Biomarker panel for Anti-TNF response in patients with crohn's disease

Abstract

Disclosed are methods of characterizing, diagnosing, monitoring, and / or treating an individual with Crohn's Disease (CD) comprising detecting, in a biological sample obtained from the individual, a plurality of biomarkers. The biomarkers may be used for one or more of predicting remission of CD in an individual, determining longitudinal assessment of response to a biologic therapy, predicting or determining response status of the individual to an anti-tumor necrosis factor (TNF) therapy. Further disclosed are systems and compositions for use with the disclosed methods.

Description

BIOMARKER PANEL FOR ANTI-TNF RESPONSE IN PATIENTS WITH CROHN’SDISEASECROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This patent application claims priority from U.S. Provisional Application No. 63 / 649,549 filed May 20, 2024, which is incorporated herein by reference in its entirety.STATEMENT REGARDING FEDERALLY-SPONSORED RESEARCH

[0002] This invention was made with government support under DK 132408 awarded by the National Institutes of Health. The government has certain rights in the invention.BACKGROUND

[0003] Current evidence suggests the inflammatory bowel diseases, Crohn’s disease (CD) and ulcerative colitis, are caused by an exaggerated immune response to gut microbiota in a genetically susceptible host.1CD is best characterized as a relapsing and remitting inflammatory disorder of the intestinal tract that without adequate control will lead to progressive intestinal damage.2, 3As a cure for CD remains elusive, the primary therapeutic target is the heightened immune response. For more than twenty-five years, the inhibitors of tumor necrosis factor-a (anti-TNF) have been approved by the United States Food and Drug Administration for the management of moderate to severe CD, which includes infliximab and adalimumab, for children >6 years old. With the early use of anti-TNF to treat children and adults with moderate to severe CD, along with innovative strategies to optimize drug exposure, there has been a reduction in CD-related surgeries and improved rates of sustained steroid-free remission.4-6The initial response rate to anti-TNF is high (>80%), but despite dose optimization and early use, the rate of intestinal healing remains less than 40% and the yearly loss of response rate is 10- 15%.7,8Given a gradual decline in anti-TNFdurability, many children with CD are switched to an alternative advanced therapy (off-label use) with a different mechanism of action.9Interestingly, emerging data suggests that response rates decline with each subsequent advanced therapy, underscoring the critical need for biomarkers or companion diagnostics to guide treatment selection within the expanding armamentarium of advanced therapies.10[00041 The anti-TNF biologies have been historically used as the first-line biologic for children and adults with a more severe CD phenotype (stricturing, internal penetrating or perianal disease), extensive bowel inflammation or for children with growth failure.11, 12Rather than relying on disease phenotype to drive therapy selection, several biomarker investigations suggest that patients with CD exhibit distinct molecular endotypes that can be used to personalize therapy selection. For example, Martin et al. conducted single-cell RNA sequencing to identify a pathogenic cellular module in anti-TNF nonresponders.13The module was characterized by inflammatory fibroblasts, activated monocytes and specific T cell subsets that could be used as both biomarkers or targets to improve treatment response.13Moreover, by analyzing the serum proteome of newly diagnosed children with CD, Ungaro et al. discovered specific protein signatures associated with disease progression.14

[0005] Implementation of these and other novel biomarker discoveries in real-world practice, however, has been hindered by challenges such as a lack of external validation, ease of collection (blood compared to tissue based biomarkers), regulatory constraints, reimbursement issues and poor performance in controlled trials.6Specifically, Noor et al. conducted a randomized, openlabel trial to compare the use of early, top-down therapy (infliximab plus immunomodulator) versus an accelerated step-up approach to achieve steroid-free and surgery free remission in patients with newly diagnosed CD.6In this trial, randomization was stratified based on the resultsof a 17-gene blood-based biomarker (PredictSURE-IBD assay).6The study found that sustained steroid-free and surgery-free remission occurred significantly more often in the top-down group.However, there was no interaction between the biomarker results and treatment strategy.6

[0006] Traditional peripheral blood biomarkers such as c-reactive protein, erythrocyte sedimentation rate or serum albumin appear to be far superior markers of the biologies’ pharmacokinetics than indicators of a specific inflammatory endotype to base treatment selection.15Until recently, the use of plasma proteomics for biomarker discovery has been limited to small cohorts, use of more traditional protein quantification (such as mass spectrometry) or use of small-scale protein (commercial) panels.16"19Rather, the recent development of more comprehensive plasma proteomic platforms allows for a more complete analysis of the patients inflammatory endotype with a discovery of a panel of serum proteins that could predict the onset of inflammatory bowel disease up to five years before diagnosis.17

[0007] With an emerging list of advanced therapies to manage patients with CD, there is a critical need for individualized treatment selection based on an integrated analysis of the patient’s molecular endotype and the therapeutic’s mechanism of action. This novel, personalized approach to treatment selection could be a gamechanger in the field and an excellent alternative to the current, nonspecific trial and error approach. Moreover, the use of longitudinal biomarker assessments during treatment could be used to identify an inflammatory endotype during secondary nonresponse that may signal an alternative advanced therapy with a different mechanism of action or suggest the use of combination advanced therapy to improve intestinal healing.

[0008] In sum, while the number of advanced therapies for managing Crohn’s disease (CD) has grown significantly, selection of first- line therapy remains largely trial and error due to thelack of biomarkers that integrate inflammatory endotype with mechanism of action. The present disclosure seeks to address one or more of the aforementioned needs in the art.BRIEF SUMMARY

[0009] Disclosed are methods of characterizing, diagnosing, monitoring, and / or treating an individual with Crohn’s Disease (CD) comprising detecting, in a biological sample obtained from the individual, a plurality of biomarkers. The biomarkers may be used for one or more of predicting remission of CD in an individual, determining longitudinal assessment of response to a biologic therapy, predicting or determining response status of the individual to an anti-tumor necrosis factor (TNF) therapy. Further disclosed are systems and compositions for use with the disclosed methods.BRIEF DESCRIPTION OF THE DRAWINGS

[0010] This application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0011] Those of skill in the art will understand that the drawings, described below, are for illustrative purposes only. The drawings are not intended to limit the scope of the present teachings in any way.

