WO2025177214A1 - Therapeutic methods - Google Patents

Abstract

The invention provides methods related to combination therapies comprising an anti- α4β7 antibody, e.g., vedolizumab, and a JAK inhibitor for treating inflammatory bowel disease.

Description

THERAPEUTIC METHODS

RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Appln. No. 63/555,539, filed on February 20, 2024. The contents of the foregoing applications are hereby incorporated by reference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on February 6, 2025, is named T103022_1380WO_SL.xml and is 56,932 bytes in size.

FIELD OF THE INVENTION

The present invention relates to a combination therapy comprising an anti-a4p7 antibody (e.g., vedolizumab), and a JAK inhibitor.

BACKGROUND OF THE INVENTION

Inflammatory bowel disease is a chronic, relapsing, inflammatory disorder of the gastrointestinal (GI) tract. Examples of IBD include ulcerative colitis and Crohn’s disease.

Ulcerative colitis is characterized by superficial, continuous mucosal inflammation and ulcers limited to the colon. The prevalence of UC is approximately 200/100,000 of the United States (US) population (Loftus, E. V., Jr. 2004. Clinical epidemiology of inflammatory bowel disease: Incidence, prevalence, and environmental influences. Gastroenterology, 126, 1504-17.).

The pathophysiology of UC is complex with multiple environmental and genetic factors interacting to cause expression of disease. Untreated, the clinical course of UC is unpredictable and marked by alternating periods of exacerbation and remission. Approximately 15% of patients experience an aggressive disease course, and 20% of these patients may require hospitalization for severe disease activity. The 5- and 10-year cumulative risk of colectomy is 10% to 15%, primarily limited to patients with moderate to severe disease activity; a subset of hospitalized patients with acute severe UC have short-term colectomy rates of 25% to 30% (Feuerstein, J. D., et al. 2020. AGA Clinical Practice Guidelines on the Management of Moderate to Severe Ulcerative Colitis. Gastroenterology, 158, 1450-1461.; and Zafer, M., Zhang, H., Dwadasi, S., Goens, D., Paknikar, R., Dalal, S., Cohen, R. D., Pekow, J., Rubin, D. T., Sakuraba, A. & Micic, D. 2022. A Clinical Predictive Model for One-year Colectomy in Adults Hospitalized for Severe Ulcerative Colitis. Crohns Colitis 360, 4, otab082.).

Treatment of UC is chosen according to disease activity and the extent of colonic involvement. There are a number of different drug classes for long-term management of moderate to severe UC, including TNF-a antagonists (infliximab, adalimumab, golimumab), anti-integrin agents (vedolizumab (VDZ)), Janus kinase inhibitors (tofacitinib (TOF)), interleukin 12/23 antagonist (ustekinumab), and immunomodulators (thiopurines, methotrexate (MTX)) (Burri, E., et al, 2020. Treatment Algorithm for Mild and Moderate-to- Severe Ulcerative Colitis: An Update. Digestion, 101 Suppl 1, 2-15.). Corticosteroids are recommended for short-term use due to the potential for serious adverse effects. Immunomodulators, although effective for the maintenance (but not induction) of remission, are also associated with serious adverse effects. They do, however, demonstrate a synergistic effect with biologies (Hashash, J. G., Fadel, C. G. A., Rimmani, H. H. & Sharara, A. I. 2021. Biologic monotherapy versus combination therapy with immunomodulators in the induction and maintenance of remission of Crohn's disease and ulcerative colitis. Ann Gastroenterol, 34, 612-624.). Advanced treatments for UC typically result in clinical remission rates at 1 year of 30% to 50%, suggesting there may be a therapeutic ceiling for the use of single agents (Danese, S., et al, L. 2022. The future of drug development for inflammatory bowel disease: the need to ACT (advanced combination treatment). Gut, 71, 2380-2387.).

Short- and long-term (1-year) clinical remission rates in Crohn’s disease (CD) currently range between 30% and 50%, suggesting that a therapeutic ceiling has been reached with single biologic agents including vedolizumab (Berinstein, E. M., et al. 2023. Efficacy and Safety of Dual Targeted Therapy for Partially or Non-responsive Inflammatory Bowel Disease: A Systematic Review of the Literature. Dig Dis Sci, 68(6), 2604-23).

