Methods of treating a patient having inflammatory bowel disease

Abstract

The present disclosure relates generally to methods for treating inflammatory bowel disease ("IBD", e.g., Crohn's disease or ulcerative colitis) in a patient. More specifically, the present disclosure relates to methods for selecting a therapy for treating a patient having IBD. In embodiments, the foregoing can also be used to assess the severity of the IBD. The predictive aspects of the methods can facilitate and accelerate the identification and stratification of a population of IBD patients who are responsive to treatment with a RIPK2 inhibitor. The foregoing methods can further comprise treating the IBD by administering a RIPK2 inhibitor to the patient.

Description

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 545,100, filed October 20, 2023, the entire contents of which are incorporated herein by reference. Technical Field

[0002] This disclosure relates generally to methods for treating patients with inflammatory bowel disease (“IBD”, such as Crohn’s disease or ulcerative colitis). More specifically, this disclosure relates to methods for selecting therapies for treating patients with IBD. In one aspect, this disclosure provides methods for predicting whether a patient with suspected, diagnosed, or previously diagnosed IBD will be eligible for treatment with a RIPK2 inhibitor. In some embodiments, these methods include determining whether IBD is RIPK2 inhibitor-sensitive IBD, for example, by: (i) measuring the expression levels of one or more RIPK2-related biomarkers (e.g., a set of gene signatures or a subset thereof) in a sample obtained from the patient; (ii) comparing the expression levels determined in (i) with predetermined reference levels; 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 accelerate the identification and stratification of a population of IBD patients who are responsive to RIPK2 inhibitor treatment. The aforementioned method may also include treating the IBD by administering a RIPK2 inhibitor to the patient. Background Technology

[0003] Ulcerative colitis (UC) and Crohn's disease (CD) are major chronic inflammatory bowel diseases in humans. These conditions are inherently autoimmune and occur in the absence of infection. IBD affects up to 2,000,000 Americans (an increase of about 15% annually) and is associated with unacceptably high morbidity and mortality rates. IBD also places a significant burden on the US healthcare system because the most effective treatments are rather expensive biological drugs.

[0004] IBD occurs in genetically susceptible individuals due to an inappropriate immune response mediated by complex interactions between environmental stimuli, microbial factors, and the gut-immune system. A hallmark of IBD is an excessive immune response that directly or indirectly mediates damage to gastrointestinal tissues through the release of soluble pro-inflammatory mediators.

[0005] Although different forms of IBD exhibit pathophysiological and clinical differences, the treatment methods used to manage IBD share many common elements. Medical management of IBD is primarily empirical, employing anti-inflammatory or immunosuppressive drugs. If disease remission can be achieved, sulfasalazine and 5-aminosalicylic acid are used to treat mild IBD and as maintenance therapy; however, these therapies have generally shown ineffectiveness in patients with moderate to severe disease.

[0006] NOD1 and NOD2 (nucleotide-binding oligomerization domains 1 and 2) are members of the NOD-like receptor (NLR) family, representing important components of the mammalian innate immune system and acting as intracellular receptors for peptidoglycan (PGN), a component of the bacterial cell wall. NOD1 and NOD2 detect the presence of intracellular bacteria by binding to PGN fragments. Genetic polymorphisms in the genes encoding NOD1 and NOD2 have been associated with inflammatory conditions. Once activated, NOD signaling leads to the activation of NF-κB and MAP kinases, resulting in the transcription of pro-inflammatory kinases and the induction of autophagy.

[0007] NOD1 and NOD2 require RIPK2 as a common scaffold (adaptor) protein to propagate downstream signals that lead to aberrant pro-inflammatory innate immune activation. In particular, RIPK2 is crucial for NF-κB activation and subsequent cytokine production. Inhibition of RIPK2 can alleviate aberrant inflammatory states, such as intestinal inflammation. Therefore, inhibitors of RIPK2 have the potential to be used as therapeutic agents, for example, to reduce or alleviate inflammation in inflammatory conditions such as inflammatory bowel diseases (including Crohn's disease and ulcerative colitis). Summary of the Invention

[0008] This disclosure relates generally to methods for treating patients with inflammatory bowel disease (“IBD”, such as Crohn’s disease or ulcerative colitis). More specifically, this disclosure relates to methods for selecting therapies for treating patients with IBD. In one aspect, this disclosure provides methods for predicting whether a patient with suspected, diagnosed, or previously diagnosed IBD will be eligible for treatment with a RIPK2 inhibitor. In some embodiments, these methods include determining whether IBD is RIPK2 inhibitor-sensitive IBD, for example, by: (i) measuring the expression levels of one or more RIPK2-related biomarkers (e.g., a set of gene signatures or subsets thereof) in a sample obtained from the patient; (ii) comparing the expression levels determined in (i) with predetermined reference levels; 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 accelerate the identification and stratification of a population of IBD patients who are responsive to RIPK2 inhibitor treatment. The aforementioned method may also include treating the IBD by administering a RIPK2 inhibitor to the patient.

[0009] In one aspect, this disclosure provides methods for predicting whether a patient with inflammatory bowel disease will be eligible for treatment with a RIPK2 inhibitor. These methods include: (a) Measure the expression levels of multiple biomarkers in samples obtained from the patient, wherein the expression level of each of the biomarkers is increased by activation of RIPK2; (b) Compare the expression levels of each of the biomarkers determined in step (a) with a corresponding predetermined reference value; and (c) When one or more of the expression levels determined in step (a) are higher than one or more of the corresponding predetermined reference values, the patient is deemed eligible for treatment with the RIPK2 inhibitor.

[0010] In some implementations, these methods further include (d) identifying the patient as ineligible for treatment with the RIPK2 inhibitor when one or more of the expression levels determined in step (a) are below one or more of the corresponding predetermined reference values.

[0011] On the other hand, this disclosure provides methods for treating inflammatory bowel disease with RIPK2 inhibitors in eligible patients in need. These methods include: (a) Measure the expression levels of multiple biomarkers in samples obtained from the patient, wherein the expression level of each of the biomarkers is increased by activation of RIPK2; (b) Compare each of the biomarker expression levels determined in step (a) with a corresponding predetermined reference value; (c) When one or more of the expression levels determined in step (a) are higher than one or more of the corresponding predetermined reference values, the patient is deemed eligible for treatment with the RIPK2 inhibitor; and (d) Administer a therapeutically effective amount of the RIPK2 inhibitor to the patient identified as eligible for treatment in step (c).

[0012] In some implementations, these methods further include (e) identifying the patient as ineligible for treatment with the RIPK2 inhibitor when one or more of the expression levels determined in step (a) are below one or more of the corresponding predetermined reference values; and not administering a therapeutically effective amount of the RIPK2 inhibitor to the patient.

