieved by use of a slowly-eroding wax plug (e.g., various PEGS, including for example, PEG6000) or pectin. In embodiments, the present disclosure contemplates the use of a delayed-release coating that degrade as a function of time which comprises a swell layer comprising croscarmellos sodium and hydroxyproplycellulose. In such embodiments, the formulation may further include an osmotic rupture coating that comprises ethylcellulose such as ethylcellulose dispersions.

Alternatively, the stability of the modified-release formulation can be enzyme-dependent. Delayed-release coatings that are enzyme dependent will be substantially stable in fluid that does not contain a particular enzyme and substantially unstable in fluid containing the enzyme. The delayed-release coating will essentially disintegrate or dissolve in fluid containing the appropriate enzyme. Enzyme-dependent control can be brought about, for example, by using materials which release the active ingredient only on exposure to enzymes in the intestine, such as galactomannans. Also, the stability of the modified-release formulation can be dependent on enzyme stability in the presence of a microbial enzyme present in the gut flora. For example, In embodiments, the delayed-release coating may be degraded by a microbial enzyme present in the gut flora. In embodiments, the delayed-release coating may be degraded by bacteria present in the small intestine. In embodiments, the delayed-release coating may be degraded by bacteria present in the large intestine.

In embodiments, the modified release formulation is designed for release in the colon. Various colon-specific delivery approaches may be utilized. For example, the modified release formulation may be formulated using a colon-specific drug delivery system (CODES) as described for example, in Li et al., AAPS PharmSciTech (2002), 3(4): 1-9, the entire contents of which are incorporated herein by reference. Drug release in such a system is triggered by colonic microflora coupled with pH-sensitive polymer coatings. For example, the formulation may be designed as a core tablet with three layers of polymer. The first coating is an acid-soluble polymer (e.g., EUDRAGIT E), the outer coating is enteric, along with a hydroxypropyl methylcellulose barrier layer interposed in between. In embodiments, colon delivery may be achieved by formulating the alkaline phosphatase (and/or additional agent) with specific polymers that degrade in the colon such as, for example, pectin. The pectin may be further gelled or crosslinked with a cation such as a zinc cation. In embodiments, the formulation is in the form of ionically crosslinked pectin beads which are further coated with a polymer (e.g., EUDRAGIT polymer). Additional colon specific formulations include, but are not limited to, pressure-controlled drug delivery systems (prepared with, for example, ethylcellulose) and osmotic controlled drug delivery systems (i.e., ORDS-CT).

Formulations for colon specific delivery of the IAP and/or composition comprising Bacteroides acidifaciens (and/or additional agents), as described herein, may be evaluated using, for example, in vitro dissolution tests. For example, parallel dissolution studies in different buffers may be undertaken to characterize the behavior of the formulations at different pH levels. Alternatively, in vitro enzymatic tests may be carried out. For example, the formulations may be incubated in fermenters containing suitable medium for bacteria, and the amount of drug released at different time intervals is determined. Drug release studies can also be done in buffer medium containing enzymes or rat or guinea pig or rabbit cecal contents and the amount of drug released in a particular time is determined. In embodiments, in vivo evaluations may be carried out using animal models such as dogs, guinea pigs, rats, and pigs. Further, clinical evaluation of colon specific drug delivery formulations may be evaluated by calculating drug delivery index (DDI) which considers the relative ratio of ROE (relative colonic tissue exposure to the drug) to RSC (relative amount of drug in blood i.e. that is relative systemic exposure to the drug). Higher drug DDI indicates better colon drug delivery. Absorption of drugs from the colon may be monitored by colonoscopy and intubation.

In embodiments, the present formulations provide for substantial uniform dissolution of the IAP (and/or additional agent) in the area of release in the Gl tract. In embodiments, the present formulation minimizes patchy or heterogeneous release of the IAP.

In embodiments, the present disclosure provides for modified-release formulations that release multiple doses of the IAP, at different locations along the intestines, at different times, and/or at different pH. In embodiments, the modified-release formulation comprises a first dose of the IAP and a second dose of the IAP, wherein the first dose and the second dose are released at different locations along the intestines, at different times, and/or at different pH. For example, the first dose is released at the duodenum, and the second dose is released at the ileum. In another example, the first dose is released at the jejunum, and the second dose is released at the ileum. In embodiments, the first dose is released at a location along the small intestine (e.g., the duodenum), while the second dose is released along the large intestine (e.g., the ascending colon). In embodiments, the modified-release formulation may release at least one dose, at least two doses, at least three doses, at least four doses, at least five doses, at least six doses, at least seven doses, or at least eight doses of the IAP at different locations along the intestines, at different times, and/or at different pH.

In embodiments, the formulations of the present disclosure take the form of those as described in one or more of US Patent Nos. 8,535,713 and 8,9117,77 and US Patent Publication Nos. 20120141585, 20120141531 , 2006/001896, 2007/0292523, 2008/0020018, 2008/0113031 , 2010/0203120, 2010/0255087, 2010/0297221, 2011/0052645, 2013/0243873, 2013/0330411, 2014/0017313, and 2014/0234418, the contents of which are hereby incorporated by reference in their entirety.

In embodiments, the formulations of the present disclosure take the form of those described in one or more of US Patent Nos. 4,196,564; 4,196,565; 4,247,006; 4,250,997; 4,268,265; 5,317,849; 6,572,892; 7,712,634; 8,074,835; 8,398,912; 8,440,224; 8,557,294; 8,646,591 ; 8,739,812; 8,810,259; 8,852,631 ; and 8,911,788 and US Patent Publication Nos. 2014/0302132; 2014/0227357; 20140088202; 20130287842; 2013/0295188; 2013/0307962; and 20130184290, the contents of which are hereby incorporated by reference in their entirety.

In embodiments, the process of formulating the IAP is sufficiently gentle such that the tertiary structure of the IAP (e.g., dimeric structure) is substantially intact. In embodiments, the process of formulating the IAP includes a step of refolding the IAP. In such embodiments, the step of refolding the IAP may include the addition of magnesium and/or cyclodextrin.

In embodiments, the modified-release formulation is a modified-release powder formulation.

In embodiments, the modified-release formulation including lAPs described herein, and variants thereof, and/or additional agents is administered orally.

Suitable dosage forms for oral use include, for example, solid dosage forms such as tablets, capsules, powders, and granules. In embodiments, the modified-release formulation is in the form of powders. In embodiments, the powdered formulations of the present disclosure can be added to food (e.g. juices, strained and/or pureed foods (e.g. fruits, vegetables), sauces, infant formulas, milk, etc.). In embodiments, the modified-release formulation is packaged in the form of a sachet. In embodiments, the modified-release formulation is in the form of tablets. In embodiments, the modified-release formulation is in the form of tablets comprising powders. In embodiments, the modified-release formulation is in the form of capsules. In embodiments, the modified-release formulation is in the form of capsules comprising powders. In embodiments, the modified-release formulation of the disclosure is in the form of powders. In embodiments, the powders are formed by spray drying and/or by spray-dried dispersion (SDD) technology. In embodiments, the powders comprising lAPs are formed by dissolving lAPs and polymers in a solvent and then spray-drying the solution. The resulting powder comprises the lAPs dispersed within a solid polymeric matrix.

Various types of polymers may be used for the modified-release formulation of the disclosure. In embodiments, the polymer is an enteric polymer that is substantially stable in acidic environments and substantially unstable in near neutral to alkaline environments. In embodiments, the enteric polymer is substantially stable in gastric fluid.

Illustrative polymers include, but are not limited to, copovidone, polyvinyl caprolactam-polyvinyl acetatepolyethyleneglycol copolymer, poly(vinylpyrrolidinone) (PVP), hydroxypropylmethylcellulose or hypromellose (HPMC), hypromellose phthalate (HPMCP), hydroxypropylmethylcellulose or hypromellose acetate succinate (HPMCAS), methacrylate/methacrylic acid copolymer, and mixtures thereof. In embodiments, the polymer is HPMCAS. In embodiments, the polymer is HPMCAS LF, LG, MF, MG, HF, or HG. In embodiments, the polymer is HPMCAS-LF.

Buffers

Various types of solvents/buffers may be used for preparation of the powders of the disclosure. In embodiments, the solvents/buffers are organic solvents/buffers. Illustrative solvents/buffers that may be used to dissolve the IAP and polymer prior to spray-drying include, but are not limited to, ethanol, methanol, acetone, I PA, tetrahydrafuran, dichloromethane, and mixtures thereof. In embodiments, the solvent used is water such as distilled DI water. In embodiments, the buffer used is monosodium phosphate monohydrate.

