WO2025085928A1 - Methods of treating a patient with an inflammatory bowel disease - Google Patents

Abstract

This disclosure relates generally to methods for treating an inflammatory bowel disease ("IBD", e.g., Crohn's disease or ulcerative colitis) in a patient. More particularly, this disclosure relates to methods for selecting a therapy for treating a patient suffering from an IBD. In embodiments, the foregoing can also be used to assess the severity of the IBD. The predictive aspects of said methods can facilitate and expedite the identification and stratification of IBD patient populations that are responsive to treatment with a RIPK2 inhibitor. The foregoing methods can further include treating the IBD by administering a RIPK2 inhibitor to the patient.

Description

METHODS OF TREATING A PATIENT WITH AN INFLAMMATORY BOWEL DISEASE

This application claims the benefit of United States Provisional Application No. 63/545,100, filed on October 20, 2023, which is incorporated herein by reference in its entirety.

TECHNICAL FIELD

This disclosure relates generally to methods for treating an inflammatory bowel disease (“IBD”, e.g., Crohn’s disease or ulcerative colitis) in a patient. More particularly, this disclosure relates to methods for selecting a therapy for treating a patient suffering from an IBD. In one aspect, this disclosure features methods for predicting whether a patient suffering from a suspected, diagnosed, or previously diagnosed IBD will be eligible for treatment with a RIPK2 inhibitor. In some embodiments, the methods include determining whether the IBD is a RIPK2 inhibitorsensitive IBD, e.g., by (i) measuring an expression level of one or more RIPK2- associated biomarkers (e.g., a gene signature set or subset thereof) in a sample obtained from the patient; (ii) comparing the expression level determined in (i) with a predetermined reference level; and (iii) determining that the patient will be eligible for treatment with a RIPK2 inhibitor when the level determined in step (i) is higher than the predetermined reference level. In embodiments, the foregoing can also be used to assess the severity of the IBD. The predictive aspects of the methods can facilitate and expedite the identification and stratification of IBD patient populations that are responsive to treatment with a RIPK2 inhibitor. The foregoing methods can further include treating the IBD by administering a RIPK2 inhibitor to the patient.

BACKGROUND

Ulcerative colitis (UC) and Crohn's disease (CD) are the predominant chronic, inflammatory bowel diseases in humans. These disorders are autoimmune in nature and occur in the absence of infection. IBD effects up to 2,000,000 Americans (increasing -15% annually) and it is associated with unacceptably high rates of morbidity and mortality. IBD is also a significant burden on the U.S. health care system as the most effective treatments are biological drugs that are quite costly. IBD occurs as the result of inappropriate immune responses in genetically susceptible individuals mediated by complex interactions between environmental stimuli, microbial factors, and the intestinal immune system. The hallmark of IBD is represented either by excessive immune responses that mediate gastrointestinal tissue damage, directly or through the release of soluble, pro-inflammatory mediators.

Although different forms of IBD show pathophysiological and clinical differences, the therapeutic approach to managing IBD shares many common elements. Medical management of IBD is largely empirical, employing anti-inflammatory or immunosuppressive drugs. Salicylazosulfapyridine and 5-aminosalicylic acid are used to treat mild IBD and as maintenance therapy if disease remission can be achieved; however, these therapies have, in general, been shown not to be effective in patients with moderate to severe disease.

NODI and NOD2 (nucleotide-binding oligomerization domains 1 and 2) are members of the NOD-like receptor (NLR) family, which represent important components of the mammalian innate immune system, serving as intracellular receptors for peptidoglycan (PGN), a component of bacterial cell walls. NODI and NOD2 detect the presence of intracellular bacteria by binding to PGN fragments. Heredity polymorphisms in the genes encoding NODI and NOD2 have been associated with inflammatory disorders. Once activated, NOD signaling leads to activation of NF-kB and MAP kinases, resulting in the transcription of pro- inflammatory kinases and the induction of autophagy.

NODI and NOD2 require RIPK2 as a common scaffolding (adaptor) protein to propagate downstream signals that lead to aberrant proinflammatory innate immune activation. In particular, RIPK2 is critical for NF-kB activation and subsequent cytokine production. Inhibition of RIPK2 resolves abnormal inflammation states such as intestinal inflammation. Accordingly, inhibitors of RIPK2 have potential to act as therapeutic agents, for example, to reduce or resolve inflammation for inflammatory disorders such as inflammatory bowel disease (including Crohn's disease and ulcerative colitis).

SUMMARY

This disclosure relates generally to methods for treating an inflammatory bowel disease (“IBD”, e.g., Crohn’s disease or ulcerative colitis) in a patient. More particularly, this disclosure relates to methods for selecting a therapy for treating a patient suffering from an IBD. In one aspect, this disclosure features methods for predicting whether a patient suffering from a suspected, diagnosed, or previously diagnosed IBD will be eligible for treatment with a RIPK2 inhibitor. In some embodiments, the methods include determining whether the IBD is a RIPK2 inhibitorsensitive IBD, e.g., by (i) measuring an expression level of one or more RIPK2- associated biomarkers (e.g., a gene signature set or subset thereof) in a sample obtained from the patient; (ii) comparing the expression level determined in (i) with a predetermined reference level; and (iii) determining that the patient will be eligible for treatment with a RIPK2 inhibitor when the level determined in step (i) is higher than the predetermined reference level. In embodiments, the foregoing can also be used to assess the severity of the IBD. The predictive aspects of said methods can facilitate and expedite the identification and stratification of IBD patient populations that are responsive to treatment with a RIPK2 inhibitor. The foregoing methods can further include treating the IBD by administering a RIPK2 inhibitor to the patient.

In one aspect, this disclosure features methods for predicting whether a patient suffering from an inflammatory bowel disease will be eligible for treatment with a RIPK2 inhibitor. The methods include:

(a) measuring an expression level of a plurality of biomarkers in a sample obtained from the patient, wherein the expression level of each of the biomarkers is increased by activation of RIPK2;

(b) comparing each of the biomarker expression levels determined at step (a) with a corresponding predetermined reference value; and

(c) identifying that the patient is eligible for treatment with the RIPK2 inhibitor when one or more of the expression levels determined at step (a) are higher than the one or more of the corresponding predetermined reference values.

In some embodiments, the methods further include (d) identifying that the patient is not eligible for treatment with the RIPK2 inhibitor when one or more of the expression levels determined at step (a) are lower than the one or more of the corresponding predetermined reference values. In another aspect, this disclosure features methods for treating an inflammatory bowel disease with a RIPK2 inhibitor in an eligible patient in need thereof. The methods include:

(a) measuring an expression level of a plurality of biomarkers in a sample obtained from the patient, wherein the expression level of each of the biomarkers is increased by activation of RIPK2;

(b) comparing each of the biomarker expression levels determined at step (a) with a corresponding predetermined reference value;

(c) identifying that the patient is eligible for treatment with the RIPK2 inhibitor when one or more of the expression levels determined at step (a) are higher than the one or more of the corresponding predetermined reference values; and

(d) administering a therapeutically effective amount of a RIPK2 inhibitor to the patient identified as being eligible for treatment in step (c).

In some embodiments, the methods further include (e) identifying that the patient is not eligible for treatment with the RIPK2 inhibitor when one or more of the expression levels determined at step (a) are lower than the one or more of the corresponding predetermined reference values; and not administering a therapeutically effective amount of a RIPK2 inhibitor to the patient.

In a further aspect, this disclosure features kits for use in predicting whether a patient suffering from an inflammatory bowel disease will be eligible for treatment with a RIPK2 inhibitor. In some embodiments, the kits include one or more of the following: an assay for a preselected gene signature set, primers for a preselected gene signature set, buffers and positive and negative controls and instructions for use.

In one aspect, this disclosure features methods that include:

(a) providing a biological sample from a patient suffering from an inflammatory bowel disease; and

(b) measuring the expression in the biological sample of at least 4 biomarkers selected from (Gene Set I):

In some embodiments, the methods include determining the expression levels of at least or exactly 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, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, or 111 of the biomarkers (Gene Set I).