[0012] FIG. 1 is a schematic representation of the overall workflow used in the examples

[0013] FIG. 2 is a heatmap of proteins significantly associated with any of the select outcomes.

[0014] FIG. 3 is a schematic showing the network of significantly enriched biological processes and pathways in proteins significantly associated with any of the select outcomes.

[0015] FIG. 4 is a schematic representation of the workflow used for selecting biomarkers.

[0016] FIG. 5 is a heatmap of the ten biomarker candidates vs the select 13 outcomes.

[0017] FIG. 6 shows predictive performance of the ten-protein biomarker panel with respect to the 13 select outcomes. Both precision-recall (left) and standard ROC (right) curves were used for measuring the performance. Each value represents the mean and the standard deviations of the area-under-the-curve (AUC) values, averaged across five random experiment runs (with different samples used for training and testing purposes).

[0018] FIG. 7 shows a network of significantly enriched biological processes and pathways in proteins significantly associated with any of the select outcomes.

[0019] FIG. 8 show a network of significantly enriched biological processes and pathways in proteins significantly associated with any of the select outcomes.

[0020] FIG. 9 depicts Phase 1 and Phase2 Participant Enrollment and Final Cohort included in the Primary ENvISION Study. The enrollment strategy for Phasel was to capture the proteome at the exact time of the diagnosis of Crohn’s disease (prior to the colonoscopy) and included 42 / 80 Crohn’s disease patients enrolled. The Phase2 strategy was used to assess the proteome immediately prior to the start of the anti-TNF biologic (not at the time of diagnostic colonoscopy) and included 38 / 80 Crohn’s disease patients enrolled.

[0021] FIG. 10 shows treatment outcomes at one year. Each outcome only includes those patients that had the evaluation performed. Abbreviations: SES-CD, simple endoscopic severity - Crohn’s disease; wPCDAI, weighted pediatric Crohn’s disease activity index; sMARIA, simplified Magnetic Resonance Index of Activity.

[0022] FIG. 11. Treatment outcomes three months after the start of the anti-TNF biologic. Each outcome only includes those patients that had the evaluation performed. Biochemical remission, defined by fecal calprotectin <250 pg / g or c-reactive protein <0.5 mg / dL; Fecal calprotectin remission, defined by fecal calprotectin <250 pg / g; Therapeutic trough, defined by an maintenance infliximab trough >5 pg / mL or adalimumab trough >7.5 pg / mL; primary biochemicalnonresponse (NR), defined by a failure to improve baseline fecal calprotectin >100 pg / g or failure to improve c-reactive protein >0.5 mg / dL (limited to patients with a baseline c-reactive protein >1.0 mg / dL); primary clinical nonresponse, defined as no change or worsening in the baseline weighted pediatric Crohn’s disease activity index (wPCDAI) or a wPCDAI >40 at month3.DETAILED DESCRIPTION[00231 Unless otherwise noted, terms are to be understood according to conventional usage by those of ordinary skill in the relevant art. In case of conflict, the present document, including definitions, will control. Exemplary methods and materials are described below, although methods and materials similar or equivalent to those described herein may be used in practice or testing of the present invention. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting.

[0024] The methods disclosed herein may comprise, consist of, or consist essentially of the elements of the compositions and / or methods as described herein, as well as any additional or optional element described herein or otherwise useful in methods of characterizing, diagnosing, monitoring, and / or treating an individual with Crohn’s Disease (CD). The disclosed methods allow for individualized treatment selection, particularly for use of anti-TNF biologies.

[0025] In one aspect, disclosed is a method of characterizing an individual with Crohn’s Disease (CD) comprising detecting, in a biological sample obtained from the individual, a plurality of biomarkers comprising, or consisting of: tubulin polymerization promoting protein, HUGO Gene Nomenclature Committee (HGNC): 24164 (TPPP), leucine rich repat containing G protein- coupled receptor 4, HGNC: 13299 LGR4), DNAJ heat shock protein family member C5 beta, HGNC: 24138 (DNAJC5B). dynein light chair roadblock-type 2, HGNC: 15467 (DYNLRB2).leukocyte receptor tyrosine kinase, HGNC: 6721 (LTK). CD320 molecule, HGNC: 16692 (CD320), glutaredoxin 2, HGNC: 16065 (GLRX2), Bcl2 modifying factor, HGNC: 24132 (BMF), lymphotoxin alphal: lymphotoxin beta2, HGNC: 6709, 6711 (LTA.LTB). checkpoint kinase 2, HGNC: 16627 (CHEK2). and combinations thereof, in which the detecting of the plurality of biomarkers can be used to characterize the individual for predicting outcome and / or treating the individual.

[0026] In one aspect, disclosed is a method of diagnosing an individual with Crohn’s Disease (CD) comprising detecting, in a biological sample obtained from the individual, a plurality of biomarkers comprising, or consisting of: TPPP, LGR4, DNAJC5B. DYNLRB2, LTK, CD320, GLRX2, BMP, LTA.LTB, CHEK2, and combinations thereof, in which the detecting of the plurality of biomarkers can be used to diagnose the individual as having, or not having CD.

[0027] In one aspect, disclosed is a method of monitoring an individual with Crohn’s Disease (CD) comprising detecting, in a biological sample obtained from the individual, a plurality of biomarkers comprising, or consisting of TPPP, LGR4, DNAJC5B. DYNLRB2, LTK, CD320, GLRX2, BMP, LTA.LTB, CHEK2 and combinations thereof, in which the detecting of the plurality of biomarkers can be used to monitor the health of the individual, or the response to a treatment of an individual with a therapy for CD.

[0028] In one aspect, disclosed is a method of treating an individual with Crohn’s Disease (CD) comprising detecting, in a biological sample obtained from the individual, a plurality of biomarkers comprising, or consisting of: TPPP, LGR4, DNAJC5B. DYNLRB2, LTK, CD320, GLRX2, BMP, LTA.LTB, CELEK2 and combinations thereof, in which the detecting of the plurality of biomarkers is used to determine a treatment for the individual having CD.