[0013] On the other hand, this disclosure provides a kit for predicting whether a patient with inflammatory bowel disease will be eligible for treatment with a RIPK2 inhibitor. In some embodiments, the kit includes one or more of the following: assay for a preselected set of genetic features, primers for the preselected set of genetic features, buffer solution, positive and negative controls, and instructions for use.

[0014] In one aspect, this disclosure provides methods including the following: (a) Providing biological samples from patients with inflammatory bowel disease; and (b) Measure the expression of at least four biomarkers selected from the following (gene set I) in the biological sample: In some embodiments, the method includes 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).

[0015] In some embodiments, the method includes determining the expression levels of at least 7, 11, or 30 of the biomarkers (gene set I).

[0016] In some embodiments, the biomarker is selected from the following (gene set II): In some embodiments, the method includes 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 genes listed in gene set II.

[0017] In some embodiments, the method includes determining the expression levels of at least 7, 11, or 30 of the genes listed in Gene Set II of Table 2.

[0018] In some embodiments, the method includes determining the expression levels of at least four biomarkers selected from the following (gene set III): In some embodiments, the method includes determining the expression levels of at least or exactly 4, 5, 6, 7, 8, 9, 10, and 11 of the biomarkers listed in gene set III.

[0019] In some embodiments, the method includes determining the expression levels of at least or exactly 7 or 11 of the biomarkers listed in gene set III.

[0020] In some implementations, the method includes determining the expression levels of at least or exactly 7 or 11 of the biomarkers listed in Gene Set IV.

[0021] Gene set 4 In some embodiments, the method includes determining the expression levels of at least or exactly 1, 2, 3, 4, 5, 6, or 7 of the biomarkers in gene set IV.

[0022] In some implementations, inflammatory bowel disease is Crohn's disease.

[0023] In some implementations, inflammatory bowel disease is ulcerative colitis.

[0024] In some implementations, the sample is an intestinal biopsy.

[0025] In some implementations, the sample is a rectal biopsy.

[0026] In some implementations, the sample is an ileal biopsy.

[0027] In some implementations, the sample includes myeloid cells.

[0028] In some implementations, the sample includes fibroblasts.

[0029] In some implementations, the sample includes inflammatory mononuclear cells.

[0030] In some implementations, the patient is a non-responder to TNF therapy.

[0031] In some implementations, the patient is a non-responder to integrin therapy.

[0032] In some implementations, the patient is a non-responder to IL-23 therapy.

[0033] In some implementations, measuring the expression level of a biomarker includes measuring the amount of mRNA.

[0034] In some embodiments, measuring the expression level of the biomarker includes performing one or more of the following assays: immunohistochemistry, ELISA, Western blot, or immunoprecipitation.

[0035] In some implementations, these methods also include calculating RIPK2 gene trait scores.

[0036] In some implementations, these methods also include determining a gene set variation analysis score based on the determined expression level.

[0037] In some implementations, the patient is being treated with anti-TNF therapy or has previously been treated with the same therapy.

[0038] In some implementations, the patient is being treated with anti-integrin therapy or has previously been treated with the same therapy.

[0039] In some implementations, the patient is being treated with anti-IL-23 therapy or has previously been treated with the same therapy.

[0040] In some implementations, patients are refractory to 5-ASA treatment.

[0041] In some implementations, these methods also include treating patients with RIPK2 inhibitors.

[0042] In some embodiments, these methods also include a RIPK2 inhibitor selected from compounds 1-622.

[0043] In some implementations, these methods also include administering a therapeutically effective amount of a second agent.

[0044] In some implementations, the second agent is an anti-inflammatory agent or an anti-autoimmune agent.

[0045] In some embodiments, the second agent is selected from anti-TNF agents, anti-IL-23 agents, anti-integrin agents, and JAK inhibitors.

[0046] As used herein, the term "sample" refers to any substance of biological origin. Examples of samples include, but are not limited to, substances obtained from intestinal biopsies, such as rectal or ileal biopsies. Substances may include, but are not limited to, whole blood, such as purified fractions of whole blood. In some embodiments, the sample includes one or more myeloid cells, one or more fibroblasts, one or more inflammatory mononuclear cells, or any combination thereof.

[0047] As used herein, the term "measuring expression levels" refers to applying gene-specific reagents, such as probes, primers, or antibodies, and / or methods to a sample to determine or quantitatively, semi-quantitatively, or qualitatively measure the amount of one or more genes present. As those skilled in the art will understand, quantitative, semi-quantitative, or qualitative measurements of the amount of one or more genes present can be determined via the detection and quantification of mRNA. In some embodiments, these methods also include isolating RNA from the sample. In some aspects, "measuring expression levels" may include performing RT-PCR, hybridization, transcriptome analysis, RNA-seq, single-cell RNA-seq, Northern blot, Western blotting, immunohistochemistry, or ELISA. For example, "measuring expression levels" may include performing array hybridization. In one aspect, transcriptome analysis may include obtaining sequence information of the expressed RNA molecules.

[0048] Other methods for mRNA detection and quantification can also be applied, such as in situ hybridization or spatial transcriptomics of mRNA in formalin-fixed, paraffin-embedded (FFPE) tissue samples or cells. (QuantiGene® ViewRNA (Affymetrix)) uses a set of probes for each mRNA that specifically bind to the amplification system to amplify the hybridization signal; 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 to measure gene expression levels in samples, and for example, to measure mRNA levels in FFPE samples. In short, TaqMan probe-based assays utilize probes that specifically hybridize to mRNA targets. The probe contains a quencher dye and a reporter dye (fluorescent molecule) attached to each end and emits fluorescence only when specific hybridization with the mRNA target occurs. During the amplification step, the exonuclease activity of the polymerase detaches the quencher dye and reporter dye from the probe, resulting in fluorescence emission. The fluorescence emission is recorded, and the signal is measured using a detection system; these signal intensities are used to calculate the abundance (gene expression) of a given transcript in the sample.

[0049] The terms “treat,” “treating,” and “treatment” mean the reduction or elimination of a symptom, disease, or illness, or one or more symptoms associated with a symptom, disease, or illness; or the slowing of the progression, spread, or worsening of a symptom, disease, or illness, or one or more symptoms thereof. In some implementations, the beneficial effects obtained by a subject from a treatment do not result in a complete cure of the disease, symptom, or illness.

[0050] As used herein, the term “predictive” refers to the probability or likelihood that a patient will respond to treatment with a RIPK2 inhibitor. As used herein, the terms “responsiveness,” etc., refer to the ability to assess the likelihood that treatment will or will not be clinically effective.