In certain embodiments, the methods include determining the expression levels of at least 7, 11, or 30 of the biomarkers (Gene Set I).

In certain embodiments, the biomarkers are selected from (Gene Set II):

In certain embodiments, the methods include determining the expression levels of at least or exactly 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, or 30 of the genes listed in Gene Set II. In certain embodiments, the methods include determining the expression levels of at least 7, 11, or 30 of the genes listed in Table 2 Gene Set II.

In certain embodiments, the methods include determining the expression levels of at least 4 biomarkers selected from (Gene Set III):

In certain embodiments, the methods include determining the expression levels of at least or exactly 4, 5, 6, 7, 8, 9, 10, 11 of the biomarkers listed in Gene Set III.

In certain embodiments, the methods include determining the expression levels of at least or exactly 7 or 11 of the biomarkers listed in Gene Set III.

In certain embodiments, the methods include determining the expression levels of at least or exactly 7 or 11 of the biomarkers listed in Gene Set IV.

Gene Set 4

In certain embodiments, the methods include determining the expression levels of at least or exactly 1, 2, 3, 4, 5, 6, or 7 of the biomarkers in Gene Set IV.

In certain embodiments, the inflammatory bowel disease is Crohn’s disease.

In certain embodiments,, wherein the inflammatory bowel disease is ulcerative colitis. In certain embodiments, the sample is an intestinal biopsy.

In certain embodiments,, wherein the sample is a rectal biopsy.

In certain embodiments,, wherein the sample is an ileal biopsy.

In certain embodiments, the sample includes a myeloid cell.

In certain embodiments, the sample includes a fibroblast.

In certain embodiments, the sample includes an inflammatory monocyte.

In certain embodiments, the patient is a non-responder to anti-TNF therapy.

In certain embodiments, the patient is a non-responder to anti-Integrin therapy.

In certain embodiments, the patient is a non-responder to anti-IL-23 therapy.

In certain embodiments, measuring the expression level the biomarkers comprises measuring an amount of mRNA.

In certain embodiments, measuring the expression level the biomarkers comprises performing one of more of the following assays: immunohistochemistry, ELISA, Western blot, or immunoprecipitation.

In certain embodiments, the methods further include calculating a RIPK2 gene signature score.

In certain embodiments, the methods further include determining a gene set variation analysis score based on the determined expression levels.

In certain embodiments, the patient is being treated or had previously been treated with an anti-TNF therapy.

In certain embodiments, the patient is being treated or had previously been treated with an anti-Integrin therapy.

In certain embodiments, the patient is being treated or had previously been treated with an anti-IL-23 therapy.

In certain embodiments, the patient is refractory to treatment with 5-ASA.

In certain embodiments, the methods further include treating the patient with a RIPK2 inhibitor.

In certain embodiments, the methods further include the RIPK2 inhibitor is selected from compounds 1-622.

In certain embodiments, the methods further include administering a therapeutically effective amount of a second agent. In certain embodiments, the second agent is an anti-inflammatory agent or an anti-autoimmune agent.

In certain embodiments, the second agent is selected from anti-TNF agent, anti-IL-23 agent, anti-integrin agent, and JAK inhibitor.

As used herein, the term “sample” refers to any substance of biological origin. Examples of samples includes, but are not limited to, substances obtained from an intestinal biopsy, e.g., a rectal biopsy or an ileal biopsy. Substances can include, without limitation, whole blood, e.g., purified components of whole blood. In some embodiments, the sample includes one or more myeloid cells, one or more fibroblasts, one or more inflammatory monocytes, or any combination thereof.

The term “measuring an expression level” as used herein refers to the application of a gene specific reagent such as a probe, primer or antibody and/or a method to a sample, for ascertaining or measuring quantitatively, semi-quantitatively or qualitatively the amount of a gene or genes that are present. As the skilled person will appreciate, the quantitative, semi-quantitative or qualitative measurement of the amount of a gene or genes that are present can be determined via the detection and quantification of mRNA. In some embodiments, the methods further include isolating RNA from the sample. In certain aspects, “measuring an expression level” can include preforming RT-PCR, a hybridization, transcriptome analysis, RNAseq, single cell RNAseq, a Northern blot, a Western blot, immunohistochemistry, or an ELISA. For example, “measuring an expression level” can include performing an array hybridization. In one aspect, transcriptome analysis can include obtaining sequence information of expressed RNA molecules.

Other methods of mRNA detection and quantification can also be applied, e.g., mRNA in situ hybridization or spatial transcriptomics in formalin-fixed, paraffin- embedded (FFPE) tissue samples or cells. (QuantiGene®ViewRNA (Affymetrix)), which uses probe sets for each mRNA that bind specifically to an amplification system to amplify the hybridization signals; these amplified signals can be visualized using a standard fluorescence microscope or imaging system. TaqMan probe-based gene expression analysis (PCR-based) can also be used for measuring gene expression levels in samples, and for example for measuring mRNA levels in FFPE samples. In brief, TaqMan probe-based assays utilize a probe that hybridizes specifically to the mRNA target. This probe contains a quencher dye and a reporter dye (fluorescent molecule) attached to each end, and fluorescence is emitted only when specific hybridization to the mRNA target occurs. During the amplification step, the exonuclease activity of the polymerase enzyme causes the quencher and the reporter dyes to be detached from the probe, and fluorescence emission can occur. This fluorescence emission is recorded and signals are measured by a detection system; these signal intensities are used to calculate the abundance of a given transcript (gene expression) in a sample.

The terms “treat,” “treating,” and “treatment,” mean alleviating or abrogating a disorder, disease, or condition, or one or more of the symptoms associated with the disorder, disease, or condition; or to slowing the progression, spread or worsening of a disease, disorder or condition or of one or more symptoms thereof. In some embodiments, the beneficial effects that a subject derives from a therapeutic agent do not result in a complete cure of the disease, disorder or condition.

As used herein, the term “predicting” refers to a probability or likelihood for a patient to respond to the treatment with a RIPK2 inhibitor. As used herein, the term “responsiveness” and the like refer to the ability to assess the likelihood that treatment will or will not be clinically effective.

As used herein, the term “predetermined reference level” refers to the expression levels of one or more RIPK2-associated biomarkers (e.g., a gene signature set or subset thereof) in samples obtained from the general population or from a selected population of IBD patients (e.g., a non-inflamed IBD patient). A “predetermined reference level” may be determined, for example, by determining the expression level of one or more RIPK2-associated biomarkers (e.g., a gene signature set or subset thereof) in a corresponding sample obtained from one or more control subject(s). When such a predetermined reference level is used, a higher or increased levels determined in a sample (i.e. a test sample obtained from the patient) is indicative for example that said patient is eligible to a treatment with a RIPK2 inhibitor. The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features and advantages of the disclosure will be apparent from the description and drawings, and from the claims.

DESCRIPTION OF DRAWINGS

FIG. 1A is a flow diagram of the workflow summarizing the process for selecting the genes included in the signature.

FIG. IB is a boxplot of the Gene Set Variation Analysis (GSVA) scores derived from a set of 111 genes (Gene Set I). These genes were selected as being muramyl dipeptide (MDP) upregulated and subsequently downregulated in a dosedependent fashion when co-treated with a small molecule RIPK2 scaffolding inhibitor 1 (a compound that inhibits binding of RIPK2 to XIAP and has a RIPK2 IC50 < 2.5 nM

). Each box indicates the GSVA scores at the indicated dose of the small molecule RIPK2 scaffolding inhibitor + MDP, MDP alone or vehicle.