[0029] The plurality of biomarkers may be detected using any method known in the art for detection of a biomarker. For example, in aspects, the biomarkers are detected as a protein. In aspects, detection of the biomarker may be qualitative. In aspects, detection of the biomarker may be quantitative. In this aspect, any method for the detection of protein may be used, for example, Enzyme-Linked Immunosorbent Assay (ELISA), chemiluminescent immunoassays (CLIA), Western blotting, Mass Spectrometry, flow cytometry, lateral flow immunoassay, multiplex assay, immunohistochemistry, radioimmunoassay (RIA), surface plasmon resonance (SPR), and the like. The detection of the biomarker protein may be carried out via any method known by one of ordinary skill in the art.

[0030] In aspects, the expression level of the biomarker is detected. In this aspect, as above, any method for detection of an RNA corresponding to the biomarker may be used. Such methods may be either qualitative or quantitative. For example, in one aspect, Quantitative Reverse Transcription PCR (qRT-PCR / RT-qPCR) may be used. In another aspect, Reverse Transcription PCR (RT-PCR) may be used. In another aspect, Digital PCR (dPCR) / Droplet Digital PCR (ddPCR) may be used. In a further aspect, a gene expression array may be used. In a yet further aspect, in situ hybridization (ISH) may be used. The detection of the biomarker RNA transcript may be carried out via any method known by one of ordinary skill in the art.

[0031] Following detection of the plurality of biomarkers, in aspects, the level of the biomarker is determined. This comprises determining a level of expression of each of the plurality of biomarkers, whether based on the level of the protein or the RNA transcript measured. The level of the biomarker may further be compared to a control value. For example, the biomarker level is compared to a baseline value in the individual. In other aspects, the biomarker level is compared to a value which is the average baseline value for a matched cohort. In aspects, the control is anexpected value for a given biomarker, which may be determined by an understanding of that level as taught in the art.

[0032] The plurality of biomarkers detected may be 10 or less of the biomarkers described herein. For example, in aspects, all ten biomarkers are detected and, optionally, quantified. In aspects, nine biomarkers are detected and, optionally, quantified. In aspects, eight biomarkers are detected and, optionally, quantified. In aspects, seven biomarkers are detected and, optionally, quantified. In aspects, six biomarkers are detected and, optionally, quantified. In aspects, five biomarkers are detected and, optionally, quantified. In aspects, four biomarkers are detected and, optionally, quantified. In aspects, three biomarkers are detected and, optionally, quantified. In aspects, two biomarkers are detected and, optionally, quantified. In aspects, a single biomarker is detected and, optionally, quantified.

[0033] In aspects, the levels of the detected biomarkers are used to predict, or alternatively, determine, a response in the individual. For example, in aspects, a statistically significant change in a level of at least one, or at least two, or at least three, or at least four, or at least five, or at least six, or at least seven, or at least eight, or at least nine, or all ten of the plurality of biomarkers, as compared to a control value, predicts endoscopic healing of the individual.

[0034] In further aspects, a statistically significant change in a level of at least one, or at least two, or at least three, or at least four, or at least five, or at least six, or at least seven, or at least eight, or at least nine, or all ten of the plurality of biomarkers, as compared to a control value, predicts remission, as measured by magnetic resonance imaging (MRI), in the individual.

[0035] In further aspects, a statistically significant change in a level of at least one, or at least two, or at least three, or at least four, or at least five, or at least six, or at least seven, or at leasteight, or at least nine, or all ten of the plurality of hiomarkers, as compared to a control value, predicts a longitudinal assessment of response to a biologic therapy.

[0036] In further aspects, a statistically significant change in a level of at least one, or at least two, or at least three, or at least four, or at least five, or at least six, or at least seven, or at least eight, or at least nine, or all ten of the plurality of hiomarkers, as compared to a control value, predicts response status of the individual to an anti-TNF therapy.

[0037] The disclosed methods may further be used to provide a treatment to an individual. For example, in aspects, based on the detection as set forth above and herein, an anti-TNF is administered to the individual.

[0038] In certain aspects, the described method includes detecting one or more of the plurality of biomarkers prior to the administration of an anti-TNF (tumor necrosis factor) biologic. This pretreatment detection may serve as a baseline to assess the subject’s initial biomarker profile, which can be used for predicting therapeutic response or stratifying patients for appropriate treatment regimens. In other aspects, the initial pre-treatment detection may be used for a baseline for comparison following a treatment.

[0039] In other aspects, the described method comprises detecting the biomarker after the administration of an anti-TNF biologic. Post-treatment detection allows for monitoring changes in biomarker expression that may correlate with therapeutic efficacy or the emergence of resistance. In further aspects, post-treatment detection may be compared to pre-treatment detection to assist with assessment of treatment response.

[0040] In further aspects, the method comprises detecting the biomarker at a specific time point, for example, about three months, or about four months, or about five months, or about six months, or up to a year following the initiation of anti-TNF therapy. This time-based detection canbe used to determine response of the subject to the treatment and can be used to inform decisions regarding continuation, modification, or cessation of therapy.

[0041] Detection of the plurality of biomarkers is carried out in a biological sample obtained from the patent. For example, the biological sample may be selected from blood, serum, plasma, tissue, or combinations thereof. In one aspect, the biological sample is serum.

[0042] In aspects, the anti-TNF therapy is a biological therapy specifically indicated for the treatment of inflammatory bowel disease (IBD), including Crohn’s Disease and ulcerative colitis. The method may further specify that the anti-TNF therapy is selected from infliximab (IFX), adalimumab (AD AL), certolizumab-pegol, golimumab, or combinations thereof, each of which is an FDA-approved biologic agent targeting TNF-alpha.

[0043] The method may be applied to individuals of various age groups. In one embodiment, the individual is a pediatric patient, allowing for early intervention and personalized treatment strategies in younger populations. In another embodiment, the individual is an adult patient, reflecting the broader applicability of the method across age demographics.

