Compositions and methods for selecting a subject for inflammatory bowel disease treatment

Abstract

The disclosure provides methods for predicting response in a subject having IBD to anti-IL12 and / or anti-IL23-based therapies, and selecting an effective therapy.

Description

[0001] COMPOSITIONS AND METHODS FOR SELECTING A SUBJECT FOR INFLAMMATORY BOWEL DISEASE TREATMENT

[0002] RELATED APPLICATIONS

[0003] This application claims the benefit under 35 U.S.C. § 119(e) to U.S. Provisional Application No. 63 / 542,863, filed October 6, 2023, and U.S. Provisional Application No. 63 / 622,982, filed January 19, 2024, the contents of each of which are incorporated herein in their entireties.

[0004] FEDERALLY SPONSORED RESEARCH

[0005] This invention was made with government support under Grant Number DK118640, awarded by the National Institutes of Health. The Government has certain rights in the invention.

[0006] BACKGROUND

[0007] Inflammatory bowel disease (IBD) is a chronic disease characterized by long-lasting intestinal inflammation that affects over one million people in the U.S. The two main subtypes of inflammatory bowel disease are Crohn’s disease and ulcerative colitis. Crohn’s disease involves transmural inflammation at any location in the gastrointestinal tract and can be associated with fistulizing or stenotic complications. Ulcerative colitis is characterized by contiguous superficial mucosal inflammation of the colon. In some cases, the two subtypes can be differentiated by macroscopic inflammation patterns, but obtaining such data requires invasive techniques. Even with macroscopic inflammation data, differentiating between Crohn’s disease or ulcerative colitis can remain difficult or impossible in some cases, which can lead to a less specific diagnosis of “unclassified” inflammatory bowel disease. While several pharmaceutical options are available, the inflammatory bowel disease is heterogenous, and an accurate diagnosis is needed for proper treatment. More accurate diagnostic techniques would allow for more personalized and effective treatments. Thus, there is a demonstrated and urgent need for improved methods for differentiating inflammatory bowel disease subtypes.

[0008] Very early onset IBD (VEOIBD) is defined as disease onset at age <6 years and has a monogenic cause in >5% of cases. IL10R deficiency is among the most common causes of monogenic IBD and presents with severe disease refractory to conventional therapies. For most VEOIBD patients, the molecular basis of disease is unknown, and effective therapies have yet to be identified.

[0009] SUMMARY

[0010] The present disclosure provides methods for predicting whether an adult subject and / or pediatric subject having IBD will respond to an anti-IL12 and / or anti-IL23-based therapy, and selecting an effective therapy.

[0011] In an aspect, the present disclosure provides a method of treating a selected subject having Inflammatory Bowel Disease (IBD). The method involves administering an agent selected from the group consisting of: ustekinumab; risankizumab; mirikizumab; brazikumab; and guselkumab, to a subject. The subject is selected as having defective phosphorylation of STAT3 or a reduction in a JAK / STAT signalling pathway in response to IL10 and / or IL21 stimulation.

[0012] In another aspect, the present disclosure provides a panel for assessing downregulation of IL6-JAK-STAT3 signaling in a subject. The panel includes and / or consists of markers IL6, ILlb, IL1R1, CXCL8, MMP9, JAK3, and / or STAT3.

[0013] In another aspect, the present disclosure provides a kit. The kit includes the panel of any of the above aspects, or embodiments thereof, and instructions for use of the panel in the method of any of the above aspects, or embodiments thereof.

[0014] In another aspect, the present disclosure provides a panel of any of the above aspects, or embodiments thereof, for use in a method for treating a selected subject having Inflammatory Bowel Disease (IBD).

[0015] In any of the above aspects, or embodiments thereof, phosphorylation of STAT3 is detected by flow cytometry or Western blot.

[0016] In any of the above aspects, or embodiments thereof, the subject is an adult or a pediatric subject. In any of the above aspects, or embodiments thereof, the pediatric subject is less than about 10 years of age. In any of the above aspects, or embodiments thereof, the pediatric subject is less than about 5 years of age. In any of the above aspects, or embodiments thereof, the pediatric subject is less than about 2 years of age.

[0017] In any of the above aspects, or embodiments thereof, the subject displays infantileonset disease, severe colonic inflammation, and / or refractoriness to anti-Tumor Necrosis Factor (TNF) therapy.

[0018] In any of the above aspects, or embodiments thereof, Ml macrophages of the subject were enriched in a signature characterized by downregulation of IL6-JAK-STAT3 signaling. In any of the above aspects, or embodiments thereof, downregulation of IL6-JAK-STAT3 signaling comprises a reduction in STAT3 phosphorylation, a reduction in JAK activity, a reduction in MAP / ERK phosphorylation, a reduction in PI3K / AKT phosphorylation, and / or a reduction in NF-KB activation.

[0019] In any of the above aspects, or embodiments thereof, each of the markers specifically bind to a separate capture reagent. In any of the above aspects, or embodiments thereof, the capture reagents are attached to a solid support. In any of the above aspects, or embodiments thereof, the solid support is a plate, chip, beads, microfluidic platform, membrane, planar microarray, or suspension array. In any of the above aspects, or embodiments thereof, the capture reagent is an antibody, aptamer, Affibody, hybridization probe and / or fragments thereof.

[0020] Compositions and articles defined by the invention were isolated or otherwise manufactured in connection with the examples provided below. Other features and advantages of the invention will be apparent from the detailed description, and from the claims.

[0021] Definitions

[0022] Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which this invention belongs. The following references provide one of skill with a general definition of many of the terms used in this invention: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991). As used herein, the following terms have the meanings ascribed to them below, unless specified otherwise.

[0023] By “agent” is meant a peptide, antibody, nucleic acid molecule, or small compound. In some embodiments, the agent is ustekinumab, risankizumab, mirikizumab, brazikumab, or guselkumab.

[0024] By “ameliorate” is meant decrease, suppress, attenuate, diminish, arrest, or stabilize the development or progression of a disease. In some embodiments, treatment with an agent described herein decreases a symptom of IBD (e.g., VEOIBD).

[0025] By "alteration" is meant a change (increase or decrease) in the expression levels, structure, or activity of a gene or polypeptide as detected by standard art known methods such as those described herein. As used herein, an alteration includes a 10% change in expression levels, a 25% change, a 40% change, or a 50% or greater change in expression levels. In some embodiments, the alteration is in the expression of any of the markers disclosed herein, relative to a reference level of expression.

[0026] By "analog" is meant a molecule that is not identical, but has analogous functional or structural features. For example, a polypeptide analog retains the biological activity of a corresponding naturally-occurring polypeptide, while having certain biochemical modifications that enhance the analog's function relative to a naturally occurring polypeptide. Such biochemical modifications could increase the analog's protease resistance, membrane permeability, or half-life, without altering, for example, ligand binding. An analog may include an unnatural amino acid. In some embodiments, analogs of ustekinumab, risankizumab, mirikizumab, brazikumab, or guselkumab having anti-IL12 and / or anti-IL23 activity are contemplated for use in the methods of treatment disclosed herein.