In embodiments, enzyme co-factors including zinc and magnesium are used. In embodiments, the enzyme cofactor zinc is used. In embodiments, the zinc is provided as zinc sulfate heptahydrate. In embodiments, the enzyme co-factor magnesium is used. In embodiments, the magnesium is provided as magnesium sulfate heptahydrate.

In embodiments, the formulation includes a protein stabilizer such as trehalose, sucrose, lactose, mannitol, Tween 80, or polyvinyl alcohol. In embodiments, the stabilizer is sucrose. In embodiments, the stabilizer is lactose.

In embodiments, surfactants may be included for the preparation of the powders of the disclosure. The surfactants may be used as solubilizers or emulsifying agents. Illustrative surfactants include, but are not limited to, vitamin E polyethylene glycol succinate, sorbitan monostearate - 60/80, polysorbate 20, polysorbate 80, and polyoxyl 40 hydrogenated castor oil.

In embodiments, the powders comprising lAPs becomes a gel. In embodiments, the powders comprising an IAP becomes a gel in the intestines. In embodiments, the IAP is released from the gel into one or more regions of the intestines. In embodiments, at pH values greater than about 5 (e.g. about 5, or 6, or 7, or 8, or 9) the gel transforms back into the solution phase and releases the AP enzyme. In embodiments, the gel is used to control the release of the IAP in the intestines. In embodiments, the IAP is released from the gel into one or more of the group consisting of the small intestine, duodenum, jejunum, ileum, large intestine, colon transversum, colon descendens, colon ascendens, colon sigmoidenum, cecum, and rectum.

In embodiments, the formulation of the present disclosure is in the form of powders comprising the IAP dispersed within a solid polymeric matrix. In embodiments, the powders are formed by dissolving IAP and polymers in a solvent to form a solution that is subsequently spray-dried. In embodiments, the solution for spray-drying comprises about 0.1-1% by weight of IAP. For example, the IAP may be present about 0.1 %, about 0.15%, about 0.2%, about 0.25%, about 0.3%, about 0.35%, about 0.4%, about 0.45%, about 0.5%, about 0.55%, about 0.6%, about 0.65%, about 0.7%, about 0.75%, about 0.8%, about 0.85%, about 0.9%, about 0.95%, or about 1.0% by weight. In embodiments, the solution comprises about 1-10% by weight a polymer (e.g., HPMCAS-LF). For example, the polymer may be present at about 1 %, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, or about 10% by weight. In embodiments, the solution comprises about 0.05-0.5% by weight buffer (e.g., monosodium phosphate monohydrate). For example, the buffer may be present at about 0.05%, about 0.06%, about 0.07%, about 0.08%, about 0.09%, about 0.10%, about 0.11 %, about 0.12%, about

0.13%, about 0.14%, about 0.15%, about 0.16%, about 0.17%, about 0.18%, about 0.19%, about 0.20%, about

0.25%, about 0.30%, about 0.35%, about 0.40%, about 0.45%, or about 0.50% by weight. In embodiments, the solution comprises about 0.001-0.01 % by weight zinc (e.g., zinc sulfate heptahhydrate). For example, the zinc may be present at about 0.001%, about 0.002%, about 0.003%, about 0.004%, about 0.005%, about 0.006%, about 0.007%, about 0.008%, about 0.009%, or about 0.01 % by weight. In embodiments, the solution comprises about 0.01-0.1% by weight magnesium (e.g., magnesium sulfate heptahhydrate). For example, the magnesium may be present at about 0.01%, about 0.02%, about 0.03%, about 0.04%, about 0.05%, about 0.06%, about 0.07%, about 0.08%, about 0.09%, or about 0.1% by weight. In embodiments, the solution comprises about 0.1- 1% by weight a protein stabilizer (e.g., trehalose). For example, the protein stabilizer may be present at about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, or about 1 % by weight. In embodiments, the solution comprises about 90-99.9% by weight solvent (e.g., water). For example, the solvent may be present at about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% by weight.

In embodiments, the modified-release formulation of the disclosure is in the form of tablets or capsules. In embodiments, the modified-release formulation is in the form of tablets or capsules comprising the powders of the disclosure. A variety of approaches for generating tablets or capsules may be utilized to include powders of the disclosure. In embodiments, tablets of the disclosure are generated by granulation such as dry granulation. In such embodiments, the powders are pre-compressed and the resulting tablet or slug is milled to yield granules. Alternatively, the powders are pre-compressed with pressure rolls to yield granules. In embodiments, the powders are encapsulated into capsules. In embodiments, the capsule is a gelatin capsule, such as a hard gelatin capsule. In embodiments, the capsule is a hydroxypropyl methylcellulose (HPMC) capsule. In embodiments, the tablets or capsules comprise a delayed-release coating that includes an enteric agent that is substantially stable in acidic environments and substantially unstable in near neutral to alkaline environments. In embodiments, the delayed-release coating contains an enteric agent that is substantially stable in gastric fluid. The enteric agent can be selected from, for example, solutions or dispersions of methacrylic acid copolymers, cellulose acetate phthalate, hydroxypropylmethyl cellulose phthalate, polyvinyl acetate phthalate, carboxymethylethylcellulose, and EUDRAGIT®-type polymer (poly(methacrylic acid, methylmethacrylate), hydroxypropyl methylcellulose acetate succinate, cellulose acetate trimellitate, shellac or other suitable enteric coating polymers. The polymers are described in international pharmacopeias such as Ph.Eur., USP/NF, DMF, and JPE. The EUDRAGIT®-type polymers include, for example, EUDRAGIT® FS 30D, L 30 D-55, L 100-55, L 100, L 12,5, L 12,5 P, RL 30 D, RL PO, RL 100, RL 12,5, RS 30 D, RS PO, RS 100, RS 12,5, NE 30 D, NE 40 D, NM 30 D, S 100, S 12,5, and S 12,5 P. Similar polymers include Kollicoat® MAE 30 DP and Kollicoat® MAE 100 P. In embodiments, one or more of EUDRAGIT® FS 30D, L 30 D-55, L 100-55, L 100, L 12,5, L 12,5 P RL 30 D, RL PO, RL 100, RL 12,5, RS 30 D, RS PO, RS 100, RS 12,5, NE 30 D, NE 40 D, NM 30 D, S 100, S 12,5 S 12,5 P, Kollicoat® MAE 30 DP and Kollicoat® MAE 100 P is used. In embodiments, the enteric agent may be a combination of the foregoing solutions or dispersions. In embodiments, the delayed-release coating includes the enteric agent EUDRAGIT® L 100. In embodiments, the tablet or capsule is coated with the enteric agent at a coating weight of about 1-20% such as about 1 %, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11 %, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, or about 20% coating weight.

Administration and Dosages

It will be appreciated that the actual dose of the IAP and/or composition comprising at least one commensal gastrointestinal bacteria (e.g. Bacteroides acidifaciens) to be administered according to the present disclosure will vary according to the particular compound, the particular dosage form, and the mode of administration. Many factors that may modify the action of the IAP (e.g., body weight, gender, diet, time of administration, route of administration, rate of excretion, condition of the subject, drug combinations, genetic disposition and reaction sensitivities) can be taken into account by those skilled in the art. Administration can be carried out continuously or in one or more discrete doses within the maximum tolerated dose. Optimal administration rates for a given set of conditions can be ascertained by those skilled in the art using conventional dosage administration tests.