Combination therapy for IBD consisting of a biologic with an immunomodulator drug has been studied. In a study of the biologic infliximab with the immunomodulator drug azathioprine (AZA) for the treatment of CD (SONIC), the proportion of participants who were in corticosteroid-free clinical remission after 26 weeks (primary endpoint) was higher in the combination therapy group than in either of the monotherapy groups; however, the treatment response in the combination therapy group was still incomplete, with 58% of participants achieving the primary endpoint (Colombel, J., et al. 2010. Infliximab, azathioprine, or combination Therapy for Crohn's disease. N Engl J Med, 362, 1383-95). There is, therefore, an overall unmet medical need within the IBD community for patients with moderately to severely active disease who may not respond to monotherapy or the combination of a biologic with an immunosuppressant. Dual targeted therapy (DTT) has been gaining increasing interest because of the varied cytokine pattern driving inflammatory bowel disease (IBD) (Berinstein et al. 2023; Neurath, M. F. 2014. Cytokines in inflammatory bowel disease. Nat Rev Immunol, 14(5), 329-42). Targeting more than one pathogenic pathway simultaneously may provide an additive or even synergistic benefit compared with monotherapy (Solitano, V., et al. 2023. Advanced combination treatment with biologic agents and novel small molecule drugs for inflammatory bowel disease. Gastroenterol Hepatol, 19(5), 251). For example, an exploratory double-blind, placebo-controlled study evaluated the safety and tolerability of a combination of 2 monoclonal antibodies to treat CD: infliximab, an anti-tumor necrosis factor alpha (TNF-a) agent, and natalizumab, which targets the a4 integrin (Sands et al. 2007. Safety and tolerability of concurrent natalizumab treatment for patients with Crohn's disease not in remission while receiving infliximab. Inflamm Bowel Dis, 13(1), 2-11). The participants had active disease (defined as Crohn’s Disease Activity Index [CD Al] score >150) despite ongoing infliximab treatment. Participants received infliximab with either natalizumab or placebo every 4 weeks (Q4W) for an 8-week period, with safety assessed through Week 10. Although the study was not powered to detect efficacy, several positive trends suggested improved efficacy of the combination therapy compared with infliximab alone. The safety profile of the combination therapy was similar to that of the control group (infliximab plus placebo).

Thus, combination therapies for IBD may provide additional treatments for patients in need.

SUMMARY OF THE INVENTION

In various aspects, the present disclosure provides a method of treating an inflammatory bowel disease (IBD) by administering a combination of an anti-a407 antibody, or an antigen-binding fragment thereof, and a pan-JAK inhibitor, such as tofacitinib, based on differential expression of genes. Based on the expression level of specific markers, the combination therapy described herein may include administration of both an anti -0.407 antibody and a pan-JAK inhibitor during an induction phase of treatment for IBD in a patient, followed by monotherapy comprising administration of the anti -0.407 antibody for maintenance (in the absence of the pan-JAK inhibitor). In one aspect, provided herein is a method of treating a human subject having an inflammatory bowel disease (IBD), said method comprising administering a humanized anti- 06407 antibody and administering a pan-Janus kinase (JAK) inhibitor to the human subject, wherein the human subject having IBD has an increased expression level before treatment of at least one effector marker selected from the group consisting of CCR9, FASLG, IFNG, MAPK1, MAPK3, MMP1, NFKB1, PLA2G1B, and TCF4 relative to a reference level in a human subject without IBD, and/or wherein the human subject having IBD has a decreased expression level after treatment of the at least one effector marker selected from the group consisting of CCR9, FASLG, IFNG, MAPK1, MAPK3, MMP1, NFKB1, PLA2G1B, and TCF4 relative to a baseline expression level in the human subject having IBD, and wherein the humanized anti-a4p7 antibody is an IgGl antibody and comprises a heavy chain variable region comprising a CDR3 domain as set forth in SEQ ID NO: 4, a CDR2 domain as set forth in SEQ ID NO: 3, and a CDR1 domain as set forth in SEQ ID NO: 2; and comprises a light chain variable region comprising a CDR3 domain as set forth in SEQ ID NO: 8, a CDR2 domain as set forth in SEQ ID NO: 7, and a CDR1 domain as set forth in SEQ ID NO: 6.

In another aspect, provided herein is a method of treating a human subject having an inflammatory bowel disease (IBD), said method comprising administering a humanized anti- a4p7 antibody and administering a pan-Janus kinase (JAK) inhibitor to the human subject, wherein the human subject having IBD has a decreased expression level before treatment of CDH1 relative to a reference level in a human subject without IBD, and/or wherein the human subject having IBD has an increased expression level after treatment of CDH1 relative to a baseline expression level in the human subject having IBD, and wherein the humanized anti-a4p7 antibody is an IgGl antibody and comprises a heavy chain variable region comprising a CDR3 domain as set forth in SEQ ID NO: 4, a CDR2 domain as set forth in SEQ ID NO: 3, and a CDR1 domain as set forth in SEQ ID NO: 2; and comprises a light chain variable region comprising a CDR3 domain as set forth in SEQ ID NO: 8, a CDR2 domain as set forth in SEQ ID NO: 7, and a CDR1 domain as set forth in SEQ ID NO: 6.