[0051] As used herein, the term "predetermined reference level" refers to the expression level of one or more RIPK2-related biomarkers (e.g., a set of gene signatures or a subset thereof) in samples obtained from a general population or from a selected population of IBD patients (e.g., non-inflammatory IBD patients). A "predetermined reference level" can be determined, for example, by determining the expression level of one or more RIPK2-related biomarkers (e.g., a set of gene signatures or a subset thereof) in corresponding samples obtained from one or more control subjects. When using such a predetermined reference level, a higher or increased level determined in the sample (i.e., the test sample obtained from the patient) indicates, for example, that the patient is eligible for treatment with an RIPK2 inhibitor.

[0052] Details of one or more embodiments of the present invention are set forth in the following drawings and detailed description. Other features and advantages of this disclosure will be apparent from the detailed description, the drawings, and the claims. Attached Figure Description

[0053] Figure 1A This is a flowchart outlining the methods used to select genes included in a feature.

[0054] Figure 1B This is a box plot of gene set variation analysis (GSVA) ​​scores derived from a set of 111 genes (gene set I). These genes are upregulated by muramyl dipeptide (MDP) and subsequently by a small molecule RIPK2 scaffold inhibitor 1 (a RIPK2 inhibitor that inhibits the binding of RIPK2 to XIAP and RIPK2 IC50). 50 When co-treated with compounds (< 2.5 nM), they were selected by downregulation in a dose-dependent manner.

[0055] Each box indicates the GSVA score at the indicated dose of a small molecule RIPK2 stent inhibitor + MDP, MDP alone, or mediator.

[0056] Figure 1C This is a box plot representing the upregulation of 30 gene signatures (gene set II) from intestinal biopsies of patients with inflammatory bowel disease compared to healthy tissue, within the training set (RISK cohort, rectal biopsy, GSE116925; Oxford cohort, colonic biopsy, GSE117993) used to derive gene set II (UC = ulcerative colitis; cCD = colonic Crohn's disease only; iCD = ileocolic Crohn's disease). P-values ​​were calculated using the Mann-Whitney U-test.

[0057] Figure 1DThis is a box plot showing the upregulation of 30 gene features (gene set II) in intestinal biopsies from patients with inflammatory bowel disease (UC = ulcerative colitis; CD = Crohn's disease) compared to healthy tissue (GSE193677) at different disease severity levels (UC, Mayo Endoscopic Score; CD, Simplified Endoscopic Score). The dashed line indicates the 99th percentile of the healthy distribution (t-test) and is used to assess the percentage of pathway activation in each category (GSVA cutoff for this panel: UC = .46 and CD = .42). P-values ​​were calculated using the Man Whitney U test. This feature was upregulated in multiple independent datasets (data not shown; GSE57945, GSE109142, GSE59071, GSE111889).

[0058] Figure 1E The heatmap shows that for both UC and CD patients (GSE193677), the enhancement in distinguishing disease severity provided by RIPK2 biomarker features of N=7, 11, and 30 genes (gene sets IV, III, and II) is greater than the enhancement in distinguishing disease severity provided by gene features of N=111 genes (gene set I).

[0059] Figure 1F This is a receiver operating curve (ROC) showing elevated RIPK2 gene signature scores (gene set II) in pretreatment UC and CD intestinal biopsies, predicting 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 were pretreated with infliximab and were resistant to it. The area under the curve (AUC) for each therapy-disease pair is shown. An AUC of 1 indicates a perfect predictor, while an AUC of 0.5 (shown by the dashed line) indicates no predictive ability. All therapy-disease pairs showed significant p-values ​​(p < .05), calculated by ROC analysis.

[0060] Figure 1GThis is a box plot indicating elevated RIPK2 signature (gene set II) levels in infliximab-resistant UC (NR) patients at 4–6 weeks post-treatment (left, GSE73661; anti-TNF) and elevated vedelizumab-resistant UC NR patients at 52 weeks post-treatment (right, GSE73661; anti-integrin). Vedelizumab patients were pretreated with infliximab and developed resistance to it. P-values ​​were calculated using the Mann-Whitney U test.

[0061] Figure 1H This is a UMAP representation of single-cell sequencing from myeloid cells isolated from mucosal biopsies of inflamed (right, active disease) and non-inflammatory (middle) intestinal tissue obtained from CD patients, or from biopsies of healthy controls (left) (Single Cell Portal: SCP1884). Cells were downsampled to equalize cell numbers in each combination. Mean RIPK2 gene signature expression in gene set II (log2 TPM) shows RIPK2 activation in inflammatory monocytes recruited to inflamed tissues and other myeloid cell populations as indicated.

[0062] Figure 1I It indicates RIPK2 stent inhibitor 2 (a type of inhibitor that inhibits the binding of RIPK2 to XIAP and RIPK2 IC) 50 Bar graphs showing that compounds of <2.5 nM reduced the expression of the RIPK2 gene signature (gene set II) in ex vivo processed human intestinal mucosal biopsies from patients with active IBD. In addition to the RIPK2 gene signature, other gene sets evaluated similarly include those describing inflammatory monocytes[1], TNF-mediated inflammatory pathways (KEGG), ligand-receptor signaling (IL-6, LIF, and OSM), inflammatory fibroblasts[1], and gene sets associated with Crohn's disease penetrating fibrosis[2]. Bar graphs indicate the fold change in gene set expression between treatment with RIPK2 scaffold inhibitor 2 and control (DMSO). P-values ​​were calculated using permutation tests. Error bars indicate standard deviation, and dashed lines indicate a 1.5-fold reduction in each graph.

[0063] Figure 1J The figures indicate that, compared to 30 gene features (gene set II; blue) (GSE16879, iCD, left; GSE73661, UC, middle; GSE12251, UC, right), 11 gene features (gene set III; orange) and 7 gene features (gene set IV; red) equally and powerfully predicted subject operating curves for non-response to the anti-TNF therapy infliximab (IFX) in three different publicly available datasets. The diagonal line of the dashed line indicates no predictive power (ROC=.5).

[0064] Figure 2 The chemical structure of a type I RIPK2 inhibitor is shown.

[0065] Figure 3 The chemical structure of the type II RIPK2 inhibitor is shown.