[0027] By “brazikumab” is meant a monoclonal antibody that binds to interleukin IL-23, a cytokine that modulates lymphocyte function, including the T-helper (Th) 17 cell subset.

[0028] In this disclosure, "comprises," "comprising," "containing" and "having" and the like can have the meaning ascribed to them in U.S. Patent law and can mean " includes," "including," and the like; "consisting essentially of" or "consists essentially" likewise has the meaning ascribed in U.S. Patent law and the term is open-ended, allowing for the presence of more than that which is recited so long as basic or novel characteristics of that which is recited is not changed by the presence of more than that which is recited, but excludes prior art embodiments.

[0029] By “capture reagent” is meant a molecule and / or a molecule bound to a solid support structure that binds or captures a target molecule. Non-limiting examples of capture-reagents are antibodies, aptamers, Affibodies, hybridization probes and / or fragments thereof.

[0030] By “control” is meant not having or suspected of having a disease and / or not being derived from a diseased source. For example, a control sample is a sample derived from a subject that exhibit no signs of a disease (e.g., inflammatory bowel disease, Crohn’s disease, ulcerative colitis).

[0031] By “corresponds” is meant comprising at least a fragment of a double- stranded gene, such that a strand of the double-stranded inhibitory nucleic acid molecule is capable of binding to a complementary strand of the gene.

[0032] By “decreases” is meant a reduction by at least about 5% relative to a reference level. A decrease may be by 5%, 10%, 15%, 20%, 25% or 50%, or even by as much as 75%, 85%, 95% or more and any intervening percentages. “Detect” refers to identifying the presence, absence or amount of the analyte(s) to be detected. In embodiments of the disclosure, defective activation of STAT3 is detected upon cytokine stimulation. In another embodiment, a downregulation of IL6 / JAK / STAT3 signaling is detected.

[0033] By “disease” is meant any condition or disorder that damages or interferes with the normal function of a cell, tissue, or organ. Examples of diseases include inflammatory bowel disease, Crohn’s disease, and ulcerative colitis. In particular embodiments, the disease is Very Early Onset Inflammatory Bowel Disease (VEOIBD).

[0034] The term “expression” or “expressed” as used herein in reference to a gene means the transcriptional and / or translational product of that gene. The level of expression of a DNA molecule in a cell may be determined on the basis of either the amount of corresponding mRNA that is present within the cell or the amount of protein encoded by that DNA produced by the cell (Sambrook et al., 1989 Molecular Cloning: A Laboratory Manual, 18.1-18.88). Expression of a transfected gene can occur transiently or stably in a cell. During “transient expression” the transfected gene is not transferred to the daughter cell during cell division. Since its expression is restricted to the transfected cell, expression of the gene is lost over time. In contrast, stable expression of a transfected gene can occur when the gene is cotransfected with another gene that confers a selection advantage to the transfected cell. Such a selection advantage may be a resistance towards a certain toxin that is presented to the cell.

[0035] By "effective amount" is meant the amount of a required to ameliorate the symptoms of a disease relative to an untreated patient. The effective amount of active compound(s) used to practice the present invention for therapeutic treatment of a disease varies depending upon the manner of administration, the age, body weight, and general health of the subject. Ultimately, the attending physician or veterinarian will decide the appropriate amount and dosage regimen. Such amount is referred to as an "effective" amount. In some embodiments, the effective amount is an effective amount of an anti-IL12 and / or anti-IL23-based therapy disclosed herein required to ameliorate the symptoms of an inflammatory bowel disease. In some embodiments, the effective amount is an effective amount of an anti-IL12 and / or anti- IL23-based therapy disclosed herein required to induce remission in the subject.

[0036] The invention provides a number of targets that are useful for the development of highly specific drugs to treat or a disorder characterized by the methods delineated herein. In addition, the methods of the invention provide a facile means to identify therapies that are safe for use in subjects. In addition, the methods of the invention provide a route for analyzing virtually any number of compounds for effects on a disease described herein with high-volume throughput, high sensitivity, and low complexity.

[0037] By “enriched” is meant that a cell, a gene, a molecule or a signaling pathway is more numerous in an environment or under a condition than comparable control genes, molecules, or signaling pathways.

[0038] By "fragment" is meant a portion of a polypeptide or nucleic acid molecule. This portion contains at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the entire length of the reference nucleic acid molecule or polypeptide. A fragment may contain 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 nucleotides or amino acids.

[0039] By “guselkumab” is meant a monoclonal antibody that binds to interleukin IL-23, a cytokine that modulates lymphocyte function, including the T-helper (Th) 17 cell subset. Guselkumab is available under the name Tremfya®. Adult dosage forms and strengths include an injectable solution for subcutaneous (SC) administration: lOOmg / mL (One-Press patient controlled injector) lOOmg / mL (prefilled syringe)

[0040] By “high-throughput sequencing” is meant a sequencing technique that allows for large amounts of nucleic acids to be sequenced. In some embodiments, the high throughput sequencing is RNAseq.

[0041] A “host cell” or “cell” is any prokaryotic or eukaryotic cell that contains either a cloning vector or an expression vector. This term also includes those prokaryotic or eukaryotic cells that have been genetically engineered to contain the cloned gene(s) in the chromosome or genome of the host cell.

[0042] "Hybridization" means hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleobases. For example, adenine and thymine are complementary nucleobases that pair through the formation of hydrogen bonds.

[0043] The terms "isolated," "purified," or "biologically pure" refer to material that is free to varying degrees from components which normally accompany it as found in its native state. "Isolate" denotes a degree of separation from original source or surroundings. "Purify" denotes a degree of separation that is higher than isolation. A "purified" or "biologically pure" protein is sufficiently free of other materials such that any impurities do not materially affect the biological properties of the protein or cause other adverse consequences. That is, a nucleic acid or peptide of this invention is purified if it is substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized. Purity and homogeneity are typically determined using analytical chemistry techniques, for example, polyacrylamide gel electrophoresis or high performance liquid chromatography. The term "purified" can denote that a nucleic acid or protein gives rise to essentially one band in an electrophoretic gel. For a protein that can be subjected to modifications, for example, phosphorylation or glycosylation, different modifications may give rise to different isolated proteins, which can be separately purified.

[0044] By "isolated polynucleotide" is meant a nucleic acid (e.g., a DNA) that is free of the genes which, in the naturally-occurring genome of the organism from which the nucleic acid molecule of the invention is derived, flank the gene. The term therefore includes, for example, a recombinant DNA that is incorporated into a vector; into an autonomously replicating plasmid or virus; or into the genomic DNA of a prokaryote or eukaryote; or that exists as a separate molecule (for example, a cDNA or a genomic or cDNA fragment produced by PCR or restriction endonuclease digestion) independent of other sequences. In addition, the term includes an RNA molecule that is transcribed from a DNA molecule, as well as a recombinant DNA that is part of a hybrid gene encoding additional polypeptide sequence.