FIG. 1C is a boxplot representing upregulation of the 30 gene signature (Gene Set II) across intestinal biopsies from patients with inflammatory bowel disease in the training set (RISK cohort, rectal biopsies, GSE116925; Oxford cohort, colonic biopsies, GSE117993) used to derive Gene Set II (UC = Ulcerative Colitis; eCD = colon only Crohn’s Disease; iCD = ileocolonic Crohn’s Disease) in comparison to healthy tissue. P -values are calculated with a Mann Whitney U-test

FIG. ID is a boxplot representing upregulation of the 30 gene signature (Gene Set II) in intestinal biopsies from patients with inflammatory bowel disease (UC = Ulcerative Colitis; CD = Crohn’s Disease) across disease severities (UC, Mayo Endoscopic Score; CD, simple endoscopic score, SES-CD) in comparison to healthy tissue (GSE193677). The dashed line indicated 99^th percentile for healthy distribution (t-test) and is used to assess pathway activation percentages in each category (GSVA cutoff for the panel of UC=.46 and CD=.42). P-values are calculated with a Mann Whitney U-Test. The signature is upregulated across multiple, independent datasets (data not shown; GSE57945, GSE109142, GSE59071, GSE111889)

FIG. IE is a heatmap that shows enhancement in differentiate of disease severity provided by the N=7, 11, and 30 gene RIPK2 biomarkers signatures (Gene Sets IV, III, II) is greater than enhancement in differentiate of disease severity provided by the N=lll (Gene Set I) gene signature for both UC and CD patients (GSE193677)

FIG. IF is a receiver operator curve indicating that elevated RIPK2 gene signature scores (Gene Set II) in UC and CD intestinal biopsies prior to treatment predict non-response to Infliximab (anti-TNF; CD, GSE16879 and UC, GSE12251), Vedolizumab (anti-integrin; UC, GSE73661), and Ustekinumab (anti-IL23; UC, GSE206285 and CD, GSE207022). Vedolizumab patients are pre-treated with and resistant to Infliximab. The area under the curve, AUC, for each therapy-disease pair is shown. An AUC of 1 indicates a perfect predictor and an AUC of 0.5, shown by the dashed line, indicates no predictive power. All therapy-disease pairs show significant p-values (p< 05), calculated with a ROC analysis.

FIG 1G is a boxplot indicating the RIPK2 signature (Gene Set II) is elevated in UC non-responders (NR) to Infliximab 4-6 weeks post-treatment (left, GSE73661; anti- TNF), and in UC NR to Vedolizumab 52 weeks post-treatment (right, GSE73661; anti- integrin). Vedolizumab patients are pre-treated with and resistant to Infliximab. P- values are calculated with a Mann-Whitney U-Test.

FIG. 1H is a UMAP representation from single cell sequencing of myeloid cells isolated from mucosal biopsies taken from inflamed (right, active disease) and uninflamed (middle) intestinal tissue from CD patients or biopsies from healthy controls (left) (Single Cell Portal: SCP1884). Cells are down-sampled to equalize cell number across panels. Average RIPK2 gene signature expression across Gene Set II (log2 TPM) shows activation of RIPK2 in inflammatory monocytes that are recruited to inflamed tissue as well as other myeloid cell populations as indicated.

FIG II is barplot indicating RIPK2 scaffolding inhibitor 2 (a compound that inhibits binding of RIPK2 to XIAP and has a RIPK2 IC50 < 2.5 nM ) can reduce expression of the RIPK2 gene signature (Gene Set II) in ex-vivo treated human intestinal mucosal biopsies from IBD patients with active disease. In addition to the RIPK2 gene signature, additional gene sets similarly evaluated included those that describe inflammatory monocytes [1], TNF-mediated inflammatory pathway (KEGG), ligand-receptor signaling (IL-6, LIF, and OSM), inflammatory fibroblasts [1] and a gene set associated with Crohn’s disease penetrating fibrosis [2], Barplots indicate fold change in expression of gene sets between treatment with RIPK2 scaffolding inhibitor 2 versus control (DMSO). P-values are calculated with permutation test. Error bars indicate standard deviation, and dashed line indicates 1.5- fold reduction for each plot.

FIG. 1J is a receiver operator curve indicating an 11 gene signature (Gene Set III; orange) and a 7 gene signature (Gene Set IV; red) both equally and powerfully predict non-response to anti-TNF therapy, Infliximab (IFX) across 3 different publicly available datasets in comparison to a 30 gene signature (Gene Set II; blue) (GSE16879, iCD, left; GSE73661, UC, middle; GSE12251, UC, right). The dashed diagonal line indicates no predictive capacity (ROC=.5)

FIG. 2 shows the chemical structure of type I RIPK2 inhibitors.

FIG. 3 shows the chemical structure of type II RIPK2 inhibitors.

FIG. 4 shows exemplary PROTACs for degradation of RIPK2.

DETAILED DESCRIPTION

This disclosure relates generally to methods for treating an inflammatory bowel disease (“IBD”, e.g., Crohn’s disease or ulcerative colitis) in a patient. More particularly, this disclosure relates to methods for selecting a therapy for treating a patient suffering from an IBD. In one aspect, this disclosure features methods for predicting whether a patient suffering from a suspected, diagnosed, or previously diagnosed IBD will be eligible for treatment with a RIPK2 inhibitor. In some embodiments, the methods include determining whether the IBD is a RIPK2 inhibitorsensitive IBD, e.g., by (i) determining an expression level of one or more RIPK2- associated biomarkers (e.g., a gene signature set or subset thereof) in a sample obtained from the patient; (ii) comparing the expression level determined in (i) with a predetermined reference level; and (iii) determining that the patient will be eligible for treatment with a RIPK2 inhibitor when the level determined in step (i) is higher than the predetermined reference level. In embodiments, the foregoing can also be used to assess the severity of the IBD. The predictive aspects of said methods can facilitate and expedite the identification and stratification of IBD patient populations that are responsive to treatment with a RIPK2 inhibitor. The foregoing methods can further include treating the IBD by administering a RIPK2 inhibitor to the patient.

In some embodiments, the plurality of biomarkers is a plurality of genes. In certain embodiments, the plurality of genes is a preselected gene signature set.

In some embodiments, the RIPK2 gene signature is the gene signature shown in Table 1 (Gene Set I).

Table 1

In certain of the foregoing embodiments, the methods include determining the expression levels of at least 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, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, or 111 of the genes listed in Table 1 (Gene Set I).

For example, the methods can include determining the expression levels of at least 7, 11, or 30 of the genes listed in Table 1 (Gene Set I). In some embodiments, the RIPK2 gene signature is the gene signature shown in

Table 2 (Gene Set II). Table 2

In certain of the foregoing embodiments, the methods include determining the expression levels of at least 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, or 30 of the genes listed in Table 2 (Gene Set II).

For example, the methods can include determining the expression levels of at least 7, 11, or 30 of the genes listed in Table 2 (Gene Set II).

In some embodiments, the RIPK2 gene signature is the gene signature shown in

Table 3 (Gene Set III).

Table 3

In certain of the foregoing embodiments, the methods include determining the expression levels of at least 4, 5, 6, 7, 8, 9, 10, or 11 of the genes listed in Table 3 (Gene Set III)

For example, the methods can include determining the expression levels of at least 7 or 11 of the genes listed in Table 3 (Gene Set III).

In some embodiments, the RIPK2 gene signature is the gene signature shown in Table 4 (Gene Set IV). Table 4

For example, the methods can include determining the expression levels of at least 1, 2, 3, 4, 5, 6, or 7 of the genes listed in Table 4 (Gene Set IV).

In some embodiments, the inflammatory bowel disease is Crohn’s disease. In certain embodiments, the inflammatory bowel disease is moderate Crohn’s disease. In certain embodiments, the inflammatory bowel disease is severe Crohn’s disease.

In some embodiments, the inflammatory bowel disease is ulcerative colitis. In certain embodiments, the inflammatory bowel disease is moderate ulcerative colitis. In certain embodiments, the inflammatory bowel disease is severe ulcerative colitis.

In some embodiments, the patient is suffering from an additional condition or co-morbidity. For example, the additional condition or co-morbidity can be perianal disease. As another example, the additional condition or co-morbidity can be extraintestinal manifestation.