Individual doses of the IAP and/or composition comprising at least one commensal gastrointestinal bacteria (e.g. Bacteroides acidifaciens) can be administered in unit dosage forms (e.g., tablets or capsules) containing, for example, from about 0.01 mg to about 1,000 mg, about 0.01 mg to about 900 mg, about 0.01 mg to about 800 mg, about 0.01 mg to about 700 mg, about 0.01 mg to about 600 mg, about 0.01 mg to about 500 mg, about 0.01 mg to about 400 mg, about 0.01 mg to about 300 mg, about 0.01 mg to about 200 mg, from about 0.1 mg to about 100 mg, from about 0.1 mg to about 90 mg, from about 0.1 mg to about 80 mg, from about 0.1 mg to about 70 mg, from about 0.1 mg to about 60 mg, from about 0.1 mg to about 50 mg, from about 0.1 mg to about 40 mg, from about 0.1 mg to about 30 mg, from about 0.1 mg to about 20 mg, from about 0.1 mg to about 10 mg, from about 0.1 mg to about 5 mg, from about 0.1 mg to about 3 mg, or from about 0.1 mg to about 1 mg active ingredient per unit dosage for. For example, a unit dosage form can be about 0.01 mg, about 0.02 mg, about 0.03 mg, about 0.04 mg, about 0.05 mg, about 0.06 mg, about 0.07 mg, about 0.08 mg, about 0.09 mg, about 0.1 mg, about 0.2 mg, about 0.3 mg, about 0.4 mg, about 0.5 mg, about 0.6 mg, about 0.7 mg, about 0.8 mg, about 0.9 mg, about 1 mg, about 2 mg, about 3 mg, about 4 mg, about 5 mg, about 6 mg, about 7 mg, about 8 mg, about 9 mg, about 10 mg, about 11 mg, about 12 mg, about 13 mg, about 14 mg, about 15 mg, about 16 mg, about 17 mg, about 18 mg, about 19 mg, about 20 mg, about 21 mg, about 22 mg, about 23 mg, about 24 mg, about 25 mg, about 26 mg, about 27 mg, about 28 mg, about 29 mg, about 30 mg, about 31 mg, about 32 mg, about 33 mg, about 34 mg, about 35 mg, about 36 mg, about 37 mg, about 38 mg, about 39 mg, about 40 mg, about 41 mg, about 42 mg, about 43 mg, about 44 mg, about 45 mg, about 46 mg, about 47 mg, about 48 mg, about 49 mg, about 50 mg, about 51 mg, about 52 mg, about 53 mg, about 54 mg, about 55 mg, about 56 mg, about 57 mg, about 58 mg, about 59 mg, about 60 mg, about 61 mg, about 62 mg, about 63 mg, about 64 mg, about 65 mg, about 66 mg, about 67 mg, about 68 mg, about 69 mg, about 70 mg, about 71 mg, about 72 mg, about 73 mg, about 74 mg, about 75 mg, about 76 mg, about 77 mg, about 78 mg, about 79 mg, about 80 mg, about 81 mg, about 82 mg, about 83 mg, about 84 mg, about 85 mg, about 86 mg, about 87 mg, about 88 mg, about 89 mg, about 90 mg, about 91 mg, about 92 mg, about 93 mg, about 94 mg, about 95 mg, about 96 mg, about 97 mg, about 98 mg, about 99 mg, about 100 mg, about 200 mg, about 300 mg, about 400 mg, about 500 mg, about 600 mg, about 700 mg, about 800 mg, about 900 mg, or about 1,000 mg of the IAP, inclusive of all values and ranges therebetween.

In embodiments, the IAP and/or composition comprising at least one commensal gastrointestinal bacteria (e.g. Bacteroides acidifaciens) is administered at an amount of from about 0.01 mg to about 1 ,000 mg daily, about 0.01 mg to about 900 mg daily, about 0.01 mg to about 800 mg daily, about 0.01 mg to about 700 mg daily, about 0.01 mg to about 600 mg daily, about 0.01 mg to about 500 mg daily, about 0.01 mg to about 400 mg daily, about 0.01 mg to about 300 mg daily, about 0.01 mg to about 200 mg daily, about 0.01 mg to about 100 mg daily, an amount of from about 0.1 mg to about 100 mg daily, from about 0.1 mg to about 95 mg daily, from about 0.1 mg to about 90 mg daily, from about 0.1 mg to about 85 mg daily, from about 0.1 mg to about 80 mg daily, from about 0.1 mg to about 75 mg daily, from about 0.1 mg to about 70 mg daily, from about 0.1 mg to about 65 mg daily, from about 0.1 mg to about 60 mg daily, from about 0.1 mg to about 55 mg daily, from about 0.1 mg to about 50 mg daily, from about 0.1 mg to about 45 mg daily, from about 0.1 mg to about 40 mg daily, from about 0.1 mg to about 35 mg daily, from about 0.1 mg to about 30 mg daily, from about 0.1 mg to about 25 mg daily, from about 0.1 mg to about 20 mg daily, from about 0.1 mg to about 15 mg daily, from about 0.1 mg to about 10 mg daily, from about 0.1 mg to about 5 mg daily, from about 0.1 mg to about 3 mg daily, from about 0.1 mg to about 1 mg daily, or from about 5 mg to about 80 mg daily. In embodiments, the IAP is administered at a daily dose of about 0.01 mg, about 0.02 mg, about 0.03 mg, about 0.04 mg, about 0.05 mg, about 0.06 mg, about 0.07 mg, about 0.08 mg, about 0.09 mg, about 0.1 mg, about 0.2 mg, about 0.3 mg, about 0.4 mg, about 0.5 mg, about 0.6 mg, about 0.7 mg, about 0.8 mg, about 0.9 mg, about 1 mg, about 2 mg, about 3 mg, about 4 mg, about 5 mg, about 6 mg, about 7 mg, about 8 mg, about 9 mg, about 10 mg, about 11 mg, about 12 mg, about 13 mg, about 14 mg, about 15 mg, about 16 mg, about 17 mg, about 18 mg, about 19 mg, about 20 mg, about 21 mg, about 22 mg, about 23 mg, about 24 mg, about 25 mg, about 26 mg, about 27 mg, about 28 mg, about 29 mg, about 30 mg, about 31 mg, about 32 mg, about 33 mg, about 34 mg, about 35 mg, about 36 mg, about 37 mg, about 38 mg, about 39 mg, about 40 mg, about 41 mg, about 42 mg, about 43 mg, about 44 mg, about 45 mg, about 46 mg, about 47 mg, about 48 mg, about 49 mg, about 50 mg, about 51 mg, about 52 mg, about 53 mg, about 54 mg, about 55 mg, about 56 mg, about 57 mg, about 58 mg, about 59 mg, about 60 mg, about 61 mg, about 62 mg, about 63 mg, about 64 mg, about 65 mg, about 66 mg, about 67 mg, about 68 mg, about 69 mg, about 70 mg, about 71 mg, about 72 mg, about 73 mg, about 74 mg, about 75 mg, about 76 mg, about 77 mg, about 78 mg, about 79 mg, about 80 mg, about 81 mg, about 82 mg, about 83 mg, about 84 mg, about 85 mg, about 86 mg, about 87 mg, about 88 mg, about 89 mg, about 90 mg, about 91 mg, about 92 mg, about 93 mg, about 94 mg, about 95 mg, about 96 mg, about 97 mg, about 98 mg, about 99 mg, about 100 mg, about 200 mg, about 300 mg, about 400 mg, about 500 mg, about 600 mg, about 700 mg, about 800 mg, about 900 mg, or about 1 ,000 mg, inclusive of all values and ranges therebetween.

In embodiments, a suitable dosage of the IAP and/or composition comprising at least one commensal gastrointestinal bacteria (e.g. Bacteroides acidifaciens) is in a range of about 0.01 mg/kg to about 100 mg/kg of body weight of the subject, about 0.01 mg/kg to about 90 mg/kg of body weight of the subject, about 0.01 mg/kg to about 80 mg/kg of body weight of the subject, about 0.01 mg/kg to about 70 mg/kg of body weight of the subject, about 0.01 mg/kg to about 60 mg/kg of body weight of the subject, about 0.01 mg/kg to about 50 mg/kg of body weight of the subject, about 0.01 mg/kg to about 40 mg/kg of body weight of the subject, about 0.01 mg/kg to about 30 mg/kg of body weight of the subject, about 0.01 mg/kg to about 20 mg/kg of body weight of the subject, about 0.01 mg/kg to about 10 mg/kg of body weight of the subject, for example, about 0.01 mg/kg, about 0.02 mg/kg, about 0.03 mg/kg, about 0.04 mg/kg, about 0.05 mg/kg, about 0.06 mg/kg, about 0.07 mg/kg, about 0.08 mg/kg, about 0.09 mg/kg, about 0.1 mg/kg, about 0.2 mg/kg, about 0.3 mg/kg, about 0.4 mg/kg, about 0.5 mg/kg, about 0.6 mg/kg, about 0.7 mg/kg, about 0.8 mg/kg, about 0.9 mg/kg, about 1 mg/kg, about 1.1 mg/kg, about 1.2 mg/kg, about 1.3 mg/kg, about 1.4 mg/kg, about 1.5 mg/kg, about 1.6 mg/kg, about 1.7 mg/kg, about 1 .8 mg/kg, 1.9 mg/kg, about 2 mg/kg, about 3 mg/kg, about 4 mg/kg, about 5 mg/kg, about 6 mg/kg, about 7 mg/kg, about 8 mg/kg, about 9 mg/kg, about 10 mg/kg body weight, about 20 mg/kg body weight, about 30 mg/kg body weight, about 40 mg/kg body weight, about 50 mg/kg body weight, about 60 mg/kg body weight, about 70 mg/kg body weight, about 80 mg/kg body weight, about 90 mg/kg body weight, or about 100 mg/kg body weight, inclusive of all values and ranges therebetween. In embodiments, a suitable dosage of the IAP is in a range of about 0.01 mg/kg to about 10 mg/kg of body weight, in a range of about 0.01 mg/kg to about 9 mg/kg of body weight, in a range of about 0.01 mg/kg to about 8 mg/kg of body weight, in a range of about 0.01 mg/kg to about 7 mg/kg of body weight, in a range of 0.01 mg/kg to about 6 mg/kg of body weight, in a range of about 0.05 mg/kg to about 5 mg/kg of body weight, in a range of about 0.05 mg/kg to about 4 mg/kg of body weight, in a range of about 0.05 mg/kg to about 3 mg/kg of body weight, in a range of about 0.05 mg/kg to about 2 mg/kg of body weight, in a range of about 0.05 mg/kg to about 1.5 mg/kg of body weight, or in a range of about 0.05 mg/kg to about 1 mg/kg of body weight.