[0045] By an "isolated polypeptide" is meant a polypeptide of the invention that has been separated from components that naturally accompany it. Typically, the polypeptide is isolated when it is at least 60%, by weight, free from the proteins and naturally-occurring organic molecules with which it is naturally associated. In embodiments, the preparation is at least 75%, at least 90%, or at least 99%, by weight, a polypeptide of the invention. An isolated polypeptide of the invention may be obtained, for example, by extraction from a natural source, by expression of a recombinant nucleic acid encoding such a polypeptide; or by chemically synthesizing the protein. Purity can be measured by any appropriate method, for example, column chromatography, polyacrylamide gel electrophoresis, or by HPLC analysis.

[0046] By “marker” is meant any protein or polynucleotide having an alteration in expression level or activity that is associated with a disease or disorder. In some embodiments, the markers include IL6, ILlb, IL1R1, CXCL8, MMP9, JAK3, and / or STAT3.

[0047] By “mirikizumab” is meant a monoclonal antibody that binds to interleukin IL-23, a cytokine that modulates lymphocyte function, including the T-helper (Th) 17 cell subset. Mirikizumab is available under the name Omvoh™. Adult dosage forms and strengths include:

[0048] Intravenous (IV) induction dosage

[0049] 20 mg / mL (300 mg / 15 mL single-dose vial)

[0050] Subcutaneous (SC) maintenance dosage

[0051] 100 mg / mL single-dose prefilled pen.

[0052] By “operably linked” refers to a functional linkage between a regulatory sequence and a coding sequence, where a first polynucleotide is positioned adjacent to a second polynucleotide that directs transcription of the first polynucleotide when appropriate molecules (e.g., transcriptional activator proteins) are bound to the second polynucleotide. The described components are therefore in a relationship permitting them to function in their intended manner. For example, placing a coding sequence under regulatory control of a promoter means positioning the coding sequence such that the expression of the coding sequence is controlled by the promoter.

[0053] By “panel” is meant a plurality of agents that bind target molecules, e.g, antibodies that bind marker molecules.

[0054] By “portion” is meant a fragment of a polypeptide or nucleic acid molecule. This portion contains at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the entire length of the reference nucleic acid molecule or polypeptide. A fragment may contain 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 nucleotides.

[0055] By “positioned for expression” is meant that the polynucleotide of the disclosure (e.g., a DNA molecule) is positioned adjacent to a DNA sequence that directs transcription and translation of the sequence (i.e., facilitates the production of, for example, a recombinant microRNA molecule described herein).

[0056] The term “promoter” as used herein refers to a sequence of DNA that directs the expression (transcription) of a gene. A promoter may direct the transcription of a prokaryotic or eukaryotic gene. A promoter may be “inducible”, initiating transcription in response to an inducing agent or, in contrast, a promoter may be “constitutive”, whereby an inducing agent does not regulate the rate of transcription. A promoter may be regulated in a tissue- specific or tissue-preferred manner, such that it is only active in transcribing the operable linked coding region in a specific tissue type or types.

[0057] By “refractoriness” is meant the state of being insensitive or resistant to treatment with a certain therapy. By “RNA-seq” is meant RNA sequencing for detecting and quantifying messenger RNA molecules (mRNA) in a biological sample, which, for example, may be used to study cellular responses. A related term, “scRNA-seq” is single-cell RNA sequencing, which may be, for example, a droplet-based single-cell RNA-seq or “Drop-seq,” that is a sequencing technology for analyzing RNA expression in at least hundreds of thousands of individual cells in embodiments of the disclosure, but may alternatively use any other high-throughput sequencing platform.

[0058] As used herein, “obtaining” as in “obtaining an agent” includes synthesizing, purchasing, or otherwise acquiring the agent.

[0059] "Primer set" means a set of oligonucleotides that may be used, for example, for PCR. A primer set would consist of at least 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 30, 40, 50, 60, 80, 100, 200, 250, 300, 400, 500, 600, or more primers.

[0060] By “reduces” is meant a negative alteration of at least 10%, 25%, 50%, 75%, or 100%.

[0061] By “reference” is meant a standard or control condition or profile. In some embodiments, the reference profile is an expression profile indicating downregulation of IL6 / JAK / STAT3 signaling in a subject. In some embodiments, the reference profile is an expression profile indicating no downregulation of IL6 / JAK / STAT3 signaling in a subject. In some embodiments, the reference profile is an expression profile derived from a subject or subjects who responded to anti-IL12 and / or anti-IL23-based therapies. In some embodiments, the reference profile is an expression profile derived from a subject or subjects who did not respond to anti-IL12 and / or anti-IL23 -based therapies.

[0062] A "reference sequence" is a defined sequence used as a basis for sequence comparison. A reference sequence may be a subset of or the entirety of a specified sequence; for example, a segment of a full-length cDNA or gene sequence, or the complete cDNA or gene sequence. For polypeptides, the length of the reference polypeptide sequence will generally be at least about 16 amino acids, preferably at least about 20 amino acids, more preferably at least about 25 amino acids, and even more preferably about 35 amino acids, about 50 amino acids, or about 100 amino acids. For nucleic acids, the length of the reference nucleic acid sequence will generally be at least about 50 nucleotides, preferably at least about 60 nucleotides, more preferably at least about 75 nucleotides, and even more preferably about 100 nucleotides or about 300 nucleotides or any integer thereabout or therebetween. By “risankizumab” is meant a monoclonal antibody that binds to interleukin IL-23, a cytokine that modulates lymphocyte function, including the T-helper (Th) 17 cell subset. Risankizumab is available under the name Skyrizi®. Adult dosage forms and strengths include:

[0063] SC injectable solutions

[0064] 75mg / 0.83mL (single-dose prefilled syringe) 150mg / mL (single-dose prefilled syringe or pen) 180 mg / 1.2 mL (150 mg / mL) in each single-dose prefilled cartridge 360 mg / 2.4 mL (150 mg / mL) in each single-dose prefilled cartridge (for Crohn disease)

[0065] Each syringe / pen has affixed 27-gauge 0.5-inch needle

[0066] IV injectable solution

[0067] 600mg / 10mL (single-dose vial)

[0068] By "specifically binds" is meant a compound or antibody that recognizes and binds a polypeptide of the invention, but which does not substantially recognize and bind other molecules in a sample, for example, a biological sample, which naturally includes a polypeptide of the invention.