In some embodiments, the sample is an intestinal biopsy. In certain embodiments, the sample is a rectal biopsy. In other embodiments, the sample is an ileal biopsy.

In certain embodiments, the sample includes a myeloid cell.

In certain embodiments, the sample includes a fibroblast.

In certain embodiments, the sample includes an inflammatory monocyte.

In certain embodiments, the sample includes a neutrophil. In some embodiments, the patient identified in step (c) is a non-responder to anti-TNF therapy.

In some embodiments, the patient is being treated or had previously been treated with an anti-TNF therapy.

In some embodiments, the patient identified in step (c) is a non-responder to anti-Integrin therapy.

In some embodiments, the patient is being treated or had previously been treated with an anti-Integrin therapy.

In some embodiments, measuring the expression level of a plurality of biomarkers includes measuring an amount of mRNA.

In certain embodiments, measuring the expression level of a plurality of biomarkers comprises performing one of more of the following assays: immunohistochemistry, ELISA, Western blot, or immunoprecipitation.

In some embodiments, each of the corresponding predetermined reference values is obtained from one or more non-IBD patients.

In some embodiments, each of the corresponding predetermined reference values is obtained from one or more non-inflamed IBD patients.

In some embodiments, the expression level determined in step (i) is 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more higher than the predetermined reference level; or is 1-fold, 2-fold, 3-fold, 4-fold 5, 6, 7, 8, 9, 10, or 15-fold or more.

In some embodiments, the method further includes calculating a RIPK2 gene signature score.

In another method, the gene expression values may be used to generate a weighted “signature score.” For example, each relative gene fold change may be multiplied by the coefficient from the gene signature and then the sum of these values taken as a signature score. This score may then be normalized to the sum of the absolute values of the coefficients. In some embodiments, the expression level of the plurality of biomarkers is higher in a rectal biopsy sample obtained from the patient than in an ileal biopsy sample obtained from the patient.

In certain of these embodiments, the patient is suffering from ulcerative colitis.

In some embodiments, the patient is refractory to treatment with 5-ASA. In certain of these embodiments, the patient is suffering from ulcerative colitis; e.g., the ulcerative colitis can be moderate ulcerative colitis; or the ulcerative colitis can be severe ulcerative colitis; or the ulcerative colitis can be moderate to severe ulcerative colitis.

In some embodiments, the patient is being treated with, has previously been treated with, or is refractory to an anti-TNF therapy.

In some embodiments, the patient is being treated with, has previously been treated with, or is refractory to an anti-Integrin therapy.

RIPK2 inhibitors

RIPK2 inhibitors can be selected as desired, e.g., RIPK2 scaffolding inhibitors.

A variety of RIPK2 inhibitors have been reported in the literature (Table 5). In particular, quinazoline-based GSK2983559 exhibits kinome-wide selectivity and, after optimization to a phosphate ester prodrug, entered a clinical trial in humans (NCT03358407) against inflammatory bowel disease (IBD). Some FDA-approved drugs have been identified as pan-RIPK antagonists, such as Ponatinib and Sorafenib. These inhibitors can be divided into two categories: Type I kinase inhibitors and Type II kinase inhibitors. Type I kinase inhibitors include ATP-competitors that occupy the ATP pocket of targeting kinase by mimicking the purine ring of ATP. Typically, type I kinase inhibitors contain a heterobicyclic aromatic ring that binds to the purine binding site within the active conformational state. Compounds that fall into this category are shown in FIG. 2, and include erlotinib, gefitinib, adezmapimod (SB203580) as well as gefitinib, adezmapimod, and Compound 1, reported by researchers at GlaxoSmithKline (Charnley, 2015). Type II kinase inhibitors target the inactive conformation of kinases by interacting with the catalytic site of unphosphorylated proteins. In the inactive conformation, the DFG motif of the kinase is directed away from the ATP -binding site. The exclusivity of inactive kinase conformations renders type II kinase inhibitors more selective than type I. Compounds that fall into this category are shown in FIG. 3, and include sorafenib, ponatinib, regorafenib, and compound 2, a sorafenib analog reported by Haile et al., 2016 as a screening hit (RIPK2 FP IC₅o = 794 nM).

An additional approach to modulating RIPK2 is Proteolysis-Targeting Chimera (PROTAC) compounds that hijack the ubiquitin-proteasome system (UPS) to degrade the proteins of interest, in this case RIPK2. PROTACs are heterobifunctional molecules that simultaneously bind to a POI and an E3 ligase, which leads to ubiquitination of the POI and its subsequent degradation by UPS. After the POI is degraded, PROTAC is recycled and continues to bind to a new copy of the POI. Exemplary PROTACs targeting RIP2K as reported by Bondenson and collegues (2015, Nat. Chem. Biol. 11, 611-617) are shown in FIG. 4.

Table 5. Exemplary RIPK2 inhibitors from the literature

Table 6. Exemplary RIPK2 inhibitor series

In some embodiments, the RIPK2 inhibitor is selected from any one or more of the compounds listed in Table 1, or a pharmaceutically acceptable salt thereof. In some embodiments, the RIPK2 inhibitor is selected from any one or more of the compounds listed in FIG. 3, or a pharmaceutically acceptable salt thereof. In some embodiments, the RIPK2 inhibitor is selected from any one or more of the compounds disclosed in European Journal of Medicinal Chemistry 260 (2023) 115717, i.e., 16a-16m, 17a-17i, 17k, 17m, 18an, 18ao, 18ap, 18cn, 18mn, 20a-20d, and 2 la-2 Id, or a pharmaceutically acceptable salt thereof. In some embodiments, the RIPK2 inhibitor can be any one or more of the compounds having formula (I), which are disclosed and claimed in WO2020132384, which is incorporated herein by reference in its entirety. Compounds include, but are not limited to, compounds 1-1 to I-122-ii in WO2020132384, which claims compounds of formula (I’):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the RIPK2 inhibitor can be any one or more of the compounds having formula (I), which are disclosed and claimed in WO2018052772, which is incorporated herein by reference in its entirety. Compounds include, but are not limited to, compounds 1-101 in WO2018052772, which claims compounds of formula (I):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the RIPK2 inhibitor can be any one or more of the compounds having formula (I), which are disclosed and claimed in WO2018052773, which is incorporated herein by reference in its entirety. Compounds include, but are not limited to, compounds 1-176 in WO2018052773, which claims compounds of formula (I):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the RIPK2 inhibitor can be any one or more of the compounds having formula (I), which are disclosed and claimed in USSN 63/427,317, filed on November 22, 2022, which is incorporated herein by reference in its entirety. Compounds include, but are not limited to, compounds 1-461 in 63/427,317, which claims compounds of formula (I):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the RIPK2 inhibitor can be any one or more of the compounds having formula (I), which are disclosed and claimed in USSN 63/443,760, filed on February 07, 2023, which is incorporated herein by reference in its entirety. Compounds include, but are not limited to, compounds 1-461 in 63/443,760, which claims compounds of formula (I):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the RIPK2 inhibitor can be any one or more of the compounds having formula (I), which are disclosed and claimed in USSN 63/468,591, filed on May 24, 2023, which is incorporated herein by reference in its entirety. Compounds include, but are not limited to, compounds 1-622 in 63/468,591, which claims compounds of formula (I):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the RIPK2 inhibitor can be any one or more of the compounds having formula (I), which are disclosed and claimed in WO 2024/112854, filed on November 22, 2023, which is incorporated herein by reference in its entirety. Compounds include, but are not limited to, compounds 1-622 of WO 2024/112854 which claims compounds of formula (I) or a pharmaceutically acceptable salt thereof:

wherein: R^la, R^lb, and R^lc are each independently selected from H, Ci-6 alkyl, halogen, CN, and;

R² is H or Ci-3 alkyl;