In embodiments, the IAP and/or composition comprising at least one commensal gastrointestinal bacteria (e.g. Bacteroides acidifaciens) may be administered, for example, more than once daily (e.g., about two, about three, about four, about five, about six, about seven, about eight, about nine, or about ten times per day), about once per day, about every other day, about every third day, about once a week, about once every two weeks, about once every month, about once every two months, about once every three months, about once every six months, or about once every year.

Methods of Treatment

In embodiments, IAP and/or a composition comprising commensal gastrointestinal bacteria, including but not limited to Bacteroides acidifaciens, of the present disclosure are co-administered. The co-administration can occur simultaneously or sequentially.

In one aspect, the present disclosure provides methods of preventing, treating, and/or reducing a cancer therapy-mediated side effect in a subject in need thereof, comprising administering to the subject an IAP, wherein the subject is undergoing therapy with a composition comprising at least one commensal gastrointestinal bacteria. In embodiments, the cancer therapy comprises immune checkpoint inhibitor (CPI) immunotherapy, chemotherapy, and/or radiotherapy.

In aspects, the present disclosure provides methods for improving and/or increasing and/or enhancing efficacy of a cancer therapy relative to the treatment before or in the absence of IAP, comprising administering to the subject an IAP, wherein the subject is undergoing therapy with a composition comprising at least one commensal gastrointestinal bacteria. In embodiments, the cancer therapy comprises immune checkpoint inhibitor (CPI) immunotherapy, chemotherapy, and/or radiotherapy.

In aspects, the present disclosure provides methods for preventing and/or treating an immune checkpoint inhibitor (CPI)-mediated gastrointestinal (Gl) side effect in a subject in need thereof, comprising administering to the subject an IAP and an CPI selected from an agent that modulates one or more of PD-1 , PD-L1, PD-L2, ICOS, ICOSL, and CTLA-4.

In embodiments, the methods and uses of the present disclosure include use of IAP and/or a composition comprising commensal gastrointestinal bacteria, including but not limited to Bacteroides acidifaciens, as an adjuvant to any of these initial and/or adjunctive therapies (including co-administration or sequential administration). In embodiments, the methods and uses of the present disclosure include administration of the IAP and/or composition comprising commensal gastrointestinal bacteria, including but not limited to Bacteroides acidifaciens, described herein to a subject undergoing initial and/or adjunctive therapies.

In embodiments, the terms “patient” and “subject” are used interchangeably. In embodiments, the subject and/or animal is a mammal, e.g., a human, mouse, rat, guinea pig, dog, cat, horse, cow, pig, rabbit, sheep, or non- human primate, such as a monkey, chimpanzee, or baboon. In embodiments, the subject and/or animal is a nonmammal, such, for example, a zebrafish.

In embodiments, methods of the disclosure are useful in treatment a human subject. In embodiments, the human is a pediatric human. In embodiments, the human is an adult human. In embodiments, the human is a geriatric human. In embodiments, the human is a female. In embodiments, the human is a male.

In embodiments, the human subject has an age in a range of from about 1 to about 18 months old, from about 18 to about 36 months old, from about 1 to about 5 years old, from about 5 to about 10 years old, from about 10 to about 15 years old, from about 15 to about 20 years old, from about 20 to about 25 years old, from about 25 to about 30 years old, from about 30 to about 35 years old, from about 35 to about 40 years old, from about 40 to about 45 years old, from about 45 to about 50 years old, from about 50 to about 55 years old, from about 55 to about 60 years old, from about 60 to about 65 years old, from about 65 to about 70 years old, from about 70 to about 75 years old, from about 75 to about 80 years old, from about 80 to about 85 years old, from about 85 to about 90 years old, from about 90 to about 95 years old or from about 95 to about 100 years old.

Additional Agents and Combination Therapy

Administration of the present compositions and formulations comprising the IAP and/or commensal gastrointestinal bacteria, including but not limited to Bacteroides acidifaciens, may be combined with additional agents. In embodiments, the agents are useful to preventing, treating, and/or reducing side effects, such as but not limited to cancer therapy-mediated side effects, in a subject. Co-administration of the additional agent and the present compositions/formulations may be simultaneous or sequential. Further, the present compositions/formulations may comprise an additional agent (e.g. via co-formulation). For example, the additional agent and the IAP and/or composition comprising commensal gastrointestinal bacteria, including but not limited to Bacteroides acidifaciens, may be combined into a single formulation. Alternatively, the additional agent and the IAP and/or composition comprising commensal gastrointestinal bacteria, including but not limited to Bacteroides acidifaciens, may be formulated separately.

In embodiments, the additional agent and the IAP and/or composition comprising commensal gastrointestinal bacteria, including but not limited to Bacteroides acidifaciens, are administered to a subject simultaneously. The term “simultaneously” as used herein, means that the additional agent and the IAP and/or composition comprising commensal gastrointestinal bacteria, including but not limited to Bacteroides acidifaciens, are administered with a time separation of no more than about 60 minutes, such as no more than about 30 minutes, no more than about 20 minutes, no more than about 10 minutes, no more than about 5 minutes, or no more than about 1 minute. Administration of the additional agent and the IAP and/or composition comprising commensal gastrointestinal bacteria, including but not limited to Bacteroides acidifaciens, can be by simultaneous administration of a single formulation (e.g., a formulation comprising the additional agent and the IAP and/or composition comprising Bacteroides acidifaciens) or of separate formulations (e.g., a first formulation including the additional agent and a second formulation including the IAP and/or composition comprising Bacteroides acidifaciens).

In embodiments, the additional agent and the IAP and/or composition comprising commensal gastrointestinal bacteria, including but not limited to Bacteroides acidifaciens, are administered to a subject simultaneously but the release of the additional agent and the IAP and/or composition comprising commensal gastrointestinal bacteria, including but not limited to Bacteroides acidifaciens, from their respective dosage forms (or single unit dosage form if co-formulated) may occur sequentially.

Co-administration does not require the additional agent and the IAP and/or composition comprising commensal gastrointestinal bacteria, including but not limited to Bacteroides acidifaciens, to be administered simultaneously, if the timing of their administration is such that the pharmacological activities of the additional agent and the IAP and/or composition comprising commensal gastrointestinal bacteria, including but not limited to Bacteroides acidifaciens, overlap in time. For example, the additional agent and the IAP and/or composition comprising commensal gastrointestinal bacteria, including but not limited to Bacteroides acidifaciens, can be administered sequentially. The term “sequentially” as used herein means that the additional agent and the IAP and/or composition comprising commensal gastrointestinal bacteria, including but not limited to Bacteroides acidifaciens, are administered with a time separation of more than about 60 minutes. For example, the time between the sequential administration of the additional agent and the IAP and/or composition comprising commensal gastrointestinal bacteria, including but not limited to Bacteroides acidifaciens, can be more than about 60 minutes, more than about 2 hours, more than about 5 hours, more than about 10 hours, more than about 1 day, more than about 2 days, more than about 3 days, or more than about 1 week apart. The optimal administration times will depend on the rates of metabolism, excretion, and/or the pharmacodynamic activity of the additional agent and the IAP and/or composition comprising commensal gastrointestinal bacteria, including but not limited to Bacteroides acidifaciens, being administered. Either the additional agent or the IAP and/or composition comprising commensal gastrointestinal bacteria, including but not limited to Bacteroides acidifaciens, may be administered first.