[0066] Figure 4 An exemplary PROTAC for degrading RIPK2 is shown. Detailed Implementation

[0067] This disclosure relates generally to methods for treating patients with inflammatory bowel disease (“IBD”, such as Crohn’s disease or ulcerative colitis). More specifically, this disclosure relates to methods for selecting therapies for treating patients with IBD. In one aspect, this disclosure provides methods for predicting whether a patient with suspected, diagnosed, or previously diagnosed IBD will be eligible for treatment with a RIPK2 inhibitor. In some embodiments, these methods include determining whether IBD is RIPK2 inhibitor-sensitive IBD, for example, by: (i) determining the expression levels of one or more RIPK2-related biomarkers (e.g., a set of gene signatures or subsets thereof) in a sample obtained from the patient; (ii) comparing the expression levels determined in (i) with predetermined reference levels; 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 accelerate the identification and stratification of a population of IBD patients who are responsive to RIPK2 inhibitor treatment. The aforementioned method may also include treating the IBD by administering a RIPK2 inhibitor to the patient.

[0068] In some implementations, multiple biomarkers are multiple genes.

[0069] In some implementations, multiple genes are a pre-selected set of gene features.

[0070] In some implementations, the RIPK2 gene signature is the gene signature shown in Table 1 (Gene Set I).

[0071] Table 1 In some of the foregoing embodiments, the method includes 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 genes listed in Table 1 (Gene Set I).

[0072] For example, the method may include determining the expression levels of at least 7, 11, or 30 of the genes listed in Table 1 (Gene Set I).

[0073] In some implementations, the RIPK2 gene signature is the gene signature shown in Table 2 (Gene Set II).

[0074] Table 2 In some of the foregoing embodiments, the method includes 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).

[0075] For example, the method may include determining the expression levels of at least 7, 11, or 30 of the genes listed in Table 2 (Gene Set II).

[0076] In some implementations, the RIPK2 gene signature is the gene signature shown in Table 3 (Gene Set III).

[0077] Table 3 In some of the foregoing embodiments, the method includes 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).

[0078] For example, the method may include determining the expression levels of at least 7 or 11 of the genes listed in Table 3 (Gene Set III).

[0079] In some implementations, the RIPK2 gene signature is the gene signature shown in Table 4 (Gene Set IV).

[0080] Table 4 For example, the method may 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).

[0081] In some implementations, inflammatory bowel disease is Crohn's disease. In some implementations, inflammatory bowel disease is moderate Crohn's disease. In some implementations, inflammatory bowel disease is severe Crohn's disease.

[0082] In some implementations, inflammatory bowel disease is ulcerative colitis. In some implementations, inflammatory bowel disease is moderate ulcerative colitis. In some implementations, inflammatory bowel disease is severe ulcerative colitis.

[0083] In some implementations, the patient has additional symptoms or comorbidities. For example, additional symptoms or comorbidities could be perianal diseases. As another example, additional symptoms or comorbidities could be extraintestinal manifestations.

[0084] In some embodiments, the sample is an intestinal biopsy. In some embodiments, the sample is a rectal biopsy. In other embodiments, the sample is an ileal biopsy.

[0085] In some implementations, the sample includes myeloid cells.

[0086] In some implementations, the sample includes fibroblasts.

[0087] In some implementations, the sample includes inflammatory mononuclear cells.

[0088] In some implementations, the sample includes neutrophils.

[0089] In some implementations, the patient identified in step (c) is a non-responder to anti-TNF therapy.

[0090] In some implementations, the patient is being treated with anti-TNF therapy or has previously been treated with the same therapy.

[0091] In some implementations, the patient identified in step (c) is a non-responder to anti-integrin therapy.

[0092] In some implementations, the patient is being treated with anti-integrin therapy or has previously been treated with the same therapy.

[0093] In some implementations, measuring the expression levels of multiple biomarkers includes measuring the amount of mRNA.

[0094] In some implementations, measuring the expression levels of multiple biomarkers includes performing one or more of the following assays: immunohistochemistry, ELISA, Western blotting, or immunoprecipitation.

[0095] In some implementations, each of the corresponding predetermined reference values ​​is obtained from one or more non-IBD patients.

[0096] In some implementations, each of the corresponding predetermined reference values ​​is obtained from one or more non-inflammatory IBD patients.

[0097] In some implementations, the expression level determined in step (i) is 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more higher than a predetermined reference level; or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 15 times or more higher.

[0098] In some implementations, these methods also include calculating RIPK2 gene trait scores.

[0099] In another approach, gene expression values ​​can be used to generate a weighted “feature score.” For example, each relative fold change can be multiplied by a coefficient from the gene trait, and the sum of these values ​​is then used as the feature score. This score can then be normalized to the sum of the absolute values ​​of the coefficients.

[0100] In some implementations, the expression levels of the plurality of biomarkers in rectal biopsy samples obtained from the patient are higher than their expression levels in ileal biopsy samples obtained from the patient.

[0101] In some implementations, the patient has ulcerative colitis.

[0102] In some implementations, the patient is refractory to 5-ASA treatment. In some implementations, the patient has ulcerative colitis; for example, the ulcerative colitis may be moderate; or the ulcerative colitis may be severe; or the ulcerative colitis may be moderate to severe.

[0103] In some implementations, the patient is being treated with anti-TNF therapy, either because they have previously been treated with this therapy or because the disease is refractory to it.

[0104] In some implementations, the patient is being treated with anti-integrin therapy, either because they have previously been treated with this therapy or because the condition is refractory to it.

[0105] RIPK2 inhibitors RIPK2 inhibitors, such as RIPK2 stent inhibitors, can be selected as needed.

[0106] Several RIPK2 inhibitors have been reported in the literature (Table 5). In particular, the quinazoline-based GSK2983559 exhibits kinome-wide selectivity and, after optimization as a phosphate prodrug, entered a human clinical trial for inflammatory bowel disease (IBD) (NCT03358407). Several 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 the target 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 in its active conformation. Compounds belonging to this category... Figure 2 The list includes erlotinib, gefitinib, adezmapimod (SB203580), and compound 1 reported by researchers at GlaxoSmithKline (Charnley, 2015). Type II kinase inhibitors target the inactive conformation of the kinase by interacting with the catalytic site of the unphosphorylated protein. In the inactive conformation, the DFG motif of the kinase is directed away from the ATP-binding site. The exclusivity of the inactive kinase conformation makes type II kinase inhibitors more selective than type I. Compounds belonging to this class are listed in... Figure 3 The study shows, and includes, sorafenib, ponatinib, regorafenib, and compound 2 (a sorafenib analog reported by Haile et al., 2016, as a screening hit (RIPK2 FP IC)). 50 = 794 nM).