R³ is selected from S(=O)2R⁵, halogen, 4- to 10-membered heterocyclyl, 5-12 membered heteroaryl, S(=O)(=NR⁶)(R⁷), QR⁷, C(=O)NR⁸R⁹, NH(C=O)R⁵, CN, NR⁸R⁹, P(=O)R^8aR^9a;

R⁴ is selected from H, halogen, Ci-6 alkyl, and Ci-6 alkoxy;

R⁵ is selected from Ci-6 alkyl, NR¹⁰Rⁿ, C3-6 cycloalkyl, and 4- to 10-membered heterocyclyl;

R⁶ is selected from H, CN, and C1-6 alkyl;

R⁷ is selected from C1-6 alkyl, C3-6 cycloalkyl, and 4- to 10-membered heterocyclyl, 5- 12 membered heteroaryl, or

R⁶ and R⁷ taken together with the nitrogen and sulfur atoms to which they are attached form 4- to 10-membered heterocyclyl;

Q is selected from O, S, -S(=O)-, and -C(=O)-;

R⁸ and R⁹ are each independently selected from H, C1-6 alkyl, C1-6 deuteroalkyl, C3-6 cycloalkyl, and 4- to 10-membered heterocyclyl, or

R⁸ and R⁹ taken together with the nitrogen atom to which they are attached form 4- to 10-membered heterocyclyl;

R^8a and R^9b are each independently C1-6 alkyl, or

R^8a and R^9a taken together with the phosphorus atom to which they are attached form 4- to 10-membered heterocyclyl;

R¹⁰ and R¹¹ are each independently H or C1-6 alkyl, or

R¹⁰ and R¹¹ taken together with the nitrogen to which they are attached form 4- to 10- membered heterocyclyl;

W is selected from a bond, O, NR², O(Ci-2 alkylene), NH(CI-2 alkylene), C1-2 alkylene, and C3-6 cycloalkylene;

X is a moiety represented by one of the following structural formulas:

Y² and Y³ are each independently CR⁴ or N;

U is CR^12b or N;

Z is CR^lb or N;

L, M, and J are each independently selected from N, O, or S, provided that two of L, M, and J are N;

R¹² is selected from C3-6 alkyl, C3-6 cycloalkyl, C5-12 bridged bicyclic carbocyclyl, and 4- to 10-membered heterocyclyl;

R^12a is selected from C1-6 alkyl, C1-6 deuteroalkyl, C3-6 cycloalkyl, C5-12 bridged bicyclic carbocyclyl, and 4- to 10-membered heterocyclyl;

R^12b and R¹³ are each independently H or C1-6 alkyl; and

1 1 1

1 1 is a single bond or a double bond, wherein each C1-6 alkyl, C1-3 alkyl, C1-2 alkylene, C3-6 alkyl, C3-6 cycloalkyl, C1-6 alkoxy, C5-12 bridged bicyclic carbocyclyl, 5-12 membered heteroaryl, and 4- to 10-membered heterocyclyl is optionally substituted with 1 to 3 substituents independently selected from deuterium, oxo, F, Cl, Br, CN, OR¹⁴, SR¹⁵, NR¹⁶R¹⁷, S(O)R¹⁸, S(O)₂R^18a, NR¹⁹S(=O)R²⁰, C(=O)OR^20a, C(=O)NR²¹R²², NR²³C(=O)R²⁴, C(=S)NR²⁵R²⁶, C(=O)R²⁷, C1-6 alkyl, Ci-₆ deuteroalkyl, C3-8 cycloalkyl, C2-6 alkenyl, halo(Ci-6)alkyl, C1-3 alkylsulfonylaminoalkyl, hydroxy(Ci-6)alkyl, amino(Ci-6)alkyl, (Ci-e) alkylamino(Ci-6)alkyl, cyano(Ci- e)alkyl, C1-3 alkylcarbonylamino(Ci-6)alkyl, C1-3 alkoxy, halo(Ci-3)alkoxy, Ci- 6 alkoxy(Ci-3)alkyl, Ce-12 aryl, 4- to 8-membered heterocyclyl, and 5- to 12- membered heteroaryl, wherein

R¹⁴ R¹⁵, R¹⁸, R^18a, R²⁰. R^20a R²⁴ and R²⁷ are each independently hydrogen or C1-6 alkyl;

R¹⁶ and R¹⁷ are each independently selected from hydrogen, C1-6 alkyl, hydroxy(Ci- e)alkyl, and halo(Ci-6)alkyl;

R¹⁹ and R²³ are each independently C1-6 alkyl or halo(Ci-6)alkyl;

R²¹ R²² R²5 _ancj R26 _{are eac} i_n(iep_en(ien ly selected from H, C1-6 alkyl, C1-3 alkoxy(Ci-6)alkyl, hydroxy(Ci-e)alkyl, cyano(Ci-e)alkyl, amino(Ci-6)alkyl, C1-3 alkylamino(Ci-6)alkyl, and di(Ci-3)alkylamino(Ci-6)alkyl; or

R²¹ and R²² or R²⁵ and R²⁶, together with the nitrogen to which they are attached, form a 3-8 membered ring optionally substituted with 1 to 3 substituents independently selected from deuterium, oxo, F, Cl, Br, CN, OR¹⁴, SR¹⁵, NR¹⁶R¹⁷, S(O)R¹⁸, S(O)₂R^18a, NR¹⁹S(=O)R²⁰, C(=O)OR^20a, C(=O)NR²¹R²², NR²³C(=O)R²⁴, C(=S)NR²⁵R²⁶, C(=O)R²⁷, CI-6 alkyl, C3-8 cycloalkyl, C2-6 alkenyl, halo(Ci-6)alkyl, C1-3 alkylsulfonylaminoalkyl, hydroxy(Ci-6)alkyl,

5 amino(Ci-6)alkyl, cyano(Ci-e)alkyl, C1-3 alkylcarbonylamino(Ci-6)alkyl, C1-3 alkoxy, halo(Ci-3)alkoxy, C1-6 alkoxy(Ci-3)alkyl, Ce-12 aryl, 4- to 10-membered heterocyclyl, and 5- to 12-membered heteroaryl. provided that when Y² is CH substituted with R⁴ and R⁴ is optionally substituted C1-6 alkoxy, W-R³ is not CN or optionally substituted C1-6 alkoxy; and

10 provided that when Y¹, Y² and Y³ are each CH, then W-R³ is not F.

In some case, these compounds include any of:

In some embodiments, the RIPK2 inhibitor can be any one or more of the compounds having formula (I), which are disclosed and claimed in USSN 63/521,550, filed on June 16, 2023, which is incorporated herein by reference in its entirety. Compounds include, but are not limited to, compounds 1-251 in 63/521,550, which claims compounds of formula (I):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the RIPK2 inhibitor can be any one or more of the compounds having formula (I), which are disclosed and claimed in USSN 63/521,538, filed on June 16, 2023, which is incorporated herein by reference in its entirety. Compounds include, but are not limited to, compounds 1-276 in 63/521,538, which claims compounds of formula (I’):

or a pharmaceutically acceptable salt thereof.

In some embodiments, a RIPK2 inhibitor, or a pharmaceutically acceptable salt, and/or hydrate, and/or cocrystal, and/or drug combination thereof) is administered as a pharmaceutical composition that includes the RIPK2 inhibitor and one or more pharmaceutically acceptable excipients, and optionally one or more additional therapeutic agents as described herein. The term “pharmaceutically acceptable salt” refers to a formulation of a compound that does not cause significant irritation to an organism to which it is administered and does not abrogate the biological activity and properties of the compound.