[0044] In further aspects, a use of a biomarker panel comprising TPPP, LGR4, DNAJC5B, DYNLRB2, LTK, CD320, GLRX2, BMF, LTA.LTB. CHEK2, for predicting response to anti-TNF therapy in a subject diagnosed with Crohn’s Disease (CD) is disclosed. This panel may be used to stratify patients based on their likelihood of responding to treatment, thereby enhancing clinical decision-making.

[0045] In further aspects, a system is also provided, comprising a processor configured to receive biomarker expression data and to output a predicted therapeutic response based on a trained classifier. The biomarkers used in this system may be selected from one or more of: TPPP TPPP, LGR4, DNAJC5B, DYNLRB2, LTK, CD320, GLRX2, BMF, LTA.LTB, CHEK2,AGRN, SLC16A3,QS0 2, and ADAMTS13. The system may be implemented in a clinical decision support tool to assist healthcare providers in optimizing treatment plans.

[0046] In yet further aspects, a non-transitory computer-readable medium is disclosed, storing instructions that, when executed by a processor, perform the method as described herein. The non- transitory computer-readable medium allows for the use of the method in software applications for clinical or research use.

[0047] In certain aspects, a composition for use in the described methods is disclosed. For example, disclosed is a composition comprising a solid support or solution that includes a plurality of binding agents, such as antibodies, antibody fragments, or other affinity reagents, each specific for a biomarker. The plurality of binding agents include binding agents specific for protein, as well as binding reagents (such as complementary nucleotides) specific for a transcript encoding a biomarker as disclosed herein. The plurality of binding agents may be used to detect one or more biomarkers selected from tubulin polymerization promoting protein TPPP leucine rich repeat containing G protein-coupled receptor 4 (LGR4). DNAJ heat shock protein family member C5 beta DNAJC5B), dynein light chain roadblock-type 2 (DYNLRB2), leukocyte receptor tyrosine kinase (LTK), CD320 molecule (CD320). glutaredoxin 2 (GLRX2). Bcl2 modifying factor (BMF). lymphotoxin alphal: lymphotoxin beta2 (LTA.LTB). and checkpoint kinase 2 (CHEK2). The solid support may include, but is not limited to, microarray slides, beads (e.g., magnetic or polystyrene), membranes, or multi-well plates. In aspects, each binding agent is immobilized in a spatially addressable format to enable multiplexed detection. In further aspects, the binding agents may be suspended in a buffered aqueous medium suitable for maintaining binding activity and enabling simultaneous or sequential detection of the biomarkers. The composition may be used inimmunoassays, such as ELISA, flow cytometry, or bead-based multiplex assays, to facilitate the qualitative or quantitative detection of the biomarkers in a biological sample.

[0048] Definitions

[0049] As used herein and in the appended claims, the singular forms “a,” “and,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a method” includes a plurality of such methods and reference to “a dose” includes reference to one or more doses and equivalents thereof known to those skilled in the art, and so forth.

[0050] The term “about” or “approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, e.g., the limitations of the measurement system. For example, “about” may mean within 1 or more than 1 standard deviation, per the practice in the art. Alternatively, “about” may mean a range of up to 20%, or up to 10%, or up to 5%, or up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term may mean within an order of magnitude, preferably within 5 -fold, and more preferably within 2- fold, of a value. Where particular values are described in the application and claims, unless otherwise stated the term “about” meaning within an acceptable error range for the particular value should be assumed.

[0051] The terms “individual,” “host,” “subject,” and “patient” are used interchangeably to refer to an animal that is the object of treatment, observation and / or experiment. Generally, the term refers to a human patient, but the methods and compositions may be equally applicable to non-human subjects such as other mammals. In some aspects, the terms refer to humans. In further aspects, the terms may refer to children.

[0052] The following non-limiting examples are provided to further illustrate embodiments of the invention disclosed herein. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent approaches that have been found to function well in the practice of the invention, and thus may be considered to constitute examples of modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes may be made in the specific embodiments that are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

[0053] Example 1

[0054] Background and aims: The number of advanced therapies for managing Crohn’s disease (CD) has grown significantly. However, selection of first-line therapy remains largely trial and error due to the lack of biomarkers that integrate inflammatory endotype with mechanism of action. The global aim was to perform a comprehensive proteomic evaluation to identify novel candidate bio markers associated with anti-TNF outcomes.

[0055] Methods: This was a multicenter, observational cohort of children and young adults with CD who were enrolled prior to starting infliximab or adalimumab with longitudinal biospecimens obtained for one year. The SOMAscan® (Somalogic, Inc, Boulder, CO, 7322 analytes) was performed on plasma obtained before initiating anti-TNF therapy, as well as at three months and twelve months after starting the biologic.

[0056] In this study, the SOMAscan™ (SomaLogic, Boulder, CO) platform was used to investigate the abundance of >7300 plasma proteins from CD patients starting either infliximab or adalimumab. The primary aim was to perform a comprehensive proteomic evaluation with SOMAscan™ to identify novel candidate biomarkers associated with anti-TNF outcomes. Aprotein panel of 10 proteins was discovered, serving as a platform for personalizing the selection of advanced therapies in patients with CD.

[0057] Materials and Methods

[0058] Cohort. The Precision Crohn’s Disease Management Utilizing Predictive Protein Panels (ENvISION) trial (NCT04131504) was a multicenter, observational study of children and young adults (<22 years old) with CD starting an anti-TNF biologic (infliximab or adalimumab). Patients were enrolled at one of two timepoints, either at Phase 1 (cross-sectional phase) or Phase2 (longitudinal phase). Phase 1 patients included those who were suspected to have inflammatory bowel disease and underwent a diagnostic colonoscopy. Phasel patients found to have CD and referred to start an anti-TNF biologic were approached for Phase2. Phase2 also included patients who were referred to start an anti-TNF that were not enrolled in Phasel. Baseline demographic, disease severity and phenotype were collected while clinical, biochemical, endoscopic and radiologic outcomes were monitored up to one year or until the biologic was stopped or a patient had a CD-related surgery. Patients with CD were enrolled with an Institutional Review Board approved research protocol at Cincinnati Children’s Hospital Medical Center, Connecticut Children’s Hospital, Nationwide Children’s Hospital, and at the Medical College of Wisconsin between October 2019 and December 2021.