[0069] Nucleic acid molecules useful in the methods of the invention include any nucleic acid molecule that encodes a polypeptide of the invention or a fragment thereof. Such nucleic acid molecules need not be 100% identical with an endogenous nucleic acid sequence, but will typically exhibit substantial identity. Polynucleotides having “substantial identity” to an endogenous sequence are typically capable of hybridizing with at least one strand of a double- stranded nucleic acid molecule. Nucleic acid molecules useful in the methods of the invention include any nucleic acid molecule that encodes a polypeptide of the invention or a fragment thereof. Such nucleic acid molecules need not be 100% identical with an endogenous nucleic acid sequence, but will typically exhibit substantial identity. Polynucleotides having “substantial identity” to an endogenous sequence are typically capable of hybridizing with at least one strand of a double- stranded nucleic acid molecule. By "hybridize" is meant pair to form a double- stranded molecule between complementary polynucleotide sequences (e.g., a gene described herein), or portions thereof, under various conditions of stringency. (See, e.g., Wahl, G. M. and S. L. Berger (1987) Methods Enzymol. 152:399; Kimmel, A. R. (1987) Methods Enzymol. 152:507).

[0070] For example, stringent salt concentration will ordinarily be less than about 750 mM NaCl and 75 mM trisodium citrate, preferably less than about 500 mM NaCl and 50 mM trisodium citrate, and more preferably less than about 250 mM NaCl and 25 mM trisodium citrate. Low stringency hybridization can be obtained in the absence of organic solvent, e.g., formamide, while high stringency hybridization can be obtained in the presence of at least about 35% formamide, and more preferably at least about 50% formamide. Stringent temperature conditions will ordinarily include temperatures of at least about 30° C, more preferably of at least about 37° C, and most preferably of at least about 42° C. Varying additional parameters, such as hybridization time, the concentration of detergent, e.g., sodium dodecyl sulfate (SDS), and the inclusion or exclusion of carrier DNA, are well known to those skilled in the art. Various levels of stringency are accomplished by combining these various conditions as needed. In a preferred: embodiment, hybridization will occur at 30° C in 750 mM NaCl, 75 mM trisodium citrate, and 1% SDS. In a more preferred embodiment, hybridization will occur at 37° C in 500 mM NaCl, 50 mM trisodium citrate, 1% SDS, 35% formamide, and 100 .mu.g / ml denatured salmon sperm DNA (ssDNA). In a most preferred embodiment, hybridization will occur at 42° C in 250 mM NaCl, 25 mM trisodium citrate, 1% SDS, 50% formamide, and 200 pg / ml ssDNA. Useful variations on these conditions will be readily apparent to those skilled in the art.

[0071] For most applications, washing steps that follow hybridization will also vary in stringency. Wash stringency conditions can be defined by salt concentration and by temperature. As above, wash stringency can be increased by decreasing salt concentration or by increasing temperature. For example, stringent salt concentration for the wash steps will preferably be less than about 30 mM NaCl and 3 mM trisodium citrate, and most preferably less than about 15 mM NaCl and 1.5 mM trisodium citrate. Stringent temperature conditions for the wash steps will ordinarily include a temperature of at least about 25° C, more preferably of at least about 42° C, and even more preferably of at least about 68° C. In a preferred embodiment, wash steps will occur at 25° C in 30 mM NaCl, 3 mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, wash steps will occur at 42 C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, wash steps will occur at 68° C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. Additional variations on these conditions will be readily apparent to those skilled in the art. Hybridization techniques are well known to those skilled in the art and are described, for example, in Benton and Davis (Science 196:180, 1977); Grunstein and Hogness (Proc. Natl. Acad. Sci., USA 72:3961, 1975); Ausubel et al. (Current Protocols in Molecular Biology, Wiley Interscience, New York, 2001); Berger and Kimmel (Guide to Molecular Cloning Techniques, 1987, Academic Press, New York); and Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York.

[0072] By "substantially identical" is meant a polypeptide or nucleic acid molecule exhibiting at least 50% identity to a reference amino acid sequence (for example, any one of the amino acid sequences described herein) or nucleic acid sequence (for example, any one of the nucleic acid sequences described herein). Preferably, such a sequence is at least 60%, more preferably 80% or 85%, and more preferably 90%, 95% or even 99% identical at the amino acid level or nucleic acid to the sequence used for comparison.

[0073] Sequence identity is typically measured using sequence analysis software (for example, Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705, BLAST, BESTFIT, GAP, or PILEUP / PRETTYBOX programs). Such software matches identical or similar sequences by assigning degrees of homology to various substitutions, deletions, and / or other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. In an exemplary approach to determining the degree of identity, a BLAST program may be used, with a probability score between e'3and e'100indicating a closely related sequence.

[0074] By "subject" is meant a mammal, including, but not limited to, a human or nonhuman mammal, such as a bovine, equine, canine, ovine, or feline. In some embodiments, a subject is a pediatric subject (e.g., a child under the age of about 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1). In some embodiments, a subject is a subject having an inflammatory bowel disease.

[0075] Ranges provided herein are understood to be shorthand for all of the values within the range. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.

[0076] As used herein, the terms “treat,” treating,” “treatment,” and the like refer to reducing or ameliorating a disorder and / or symptoms associated therewith. It will be appreciated that, although not precluded, treating a disorder or condition does not require that the disorder, condition or symptoms associated therewith be completely eliminated.

[0077] By “ustekinumab” is meant a monoclonal antibody that binds to interleukins (IL)- 12 and IL-23, cytokines that modulate lymphocyte function, including T-helper (Th) 1 and Th 17 cell subsets. See, for example, Benson et al., MAbs. 2011 Nov-Dec; 3(6): 535-545, which is incorporated herein in its entirety. Ustekinumab is available under the name Stelara. Adult dosage forms and strengths include an injectable solution for subcutaneous (SC) administration:

[0078] 45mg / 0.5mL (prefilled syringe or single-dose vial)

[0079] 90mg / mL (prefilled syringe) injectable solution for IV infusion

[0080] 130mg / 26mL (5mg / mL) single-dose vial

[0081] Pediatric dosage forms and strengths include:

[0082] Injectable solution for SC

[0083] • 45mg / 0.5mL (prefilled syringe or single-dose vial)

[0084] • 90mg / mL (prefilled syringe)

[0085] Unless specifically stated or obvious from context, as used herein, the term "or" is understood to be inclusive. Unless specifically stated or obvious from context, as used herein, the terms "a", "an", and "the" are understood to be singular or plural.

[0086] Unless specifically stated or obvious from context, as used herein, the term “about” is understood as within a range of normal tolerance in the art, for example within 2 standard deviations of the mean. About can be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear from context, all numerical values provided herein are modified by the term about.

[0087] The recitation of a listing of chemical groups in any definition of a variable herein includes definitions of that variable as any single group or combination of listed groups. The recitation of an embodiment for a variable or aspect herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.

[0088] Any compositions or methods provided herein can be combined with one or more of any of the other compositions and methods provided herein.