In some embodiments, a RIPK2 inhibitor can be administered in combination with one or more conventional pharmaceutical excipients. Pharmaceutically acceptable excipients include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, self-emulsifying drug delivery systems (SEDDS) such as d-a-tocopherol polyethylene glycol 1000 succinate, surfactants used in pharmaceutical dosage forms such as Tweens, poloxamers or other similar polymeric delivery matrices, serum proteins, such as human serum albumin, buffer substances such as phosphates, tris, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium-chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethyl cellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, and wool fat. Cyclodextrins such as a-, 0, and y-cyclodextrin, or chemically modified derivatives such as hydroxyalkylcyclodextrins, including 2- and 3-hydroxypropyl-P-cyclodextrins, or other solubilized derivatives can also be used to enhance delivery of compounds described herein. Dosage forms or compositions containing a chemical entity as described herein in the range of 0.005% to 100% with the balance made up from nontoxic excipient may be prepared. The contemplated compositions may contain 0.001%- 100% of a chemical entity provided herein, in one embodiment 0.1-95%, in another embodiment 75-85%, in a further embodiment 20-80%. Actual methods of preparing such dosage forms are known, or will be apparent, to those skilled in this art; for example, see Remington: The Science and Practice of Pharmacy, 22^nd Edition (Pharmaceutical Press, London, UK. 2012).

In some embodiments, a RIPK2 inhibitor described herein or a pharmaceutical composition thereof can be administered to subject in need thereof by any accepted route of administration. Acceptable routes of administration include, but are not limited to, buccal, cutaneous, endocervical, endosinusial, endotracheal, enteral, epidural, interstitial, intra-abdominal, intra-arterial, intrabronchial, intrabursal, intracerebral, intracistemal, intracoronary, intradermal, intraductal, intraduodenal, intradural, intraepidermal, intraesophageal, intragastric, intragingival, intraileal, intralymphatic, intramedullary, intrameningeal, intramuscular, intraovarian, intraperitoneal, intraprostatic, intrapulmonary, intrasinal, intraspinal, intrasynovial, intratesticular, intrathecal, intratubular, intratumoral, intrauterine, intravascular, intravenous, nasal, nasogastric, oral, parenteral, percutaneous, peridural, rectal, respiratory (inhalation), subcutaneous, sublingual, submucosal, topical, transdermal, transmucosal, transtracheal, ureteral, urethral and vaginal. In certain embodiments, a preferred route of administration is oral.

In some cases, the patient is treated with a therapeutically effective amount of a RIPK2 inhibitor and an autoimmune agent such as, but not limited to, an anti-TNF agent (for example, an anti-TNF biologies such as Enbrel® (etanecerpt), Humira® (adalimumab), Remicade® (infliximab), Cimzia® (certolizumab), and Simponi® (golimumab).

Suitable anti-inflammatory/autoimmune agents for treatment include 5- aminosalicyclic acid and mesalamine preparations, sulfasalazine, hydroxycloroquine, thiopurines (azathioprin, mercaptopurin), methotrexate, cyclophosphamide, cyclosporine, calcineurin inhibitors (cyclosporine, pimecrolimus, tacrolimus), mycophenolic acid (CellCept®), mTOR inhibitors (temsirolimus, everolimus), JAK inhibitors (tofacitinib), (Xeljan®)), Syk inhibitors (fostamatinib), corticosteroids, particularly low-dose corticosteroids (such as prednisone (Deltasone®) and bundesonide) and anti-inflammatory biologies such as anti-IL6R mAbs (Actemra® (tocilizumab)), anti-IL6 biologies, anti-IL I (anakinra (Kineret®), canakinumab (Haris®), rilonacept (Arcalyst®)), anti-or IL12 or and IL23 biologies (ustekinumab (Stelara®)), anti-IL 17 biologies (secukinumab), anti-CD22 (epratuzumab), anti- integrin agents(natalizumab (Tysabri®)), vedolizumab (Entyvio®)), anti-IFNa (sifalimumab), anti-CD20 mAbs (rituximab (Rituxan®) and ofatumumab (Arzerra®)), and other agents, such as abatacept (Orencia®), anakinra (Kineret®), canakinumab (Haris®), rilonacept (Arcalyst®), secukinumab, epratuzumab, sifalimumab, and belimumab (Benlysta®), CD4 biologies and other cytokine inhibitors or biologies to T- cell or B-cell receptors or interleukins. Examples of suitable anti-TNF agents include the anti-TNF biologies such as Enbrel® (etanecerpt), Humira® (adalimumab), Remicade® (infliximab), Cimzia® (certolizumab), and Simponi® (golimumab).

EXAMPLE

Example 1 - Identification of Reduced Gene Signatures that stratify disease severity across independent datasets! and predict non-response to anti-TNF

Thirty mice (5 mice/group) were pre-treated twice daily for 4 days with either vehicle (PBS/saline) or with small molecule RIPK2 scaffolding inhibitor 1 (a compound that inhibits binding of RIPK2 to XIAP and has a RIPK2 IC50 < 2.5 nM) at escalating doses (1, 10, 30 and 100 mpk). Following this, mice in the treatment groups were challenged with 8mpk MDP for two hours and colon tissue was biopsied for RNA sequencing (RNA sequencing) analysis. RNA sequencing data analysis showed that 102 unique genes were inhibited by a RIPK2 scaffolding inhibitor in a dose-dependent manner. These genes were combined with a set of 49 genes that were previously published as being MDP responsive in purified mouse bone-marrow derived macrophages (GSE10182) (Kim, Y.G., et al., The cytosolic sensors Nodi and Nod2 are critical for bacterial recognition and host defense after exposure to Toll-like receptor ligands. Immunity, 2008. 28(2): p. 246-57) resulting in a total of 111 unique genes. This gene set was further tuned to human IBD patient data by utilizing two external RNA sequencing dataset (GSE166925 and GSE117993) (Friedrich, M., et al., IL-l-driven stromal-neutrophil interactions define a subset of patients with inflammatory bowel disease that does not respond to therapies. Nat Med, 2021. 27(11): p. 1970-1981; Haberman, Y., et al., Ulcerative colitis mucosal transcriptomes reveal mitochondriopathy and personalized mechanisms underlying disease severity and treatment response. Nat Commun, 2019. 10(1): p. 38), which comprises biopsies from the small intestine, large intestine, and rectum of patients diagnosed with iCD (ileum Crohn’s disease), eCD (colonic Crohn’s disease), and UC (Ulcerative Colitis). From the initial set of 111 genes (Gene Set I), a total of 30 (Gene Set II) were selected as being upregulated in disease relative to healthy patients (Mann-Whitney U test; p< 01) across all disease severities and types (Figure 1C).

Using multiple independent validation cohorts (GSE57945, GSE109142, GSE59071, GSE111889, GSE193677) (Lloyd-Price, J., et al., Multi-omics of the gut microbial ecosystem in inflammatory bowel diseases. Nature, 2019. 569(7758): p. 655-662; Friedrich, M., et al., IL-l-driven stromal-neutrophil interactions define a subset of patients with inflammatory bowel disease that does not respond to therapies. Nat Med, 2021. 27(11): p. 1970-1981; Haberman, Y., et al., Ulcerative colitis mucosal transcriptomes reveal mitochondriopathy and personalized mechanisms underlying disease severity and treatment response. Nat Commun, 2019. 10(1): p. 38; Haberman, Y., et al., Pediatric Crohn disease patients exhibit specific ileal transcriptome and microbiome signature. J Clin Invest, 2014. 124(8): p. 3617-33; Vanhove, W., et al., Strong Upregulation of AIM2 and IFI16 Inflammasomes in the Mucosa of Patients with Active Inflammatory Bowel Disease. Inflamm Bowel Dis, 2015. 21(11): p. 2673-82) the signature (Gene Set II) was found to be progressively elevated in inflamed tissues across inactive to severe disease in comparison to biopsies from healthy tissue and inactive disease (representative example, GSE193677, Figure 1D- E) (Argmann, C., et al., Biopsy and blood-based molecular biomarker of inflammation in IBD. Gut, 2023. 72(7): p. 1271-12878). The RIPK2 signature was assessed in transcriptomic datasets from colonic biopsies prior to treatment with standard of care drugs. The RIPK2 signature was elevated in non-responders and high RIPK2 signature (Gene Set II) could significantly predict eventual non-response to anti-TNF (GSE16879 and GSE12251) (Arijs, I., et al., Mucosal gene expression of antimicrobial peptides in inflammatory bowel disease before and after first infliximab treatment. PLoS One, 2009. 4(11): p. e7984; Arijs, I., et al., Mucosal gene signatures to predict response to infliximab in patients with ulcerative colitis. Gut, 2009. 58(12): p. 1612-99, 10) (Infliximab) therapy, anti-IL23 therapy (GSE206285, GSE207022) (Pavlidis, P., et al., Interleukin-22 regulates neutrophil recruitment in ulcerative colitis and is associated with resistance to ustekinumab therapy. Nat Commun, 2022. 13(1): p. 5820) (Ustekinumab), and anti-integrin (GSE73661) (Arijs, I., et al., Effect of vedolizumab (anti-alpha4beta7-integrin) therapy on histological healing and mucosal gene expression in patients with UC. Gut, 2018. 67(1): p. 43-52)