Co-administration also does not require the additional agent and the IAP and/or composition comprising commensal gastrointestinal bacteria, including but not limited to Bacteroides acidifaciens, to be administered to the subject by the same route of administration. Rather, each therapeutic agent can be administered by any appropriate route, for example, parenterally or non-parenterally.

In embodiments, the additional agents of the present disclosure include one or more of blood products, colony stimulating factors, cytokines and/or growth factors, antibiotics, diluting and/or blocking agents, mobilizing or chelating agents, stem cell transplants, antioxidants or free radicals, and radioprotectants.

In embodiments, the blood product is one or more of hematopoietic growth factors, such as filgrastim (e.g. NEUPOGEN), a granulocyte colony-stimulating factor (G-CSF), which may be optionally pegylated (e.g. NEULASTA); sargramostim; and a granulocyte-macrophage colony-stimulating factor (GM-CSF) and a KSF. In embodiments, the additional agent is one or more cytokines and/or growth factors that may confer radioprotection by replenishing and/or protecting the radiosensitive stem cell populations. Radioprotection with minimal side effects may be achieved by the use of stem cell factor (SCF, c-kit ligand), Flt-3 ligand, and interleukin-1 fragment IL-1 b-rd. Protection may be achieved through induction of proliferation of stem cells (e.g. via all mentioned cytokines), and prevention of their apoptosis (e.g. via SCF). The treatment allows accumulation of leukocytes and their precursors prior to irradiation thus enabling quicker reconstitution of the immune system after irradiation. SCF efficiently rescues lethally irradiated mice with a dose modifying factor (DMF) in range 1.3- 1.35 and is also effective against gastrointestinal syndrome. Flt-3 ligand also provides strong protection in mice and rabbits.

Several factors, while not cytokines by nature, stimulate the proliferation of the immunocytes and may be used in combination with the IAP and/or composition comprising commensal gastrointestinal bacteria, including but not limited to Bacteroides acidifaciens, at the doses and regimens described herein. For example, 5-AED (5- androstenediol) is a steroid that stimulates the expression of cytokines and increases resistance to bacterial and viral infections. Synthetic compounds, such as ammonium tri-chloro(dioxoethylene-O,O'-) tellurate (AS-101), may also be used to induce secretion of numerous cytokines and for combination with the IAP and/or composition comprising commensal gastrointestinal bacteria, including but not limited to Bacteroides acidifaciens. Growth factors and cytokines may also be used to provide protection against the gastrointestinal syndrome. Keratinocyte growth factor (KGF) promotes proliferation and differentiation in the intestinal mucosa, and increases the postirradiation cell survival in the intestinal crypts. Hematopoietic cytokine and radioprotectant SCF may also increase intestinal stem cell survival and associated short-term organism survival.

In embodiments, the IAP and/or composition comprising commensal gastrointestinal bacteria, including but not limited to Bacteroides acidifaciens, may be added to a regimen of cytokines (e.g. for FILGRASTIM (G-CSF) 2.5- 5 pg/kg/d QD s.c. (100-200 pg/m²/d); for SARGRAMOSTIM (GM-CSF) 5-10 pg/kg/d QD s.c. (200-400 pg/m²/d); and/or for PEGFILGRASTIM (pegG-CSF) 6 mg once s.c.).

In embodiments, the additional agent is an interleukin, such as IL-12 (e.g. HEMAMAX (NEUMEDICINES, INC.)).

In embodiments, the additional agent is a diluting and/or blocking agents. For example, stable iodide compounds may be used (e.g. liquid (ThyroShield) and the tablet (losat) KI (NUKEPILLS), Rad Block, I.A.A.A.M., No-Rad, Life Extension (LEF), KI4U, NukeProtect, ProKI)). A 130 mg dose of daily of oral potassium iodide (KI) may be used in conjunction with the IAP and/or composition comprising commensal gastrointestinal bacteria, including but not limited to Bacteroides acidifaciens.

In embodiments, the additional agent is a mobilizing or chelating agent. Illustrative mobilizing agents include propylthiouracil and methimazole, with may reduce the thyroid’s retention of radioactive compounds. Further the IAP and/or composition comprising commensal gastrointestinal bacteria, including but not limited to Bacteroides acidifaciens, can be used alongside increasing oral fluids to a human subject to promote excretion. Illustrative chelating agents are water soluble and excreted in urine. Illustrative chelating agents include DTPA and EDTA. Dimercaprol forms stable chelates with mercury, lead, arsenic, gold, bismuth, chromium, and nickel and therefore may be considered for the treatment of internal contamination with the radioisotopes of these elements. Penicillamine chelates copper, iron, mercury, lead, gold, and possibly other heavy metals.

In embodiments, the additional agent is a stem cell transplant (e.g. bone marrow transplant, PBSCT, MSCT). In embodiments the stem cell transplant is Remestemcel-L (Osiris) of CLT -008 (Cellerant).

In embodiments, the additional agent is an antioxidant or free radical. Antioxidants and free radical scavengers that may be used in the practice of the disclosure include, but are not limited to, thiols, such as cysteine, cysteamine, glutathione and bilirubin; amifostine (WR-2721); vitamin A; vitamin C; vitamin E; and flavonoids such as Indian holy basil (Ocimum sanctum), orientin and vicenin.

In embodiments, the additional agent may be a radioprotectant e.g. an antioxidant (e.g. amifostine and vitamin E, gamma tocotrienol (a vitamin-E moiety), and genistein (a soy byproduct)), a cytokine (e.g. a stem cell factor), a growth factor (e.g. keratinocyte growth factor), a steroid (e.g. 5-androstenediol), ammonium trichloro(dioxoethylene-O,O')tellurate, thyroid protecting agents (e.g. Potassium iodide (KI) or potassium iodate (KIO₃) (e.g. liquid (ThyroShield) and the tablet (losat) KI (NUKEPILLS), Rad Block, I.A.A.A.M., No-Rad, Life Extension (LEF), KI4U, NukeProtect, ProKI)), anti-nausea agents, anti-diarrhea agents, antiemetics ((e.g. oral prophylactic antiemetics) such as granisetron (KYTRIL), ondansetron (ZOFRAN), and 5-HT3 blockers with or without dexamethasone), analgesics, anxiolytics, sedatives, cytokine therapy, and antibiotics.

In embodiments, the antibiotic is one or more of an anti-bacterial (anti-gram positive and anti-gram negative agents), and/or anti-fungal, and/or anti-viral agent. By way of non-limiting example, in embodiments, the antibiotic may be a quinolone, e.g. ciprofloxacin, levofloxacin, a third- or fourth-generation cephalosporin with pseudomonal coverage: e.g., cefepime, ceftazidime, or an aminoglycoside: e.g. gentamicin, amikacin, penicillin or amoxicillin, acyclovir, vanomycin. In embodiments, the antibiotic targets Pseudomonas aeruginosa.

Definitions

As used herein, “a,” “an,” or “the” can mean one or more than one.

Further, the term “about” when used in connection with a referenced numeric indication means the referenced numeric indication plus or minus up to 10% of that referenced numeric indication. For example, the language “about 50%” covers the range of 45% to 55%.

An “effective amount,” when used in connection with medical uses is an amount that is effective for providing a measurable treatment, prevention, or reduction in the rate of pathogenesis of a disorder of interest.

As used herein, the term “therapeutic window” refers to the range of pharmacodynamic effects induced by a range of doses of one or more pharmaceutically active agents, providing a balance between one or more desired (positive) effect(s) and one or more adverse (negative) effect(s). In embodiments, the therapeutic window is referred to as a pharmacodynamic profile. The therapeutic window may relate to a given point in time or may span a period of time of any length, including for example minutes, hours, days or longer, shorter or to any intermediate period of time. The desirability and undesirability of an effect can be defined based on a variety of criteria, and include without limitation, medical practices, rules and regulations, cultural and demographic norms, genetic factors and personal preferences and tolerances. For example, the desirability and undesirability of an effect can be defined based on the purpose of treatment and based on generally acceptable values and optionally may take into account other parameters such as subject preference, capacity and activity. It is noted that a given effect may be regarded as desired in some cases, but be regarded as undesired in other cases, and vice versa.