[0107] Another approach to modulating RIPK2 is through proteolytically targeted chimeric (PROTAC) compounds, which hijack the ubiquitin-proteasome system (UPS) to degrade the protein of interest, in this case RIPK2. PROTACs are heterobifunctional molecules that simultaneously bind to both the POI and E3 ligase, leading to ubiquitination of the POI and its subsequent degradation by the UPS. After POI degradation, the PROTAC is recovered and continues to bind to a new copy of the POI. Exemplary PROTACs targeting RIP2K, as reported by Bondenson et al. (2015, Nat. Chem. Biol. 11, 611–617), are... Figure 4 As shown in the image.

[0108] 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... Figure 3 The RIPK2 inhibitor is selected from any one or more of the compounds listed herein, 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 21a-21d), or a pharmaceutically acceptable salt thereof.

[0109] In some embodiments, the RIPK2 inhibitor may be any one or more of the compounds having formula (I) disclosed and claimed in WO2020132384, the entire text of which is incorporated herein by reference. The compounds include, but are not limited to, compounds I-1 to I-122-ii as disclosed in WO2020132384, which claims compounds of formula (I'): (I'), Or its pharmaceutically acceptable salt.

[0110] In some embodiments, the RIPK2 inhibitor may be any one or more of the compounds having formula (I) disclosed and claimed in WO2018052772, the entire contents of which are incorporated herein by reference. The compounds include, but are not limited to, compounds 1-101 of WO2018052772, which claims compounds of formula (I): (I), Or its pharmaceutically acceptable salt.

[0111] In some embodiments, the RIPK2 inhibitor may be any one or more of the compounds having formula (I) disclosed and claimed in WO2018052773, the entire contents of which are incorporated herein by reference. The compounds include, but are not limited to, compounds 1-176 of WO2018052773, which claims compounds of formula (I): (I), Or its pharmaceutically acceptable salt.

[0112] In some embodiments, the RIPK2 inhibitor may be any one or more of the compounds having formula (I) disclosed and claimed in USSN 63 / 427,317, filed November 22, 2022, the entire contents of which are incorporated herein by reference. The compounds include, but are not limited to, compounds 1-461 of 63 / 427,317, which claims compounds of formula (I): (I), Or its pharmaceutically acceptable salt.

[0113] In some embodiments, the RIPK2 inhibitor may be any one or more of the compounds having formula (I) disclosed and claimed in USSN 63 / 443,760, filed February 7, 2023, the entire contents of which are incorporated herein by reference. The compounds include, but are not limited to, compounds 1-461 of 63 / 443,760, which claims compounds of formula (I): (I), Or its pharmaceutically acceptable salt.

[0114] In some embodiments, the RIPK2 inhibitor may be any one or more of the compounds having formula (I) disclosed and claimed in USSN 63 / 468,591, filed May 24, 2023, the entire contents of which are incorporated herein by reference. The compounds include, but are not limited to, compounds 1-622 of 63 / 468,591, which claims compounds of formula (I): (I), Or its pharmaceutically acceptable salt.

[0115] In some embodiments, the RIPK2 inhibitor may be any one or more of the compounds having formula (I) disclosed and claimed in WO 2024 / 112854, filed November 22, 2023, the entire contents of which are incorporated herein by reference. The compounds include, but are not limited to, compounds 1-622 of WO 2024 / 112854, which claims compounds of formula (I) or pharmaceutically acceptable salts thereof: (I) in: R 1a R 1b and R 1c Each is independently selected from H and C. 1-6 Alkyl, halogen, CN and; R 2 Is it H or C? 1-3 alkyl; R 3 Selected from S(=O)2R 5 Halogens, 4- to 10-membered heterocyclic groups, 5- to 12-membered heteroaryl groups, S(=O)(=NR) 6 (R) 7 ), QR 7 C(=O)NR 8 R 9 NH(C=O)R 5 CN, NR 8 R 9 P(=O)R 8a R 9a ; R 4 Selected from H, halogens, C 1-6 Alkyl and C 1-6 Alkoxy; R 5 Selected from C 1-6 Alkyl, NR 10 R 11 C 3-6 Cycloalkyl groups and 4- to 10-membered heterocyclic groups; R 6 Selected from H, CN and C 1-6 alkyl; R 7 Selected from C 1-6 Alkyl, C 3-6 Cycloalkyl and 4- to 10-membered heterocyclic groups, 5- to 12-membered heteroaryl groups, or R 6and R 7 Together with the nitrogen and sulfur atoms they are attached to, they form 4- to 10-membered heterocyclic groups; Q is selected from O, S, -S(=O)- and -C(=O)-; R 8 and R 9 Each is independently selected from H and C. 1-6 Alkyl, C 1-6 Deuterated alkyl, C 3-6 Cycloalkyl groups and 4- to 10-membered heterocyclic groups, or R 8 and R 9 Together with the nitrogen atoms they are attached to, they form 4- to 10-membered heterocyclic groups; R 8a and R 9b Each independently is C 1-6 Alkyl, or R 8a and R 9a Together with the phosphorus atoms they are attached to, they form 4- to 10-membered heterocyclic groups; R 10 and R 11 Each independently is H or C 1-6 Alkyl, or R 10 and R 11 Together with the nitrogen atoms they are attached to, they form 4- to 10-membered heterocyclic groups; W is selected from key, O, NR 2 O(C) 1-2 Alkylene), NH(C) 1-2 alkylene), C 1-2 Alkylene and C 3-6 Cycloalkylene; X is represented by one of the following structural formulas: , , , , , ; Y 1 It is CH or N; Y 2 and Y 3 Each independently for CR 4 Or N; U is CR 12b Or N; Z is CR 1b 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 Selected from C 3-6 Alkyl, C 3-6 cycloalkyl, C 5-12 Bridged bicyclic carbocyclic groups and 4- to 10-membered heterocyclic groups; R 12a Selected from C 1-6 Alkyl, C 1-6 Deuterated alkyl, C 3-6 cycloalkyl, C 5-12 Bridged bicyclic carbocyclic groups and 4- to 10-membered heterocyclic groups; R 12b and R 13 Each independently is H or C 1-6 alkyl; and Is it a single or double bond? Each C 1-6 Alkyl, C 1-3 Alkyl, C 1-2 Alkylene, C 3-6 Alkyl, C 3-6 cycloalkyl, C 1-6 Alkoxy, C 5-12 The bridged bicyclic carbocyclic group, the 5-12 membered heteroaryl group, and the 4- to 10 membered heterocyclic group are optionally substituted by 1 to 3 independent substituents selected from the following: deuterium, oxo, F, Cl, Br, CN, OR. 14 SR 15 NR 16 R 17 S(O)R 18 S(O)2R 18a NR 19 S(=O)R 20 C(=O)OR 20a C(=O)NR 21 R 22 NR 23 C(=O)R 24 C(=S)NR 25 R 26 C(=O)R 27 C 1-6 Alkyl, C 1-6 Deuterated alkyl, C 3-8 cycloalkyl, C 2-6 alkenyl, halogenated (C 1-6 )alkyl, C 1-3 alkylsulfonylaminoalkyl, hydroxyl (C 1-6 )alkyl, amino (C 1-6 )alkyl, (C 1-6 )alkylamino(C 1-6 )alkyl, cyano (C1-6 )alkyl, C 1-3 Alkyl carbonyl amino (C 1-6 )alkyl, C 1-3 Alkoxy, halogenated (C 1-3 )alkoxy, C 1-6 Alkoxy (C 1-3 )alkyl, C 6-12 Aryl, 4- to 8-membered heterocyclic and 5- to 12-membered heteroaryl, among which R 14 、 R 15 、 R 18 、 R 18a R 20 、 R 20a 、 R 24 、 and R 27 Each is independently hydrogen or C 1-6 alkyl; R 16 and R 17 Each is independently selected from hydrogen and C. 1-6 Alkyl, hydroxyl (C 1-6 )alkyl and halogen (C 1-6 )alkyl; R 19 and R 23 Each independently is C 1-6 Alkyl or halogenated (C 1-6 )alkyl; R 21 、 R 22 、 R 25 and R 26 Each is independently selected from H and C. 1-6 Alkyl, C 1-3 Alkoxy (C 1-6 )alkyl, hydroxyl (C 1-6 )alkyl, cyano (C 1-6 )alkyl, amino (C 1-6 )alkyl, C 1-3 Alkylamino (C 1-6 )alkyl and di(C 1-3 )alkylamino(C 1-6 alkyl; or R 21 and R 22 Or R 25 and R 26Together with the nitrogen atoms to which they are attached, they form 3-8 membered rings optionally substituted by 1 to 3 independently selected from the following substituents: deuterium, oxo, F, Cl, Br, CN, OR. 14 SR 15 NR 16 R 17 S(O)R 18 S(O)2R 18a NR 19 S(=O)R 20 C(=O)OR 20a C(=O)NR 21 R 22 NR 23 C(=O)R 24 C(=S)NR 25 R 26 C(=O)R 27 C 1-6 Alkyl, C 3-8 cycloalkyl, C 2-6 alkenyl, halogenated (C 1-6 )alkyl, C 1-3 alkylsulfonylaminoalkyl, hydroxyl (C 1-6 )alkyl, amino (C 1-6 )alkyl, cyano (C 1-6 )alkyl, C 1-3 Alkyl carbonyl amino (C 1-6 )alkyl, C 1-3 Alkoxy, halogenated (C 1-3 )alkoxy, C 1-6 Alkoxy (C 1-3 )alkyl, C 6-12 Aryl, 4- to 10-membered heterocyclic and 5- to 12-membered heteroaryl.