[0089] BRIEF DESCRIPTION OF THE DRAWINGS

[0090] FIG. 1A provides two graphs showing a flow cytometry analysis to assess phosphorylation of STAT3 (pSTAT3) in CD45+ PBMCs from a healthy control (HC, top), example IL10R deficient patient (IL10R, inset), and STAT3-defective patient (P, bottom). Cells were unstimulated, stimulated with IL 10 (25 ng / ml) or IL21 (100 ng / ml) for 20 mins. Representative STAT3- defective patient data shows absent right-shift in pSTAT3 with IL10 and IL21 stimulation. FIG. IB provides a Western blot demonstrating pSTAT3, STAT3, and beta-actin under unstimulated, IL10- or IL21- stimulated conditions in P and HC. The STAT3-defective patient had similar total STAT3 compared to HC, but less pSTAT3 upon IL10 and IL21 stimulation.

[0091] FIG. 2 provides a principal component analysis (PCA) of blood transcriptome data showing that the STAT3-defective and IL10R deficient patients generally clustered together.

[0092] FIG. 3A provides a plot showing hallmark pathway enrichments (negative log unadjusted p-value) in cell-type specific differentially expressed genes (DEGs) from STAT3- defective group (yellow, top row) and ILlOR-deficient groups (green, top row) compared to rest. Genes down-regulated in STAT3-defective Ml macrophages (blue arrow) were enriched in genes defining the IL6- JAK-STAT3 signaling, interferon alpha response, and interferon gamma response pathways (blue boxes), consistent with biochemical studies. FIG. 3B provides a plot showing DEGs from STAT3-defective Ml macrophages projected onto adult colonic Crohn’s disease Bayesian network and extended out a path length of 1. The resulting STAT3- defective subnetwork contains 440 genes, including the known VEOIBD genes STAT3, STAT1, TYMP, and TRIM22. STAT3 was not a DEG but rather appeared in the subnetwork as a “nearest neighbor”. An unbiased analysis revealed the top 3 enriched signaling pathways in this STAT3-defective subnetwork included IL23, IL6, and IL12. This is of significant interest given at least one STAT3-defective patient with disease refractory to anti-TNF and vedolizumab achieved remission with ustekinumab (anti-IL12 / 23).

[0093] FIG. 4 provides a diagram of the JAK / STAT signaling pathway.

[0094] DETAILED DESCRIPTION

[0095] The disclosure features compositions and methods for characterizing and treating VEOIBD in a pediatric subject.

[0096] The invention is based, at least in part, on the discovery of a biochemical and transcriptional signature in blood in 6 patients with refractory very early onset inflammatory bowel disease. Flow cytometry and bulk RNA- sequencing analysis was employed to localize pathway-level dysfunction in VEOIBD patients with unknown molecular basis. The biochemical signature is characterized by defective activation of STAT3 upon stimulation with multiple cytokines. The transcriptional signature is characterized by downregulation of IL6 / JAK / STAT3 signaling. The transcriptional signature was projected onto an adult Crohn’s disease Bayesian network to generate a “STAT3-defective” subnetwork, which was significantly enriched in IL23- and IL12-mediated signaling events. Three of the 6 STAT3- defective patients have been exposed to ustekinumab (antil-IL12 / 23) or risankizumab (anti- IL23) and have had dramatic clinical response / remission after failing multiple other biologies, indicating that the STAT3 defective signature may predict response to anti-IL12 and / or anti-IL-23 treatment. Application of this signature in IBD more broadly could identify a subset of patients who may achieve remission with ustekinumab, risankizumab, or other anti-IL12 and / or anti-IL23-based therapies prior to treatment initiation.

[0097] Accordingly, the disclosure provides compositions and methods for characterizing IBD, including VEOIBD in a pediatric subject.

[0098] JAK / STAT Pathway

[0099] Interleukin-6 (IL-6) is a member of the IL-6-type cytokines family with pro- and antiinflammatory properties. They activate the Janus tyrosine kinase (JAK) family members (JAK1, JAK2, and TYK2), leading to the activation of transcription factors of the signal transducer and activator of transcription (STAT) family. All IL-6-type cytokines potently activate STAT3 and to a minor extent STAT1, while SOCS3 is the primary inhibitor of IL-6 signaling. IL-6 is produced by a wide range of cells including monocytes / macrophages, T cells, endothelial cells, fibroblasts, and hepatocytes. A description of JAK / STAT signalling pathway is provided at FIG. 4. In embodiments of the disclosure, a reduction in JAK / STAT signalling is detected. A description of the JAK / STAT signaling pathway is described, for example by Hu, X., li, J., Fu, M. et al. The JAK / STAT signaling pathway: from bench to clinic. Sig Transduct Target Ther 6, 402 (2021). https: / / doi.org / 10.1038 / s41392-021-00791-l, which is incorporated by reference herein in its entirety.

[0100] IL-6 / IL-6 receptor (IL-6R) interaction activates Janus kinase (JAK) tyrosine kinases leading to phosphorylating the signal transducer and activator of transcription 3 (STAT3) / This interaction can phosphorylate mitogen-activated protein kinases (MAPK) / ERK. The phosphoinositol-3 kinase (PI3K) / AKT pathway is induced by IL-6 following JAK phosphorylation. The PI3K / AKT phosphorylation induces gene expression by activating NF- KB to promote anti-apoptosis. A downregulation of this pathway could include a reduction in MAP / ERK phosphorylation, a reduction in PI3K / AKT phosphorylation, and / or a reduction in NF-KB activation.

[0101] Methods for Detecting Markers

[0102] Any suitable method can be used to detect markers associated with inflammatory bowel disease (e.g., VEOIBD). The methods of the disclosure can be successfully practiced with one or a combination of methods that detect markers. In some embodiments, methods to detect markers further comprise steps to quantify the markers. In some embodiments, the methods disclosed herein detect markers in specific cells, such as immune cells. In some embodiments, the immune cells are conventional T cells, regulatory T cells, natural killer T cells, T helper cells, and T cells characterized by surface antigens (e.g., CD4, CD8, and CD45). In some embodiments, the immune cells are B cells. In some embodiments, the B cells are plasmablasts, B regulatory cells, and B cells characterized by surface antigens (e.g., CD27). In some embodiments, the immune cells are innate immune cells. In some embodiments, the innate immune cells are macrophages, monocytes, dendritic cells, plasmacytoid dendritic cells, CD123+innate cells, natural killer (NK) cells, CD161+natural killer cells, CD127+innate lymphoid cells (ILCs), or CD161+CD127+innate lymphoid cells. In some embodiments, the markers include genes which are differentially expressed by any of the specific cell types listed above. For example, in some embodiments, differentially expressed genes of interest which may be used as markers include IL6, ILlb, IL1R1, CXCL8, MMP9, JAK3, and STAT3.

[0103] Markers can be detected in a sample from a subject having or suspected of having inflammatory bowel disease. In some embodiments, the sample is a blood sample, such as a plasma, whole blood, sample. In some embodiments, the blood sample is a peripheral blood sample. In some embodiments, the sample is a tissue sample, such as a biopsy sample. In some embodiments, the tissue sample comprises mucosa tissue.