(Vedolizumab) therapy across multiple, independent UC and CD cohorts using a receiver operator curve (ROC) analysis (Figure IF). Additional studies were internalized in which transcriptomic data was collected from colonic mucosal biopsies both before and after treatment with Infliximab (GSE73661) and Vedolizumab (GSE73661) (VanDussen, K.L., et al., Abnormal Small Intestinal Epithelial Microvilli in Patients With Crohn's Disease. Gastroenterology, 2018. 155(3): p. 815-828). The RIPK2 signature (Gene Set II) remained elevated selectively in non-responders to Vedolizumab and Infliximab post-treatment (Figure 1G). Analysis of single cell RNA sequencing reveals inflammatory monocytes with high RIPK2 signature (Gene Set II) are expanded in the inflamed colon of patients with CD, as compared to healthy controls (Figure 1H) (Kong, L., et al., The landscape of immune dysregulation in Crohn's disease revealed through single-cell transcriptomic profiling in the ileum and colon. Immunity, 2023. 56(2): p. 444-458 e5).

To assess the effect of RIPK2 scaffolding inhibition on gene expression in humans, 44 colonic biopsies across 10 patients with IBD (4 CD, 6 UC) were collected and treated ex-vivo for 18 hours with RIPK2 scaffolding inhibitor 2 (a compound that inhibits binding of RIPK2 to XIAP and has a RIPK2 IC50 < 2.5 nM) or DMSO, after which spontaneous cytokine release of IL6 was quantified and RNA was processed for RNA sequencing. Biopsies were classified as responders based on a >50% downregulation of IL6 spontaneous cytokine release. From the RNA sequencing data, fold-change values in comparison to DMSO was calculated for all genes. The RIPK2 gene signature (Gene Set II) and gene signatures relating to inflammation and fibrosis were downregulated in responder biopsies after treatment with RIPK2 scaffolding inhibitor (Figure II).

To further refine the signature, these 30 genes were weighted (LI regularization) based on ability to predict non-response to TNF -therapy in cohorts of UC and CD patients (Arijs, I., et al., Mucosal gene expression of antimicrobial peptides in inflammatory bowel disease before and after first infliximab treatment. PLoS One, 2009. 4(11): p. e7984). Eleven genes were selected as having non-zero weights (Gene Set III). This set of 11 genes was able to accurately predict nonresponse to TNF therapy in independent cohorts of ileum CD (Arijs, I., et al., Mucosal gene expression of antimicrobial peptides in inflammatory bowel disease before and after first infliximab treatment. PLoS One, 2009. 4(11): p. e7984) and in two additional UC cohorts- -. ((5) Arijs, I et al, Effect of Vedolizumab (Anti-A4p7- Integrin) Therapy on Histological Healing and Mucosal Gene Expression in Patients with UC. Gut 2018, 67 (1), 43-52; Arijs, I. et al., Mucosal Gene Signatures to Predict Response to Infliximab in Patients with Ulcerative Colitis. Gut 2009, 58 (12), 1612-1619). The genes in the model can be further reduced to the top 7 genes (Gene Set IV) with the highest weights without notable compromise in performance (Figure 1 J). In addition, the list of 11 and 7 genes are able to equally stratify disease severity across independent datasets (Friedrich, M. et al. IL-l-Driven Stromal-Neutrophil Interactions Define a Subset of Patients with Inflammatory Bowel Disease That Does Not Respond to Therapies. Nat. Med. 2021, 27 (11), 1970-1981. (Figure IE)

In all analyses, Crohn’s Disease severity was characterized according to a simple endoscopic score for Crohn’s Disease (inactive, 0-2; mild, 3-6; moderate, 7- 15; severe >=16 ). A Mayo endoscopic score was used to characterize UC disease severity (inactive, 0 ; mild, 1; moderate, 2; severe, 3). Aggregate signature values across patient samples were calculated with gene set variation analysis, GSVA (Hanzelmann, S., R. Castelo, and J. Guinney, GSVA: gene set variation analysis for microarray and RNA-seq data. BMC Bioinformatics, 2013. 14: p. 7)

A number of embodiments of the invention have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims.

Claims

WHAT IS CLAIMED IS:

1. A method for treating an inflammatory bowel disease with a RIPK2 inhibitor in an eligible patient in need of such treatment, the method comprising:

(a) measuring an expression level of a plurality of biomarkers in a sample obtained from the patient, wherein the expression level of each of the biomarkers is increased by activation of RIPK2;

(b) comparing each of the biomarker expression levels determined at step (a) with a corresponding predetermined reference value;

(c) identifying that the patient is eligible for treatment with the RIPK2 inhibitor when one or more of the expression levels determined at step (b) are higher than the one or more of the corresponding predetermined reference values; and

(d) administering the RIPK2 inhibitor to the patient identified as being eligible for treatment in step (c).

2. The method of claim 1, wherein the plurality of biomarkers is a plurality of genes.

3. The method of claim 2, wherein the plurality of genes is a preselected gene signature set.

4. The method of claim 3, wherein the preselected gene signature set is a RIPK2-activated gene signature set.

5. The method of claim 4, wherein the RIPK2 gene signature is the gene signature shown in Table 1 (Gene Set I).

Table 1.

6. The method of claim 5, wherein the method comprises determining the expression levels of at least 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, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, or 111 of the genes listed in Table 1 (Gene Set I).

7. The method of claim 5 or 6, wherein the method comprises determining the expression levels of at least 7, 11, or 30 of the genes listed in Table 1 (Gene Set I).

8. The method of any one of claims 5-7, wherein the method comprises determining the expression levels of (Gene Set I) gene combinations that exhibit a Pearson correlation score that is greater than 0.60, or greater than 0.61, or greater than 0.62, or greater than 0.63, or greater than 0.64, or greater than 0.65, wherein the Pearson correlation score correlates a level of gene expression with a Mayo score.

9. The method of claim 4, wherein the RIPK2 gene signature is the gene signature shown in Table 2 (Gene Set II):

Table 2

10. The method of claim 9, wherein the method comprises determining the expression levels of at least 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, or 30 of the genes listed in Table 2 (Gene Set II).

11. The method of claim 9 or 10, wherein the method comprises determining the expression levels of at least 7, 11, or 30 of the genes listed in Table 2 (Gene Set II).

12. The method of claim 4, wherein the RIPK2 gene signature is the gene signature shown in Table 3 (Gene Set III):

Table 3

13. The method of claim 12, wherein the method comprises determining the expression levels of at least 4, 5, 6, 7, 8, 9, 10, 11 of the genes listed in Table 3 (Gene Set III)

14. The method of claim 12 or 13, wherein the method comprises determining the expression levels of at least 7 or 11 of the genes listed in Table 3 (Gene Set III).

15. The method of claim 4, wherein the RIPK2 gene signature is the gene signature shown in Table 4 (Gene Set IV).

Table 4

16. The method of claim 12, wherein the method comprises determining the expression levels of at least 1, 2, 3, 4, 5, 6, or 7 of the genes listed in Table 4 (Gene Set IV).