[0059] Healthy controls were also enrolled into Phasel. Healthy controls were age and sex- matched to the CD cohort. To be included in the proteomic study as a health control, participants had a fecal calprotectin <50 pg / g, no recent antibiotic use, not receiving care for a chronic illness and did not meet ROME IV criteria using the validated questionnaire for functional gastrointestinal disorders.22

[0060] Early (Month3) Outcomes. Early (relevant) outcomes were assessed at month3 in all participants. Outcomes of importance included fecal calprotectin remission <250 pg / g, biochemical remission (fecal calprotectin <250 pg / g and / or CRP <0.5 pg / dL), and sufficient anti- TNF exposure which was defined as an infliximab trough prior to dose4 >5 pg / mL or an adalimumab trough concentration >7.5 pg / mL. Primary biochemical nonresponse was defined as a failure to improve baseline fecal calprotectin >100 pg / g (limited to patients who had a baseline fecal calprotectin >250 pg / g) or failure to improve c-reactive protein >0.5 mg / dL (limited to patients with a baseline c-reactive protein >1.0 mg / dL) and primary clinical nonresponse defined as no improvement or a worsening in baseline wPCDAI or a month3 wPCDAI >40.

[0061] Late (Yearl) Outcomes. It was recommended that each participant undergo a research (or clinically-indicated) colonoscopy and magnetic resonance imaging with enterography (MRE) at 9-12 months from the start of the anti-TNF biologic. The primary treatment outcome for this investigation was endoscopic healing defined as a simple endoscopic severity-CD (SES-CD) score <3, with the participant remaining on the same anti-TNF biologic without a need for a CD-related surgery during the first year of therapy.23The endoscopic procedure was video-recorded and the SES-CD scoring was performed by two pediatric gastroenterologists trained in SES-CD scoring and blinded to any patient identifiers and the endoscopic report (AN, PM). Additional outcomes of interest included steroid-free clinical remission defined by a monthl2 weighted pediatric CD activity index (wPCDAI) <12.5 and off a corticosteroid for >2 months before month!2. Deep remission at month12 was defined by a wPCDAI <12.5 and a SES-CD <3. Early biologic response was defined by an improvement in baseline fecal calprotectin by >50% at month3 or a fecal calprotectin <250 pg / g (biochemical remission) and late biochemical remission was defined by a monthl2 fecal calprotectin <250 pg / g. MRE remission at monthl2 was defined by a simplifiedMagnetic Resonance Index of Activity (sMARIA) for CD <2 (inclusive of colon, ileum and jejunal assessments) with scores produced by one experience radiologist (JD).24Mucosal healing was defined as a SES-CD=0 and transmural healing included a SES-CD<3 plus a sMARIA<2.

[0062] Additional exploratory outcomes included endoscopic healing with a fecal calprotectin <250 pg / g, endoscopic healing with a wPCDAI <12.5 and fecal calprotectin <250 pg / g, endoscopic healing and a wPCDAI <12.5 for all patients enrolled (patients stopping anti-TNF or undergoing CD-related surgery included as not healed). Finally, adequate drug exposure defined as a yearl trough concentration >5 pg / mL was assessed for both infliximab and adalimumab.

[0063] Research only biospecimens (blood and stool) were collected prior to the start of anti- TNF, three months from the start of therapy and approximately 12 months from the start of the anti-TNF biologic. Intestinal biopsies for research were collected at the diagnostic colonoscopy for Phasel participants and for all Phase2 participant who underwent a monthl2 colonoscopy.

[0064] Peripheral Blood Proteomics. Peripheral blood was obtained throughout the study using the P100 blood collection system (BD Biosciences, Franklin Lakes, NJ) to stabilize human plasma proteins at the point of collection. Protein analytes were measured using SOMAscan® v4.1 (SomaLogic, Inc., Boulder, CO), performed by Washington University School of Medicine Genome Technology Access Center (GTAC), St. Louis, MO. SOMAscan v4.1 measures 7322 protein analytes. The dynamic range is highly reproducible and ranges from femtomolar to micromolar. In brief, SomaLogic’ s proprietary protein-capture reagents, called SOMAmer (Slow Off-rate Modified Aptamers), use aptamers (short single-stranded DNA sequences) that bind with high affinity and selectivity to distinct proteins with protein abundance quantified by DNA- hybridization microarrays. Protein abundance is read out as relative fluorescence units (RFU). Aprior study has found that the SOMAscan platform had excellent reproducibility and stability and was comparable to other commercially available proteomic platforms.25

[0065] Additional Biomarker Investigations. Fecal calprotectin was determined from a stool sample collected prior to the start of the anti-TNF, at month3 and month 12 using a commercial ELISA kit (Buhlmann, Switzerland).15

[0066] RESULTS

[0067] Patient cohort. In Phase 1, 111 subjects undergoing colonoscopy for suspected inflammatory bowel disease (cross-sectional arm) were enrolled. Of the 111 enrolled, 64 were found to have CD with 42 of the 64 contuining into Phase2 of the study (longitudinal arm). An additional 38 patients with CD that were naive to the anti-TNF biologies were enrolled and recommended to stall either infliximab or adalimumab into Phase2. Therefore a total 80 patients with CD were included in this observational study (FIG. 9). Sixty-three started infliximab and 17 patients were started on adalimumab. The mean (SD) age at the start of anti-TNF therapy was 13.7 (3.5) years, 42.5% were female and 90% were white (Table 1). Prior to the start of the anti-TNF biologic, 46.3% were exposed to a corticosteroid with 19 receiving prednisone and 18 patients on oral (9 mg) budesonide. Thirty healthy controls were enrolled into Phase 1.

[0068] Table 1. Baseline demographics and disease characteristics of the entire cohort and those with endoscopic healing or endoscopic active after 12 months of anti-TNF therapy.