[0104] Detection methods include, without limitation, hybridization-based methods, including those employed in mass cytometry (e.g., cytometry time-of-flight (CyTOF)),=a powerful proteomic technique in which antibodies are detectably labeled with rare earth metal isotopes instead of fluorophores. Such labeling allows for combining many different labeled antibodies without significant spillover between detection channels as is more common in visual (e.g., fluorescence-based) detection schemes. Labeled cells are then analyzed using high throughput mass spectrometry to quantify parameters at the single-cell level. In addition to detecting markers on a cell, CyTOF can be used to determine cell types present in a sample.

[0105] In some embodiments, markers may be detected through next generation sequencing techniques. For example, RNAseq may be used in some embodiments to detect markers of the present disclosure.

[0106] Other marker detection methods include, but are not limited to, biochip arrays, fluorescence assays (e.g. sandwich immunoassay), surface plasmon resonance, ellipsometry and atomic force microscopy. Expression levels of markers (e.g., polynucleotides or polypeptides) are compared by procedures well-known in the art, such as RT-PCR, Northern blotting, Western blotting, mass spectrometry, flow cytometry, immunocytochemistry, binding to magnetic and / or antibody-coated beads, in situ hybridization, fluorescence in situ hybridization (FISH), flow chamber adhesion assay, ELISA, microarray analysis, or colorimetric assays. For example, detection of phosphorylation of STAT3 can be performed using anti-phospho antibodies detected by flow cytometry and / or by Western blot.

[0107] Detection methods may include use of a biochip array. Biochip arrays contemplated in the present disclosure include protein arrays. One or more markers are captured on the biochip array and subjected to analysis to detect the level of the markers in a sample. In some embodiments, markers are captured with capture reagents immobilized to a solid support, such as a biochip, a multi- well microtiter plate, a resin, or a nitrocellulose membrane that is subsequently probed for the presence or level of a marker. Capture can be on a chromatographic surface or a biospecific surface. For example, a sample containing the markers, such as serum, is used in some embodiments to contact the active surface of a biochip for a sufficient time to allow binding. Unbound molecules are washed from the surface using a suitable eluent, such as phosphate buffered saline. In general, the more stringent the eluent, the more tightly the proteins must be bound to be retained after the wash.

[0108] Upon capture on a biochip, analytes can be detected by a variety of detection methods selected from, for example, a gas phase ion spectrometry method, an optical method, an electrochemical method, atomic force microscopy and a radio frequency method. In one embodiment, mass spectrometry is used. In some embodiments, the mass spectrometry used is LC-MS / MS. In some embodiments, the mass spectrometry is SELDI. Optical methods include, for example, detection of fluorescence, luminescence, chemiluminescence, absorbance, reflectance, transmittance, birefringence or refractive index (e.g., surface plasmon resonance, ellipsometry, a resonant mirror method, a grating coupler waveguide method or interferometry). Optical methods include microscopy (both confocal and non- confocal), imaging methods and non-imaging methods. Immunoassays in various formats (e.g., ELISA) are popular methods for detection of analytes captured on a solid phase. Electrochemical methods include voltammetry and amperometry methods. Radio frequency methods include multipolar resonance spectroscopy.

[0109] In some embodiments of the methods of the present invention, multiple markers are measured. The use of multiple markers increases the predictive value of the test and provides greater utility in diagnosis, toxicology, patient stratification, and patient monitoring. The process called “pattern recognition” detects patterns formed by multiple markers, which greatly improves the sensitivity and specificity of clinical proteomics for predictive medicine. Subtle variations in data from clinical samples indicate that certain patterns of protein expression indicates the presence or absence of inflammatory bowel disease, ulcerative colitis, or Crohn’s disease.

[0110] Diagnostic Assays

[0111] The present invention provides diagnostic assays for determining whether a subject with inflammatory bowel disease will respond to anti-IL12 and / or anti-IL23-based therapies. Expression levels of markers as well as differential abundance of certain cellular populations described herein are correlated with response in a subject having inflammatory bowel disease to the therapies disclosed herein, such as anti-IL12 and / or anti-IL23-based therapies. These markers can be used to discriminate between responders and non-responders to the therapies disclosed herein. In some embodiments of the invention, a sample (e.g., blood or biopsied tissue) from a subject is analyzed to determine levels of markers associated with response in a subject having inflammatory bowel disease to the therapies disclosed herein, such as anti- IL12 and / or anti-IL23-based therapies. Particular expression profiles are associated with subjects that are likely to respond to the therapies disclosed herein, such as anti-IL12 and / or anti-IL23-based therapies, or with non-responders to these therapies. In some embodiments, levels of the markers are indicative of a subject likely to respond to the therapies disclosed herein. In some embodiments, levels of the markers are indicative of a subject not likely to respond to the therapies disclosed herein. In some embodiments, an expression profile derived from a sample from a subject may be compared with a reference expression profile. The reference expression profile can be a profile from a subject who responded to the therapies disclosed herein, such as anti-IL12 and / or anti-IL23-based therapies, or a sample from a subject who did not respond to the therapies disclosed herein. The reference expression profile can also be derived a plurality of subjects who responded or did not respond to the therapies disclosed herein.

[0112] In some embodiments, diagnostic assays are provided that detect downregulation of IL6 / JAK / STAT3 signaling in a subject, such as a subject having inflammatory bowel disease (e.g., ulcerative colitis, Crohn’s disease). Without intending to be bound by theory, downregulation of IL6 / JAK / STAT3 signaling in a subject was discovered herein to be associated with response to anti-IL12 and / or anti-IL23-based therapies. Accordingly, in some embodiments, provided herein is an expression profile which indicates downregulation of IL6 / JAK / STAT3 signaling, which may be used as a reference expression profile. In some embodiments, an expression profile derived from a sample from a subject having inflammatory bowel disease (e.g., ulcerative colitis, Crohn’s disease) may be compared with the expression profile which indicates downregulation of IL6 / JAK / STAT3 signaling. In some embodiments, the comparison between the expression profile derived from a sample from a subject and the expression profile which indicates downregulation of IL6 / JAK / STAT3 signaling is used to predict whether IL6 / JAK / STAT3 signaling is downregulated in the subject, and / or whether the subject will respond to anti-IL12 and / or anti-IL23-based therapies.

[0113] In some embodiments, diagnostic assays are provided that detect whether a subject having inflammatory bowel disease (e.g., ulcerative colitis, Crohn’s disease) is likely to respond to the therapies disclosed herein, such as anti-IL12 and / or anti-IL23-based therapies. Diagnoses made using the compositions and methods of the present invention enable a caregiver to design an appropriate or optimized therapeutic regimen. Personalized treatment regimens, rather than generic therapies, for inflammatory bowel disease can greatly improve clinical outcome for a subject with the disease.