[0116] The prerequisite is that when Y 2 It was R 4 Replace CH and R 4 C is an optional substitute 1-6 When alkoxy is present, WR 3 Not CN or optional replacement C 1-6 alkoxy groups; and The prerequisite is that when Y 1 Y 2 and Y 3 When each is CH, then WR 3 It's not F.

[0117] In some cases, these compounds include any of the following: In some embodiments, the RIPK2 inhibitor may be any one or more of the compounds having formula (I) disclosed and claimed in USSN 63 / 521,550, filed June 16, 2023, the entire contents of which are incorporated herein by reference. The compounds include, but are not limited to, compounds 1-251 of 63 / 521,550, which claims compounds of formula (I): (I), Or its pharmaceutically acceptable salt.

[0118] In some embodiments, the RIPK2 inhibitor may be any or more of the compounds having formula (I) disclosed and claimed in USSN 63 / 521,538, filed June 16, 2023, the entire contents of which are incorporated herein by reference. The compounds include, but are not limited to, compounds 1-276 of 63 / 521,538, which claims compounds of formula (I'): (I'), Or its pharmaceutically acceptable salt.

[0119] In some embodiments, the RIPK2 inhibitor (or its pharmaceutically acceptable salt and / or hydrate and / or cocrystal and / or combination of drugs) is administered in the form of a pharmaceutical composition comprising the RIPK2 inhibitor and one or more pharmaceutically acceptable excipients, and optionally one or more additional therapeutic agents as described herein.

[0120] The term "pharmaceutically acceptable salt" refers to a formulation of a compound that will not cause significant irritation to the organism to which it is applied and will not eliminate the biological activity and properties of the compound.

[0121] In some implementations, RIPK2 inhibitors 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-α-tocopherol polyethylene glycol 1000 succinate), surfactants used in pharmaceutical dosage forms (such as Tweens, poloxamer, or other similar polymer delivery matrices), serum proteins (such as human serum albumin), buffering substances (such as phosphates, tris, glycine, sorbic acid, potassium sorbate), partially glycerol ester 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, polyvinylpyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethyl cellulose, polyacrylates, waxes, polyethylene-polyoxypropylene block polymers, and lanolin. Cyclodextrins (such as α-cyclodextrin, ... Cyclodextrins and γ-cyclodextrins, or chemically modified derivatives such as hydroxyalkyl cyclodextrins, including 2-hydroxypropyl-β-cyclodextrin and 3-hydroxypropyl-β-cyclodextrin, or other solubilizing derivatives, may also be used to enhance the delivery of the compounds described herein. Dosage forms or compositions containing from 0.005% to 100% of the chemical entities described herein and the balance consisting of non-toxic excipients can be prepared. The contemplated compositions may contain 0.001% to 100% of the chemical entities provided herein, 0.1% to 95% in one embodiment, 75% to 85% in another embodiment, and 20% to 80% in yet another embodiment. Practical methods for preparing such dosage forms are known to those skilled in the art, or will be readily apparent to them; for example, see [link to relevant documentation]. Remington: The Science and Practice of Pharmacy , 22nd edition (Pharmaceutical Press, London, UK. 2012).

[0122] In some implementations, the RIPK2 inhibitors or pharmaceutical compositions thereof described herein may be administered to subjects in need via any acceptable route of administration. Acceptable routes of administration include, but are not limited to, oral, skin, intracervical, intranasal, intratracheal, intraintestinal, epidural, interstitial, intraperitoneal, intraarterial, intrabronchial, intrabursal, intracerebral, intracisary, intracoronary, intradermal, intracatheter, intraduodenal, intradural, intraepithelial, intraepithelial, intraesophageal, intragastric, intragingival, intraileum, intralymphatic, intramedullary, intramedullary, intramuscular, intraovarian, intraperitoneal, intraprostatic, intrapulmonary, intrasinus, intraspinal, intrasynovial, intratestinal, intrasheath, intracanal, intratumoral, intrauterine, intravascular, intravenous, intranasal, intranasogastric, oral, parenteral, percutaneous, peridinal, rectal, respiratory (inhalation), subcutaneous, sublingual, submucosal, local, transdermal, transmucosal, transtracheal, ureteral, urethral, ​​and vaginal. In some embodiments, oral administration is preferred.