[0069] At one year, 93.8% (75 / 80) remained on the same anti-TNF biologic with 8.8% (7 / 80) requring a CD related surgery. Of those remaining on the same biologic, 78.7% (59 / 80) underwent a repeat colonoscopy and 58.7% (44 / 75) had a MRE. Of those who had a colonoscopy performed, 37 (62.7%) achieved endoscopic healing, 22 (37.3%) were endoscopic active and 24 (54.5%)achievd MRE remission. Additional yearl outcomes including the rate of fecal calprotectin remission (<250 g / g). mucosal healing (SES-CD-0), deep remission (SES-CD>3 plus a wPCDAI<12.5), and transmural healing (sMARIA<2 plus a SES-CD<3) are reported in FIG. 10.

[0070] Protein Discovery: Data preprocessing and Normalization. Protein abundance measurements were used to conduct a composite correlation analysis to identify candidate proteins associated with different outcomes following anti-TNF administration. Before this, a data preprocessing step was implemented to retain proteins in at least half of the samples at each time point (FIG. 1). Next, variance stabilization normalization (Vsn) was applied to remove the influence of technical noise in the final protein measurements26. The Vsn method makes the sample variances independent of their mean intensities via parametric transformations and maximum likelihood estimation27.

[0071] Correlation analysis. Vsn-normalized data from each time point were used in the composite correlation analysis. Specifically, Pearson correlations between baseline (pre-treatment) protein expression levels and relevant patient outcome variables were computed. 13 outcome variables were computed at three months (FIG. 11) and one year after anti-TNF administration, including year 1 MRE remission and endoscopic healing (Table 2). Additionally, Student’s asymptotic p-values were computed to assess the statistical significance of the correlation scores. 134 proteins were identified that were significantly associated (adjusted p-value < 0.05) with any of the chosen outcomes (FIG. 2). Among them, 49 proteins were overexpressed in CD, whereas the remaining 85 proteins were downregulated relative to the healthy controls. 113 proteins significantly associated with yearl sMARIA remission outcome were also identified (FIG. 2). Of these, 54 proteins were positively correlated and the remaining 59 proteins were negativelyassociated with sMARIA remission. Similarly, 12 proteins were significantly associated with a composite endoscopic healing outcome at year 1 (FIG. 2).

[0072] Table 2. Outcome variables used in the study.Endoscopic healing was defined as a Simple Endoscopic Score-Crohn’s disease <3. Data shown as mean (standard deviation), as a number (n) with a percentage or median (25-75% interquartile range) depending on data distribution.

[0073] The functional enrichment analysis of the 134 proteins indicated enrichment of pathways, and biological processes associated with anticoagulation, thrombosis and wound healing. (FIG. 3) Additionally, proteins that cocxprcsscd epithelial and goblet cells in the aging large intestine were identified. (FIG. 3) Single cell marker genes from distinct cell types from ileal Crohn’s disease were also enriched in the candidate list. (FIG. 3)

[0074] Biomarker analysis

[0075] To identify a protein biomarker panel and evaluate its predictive ability, generalized linear models (GLM) were trained with elastic net penalties28for each outcome variable (FIG. 4) using protein expression levels as the model covariates. 70% of the samples were used for model training and the remaining 30% to evaluate the final trained models. In addition, five independent runs were conducted, and the final model performances were averaged across all runs. Additionally, all models were adjusted for 16 different covariates, including pre-treatment exposures and other phenotypic traits. The area under the curve (AUC) values of both the precision-recall (PR) and receiver operating characteristic (ROC) curves were used to quantify the performance of the trained models.

[0076] To identify biomarkers associated with multiple outcome variables, composite ranks of proteins ranked according to their predictive performance were computed. (FIG. 5) Specifically, all 134 proteins were ranked based on the two AUC scores for each outcome, with the lowest- ranked protein being highly predictive of a given sample outcome status. The protein ranks were then aggregated across all relevant outcome variables using median and harmonic mean computations. Finally, protein candidates were identified (TPPP, LGR4, DNAJC5B, DYNLRB2, LTK, CD320, GLRX2, BMP, LTA.LTB, and CHEK2) that were highly ranked (top 10) using both approaches. All but one protein (DNAJC5B) was downregulated in CD compared with healthy controls. (FIG. 5)

[0077] Finally, the combined model using all ten biomarker candidates as the covariates achieved impressive results in predicting both composite endoscopic healing (AUPRC = 0.91 ± 0.054; AUROC - 0.865 ± 0.08) and sMARIA remission (AUPRC - 0.852 ± 0.041; AUROC= 0.776 ± 0.056) outcomes. These results were averaged over five runs (FIG. 6).

[0078] To validate the efficacy of these biomarker candidates, the PANTS study29was used, a UK- wide prospective observational cohort study of anti-TNF therapy outcomes. Blood gene expression data from 814 samples (324 unique patients) collected across four different time points were used in this step. Regularized logistic regression models were trained to predict the primary response status of the patient to anti-TNF therapy using the 10 biomarker genes as covariates. At each time point, 70% of the samples were used for model training, and the remaining 30% were used for evaluation. In addition, k-fold cross-validation (k = 5) was used to tune the regularization parameter. In these experiments, an incremental increase in model performance relative to the number of study visits was observed. At baseline (week 0), the trained biomarker model achieved an AUPRC score of 0.709 (70.9%), whereas three months after treatment initiation, it increased to 0.819 (81.9%). (FIG. 7) Similarly high model performances in predicting anti-TNF therapy response at week 30 (AUPRC = 0.878) and week 54 (AUPRC = 0.889, FIG. 8) were observed. Again, all models trained using data from the PANTS study were adjusted for both clinical and demographic patient data, including age, gender, disease duration, and BMI.

[0079] Discussion

[0080] Precision guided treatment selection based on biomarker panels or endotypes would be a significant advancement for both adults and children newly diagnosed with CD or currently failing an advanced therapy. In this study, a panel of ten biomarker candidates was found to achieve impressive results in predicting endoscopic healing and sMARlA remission. Furthermore, these biomarker candidates were validated using an external cohort of adult patients with CD. The implications of these findings is that clinicians can use this unique protein signature to not only identify patients who are likely to respond to the anti-TNF biologies, but also to monitor the change in proteins over time as a non-invasive measure of treatment response.