[0114] In some embodiments, methods are provided for identifying cell types in a sample from a subject having inflammatory bowel disease. Based on the identified cell types, a profile of the sample can be generated for the sample and compared to a reference profile. The reference profile can be the profile of a sample from a subject who responded to the therapies disclosed herein, such as anti-IL12 and / or anti-IL23-based therapies, or a sample from a subject who did not respond to the therapies disclosed herein.

[0115] The cell types that comprise a profile for a subject likely to respond to the therapies disclosed herein, such as anti-IL12 and / or anti-IL23-based therapies, or a subject not likely to respond to the therapies disclosed herein, can be any cell type for which there is a significant difference between the responder types. For example, a profile for a subject likely to respond to the therapies disclosed herein, such as anti-IL12 and / or anti-IL23-based therapies, is provided herein.

[0116] Marker expression can be detected and / or measured at the transcript or protein level by methods well-known in the art. Detecting expressed transcripts in a sample generally involves contacting the sample with a nucleic acid molecule comprising a nucleotide sequence that is at least partially complementary to the nucleic acid sequence of the transcript to be measured under conditions suitable for hybridization. In some embodiments, the nucleic acid molecule is labeled to allow visualization of the molecule after contacting the sample. In some embodiments, the nucleic acid molecule is present on the surface of a substrate. In some embodiments, the substrate comprises a microarray.

[0117] Marker expression can also be detected, and quantified in some embodiments, at the protein level. Antibodies that specifically bind a marker described herein or any other method known in the art can be used to monitor expression of a marker of interest. Detection of an alteration relative to a normal, reference sample can be used as a diagnostic indicator of whether a subject is predicted to be a responder or a non-responder to the therapies disclosed herein. Similarly, detection of alterations or similarities relative to a reference sample of a subject who responded to the therapies disclosed herein, such as anti-IL12 and / or anti-IL23- based therapies, may be used as a diagnostic indicator which predicts whether a subject is a responder or a non-responder to the therapies disclosed herein.

[0118] The methods described herein can be used to diagnose an individual or to confirm the results of another diagnostic method.

[0119] The markers detected using the methods described herein can be used individually to predict whether a subject will respond to the therapies disclosed herein, such as anti-IL12 and / or anti-IL23-based therapies. In some embodiments, the individual markers are used in combination with other identified markers, or with other markers known in the art that are associated with response to the therapies disclosed herein. Individual markers, or combinations thereof, are differentially expressed in a subject and, therefore, differentially present in samples from a subject predicted to respond to the therapies disclosed herein, such as anti-IL12 and / or anti-IL23-based therapies, or predicted to not respond to the therapies disclosed herein.

[0120] Analyzing a combination of markers can provide greater predictive value than a single marker in some cases. Additionally, characterizing a combination of markers can enhance the confidence in observed results. For example, detecting the expression of a plurality of markers in a sample can decrease erroneous conclusions resulting from false positives and negatives. One skilled in the art will understand that the methods disclosed herein can be adjusted or optimized to increase the sensitivity or specificity of the diagnostic assay.

[0121] Also provided herein are panels comprising markers of the present disclosure used to predict whether a subject having inflammatory bowel disease will respond to the therapies disclosed herein, such as anti-IL12 and / or anti-IL23-based therapies. In some embodiments, the panels include one or more of: IL6, ILlb, IL1R1, CXCL8, MMP9, JAK3, and STAT3. In some embodiments, the panels comprising markers of the present disclosure are measured and / or characterized in a subject, such as in a sample of the subject. In some embodiments, multiple panels of markers are measured and / or characterized in a subject.

[0122] In some embodiments, the characterization of a panel of biomarkers in a subject or a sample of the subject determines a score which identifies the subject as being a responder to the therapies disclosed herein, such as anti-IL12 and / or anti-IL23-based therapies, or as a non-responder to the therapies disclosed herein. In some embodiments, a subject identified as a responder to anti-IL12 and / or anti-IL23-based therapies, is further identified as needing anti-IL12 and / or anti-IL23-based therapies, such as ustekinumab, risankizumab, mirikizumab, brazikumab, or guselkumab.

[0123] Methods of Treatment

[0124] The present disclosure provides methods of identifying subsets of patients with inflammatory bowel disease (e.g., VEOIBD) that may respond to a specific type of therapy (e.g., ustekinumab, risankizumab, mirikizumab, brazikumab, or guselkumab). In some embodiments, these methods involve detecting the expression level of at least one marker (e.g., phosphorylated STAT3 (pSTAT3)) as disclosed above to predict whether the subject will respond to a specific type of therapy (e.g., anti-IL12 and / or anti-IL23-based therapies). In some embodiments, the method further involves administering a therapeutically effective amount of a pharmaceutical composition that is suitable for treating the subject’s particular type of inflammatory bowel disease (e.g., STAT3 defective VEOIBD). In some embodiments, the level of at least one marker (or a combination of markers) is determined for a subject before treatment commences. The marker’s pretreatment level can be compared to the marker’s level in the subject during and / or after treatment to monitor treatment efficacy. In some embodiments, the level of the marker is measured by any suitable method as described herein.

[0125] Response to the therapies disclosed herein (e.g., anti-IL12 and / or anti-IL23 -based therapies), includes amelioration of inflammatory bowel disease (e.g., UC, Crohn’s Disease) or any symptom thereof, in a subject having an inflammatory bowel disease. In some embodiments, the response is remission of the inflammatory bowel disease.

[0126] Kits for characterizing markers associated with inflammatory bowel disease

[0127] Some aspects of the present disclosure provide kits for detecting markers, such as a reduction in pSTAT3, associated with response in a subject having an inflammatory bowel disease to therapies disclosed herein (e.g., anti-IL12 and / or anti-IL23-based therapies) in a sample of the subject. The invention provides kits for predicting whether a subject having an inflammatory bowel disease (e.g., VEOIBD, UC, Crohn’s Disease) will respond to the therapies disclosed herein (e.g., anti-IL12 and / or anti-IL23-based therapies).

[0128] In some embodiments, the kit comprises a sterile container. Such containers can be boxes, ampoules, bottles, vials, tubes, bags, pouches, blister-packs, or other suitable container forms known in the art. Such containers can be made of plastic, glass, laminated paper, metal foil, or other materials suitable for holding reagents.

[0129] If desired the kit is provided together with instructions for using the kit to detect the markers of interest (pSTAT3). The instructions will generally include information about the use of the kit for the prediction of response in a subject having inflammatory bowel disease (e.g., VEOIBD, UC, Crohn’s Disease) to the therapies disclosed herein (e.g., anti-IL12 and / or anti-IL23-based therapies). In other embodiments, the instructions include at least one of the following: precautions; warnings; clinical studies; and / or references. The instructions may be printed directly on the container (when present), or as a label applied to the container, or as a separate sheet, pamphlet, card, or folder supplied in or with the container. In a further embodiment, a kit can comprise instructions in the form of a label or separate insert (package insert) for suitable operational parameters. In yet another embodiment, the kit can comprise one or more containers with appropriate positive and negative controls or control samples, to be used as standard(s) for detection, calibration, or normalization.