[0123] In some cases, patients are treated with therapeutically effective amounts of RIPK2 inhibitors and autoimmune agents, such as, but not limited to, anti-TNF agents, such as anti-TNF biologics like Enbrel® (etanecerpt), Humira® (adalimumab), Remicade® (infliximab), Cimzia® (certolizumab), and Simponi® (golimumab).

[0124] Suitable anti-inflammatory / autoimmune agents for treatment include 5-aminosalicylic acid and mesalamine preparations, sulfasalazine, hydroxychloroquine, thiopurines (azathioprin, mecaptopurin), methotrexate, cyclophosphamide, cyclosporine, calcineurin inhibitors (cyclosporine, pimecrolimus, tacrolimus), and mycophenolic acid. CellCept®, mTOR inhibitors (temsirolimus, everolimus), JAK inhibitors (tofacitinib, Xeljan®), Syk inhibitors (fostamatinib), corticosteroids, especially low-dose corticosteroids (such as prednisone (Deltasone®) and budesonide), and anti-inflammatory biologics, such as anti-IL6R mAb (Actemra®, tocilizumab), anti-IL6 biologics, anti-IL I (anakinra (Kineret®), canakinumab (Ilaris®), rilonacept (Arcalyst®), anti-IL12 or IL23 biologics (ustekinumab (Stelara®), anti-IL... 17 Biologics (secukinumab), anti-CD22 (epratuzumab), anti-integrin agents (natalizumab (Tysabri®), vedalizumab (Entyvio®), anti-IFNa (sifalimumab), anti-CD20 mAb (rituximab (Rituxan®) and ofatumumab (Arzerra®)) and other agents,Examples of suitable anti-TNF agents include abatacept (Orencia®), kinetetin (Kineret®), cannabinoids (Ilaris®), linacipide (Arcalyst®), secukinumab, epazolizumab, cifamumab, and belimumab (Benlysta®), CD4 biologics, and other cytokine inhibitors or biologics targeting T-cell, B-cell receptors, or interleukins. Examples of suitable anti-TNF agents include anti-TNF biologics such as Enbrel® (etanercept), Humira® (adalimumab), Remicade® (infliximab), Cimzia® (cetuzumab), and Simponi® (golimumab).

[0125] Example Example 1 - Identifying disease severity stratification and predicting anti-TNF non-responsiveness in independent dataset 2 Simplified genetic characteristics of the answer Thirty mice (n=5 per group) were pretreated twice daily for four days with either a medium (PBS / saline) or with escalating doses (1, 10, 30, and 100 mpk) of a small molecule RIPK2 scaffold inhibitor 1 (a compound that inhibits the binding of RIPK2 to XIAP and has an IC50 <2.5 nM). Subsequently, mice in the treated groups were challenged with 8 mpk MDP for two hours, and colon tissue biopsies were performed for RNA sequencing analysis. Analysis of the RNA sequencing data showed that the RIPK2 scaffold inhibitor inhibited 102 unique genes in a dose-dependent manner. These genes were combined with a group of 49 genes previously reported to be MDP-responsive in purified mouse bone marrow-derived macrophages (GSE10182) (Kim, YG et al., The cytosolic sensors Nod1 and Nod2 are critical for bacterial recognition and host defense after exposure to Toll-like receptor ligands. Immunity, 2008. 28(2): 246-57) to generate a total of 111 unique genes. By utilizing two external RNA sequencing datasets (GSE166925 and GSE117993) (Friedrich, M. et al., IL-1-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; Haberman, Y. et al., Ulcerative colitis mucosal transcriptomes reveal mitochondriopathy and personalized mechanisms underlying disease severity and treatment response. Nat Commun, 2019. 10(1): 38), this dataset was further adapted based on human IBD patient data. These RNA sequencing datasets contained biopsies of the small intestine, large intestine, and rectum from patients diagnosed with iCD (ileal Crohn's disease), cCD (colonic Crohn's disease), and UC (ulcerative colitis).From the initial set of 111 genes (gene set I), a total of 30 genes (gene set II) were selected because they were upregulated in all disease severity and type relative to healthy patients (Mann-Whitney U test; p<.01). Figure 1C ).

[0126] 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): 655-662; Friedrich, M. et al., IL-1-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; Haberman, Y. et al., Ulcerative colitismucosal transcriptomes reveal mitochondriopathy and personalized mechanisms underlying disease severity and treatment response. Nat Commun, 2019. 10(1): 38; Haberman, Y. et al., Pediatric Crohn disease patients exhibit specificileal transcriptome and microbiome signature. J Clin Invest, 2014. 124(8): 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): 2673-82), found that this feature (gene set II) gradually increased in inflamed tissues from inactive to severe disease compared with biopsies from healthy tissues and inactive disease (representative example GSE193677, Figures 1D-1E(Argmann, C. et al., Biopsy and blood-based molecular biomarker of inflammation in IBD. Gut, 2023. 72(7): 1271-12878). RIPK2 characterization was evaluated in transcriptome datasets from colon biopsies prior to standard treatment. RIPK2 signatures were elevated in non-responders, and high RIPK2 signatures (gene set II) significantly predicted eventual anti-TNF (GSE16879 and GSE12251) in multiple independent UC and CD cohorts using receiver operating characteristic (ROC) analysis (Arijs, I. et al., Mucosal gene expression of antimicrobial peptides ininflammatory bowel disease before and after first infliximab treatment. PLoSOne, 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): 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): 43-52) Non-response to (vedelizumab) therapy Figure 1F ). This included additional studies in which transcriptomic data were collected from colonic mucosal biopsies before and after treatment with infliximab (GSE73661) and vedelizumab (GSE73661) (VanDussen, KL et al., Abnormal Small Intestinal Epithelial Microvilliin Patients With Crohn's Disease. Gastroenterology, 2018. 155(3): 815-828). RIPK2 signature (gene set II) remained selectively elevated in non-responders to vedelizumab and infliximab after treatment. Figure 1G Single-cell RNA sequencing analysis revealed that inflammatory monocytes with high RIPK2 signatures (gene set II) expanded in the inflamed colon of patients with CD compared to healthy controls. Figure 1H ) (Kong, L. et al., Thelandscape of immune dysregulation in Crohn's disease revealed through single-cell transcriptomic profiling in the ileum and colon. Immunity, 2023. 56(2): pp. 444-458e5).