[0081] The IBD research priorities for the next five years were recently proposed by the Crohn’s and Colitis Foundation of America (CCFA) multidisciplinary panel of researchers.30The first global research priority identified was to “define clinically relevant subsets of patients with IBD using genetic, immunologic, microbial, tissue expression, and clinical profiles that will predict aggressiveness of disease, complications, and response to treatment.”30The panel aimed to accomplish this priority with (1) “prospective cohort studies of pediatric and adult patients with IBD with serial biospecimens collected throughout the course of their diseases” and (2) “improved tools for measuring disease activity in IBD.”30In addition, the North American Society of Pediatric Gastroenterology, Hepatology and Nutrition (NASPGHAN) researchers prepared a pediatric IBD research agenda identifying the following priorities: (1) developing new markers of IBD activity and (2) identifying novel, alternative therapies for pediatric IBD with fewer side effects.31

[0082] In a small study of seven pediatric IBD patients receiving infliximab, the 1300 SOMAscan platform was used to identify 18 proteins that were associated with response to infliximab and prednisone.16In a larger cohort of healthy adults who later developed IBD, Torres et al. identified a panel of 51 peripheral blood proteins that were highly predictive of CD within 5 years using the same discovery platform.17

[0083] The extensive search for biomarkers of anti-TNF response has included investigations of genetics, tissue transcriptomics, microbial and metabolomic signatures and single cell transcriptomics. Here, a 10-protein biomarker endotype from peripheral blood has been developed that will be used to non-invasively assess the likelihood of response to a class of biologies and provide a longitudinal assessment of response during the maintenance phase of treatment.

[0084] EXAMPLE 2.

[0085] Background: Given the variable response to anti-TNF therapy in patients with Crohn’ s disease (CD), it was hypothesized that there is a core biologic (inflammatory) signature associated with early response and endoscopic healing (EH). The primary objective was to identify plasma candidate proteins (a CD proteome) for anti-TNF naive patients and EH in children receiving anti- TNF therapy.

[0086] Methods: CD patients were enrolled at four pediatric centers with longitudinal blood and stool specimens collected prior to the start of either infliximab (IFX) or adalimumab (AD AL) and throughout this observational study. At month3 (M3), fecal calprotectin remission (fCalRem) was defined as a fCal <250 pg / g and BioRem was defined as a fCal<250pg / g or CRP<05g / dL. At monthl2 (M12), a research-only / mucosal healing colonoscopy was recommended and EH was defined as a Simple Endoscopic Score-CD (SES-CD)<3. Plasma protein abundance was determined by the aptamer based SOMAscanTM (SomaLogic, Boulder, CO) at baseline (BL, n=80), M3 (n=78), and at M12 (n=79) from the CD patients and 30 healthy controls (HC).

[0087] Results: Eighty CD (63 IFX, 17 ADAL) were enrolled. During the one-year study, 58 patients had a colonoscopy, 10 declined endoscopy, 6 had surgery, and 5 stopped anti-TNF therapy. EH was achieved by 37 / 59 (62.7%, no difference by biologic). Aimla was to identify a core CD proteome. Starling with 7322 protein analytes, applicant identified 2999 differently abundant proteins (1596, up, 1403 down) between the 58 steroid-naive CD patients and the 30 HC (FDR<0.05). In slides 8-10, the top / down abundant proteins and the biological processes and pathways for the CD proteome are highlighted. Aim 1 b was to identify a subset of BL, M3 or delta (M3-BL) proteins (and pathways) that were differentially abundant between patients who achieved Mfrom BL CD core proteins for M3 and M12 outcomes. As M3 BioRem was determined for all80 12 EH (SES-CD<3). In this abstract, the results are reported in the delta protein abundance(from M3-BL) for those who achieved EH vs. who had endoscopic activity (EA, SES-CD>3). Starting with all 7322, proteins in the SOMAscanTM assay, 122 were found to be differentially abundant for those who achieved EH (using a cut point of <0.01). Biological processes and pathways that were associated with this 122 protein set were then explored. Patients with EA were found to have enrichment for proinflammatory proteins (IL21, IL22, Il lb, IL36a, and IFIH1), pro- fibrotic proteins (MMP12) and complement proteins (slide 15).

[0088] Given the strength of significance for the complement pathway, it was further found that there were 9 complement proteins in this candidate signature. All 9 (CRP, CPN1, CPI, APC5, C5, CFHR, CFB, C9 and C2) had similar abundance at baseline in the EA and EH patients but were significantly lower at M3 in the patients achieving EH. By performing a classification analysis to define a cut-point for complement activation (sustained vs. decreased, PC1=O), we found those patients with a high sustained complement at M3 had a low probability for EH at M12 (OR=0.09, p=0.002, AUC=0.752).

[0089] Conclusions: A core CD proteome and a protein candidate signature at M3 (delta M3- BL) that was strongly associated with M 12 EH was found. Additional analyses will aim to validate these findings in a separate cohort.

[0090] References

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[0097] 7. van Rheenen H, van Rheenen PF. Long-Term Efficacy of Anti-Tumor NecrosisFactor Agents in Pediatric Luminal Crohn's Disease: A Systematic Review of Real-World Evidence Studies. Pediatr Gastroenterol Hepatol Nutr 2020;23:121-131.

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[0122] It should be understood that every maximum numerical limitation given throughout this specification includes every lower numerical limitation, as if such lower numerical limitations were expressly written herein. Every minimum numerical limitation given throughout this specification will include every higher numerical limitation, as if such higher numerical limitations were expressly written herein. Every numerical range given throughout this specification will include every narrower numerical range that falls within such broader numerical range, as if such narrower numerical ranges were all expressly written herein.

[0123] The dimensions and values disclosed herein are not to be understood as being strictly limited to the exact numerical values recited. Instead, unless otherwise specified, each such dimension is intended to mean both the recited value and a functionally equivalent range surrounding that value. For example, a dimension disclosed as “20 mm” is intended to mean “about 20 mm.”