As used herein, something is “decreased” if a read-out of activity and/or effect is reduced by a significant amount, such as by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, or more, up to and including at least about 100%, in the presence of an agent or stimulus relative to the absence of such modulation. As will be understood by one of ordinary skill in the art, in embodiments, activity is decreased and some downstream read-outs will decrease but others can increase.

Conversely, activity is “increased” if a read-out of activity and/or effect is increased by a significant amount, for example by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, or more, up to and including at least about 100% or more, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 50-fold, at least about 100-fold, in the presence of an agent or stimulus, relative to the absence of such agent or stimulus.

As referred to herein, all compositional percentages are by weight of the total composition, unless otherwise specified. As used herein, the word “include,” and its variants, is intended to be non-limiting, such that recitation of items in a list is not to the exclusion of other like items that may also be useful in the compositions and methods of this technology. Similarly, the terms “can” and “may” and their variants are intended to be nonlimiting, such that recitation that an embodiment can or may comprise certain elements or features does not exclude other embodiments of the present technology that do not contain those elements or features.

Although the open-ended term “comprising,” as a synonym of terms such as including, containing, or having, is used herein to describe and claim the disclosure, the present disclosure, or embodiments thereof, may alternatively be described using alternative terms such as “consisting of” or “consisting essentially of.”

As used herein, the words “preferred” and “preferably” refer to embodiments of the technology that afford certain benefits, under certain circumstances. However, other embodiments may also be preferred, under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful, and is not intended to exclude other embodiments from the scope of the technology. The amount of compositions described herein needed for achieving a therapeutic effect may be determined empirically in accordance with conventional procedures for the particular purpose. Generally, for administering therapeutic agents (e.g., microbiome-modulating agents and/or additional agents described herein) for therapeutic purposes, the therapeutic agents are given at a pharmacologically effective dose. A “pharmacologically effective amount,” “pharmacologically effective dose,” “therapeutically effective amount,” or “effective amount” refers to an amount sufficient to produce the desired physiological effect or amount capable of achieving the desired result, particularly for treating the disorder or disease. An effective amount as used herein would include an amount sufficient to, for example, delay the development of a symptom of the disorder or disease, alter the course of a symptom of the disorder or disease (e.g., slow the progression of a symptom of the disease), reduce or eliminate one or more symptoms or manifestations of the disorder or disease, and reverse a symptom of a disorder or disease. Therapeutic benefit also includes halting or slowing the progression of the underlying disease or disorder, regardless of whether improvement is realized.

Effective amounts, toxicity, and therapeutic efficacy can be determined by standard pharmaceutical procedures in cell cultures, tissue samples, tissue homogenates or experimental animals, e.g., for determining the LD50 (the dose lethal to about 50% of the population) and the ED50 (the dose therapeutically effective in about 50% of the population). The dosage can vary depending upon the dosage form employed and the route of administration utilized. The dose ratio between toxic and therapeutic effects is the therapeutic index and can be expressed as the ratio LD50/ED50. In embodiments, compositions and methods that exhibit large therapeutic indices are preferred. A therapeutically effective dose can be estimated initially from in vitro assays, including, for example, cell culture assays or measurements or methane production in stool samples. Also, a dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 as determined in cell culture, or in an appropriate animal model. Levels of the described compositions in plasma can be measured, for example, by high performance liquid chromatography. The effects of any particular dosage can be monitored by a suitable bioassay. The dosage can be determined by a physician and adjusted, as necessary, to suit observed effects of the treatment.

In certain embodiments, the effect will result in a quantifiable change of at least about 10%, at least about 20%, at least about 30%, at least about 50%, at least about 70%, or at least about 90%. In embodiments, the effect will result in a quantifiable change of about 10%, about 20%, about 30%, about 50%, about 70%, or even about 90% or more. Therapeutic benefit also includes halting or slowing the progression of the underlying disease or disorder, regardless of whether improvement is realized.

As used herein, “methods of treatment” are equally applicable to use of a composition for treating the diseases or disorders described herein and/or compositions for use and/or uses in the manufacture of a medicaments for treating the diseases or disorders described herein.

EQUIVALENTS

While the disclosure has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the disclosure following, in general, the principles of the disclosure and including such departures from the present disclosure as come within known or customary practice within the art to which the disclosure pertains and as may be applied to the essential features hereinbefore set forth and as follows in the scope of the appended claims.

Those skilled in the art will recognize, or be able to ascertain, using no more than routine experimentation, numerous equivalents to the specific embodiments described specifically herein. Such equivalents are intended to be encompassed in the scope of the following claims.

INCORPORATION BY REFERENCE All patents and publications referenced herein are hereby incorporated by reference in their entireties.

The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present disclosure is not entitled to antedate such publication by virtue of prior disclosure.

As used herein, all headings are simply for organization and are not intended to limit the disclosure in any manner. The content of any individual section may be equally applicable to all sections.

Claims

1. A method for preventing, treating, and/or reducing a cancer therapy-mediated side effect in a subject in need thereof, comprising administering to the subject an intestinal alkaline phosphatase (IAP).

2. A method for improving and/or increasing and/or enhancing efficacy of a cancer therapy relative to the treatment before or in the absence of IAP, comprising administering to the subject an intestinal alkaline phosphatase (IAP).

3. A method for preventing, treating, and/or reducing a cancer therapy-mediated side effect in a subject in need thereof, comprising administering to the subject an intestinal alkaline phosphatase (IAP), wherein the subject is undergoing therapy with a composition comprising at least one commensal gastrointestinal bacteria.

4. A method for improving and/or increasing and/or enhancing efficacy of a cancer therapy relative to the treatment before or in the absence of IAP, comprising administering to the subject an intestinal alkaline phosphatase (IAP), wherein the subject is undergoing therapy with a composition comprising at least one commensal gastrointestinal bacteria.

5. The method of any one of claim 1-4, wherein the cancer therapy comprises immune checkpoint inhibitor (CPI) immunotherapy.

6. The method of claim 5, wherein the immune checkpoint inhibitor immunotherapy is selected from an agent that modulates one or more of programmed cell death protein-1 (PD-1), programmed death-ligand 1 (PD- L1), programmed death-ligand 2 (PD-L2), inducible T-cell costimulator (ICOS), inducible T-cell costimulator ligand (ICOSL), and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4).

7. The method of claim 6, wherein the agent that modulates PD-1 is an antibody or antibody format specific for PD-1 .

8. The method of claim 7, wherein the antibody or antibody format specific for PD-1 is selected from one or more of a monoclonal antibody, polyclonal antibody, antibody fragment, Fab, Fab', Fab'-SH, F(ab')2, Fv, single chain Fv, diabody, linear antibody, bispecific antibody, multispecific antibody, chimeric antibody, humanized antibody, human antibody, and fusion protein comprising the antigen-binding portion of an antibody.

9. The method of claim 7, wherein the antibody or antibody format specific for PD-1 is selected from nivolumab, pembrolizumab, and pidilizumab.

10. The method of claim 6, wherein the agent that modulates PD-L1 is an antibody or antibody format specific for PD-L1.

11. The method of claim 10, wherein the antibody or antibody format specific for PD-L1 is selected from one or more of a monoclonal antibody, polyclonal antibody, antibody fragment, Fab, Fab', Fab'-SH, F(ab')2, Fv, single chain Fv, diabody, linear antibody, bispecific antibody, multispecific antibody, chimeric antibody, humanized antibody, human antibody, and fusion protein comprising the antigen-binding portion of an antibody.

12. The method of claim 10, wherein the antibody or antibody format specific for PD-L1 is selected from BMS-936559, atezolizumab, avelumab and durvalumab.

13. The method of claim 6, wherein the agent that modulates PD-L2 is an antibody or antibody format specific for PD-L2.

14. The method of claim 13, wherein the antibody or antibody format specific for PD-L2 is selected from one or more of a monoclonal antibody, polyclonal antibody, antibody fragment, Fab, Fab', Fab'-SH, F(ab')2, Fv, single chain Fv, diabody, linear antibody, bispecific antibody, multispecific antibody, chimeric antibody, humanized antibody, human antibody, and fusion protein comprising the antigen-binding portion of an antibody.