17. The method of claim 9-16, wherein the enhancement in differentiate of disease severity provided by (Gene Set II, III, or IV) is greater than the enhancement in differentiate of disease severity provided by (Gene Set I).

18. The method of claim 9-16, wherein the enhancement in specificity to pro-inflammatory monocytes/macrophages provided by (Gene Set II, III, or IV) is greater than the enhancement in specificity to pro-inflammatory monocytes/macrophages provided by (Gene Set I).

19. The method of claim 9-16, wherein the enhancement in detection of IBD disease activities provided by (Gene Set II, III, or IV) is greater than the enhancement in detection of IBD disease activities provided by (Gene Set I).

20. The method of claim 9-16, wherein the enhancement in predicting patient responses to anti-TNF therapy provided by (Gene Set II, III, or IV) is greater than the enhancement in specificity to pro-inflammatory monocytes/macrophages provided by (Gene Set I).

21. The method of claim 1, wherein the inflammatory bowel disease is Crohn’s disease.

22. The method of claim 1, wherein the inflammatory bowel disease is ulcerative colitis.

23. The method of claim 1, wherein the sample is an intestinal biopsy.

24. The method of claim 23, wherein the sample is a rectal biopsy.

25. The method of claim 23, wherein the sample is an ileal biopsy.

26. The method of any one of claims 23-25, wherein the sample comprises a myeloid cell.

27. The method of any one of claims 23-26, wherein the sample comprises a fibroblast.

28. The method of any one of claims 23-27, wherein the sample comprises an inflammatory monocyte.

29. The method of any one of claims 1-28, wherein the patient identified in step (c) is a non-responder to anti-TNF therapy.

30. The method of any one of claims 1-29, wherein the patient identified in step (c) is a non-responder to anti-Integrin therapy.

31. The method of any one of claims 1-30, wherein measuring the expression level of a plurality of biomarkers comprises measuring an amount of mRNA.

32. The method of any one of claims 1-31, wherein measuring the expression level of a plurality of biomarkers comprises performing one of more of the following assays: immunohistochemistry, ELISA, Western blot, or immunoprecipitation.

33. The method of any one of claims 1-32, wherein each of the corresponding predetermined reference values is obtained from one or more non-IBD patients.

34. The method of any one of claims 1-33, wherein each of the corresponding predetermined reference values is obtained from one or more noninflamed IBD patients.

35. The method of any one of claims 1-34, wherein the method further comprises calculating a RIPK2 gene signature score.

36. The method of claim 35, where a Pearson correlation coefficient between the RIPK2 signature score and a histological inflammation score is about 0.40 to about 0.60.

37. The method of claim 35, where a Pearson correlation coefficient between the RIPK2 signature score and a histological inflammation score is about 0.50 to about 0.60.

38. The method of claim 35, where a Pearson correlation coefficient between the RIPK2 signature score and a histological inflammation score is about 0.60 to about 0.66.

39. The method of any one of claims 1-38, wherein the expression level of the plurality of biomarkers is higher in a rectal biopsy sample obtained from the patient than in an ileal biopsy sample obtained from the patient.

40. The method of claim 39, wherein the patient is suffering from ulcerative colitis.

41. The method of claim 1, wherein the patient is being treated or had previously been treated with an anti-TNF therapy.

42. The method of claim 1, wherein the patient is being treated or had previously been treated with an anti-Integrin therapy.

43. The method of claim 1, wherein the patient is refractory to treatment with 5 -ASA.

44. The method of claim 43, wherein the patient is suffering from ulcerative colitis.

45. The method of claim 44, wherein from ulcerative colitis is moderate ulcerative colitis.

46. The method of claim 44, wherein from ulcerative colitis is sever ulcerative colitis.

47. The method of claim 44, wherein from ulcerative colitis is moderate to severe ulcerative colitis.

48. The method of claim 1, wherein the patient is being treated or had previously been treated with an anti-TNF therapy.

49. The method of claim 1, wherein the patient is being treated or had previously been treated with an anti-Integrin therapy.

50. A method comprising:

(a) providing a biological sample from a patient suffering from an inflammatory bowel disease; and

(b) measuring the expression in the biological sample of at least 4 biomarkers selected from (Gene Set I):

51. The method of claim 50, wherein the method comprises determining the expression levels of at least or exactly 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, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, or 111 of the biomarkers (Gene Set I).

52. The method of claim 50 or 51, wherein the method comprises determining the expression levels of at least 7, 11, or 30 of the biomarkers (Gene Set I).

53. The method of claim 50, wherein the biomarkers are selected from

(Gene Set II):

54. The method of claim 53, wherein the method comprises determining the expression levels of at least or exactly 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, or 30 of the genes listed in Gene Set II.

55. The method of claim 53, wherein the method comprises determining the expression levels of at least 7, 11, or 30 of the genes listed in Table 2 Gene Set II.

56. The method of claim 50 wherein the method comprises determining the expression levels of at least 4 biomarkers selected from (Gene Set III):

57. The method of claim 56, wherein the method comprises determining the expression levels of at least or exactly 4, 5, 6, 7, 8, 9, 10, 11 of the biomarkers listed in Gene Set III.

58. The method of claim 56, wherein the method comprises determining the expression levels of at least or exactly 7 or 11 of the biomarkers listed in Gene Set III

59. The method of claim 50, wherein the method comprises determining the expression levels of at least or exactly 7 or 11 of the biomarkers listed in Gene Set IV. Gene Set 4

60. The method of claim 59, wherein the method comprises determining the expression levels of at least or exactly 1, 2, 3, 4, 5, 6, or 7 of the biomarkers in Gene Set IV.

61. The method of claim 50, wherein the inflammatory bowel disease is Crohn’s disease.

62. The method of claim 50, wherein the inflammatory bowel disease is ulcerative colitis.

63. The method of claim 50, wherein the sample is an intestinal biopsy.

64. The method of claim 50, wherein the sample is a rectal biopsy.

65. The method of claim 50, wherein the sample is an ileal biopsy.

66. The method of any one of claims 50-65, wherein the sample comprises a myeloid cell.

67. The method of any one of claims 50-65, wherein the sample comprises a fibroblast.

68. The method of any one of claims 65, wherein the sample comprises an inflammatory monocyte.

69. The method of any one of claims 50-68, wherein the patient is a nonresponder to anti-TNF therapy.

70. The method of any one of claims 50-68, wherein the patient is a nonresponder to anti-Integrin therapy.

71. The method of any one of claims 50-68, wherein the patient is a nonresponder to anti-IL-23 therapy.

72. The method of any one of claims 50-71, wherein measuring the expression level the biomarkers comprises measuring an amount of mRNA.

73. The method of any one of claims 50-72, wherein measuring the expression level the biomarkers comprises performing one of more of the following assays: immunohistochemistry, ELISA, Western blot, or immunoprecipitation.

74. The method of any one of claims 50-73, wherein the method further comprises calculating a RIPK2 gene signature score.

75. The method of any of claims 50-72 further comprising determining a gene set variation analysis score based on the determined expression levels.

76. The method of claim 50, wherein the patient is being treated or had previously been treated with an anti-TNF therapy.

77. The method of claim 50, wherein the patient is being treated or had previously been treated with an anti-Integrin therapy.

78. The method of claim 50, wherein the patient is being treated or had previously been treated with an anti-IL-23 therapy.

79. The method of claim 50, wherein the patient is refractory to treatment with 5 -ASA.

80. The method of any of claims 50-79 further comprising treating the patient with a RIPK2 inhibitor.

81. The method of claim 80 wherein the RIPK2 inhibitor is selected from compounds 1-622.

82. The method of any one of claims 80-81, further comprising administering a therapeutically effective amount of a second agent.

83. The method of claim 82, wherein the second agent is an anti- inflammatory agent or an anti-autoimmune agent.

84. The method of claim 82, wherein the second agent is selected from anti-TNF agent, anti-IL-23 agent, anti-integrin agent, and JAK inhibitor.