[0127] To assess the effects of RIPK2 stent inhibition on human gene expression, 44 colon biopsies from 10 patients with IBD (4 CD, 6 UC) were collected and treated ex vivo for 18 hours with either RIPK2 stent inhibitor 2 (a compound that inhibits the binding of RIPK2 to XIAP and has an RIPK2 IC50 < 2.5 nM) or DMSO. Spontaneous IL6 cytokine release was then quantified, and RNA was processed for RNA sequencing. Biopsies were classified as responders based on a downregulation of >50% in spontaneous IL6 cytokine release. The fold change relative to DMSO was calculated for all genes based on RNA sequencing data. Following treatment with the RIPK2 stent inhibitor, RIPK2 gene signatures (gene set II) and gene signatures associated with inflammation and fibrosis were downregulated in responder biopsies. Figure 1I ).

[0128] To further refine this characteristic, the 30 genes were weighted (L1 regularized) based on their ability to predict TNF therapy non-response in UC and CD patient cohorts (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 due to their non-zero weight (gene set III). This set of 11 genes was found in an independent cohort of ileal 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 two additional UC cohorts. 56 Accurately predict non-response to TNF therapy. ((5) Arijs, I et al., Effect of Vedolizumab (Anti-A4β7-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 could be further reduced to the top 7 genes with the highest weights and no significant performance degradation (gene set IV). Figure 1JFurthermore, lists of 11 and 7 genes can equally stratify disease severity in independent datasets (Friedrich, M. et al. IL-1-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 1E ) In all analyses, Crohn's disease severity was characterized by the Simplified Endoscopic Score (SOS) for Crohn's disease (inactive, 0–2; mild, 3–6; moderate, 7–15; severe, >=16). The Mayo Clinic Endoscopic Score (SOS) was used to characterize UC disease severity (inactive, 0; mild, 1; moderate, 2; severe, 3). Gene set variation analysis (GSVA) ​​was used to calculate aggregated eigenvalues ​​for patient samples (Hanzelmann, S., R. Castelo, and J. Guinney, GSVA: gene set variation analysis for microarray and RNA-seq data. BMC Bioinformatics, 2013. 14: p. 7).

[0129] Several embodiments of the invention have been described. However, it should be understood that various modifications can be made without departing from the spirit and scope of the invention. Therefore, other embodiments are within the scope of the following claims.

Claims

1. A method for treating inflammatory bowel disease with a RIPK2 inhibitor in eligible patients requiring such treatment, the method comprising: (a) Measure the expression levels of multiple biomarkers in samples obtained from the patient, wherein the expression level of each of the biomarkers is increased by activation of RIPK2; (b) Compare each of the biomarker expression levels determined in step (a) with a corresponding predetermined reference value; (c) When one or more of the expression levels determined in step (b) are higher than one or more of the corresponding predetermined reference values, the patient is deemed eligible for treatment with the RIPK2 inhibitor; and (d) Administer the RIPK2 inhibitor to the patient identified as eligible for treatment in step (c).

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

3. The method of claim 2, wherein the plurality of genes is a pre-selected set of gene features.

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

5. The method of claim 4, wherein the RIPK2 gene characteristics are those shown in Table 1 (Gene Set I). Table 1. 。 6. The method of claim 5, wherein the method comprises determining the expression level 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 genes listed in Table 1 (Gene Set I).

7. The method of claim 5 or 6, wherein the method comprises determining the expression level 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 to 7, wherein the method comprises determining the expression level of a gene combination (gene set I) that exhibits a Pearson correlation score 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 the gene expression level with the Mayo score.

9. The method of claim 4, wherein the RIPK2 gene characteristics are those 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 level 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, and 11 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 claims 9 to 16, wherein the enhancement in distinguishing disease severity provided by (gene set II, III, or IV) is greater than the enhancement in distinguishing disease severity provided by (gene set I).

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

19. The method of claims 9 to 16, wherein the enhancement in detecting IBD disease activity provided by (gene set II, III, or IV) is greater than the enhancement in detecting IBD disease activity provided by (gene set I).

20. The method of claims 9 to 16, wherein the enhancement provided by (gene set II, III, or IV) in predicting a patient's response to anti-TNF therapy is greater than the enhancement provided by (gene set I) in specificity for pro-inflammatory monocytes / macrophages.

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 to 25, wherein the sample comprises myeloid cells.

27. The method of any one of claims 23 to 26, wherein the sample comprises fibroblasts.

28. The method of any one of claims 23 to 27, wherein the sample comprises inflammatory mononuclear cells.

29. The method of any one of claims 1 to 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 to 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 to 30, wherein measuring the expression levels of the plurality of biomarkers includes measuring the amount of mRNA.

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

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

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

36. The method of claim 35, wherein the Pearson correlation coefficient between the RIPK2 feature score and the histological inflammation score is about 0.40 to about 0.

60.

37. The method of claim 35, wherein the Pearson correlation coefficient between the RIPK2 feature score and the histological inflammation score is about 0.50 to about 0.

60.

38. The method of claim 35, wherein the Pearson correlation coefficient between the RIPK2 feature score and the histological inflammation score is about 0.60 to about 0.

66.

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

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

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

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

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

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

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

46. ​​The method of claim 44, wherein the ulcerative colitis is severe ulcerative colitis.

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

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

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

50. A method comprising: (a) Provide biological samples from patients with inflammatory bowel disease; and (b) Measure the expression of at least four biomarkers selected from the following (gene set I) in the biological sample: 。 51. The method of claim 50, wherein the method comprises determining the expression level 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 level of at least 7, 11 or 30 of the biomarkers (gene set I).

53. The method of claim 50, wherein the biomarker is selected from the following (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 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 Gene Set II of Table 2.

56. The method of claim 50, wherein the method comprises determining the expression levels of at least four biomarkers selected from the following (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, and 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 includes 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 to 65, wherein the sample comprises myeloid cells.

67. The method of any one of claims 50 to 65, wherein the sample comprises fibroblasts.

68. The method of any one of claims 65, wherein the sample comprises inflammatory mononuclear cells.

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

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

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

72. The method of any one of claims 50 to 71, wherein measuring the expression level of the biomarker includes measuring the amount of mRNA.

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

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

75. The method of any one of claims 50 to 72, further comprising determining a gene set variation analysis score based on the determined expression level.

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

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

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

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

80. The method of any one of claims 50 to 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 to 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 agents, anti-IL-23 agents, anti-integrin agents, and JAK inhibitors.

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