15. The method of claim 6, wherein the agent that modulates ICOS is an antibody or antibody format specific for ICOS.

16. The method of claim 15, wherein the antibody or antibody format specific for ICOS is selected from one or more of a monoclonal antibody, polyclonal antibody, antibody fragment, Fab, Fab', Fab'-SH, F(ab')2, Fv, single chain Fv, diabody, linear antibody, bispecific antibody, multispecific antibody, chimeric antibody, humanized antibody, human antibody, and fusion protein comprising the antigen-binding portion of an antibody.

17. The method of claim 15, wherein the antibody or antibody format specific for ICOS comprises JTX- 2011.

18. The method of claim 6, wherein the agent that modulates ICOSL is an antibody or antibody format specific for ICOS L.

19. The method of claim 18, wherein the antibody or antibody format specific for ICOSL is selected from one or more of a monoclonal antibody, polyclonal antibody, antibody fragment, Fab, Fab', Fab'-SH, F(ab')2, Fv, single chain Fv, diabody, linear antibody, bispecific antibody, multispecific antibody, chimeric antibody, humanized antibody, human antibody, and fusion protein comprising the antigen-binding portion of an antibody.

20. The method of claim 6, wherein the agent that modulates CTLA-4 is an antibody or antibody format specific for CTLA-4.

21. The method of claim 20, wherein the antibody or antibody format specific for CTLA-4 is selected from one or more of a monoclonal antibody, polyclonal antibody, antibody fragment, Fab, Fab', Fab'-SH, F(ab')2, Fv, single chain Fv, diabody, linear antibody, bispecific antibody, multispecific antibody, chimeric antibody, humanized antibody, human antibody, and fusion protein comprising the antigen-binding portion of an antibody.

22. The method of claim 20, wherein the antibody or antibody format specific for CTLA-4 is selected from tremelimumab and ipilimumab.

23. The method of any one of claims 1-22, wherein the cancer therapy-mediated side effect comprises a CPI-mediated gastrointestinal (Gl) side effect.

24. The method of claim 23, wherein the CPI-mediated Gl side effect comprises diarrhea and/or colitis.

25. The method of any one of claims 1-4, wherein the cancer therapy comprises chemotherapy.

26. The method of claim 25, wherein the chemotherapy comprises one or more of alkylating agents such as thiotepa and CYTOXAN cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethiylenethiophosphoramide and trimethylolomelamine; acetogenins (e.g., bullatacin and bullatacinone); a camptothecin (including the synthetic analogue topotecan); bryostatin; cally statin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); cryptophycins (e.g., cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189 and CB 1-TM1); eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics such as the enediyne antibiotics (e.g., calicheamicin, especially calicheamicin gammall and calicheamicin omegall; dynemicin, including dynemicin A; bisphosphonates, such as clodronate; an esperamicin; as well as neocarzi nostatin chromophore and related chromoprotein enediyne antibiotic chromophores), aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L- norleucine, ADRIAMYCIN doxorubicin (including morpholino- doxorubicin, cyanomorpholino-doxorubicin, 2- pyrrolino-doxorubicin and deoxy doxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals such as minoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; demecolcine; diaziquone; elformithine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK polysaccharide complex; razoxane; rhizoxin; sizofuran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2"-trichlorotriethylamine; trichothecenes (e.g., T-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxoids, e.g., TAXOL paclitaxel, ABRAXANE Cremophor-free, albumin-engineered nanoparticle formulation of paclitaxel, and TAXOTERE doxetaxel; chloranbucil; GEMZAR gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin, oxaliplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; NAVELBINE, vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; irinotecan (Camptosar, CPT-11) (including the treatment regimen of irinotecan with 5-FU and leucovorin); topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid; capecitabine; combretastatin; leucovorin (LV); oxaliplatin, including the oxaliplatin treatment regimen (FOLFOX); lapatinib (Tykerb); inhibitors of PKC-a, Raf, H-Ras, EGFR (e.g., erlotinib (Tarceva)) and VEGF-A that reduce cell proliferation and pharmaceutically acceptable salts, acids or derivatives of any of the above.

27. The method of claim 25 or 26, wherein the chemotherapy-mediated side effect comprises one or more selected from alopecia, myelosuppression, renal toxicity, weight lossr pain, nausea, vomiting, diarrhea, constipation, anemia, malnutrition, hair loss, numbness, changes in tastes, loss of appetite, thinned or brittle hair, mouth sores, memory loss, hemorrhage, cardiotoxicity, hepatotoxicity, ototoxicity, and post-chemotherapy cognitive impairment.

28. The method of claim 25 or 26, wherein the chemotherapy-mediated side effect comprises one or more selected from stomatitis, esophagopharyngitis, leukopenia and/or reduced white blood count, reduced neutrophils, reduced lymphocytes, reduced platelets, increased risk of infection, vomiting, diarrhea, nausea, poor appetite, gastrointestinal ulceration and/or bleeding, thrombocytopenia, hand-foot syndrome, alopecia, mucositis, impaired hepatic function, impaired hepatic function, and hemorrhage.

29. The method of any one of claims 1-4, wherein the cancer therapy comprises a radiotherapy.

30. The method of claim 29, wherein the radiotherapy is delivered as one or more of external-beam radiation therapy, brachytherapy, and systemic radiation therapy.

31 . The method of claim 29 or 30, wherein the radiotherapy is an external-beam radiation therapy, selected from 3-dimensional conformal radiation therapy (3D-CRT), intensity-modulated radiation therapy (IMRT, e.g. RAPIDARC), image-guided radiation therapy (IGRT), electromagnetic-guided radiation therapy (e.g. CALYPSO) tomotherapy, stereotactic radiosurgery (SRS), stereotactic body radiation therapy (SBRT, e.g. CYBERKNIFE, GAMMAKNIFE, X-KNIFE, CLINAC), Intraoperative radiation therapy (IORT), and proton therapy.

32. The method of claim 29 or 30, wherein the radiotherapy is a brachytherapy selected from interstitial brachytherapy, intracavitary brachytherapy, and episcleral brachytherapy.

33. The method of claim 29 or 30, wherein the radiotherapy is a systemic radiation therapy, selected from a radioactive iodine and a radioactive biologic.

34. The method of any one of claims 29-33, wherein the radiation-mediated side effect comprises one or more selected from fatigue, nausea and vomiting, damage to the epithelial surfaces (e.g., without limitation, moist desquamation), mouth, throat and stomach sores, intestinal discomfort (e.g., without limitation, soreness, diarrhea, and nausea), swelling, infertility, fibrosis, epilation, dryness (e.g. without limitation, dry mouth (xerostomia) and dry eyes (xerophthalmia), and dryness of the armpit and vaginal mucosa), lymphedema, heart disease, cognitive decline, radiation enteropathy (e.g. without limitation, atrophy, fibrosis and vascular changes, which may produce malabsorption, diarrhea, steatorrhea and bleeding with bile acid diarrhea and vitamin B12 malabsorption commonly found due to ileal involvement), pelvic radiation disease including radiation proctitis, producing bleeding, diarrhea and urgency, and radiation cystitis.

35. The method of any of the preceding claims, wherein the method increases a therapeutic window of the cancer therapy relative to the treatment before or in the absence of IAP.

36. The method of claim 35, wherein the increased therapeutic window of the cancer therapy comprises one or more of increasing the subject’s likelihood of receiving maintenance therapy of the cancer therapy relative to the treatment before or in the absence of IAP; increasing the subject’s likelihood of receiving a complete regimen of the cancer therapy relative to the treatment before or in the absence of IAP; increasing the subject’s likelihood of receiving more than a complete regimen of the cancer therapy relative to the treatment before or in the absence of IAP; and increasing the dose or length of the cancer therapy relative to the treatment before or in the absence of IAP.

37. The method of any of the preceding claims, wherein the method reduces the likelihood of the subject requiring a transition to palliative care.

38. The method of any of the preceding claims, wherein the method improved and/or increased and/or enhanced efficacy of the cancer therapy relative to the treatment before or in the absence of IAP allows for one or more of dose reduction, treatment duration reduction, and usage of an incomplete regimen of the cancer therapy.

39. The method of any of the preceding claims, wherein the method makes the subject suitable for treatment with a combination therapy of more than one cancer therapies.

40. The method of any one of claims 1-39, wherein the composition comprising at least one commensal gastrointestinal bacteria is a fecal microbiota transplant comprising at least one commensal gastrointestinal bacteria.

41 . The method of claim 40, wherein the commensal gastrointestinal bacteria is Bacteroides acidifaciens.