[0130] The practice of the present invention employs, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are well within the purview of the skilled artisan. Such techniques are explained fully in the literature, such as, “Molecular Cloning: A Laboratory Manual”, second edition (Sambrook, 1989); “Oligonucleotide Synthesis” (Gait, 1984); “Animal Cell Culture” (Freshney, 1987); “Methods in Enzymology” “Handbook of Experimental Immunology” (Weir, 1996); “Gene Transfer Vectors for Mammalian Cells” (Miller and Calos, 1987); “Current Protocols in Molecular Biology” (Ausubel, 1987); “PCR: The Polymerase Chain Reaction”, (Mullis, 1994); “Current Protocols in Immunology” (Coligan, 1991). These techniques are applicable to the production of the polynucleotides and polypeptides of the invention, and, as such, may be considered in making and practicing the invention. Particularly useful techniques for particular embodiments will be discussed in the sections that follow.

[0131] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the assay, screening, and therapeutic methods of the invention, and are not intended to limit the scope of what the inventors regard as their invention.

[0132] EXAMPLES

[0133] Example 1: Characterization of VEOIBD

[0134] VEOIBD patients prospectively enrolled in a biorepository were screened for IL10R deficiency using a flow cytometry assay. Patient peripheral blood mononuclear cells (PBMCs) were stimulated with IL10 and control cytokine IL21 and downstream pSTAT3 activation was measured. RNA-sequencing analysis was performed on 115 whole blood samples, comprising VEOIBD patients with either confirmed (n=35, IL10R deficient n=4) or unknown monogenic causes (n=70) and controls (n=10). Transcriptome analysis focused on patients with abnormal STAT3 activation as determined by flow cytometry.

[0135] 6 patients with refractory IBD were identified who displayed defective phosphorylation of STAT3 upon stimulation with both IL10 and IL21 by flow cytometry (FIG. 1A), a result confirmed by Western blot (FIG. IB). All 6 “STAT3-defective” patients lacked deleterious mutations in known VEOIBD genes (including IL10RA / B and STAT3). STAT3-defective patients tended to have infantile-onset disease (onset <2 years), severe colonic inflammation, and refractoriness to anti-Tumor Necrosis Factor (TNF). Transcriptome analysis in blood identified that estimated Ml macrophages in STAT3-defective patients were enriched in a signature characterized by downregulation of IL6-JAK-STAT3 signaling (p=0.01), consistent with biochemical data (FIG. 3A). Differentially expressed genes in STAT3-defective Ml macrophages were projected onto an adult colonic Crohn’s disease Bayesian network to create a STAT3-defective subnetwork (FIG. 3B). This subnetwork was significantly enriched in IL23, IL6, and IL12 signaling pathways.

[0136] 3 of the patients with refractory IBD who displayed defective phosphorylation of STAT3 were treated with either ustekinumab or risankizumab. Patients receiving ustekinumab were treated with an IV loading dose of 6 mg / kg. They were then initiated on subcutaneous maintenance dosing at 45 mg or 90 mg every 4-8 weeks, per primary clinician's judgement. Patients receiving risankizumab were treated with IV loading doses of 600 mg at weeks 0, 4, and 8. Subcutaneous maintenance dosing at 180 mg or 360 mg was initiated at week 12 and every 8 weeks thereafter.

[0137] Validated disease activity indices, such as Pediatric Crohn's Disease Activity Index (PCD Al) or Pediatric Ulcerative Colitis Activity Index (PUCAI); biomarkers (hemoglobin, ESR, CRP, albumin, calprotectin); and endoscopic re-assessment (where available) were monitored in assessment of response.

[0138] All 3 of the patients that received ustekinumab or Risankizumab achieved remission.

[0139] These results indicate that the STAT3-defective subnetwork identified in this study is of high interest given all STAT3-defective patients exposed to ustekinumab or Risankizumab achieved remission.

[0140] Other Embodiments

[0141] From the foregoing description, it will be apparent that variations and modifications may be made to the invention described herein to adopt it to various usages and conditions.

[0142] Such embodiments are also within the scope of the following claims.

[0143] The recitation of a listing of elements in any definition of a variable herein includes definitions of that variable as any single element or combination (or subcombination) of listed elements. The recitation of an embodiment herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.

[0144] All patents and publications mentioned in this specification are herein incorporated by reference to the same extent as if each independent patent and publication was specifically and individually indicated to be incorporated by reference.

Claims

CLAIMSWhat is claimed is:

1. A method of treating a subject having Inflammatory Bowel Disease (IBD), the method comprising administering an agent selected from the group consisting of: ustekinumab; risankizumab; mirikizumab; brazikumab; and guselkumab, to a subject, wherein the subject is selected as having defective phosphorylation of STAT3 or a reduction in a JAK / STAT signalling pathway in response to IL10 and / or IL21 stimulation.

2. The method of claim 1, wherein phosphorylation of STAT3 is detected by flow cytometry or Western blot.

3. The method of claim 1, wherein the subject is an adult or a pediatric subject.

4. The method of claim 1, wherein the pediatric subject is less than about 10 years of age.

5. The method of claim 1, wherein the pediatric subject is less than about 5 years of age.

6. The method of claim 1, wherein the pediatric subject is less than about 2 years of age.

7. The method of claim 1, wherein the subject displays infantile-onset disease, severe colonic inflammation, and / or refractoriness to anti-Tumor Necrosis Factor (TNF) therapy.

8. The method of claim 1, wherein Ml macrophages of the subject were enriched in a signature characterized by a downregulation of IL6-JAK-STAT3 signaling.

9. The method of claim 8, wherein the downregulation of IL6-JAK-STAT3 signaling comprises a reduction in STAT3 phosphorylation, a reduction in JAK activity, a reduction in MAP / ERK phosphorylation, a reduction in PI3K / AKT phosphorylation, and / or a reduction in NF-KB activation.

10. A panel for assessing downregulation of IL6-JAK-STAT3 signaling in a subject, the panel comprising markers IL6, ILlb, IL1R1, CXCL8, MMP9, JAK3, and / or STAT3.

11. The panel of claim 10, wherein each of the markers specifically bind to a separate capture reagent.

12. The panel of claim 11, wherein the capture reagents are attached to a solid support.

13. The panel of claim 12, wherein the solid support is a plate, chip, beads, microfluidic platform, membrane, planar microarray, or suspension array.

14. The panel of any one of claims 11-13, wherein the capture reagent is an antibody, aptamer, Affibody, hybridization probe and / or fragments thereof.

15. The panel of any one of claims 10-14 for use in a method for treating a selected subject having Inflammatory Bowel Disease (IBD).

16. A kit comprising the panel of any one of claims 10-14, and instructions for use of the panel in the method of any of claims 1-9.

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