[0124] Every document cited herein, including any cross referenced or related patent or application, is hereby incorporated herein by reference in its entirety unless expressly excluded or otherwise limited. All accessioned information (e.g., as identified by PUBMED, PUBCHEM, NCBI, UNIPROT, or EBI accession numbers) and publications in their entireties are incorporated into this disclosure by reference in order to more fully describe the state of the art as known to those skilled therein as of the date of this disclosure. The citation of any document is not an admission that it is prior art with respect to any invention disclosed or claimed herein or that it alone, or in any combination with any other reference or references, teaches, suggests or discloses any such invention. Further, to the extent that any meaning or definition of a term in this document conflicts with any meaning or definition of the same term in a document incorporated by reference, the meaning or definition assigned to that term in this document shall govern.

[0125] While particular embodiments of the present invention have been illustrated and described, it would be obvious to those skilled in the art that various other changes and modifications may be made without departing from the spirit and scope of the invention. It is therefore intended to cover in the appended claims all such changes and modifications that are within the scope of this invention.

Claims

CLAIMSWhat is claimed is:

1. A method of characterizing, diagnosing, monitoring, and / or treating an individual with Crohn’s Disease (CD) comprising detecting, in a biological sample obtained from the individual, at least one of a plurality of biomarkers comprising, or consisting of: tubulin polymerization promoting protein (TPPP), leucine rich repat containing G protein- coupled receptor 4 (LGR4), DNAJ heat shock protein family member C5 beta (DNAJC5B). dynein light chair roadblock-type 2 (DYNLRB2). leukocyte receptor tyrosine kinase (LTK), CD320 molecule (CD320), glutaredoxin 2 (GLRX2 Bcl2 modifying factor (BMF), lymphotoxin alphal: lymphotoxin beta2 (LTA.LTBj'. checkpoint kinase 2 (CHEK2). and combinations thereof.

2. The method of claim 1 , further comprising determining a level of expression of each of the plurality of biomarkers.

3. The method of claim 2, further comprising comparing the level of expression of each of the plurality of biomarkers to a control value.

4. The method of claim 3, the control value being a baseline value in the individual.

5. The method of claim 3, the control value being an average baseline value for a matched cohort.

6. The method of any one of claims 1 through 5, wherein a statistically significant change in a level of at least one, or at least two, or at least three, or at least four, or at least five, or at least six, or at least seven, or at least eight, or at least nine, or all ten of the plurality of biomarkers, as compared to a control value, predicts endoscopic healing of the individual.

7. The method of any one of claims 1 through 5, wherein a statistically significant change in a level of at least one, or at least two, or at least three, or at least four, or at least five, or at least six, or at least seven, or at least eight, or at least nine, or all ten of the plurality of biomarkers, as compared to a control value, predicts remission of CD in the individual.

8. The method of any one of claims 1 through 5, wherein a statistically significant change in a level of at least one, or at least two, or at least three, or at least four, or at least five, or at least six, or at least seven, or at least eight, or at least nine, or all ten of the plurality ofbiomarkers, as compared to a control value, determines a longitudinal assessment of response to a biologic therapy.

9. The method of any one of claims 1 through 5, wherein a statistically significant change in a level of at least one, or at least two, or at least three, or at least four, or at least five, or at least six, or at least seven, or at least eight, or at least nine, or all ten of the plurality of biomarkers, as compared to a control value, predicts response status of the individual to an anti-TNF therapy.

10. The method of any one of claims 1 through 9, further comprising administering an anti- TNF therapy to the individual.

11. The method of claim 10, wherein the detecting is carried out prior to initiation of the anti- TNF therapy.

12. The method of claim 10, wherein the detecting is carried out after initiation of an anti- TNF biologic.

13. The method of claim 10, wherein the detecting is earned out about three months after initiation of an anti-TNF treatment.

14. The method of any preceding claim, wherein the detecting of the at least one of the plurality of biomarkers is detection of a protein of the biomarker.

15. The method of any preceding claim, wherein detection of the at least one of a plurality of biomarkers comprises detection of an mRNA transcript of the biomarker.

16. The method of any preceding claim, wherein the biological sample is selected from blood, serum, plasma, and tissue.

17. The method of any preceding claim, wherein the biological sample is serum.

18. The method of any one of claims 9 through 17, wherein the anti-TNF therapy is a biological therapy for treatment of IBD.

19. The method of any one of claims 9 through 17, wherein the anti-TNF therapy is selected from infliximab (IFX), adalimumab (AD AL), certolizumab-pegol, golimumab, and combinations thereof.

20. The method of any preceding claim, wherein the individual is a pediatric patient.

21. The method of any preceding claim, wherein the individual is an adult patient.

22. Use of a biomarker panel comprising TPPP, LGR4, DNAJC5B, DYNLRB2, LTK, CD320, GLRX2, BMF, LTA.LTB, CHEK2, for predicting response to anti-TNF therapy in a subject with Crohn’s Disease (CD).

23. A system comprising a processor configured to receive biomarker expression data and output a predicted therapeutic response based on a trained classifier, the biomarkers being selected from one or more of TPPP, LGR4, DNAJC5B, DYNLRB2, LTK, CD320, GLRX2, BMF, LTA.LTB, CHEK2, AGRN, SLC16A3, QSOX2, and ADAMTS13.

24. A non-transitory computer-readable medium storing instructions that, when executed, perform the method of any one of claims 1 to 21.

25. A composition comprising a plurality of binding agents for detection of a plurality of biomarkers comprising, or consisting of: tubulin polymerization promoting protein TPPP), leucine rich repat containing G protein-coupled receptor 4 U.GR4). DNAJ heat shock protein family member C5 beta (DNAJC5B). dynein light chair roadblock- type 2 (DYNLRB2). leukocyte receptor tyrosine kinase (LTK), CD320 molecule (CD320), glutaredoxin 2 (GLRX2), Bcl2 modifying factor BMF), lymphotoxin alphal: lymphotoxin beta2 (LTA.LTB), checkpoint kinase 2 (CHEK2), and combinations thereof.

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