42. The method of claim 40 or 41, wherein the fecal transplant is stool or a derivative thereof.

43. The method of any one of claims 40-42, wherein the fecal transplant is derived from a human donor.

44. The method of any one of claims 40-43, wherein the fecal transplant is administered via one or more of oral administration, colonoscopy, sigmoidoscopy, enema, naso-gastric intubation, naso-duodenal intubation, and naso-jejunal intubation.

45. The method of any one of claims 1-44, wherein the composition comprising at least one commensal gastrointestinal bacteria is an isolated bacterial composition.

46. The method of claim 45, wherein the bacteria are isolated from one or more of human stool, the human Gl tract, and the human gut.

47. The method of claim 45 or 46, wherein the isolated commensal gastrointestinal bacteria is grown in pure or mixed cultures.

48. The method of any one of claims 1-47, wherein the isolated commensal gastrointestinal bacteria is formulated as one or more of tablets, pills, powders, capsules, lyophilized compositions, and aqueous formulations.

49. The method of any one of claims 1-48, wherein the IAP is bovine IAP (blAP).

50. The method of any one of claims 1-48, wherein the IAP comprises an amino sequence having at least about 90%, or about 95%, or about 97%, or about 98%, or about 99% sequence identity with any one of SEQ ID NOs: 1-6 and 10-14.

51. The method of claim 49 or 50, wherein the IAP comprises an amino sequence having at least about 97% sequence identity to SEQ ID NO: 11 .

52. The method of any one of the preceding claims, wherein the IAP is administered orally.

53. The method of any one of claims 45-52, wherein the composition comprising isolated commensal gastrointestinal bacteria is administered orally.

54. The method of any one of the preceding claims, wherein the method increases or preserves the number of commensal bacteria and/or composition of the gastrointestinal microbiome of the subject relative to pretreatment.

55. The method of any one of the preceding claims, wherein the method inhibits the growth of or decreases the number of pathogenic bacteria in the gastrointestinal microbiome of the subject relative to pre-treatment.

56. The method of any one of the preceding claims, wherein the method preserves the existing microbiome of the subject relative to pre-treatment.

57. A method for preventing and/or treating an immune checkpoint inhibitor (CPI)-mediated gastrointestinal (Gl) side effect in a subject in need thereof, comprising administering to the subject an intestinal alkaline phosphatase (IAP) and an CPI selected from an agent that modulates one or more of PD-1 , PD-L1, PD-L2, ICOS, ICOSL, and CTLA-4.

58. The method of claim 57, wherein the CPI-mediated Gl side effect comprises diarrhea and/or colitis.

59. The method of any one of claims 57 or 58, wherein the immune checkpoint inhibitor immunotherapy is selected from an agent that modulates one or more of programmed cell death protein-1 (PD-1), programmed death-ligand 1 (PD-L1), programmed death-ligand 2 (PD-L2), inducible T-cell costimulator (ICOS), inducible T- cell costimulator ligand (ICOSL), and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4).

60. The method of any one of claims 57-59, wherein the agent that modulates PD-1 is an antibody or antibody format specific for PD-1.

61. The method of claim 60, wherein the antibody or antibody format specific for PD-1 is selected from one or more of a monoclonal antibody, polyclonal antibody, antibody fragment, Fab, Fab', Fab'-SH, F(ab')2, Fv, single chain Fv, diabody, linear antibody, bispecific antibody, multispecific antibody, chimeric antibody, humanized antibody, human antibody, and fusion protein comprising the antigen-binding portion of an antibody.

62. The method of claim 60 wherein the antibody or antibody format specific for PD-1 is selected from nivolumab, pembrolizumab, and pidilizumab.

63. The method of any one of claims 57-59, wherein the agent that modulates PD-L1 is an antibody or antibody format specific for PD-L1 .

64. The method of claim 63, wherein the antibody or antibody format specific for PD-L1 is selected from one or more of a monoclonal antibody, polyclonal antibody, antibody fragment, Fab, Fab', Fab'-SH, F(ab')2, Fv, single chain Fv, diabody, linear antibody, bispecific antibody, multispecific antibody, chimeric antibody, humanized antibody, human antibody, and fusion protein comprising the antigen-binding portion of an antibody.

65. The method of claim 63, wherein the antibody or antibody format specific for PD-L1 is selected from BMS-936559, atezolizumab, avelumab and durvalumab.

66. The method of any one of claims 57-59, wherein the agent that modulates PD-L2 is an antibody or antibody format specific for PD-L2.

67. The method of claim 66, wherein the antibody or antibody format specific for PD-L2 is selected from one or more of a monoclonal antibody, polyclonal antibody, antibody fragment, Fab, Fab', Fab'-SH, F(ab')2, Fv, single chain Fv, diabody, linear antibody, bispecific antibody, multispecific antibody, chimeric antibody, humanized antibody, human antibody, and fusion protein comprising the antigen-binding portion of an antibody.

68. The method of any one of claims 57-59, wherein the agent that modulates ICOS is an antibody or antibody format specific for ICOS.

69. The method of claim 68, wherein the antibody or antibody format specific for ICOS is selected from one or more of a monoclonal antibody, polyclonal antibody, antibody fragment, Fab, Fab', Fab'-SH, F(ab')2, Fv, single chain Fv, diabody, linear antibody, bispecific antibody, multispecific antibody, chimeric antibody, humanized antibody, human antibody, and fusion protein comprising the antigen-binding portion of an antibody.

70. The method of claim 68, wherein the antibody or antibody format specific for ICOS comprises JTX- 2011.

71. The method of any one of claims 57-59, wherein the agent that modulates ICOSL is an antibody or antibody format specific for ICOSL.

72. The method of claim 71, wherein the antibody or antibody format specific for ICOSL is selected from one or more of a monoclonal antibody, polyclonal antibody, antibody fragment, Fab, Fab', Fab'-SH, F(ab')2, Fv, single chain Fv, diabody, linear antibody, bispecific antibody, multispecific antibody, chimeric antibody, humanized antibody, human antibody, and fusion protein comprising the antigen-binding portion of an antibody.

73. The method of any one of claims 57-59, wherein the agent that modulates CTLA-4 is an antibody or antibody format specific for CTLA-4.

74. The method of claim 73, wherein the antibody or antibody format specific for CTLA-4 is selected from one or more of a monoclonal antibody, polyclonal antibody, antibody fragment, Fab, Fab', Fab'-SH, F(ab')2, Fv, single chain Fv, diabody, linear antibody, bispecific antibody, multispecific antibody, chimeric antibody, humanized antibody, human antibody, and fusion protein comprising the antigen-binding portion of an antibody.

75. The method of claim 73, wherein the antibody or antibody format specific for CTLA-4 is selected from tremelimumab or ipilimumab.

76. The method of any one of claims 57-75, further comprising administering a fecal microbiota transplant comprising at least one commensal gastrointestinal bacteria.

77. The method of claim 76, wherein the commensal gastrointestinal bacteria is Bacteroides acidifaciens.

78. The method of any one of claims 76 or 77, wherein the fecal transplant is stool or a derivative thereof.

79. The method of any one of claims 76-78, wherein the fecal transplant is derived from a human donor.

80. The method of any one of claims 76-79, wherein the fecal transplant is administered via one or more of oral administration, colonoscopy, sigmoidoscopy, enema, naso-gastric intubation, naso-duodenal intubation, and naso-jejunal intubation.

81. The method of any one of claims 57-80, wherein the composition comprising at least one commensal gastrointestinal bacteria is an isolated bacterial composition.

82. The method of claim 81, wherein the bacteria are isolated from one or more of human stool, the human Gl tract, and the human gut.

83. The method of claim 81 or 82, wherein the isolated commensal gastrointestinal bacteria is grown in pure or mixed cultures.

84. The method of any one of claims 81-83, wherein the isolated commensal gastrointestinal bacteria is formulated as one or more of tablets, pills, powders, capsules, lyophilized compositions, and aqueous formulations.

85. The method of any one of claims 57-84, wherein the IAP is bovine IAP (blAP).

86. The method of any one of claims 57-84, wherein the IAP comprises an amino sequence having at least about 90%, or about 95%, or about 97%, or about 98%, or about 99% sequence identity to any one of SEQ ID NOs: 1-6 and 10-14.

87. The method of claim 85 or 86, wherein the IAP comprises an amino sequence having at least about 97% sequence identity to SEQ ID NO: 11 .