In another aspect, provided herein is a method of treating a human subject having an inflammatory bowel disease (IBD), said method comprising administering a humanized anti- a4p7 antibody and administering a pan-Janus kinase (JAK) inhibitor to the human subject, wherein the human subject having IBD has an increased expression level before treatment of MAPK1 and/or MAPK3 relative to a reference level in a human subject without IBD, and/or wherein the human subject having IBD has a decreased expression level after treatment of MAPK1 and/or MAPK3 relative to a baseline expression level in the human subject having IBD, and wherein the humanized anti-a4p7 antibody is an IgGl antibody and comprises a heavy chain variable region comprising a CDR3 domain as set forth in SEQ ID NO: 4, a CDR2 domain as set forth in SEQ ID NO: 3, and a CDR1 domain as set forth in SEQ ID NO: 2; and comprises a light chain variable region comprising a CDR3 domain as set forth in SEQ ID NO: 8, a CDR2 domain as set forth in SEQ ID NO: 7, and a CDR1 domain as set forth in SEQ ID NO: 6.

In another aspect, provided herein is a method of treating a human subject having an inflammatory bowel disease (IBD), said method comprising administering a humanized anti- 06407 antibody and administering a pan-Janus kinase (JAK) inhibitor to the human subject, wherein the human subject having IBD has an increased expression level before treatment of at least one effector marker selected from the group consisting of TCF4, NFKB1, MMP1, and IFNG relative to a reference level in a human subject without IBD, and/or wherein the human subject having IBD has a decreased expression level after treatment of the at least one effector marker selected from the group consisting of TCF4, NFKB1, MMP1, and IFNG relative to a baseline expression level in the human subject having IBD, and wherein the humanized anti-a4p7 antibody is an IgGl antibody and comprises a heavy chain variable region comprising a CDR3 domain as set forth in SEQ ID NO: 4, a CDR2 domain as set forth in SEQ ID NO: 3, and a CDR1 domain as set forth in SEQ ID NO: 2; and comprises a light chain variable region comprising a CDR3 domain as set forth in SEQ ID NO: 8, a CDR2 domain as set forth in SEQ ID NO: 7, and a CDR1 domain as set forth in SEQ ID NO: 6.

In one embodiment, the IBD is Crohn’s disease. In a further embodiment, the Crohn’s disease is moderately to severely active Crohn’s disease.

In one embodiment, the human subject has active inflammation on ileocolonoscopy.

In one embodiment, the human subject is biologic-naive.

In one embodiment, the human subject had a lack of an adequate response with, lost response to, or was intolerant to treatment with at least one of an immunomodulator, a tumor necrosis factor-alpha antagonist or combinations thereof.

In one embodiment, the human subject is intravenously administered a first dose of 300 mg of the humanized anti-a4p7 antibody at week 0, followed by a second dose of 300 mg of the humanized anti-a4p7 antibody at week 2. In a further embodiment, the method comprises intravenously administering a third dose of 300 mg of the humanized anti-a4p7 antibody at week 6, followed by a 300 mg dose of the humanized anti-a4p7 antibody every four or eight weeks thereafter.

In a further embodiment, the human subject is subcutaneously administered a dose of 108 mg of the humanized anti-a4p7 antibody at week 6, followed by a 108 mg dose of the humanized anti-a4p7 antibody every two weeks thereafter.

In a further embodiment, the method comprises intravenously administering a third dose of 300 mg of the humanized anti-a4p7 antibody at week 6, wherein the human patient is subcutaneously administered a dose of 108 mg of the anti-a4p7 antibody at week 14, followed by a 108 mg dose of the humanized anti-a4p7 antibody every two weeks thereafter.

In one embodiment, the dose of 108 mg is self-administered.

In one embodiment, the humanized anti-a4p7 antibody and the pan-JAK inhibitor are administered to the human subject during an induction phase.

In one embodiment, the induction phase is followed by a maintenance phase comprising administration of the humanized anti-a4p7 antibody as a monotherapy. In a further embodiment, the maintenance phase begins when the human subject achieves clinical remission and/or an endoscopic response.

In one embodiment, the pan-JAK inhibitor is tofacitinib. In a further embodiment, a 10 mg dose of tofacitinib is administered orally twice daily.

In another aspect, provided herein is a method of treating a human subject having an inflammatory bowel disease (IBD), said method comprising administering a humanized anti- a4p7 antibody to the human patient, wherein the human subject having IBD has an increased or decreased expression level before treatment of at least one effector marker selected from the group consisting of NFKB2, CLDN1, CLDN2, CLDN3, OCLN, TJP1, ITGA4, ITGB7, PDGFB, IL5, IL6, IGF2, and MMP9 relative to a reference level in a human subject without IBD, and/or wherein the human subject having IBD has a reversion after treatment in the expression level of the at least one effector marker selected from the group consisting of NFKB2, CLDN1, CLDN2, CLDN3, OCLN, TJP1, ITGA4, ITGB7, PDGFB, IL5, IL6, IGF2, and MMP9 relative to a baseline expression level in the human subject having IBD, and wherein the humanized anti-a4p7 antibody is an IgGl antibody and comprises a heavy chain variable region comprising a CDR3 domain as set forth in SEQ ID NO: 4, a CDR2 domain as set forth in SEQ ID NO: 3, and a CDR1 domain as set forth in SEQ ID NO: 2; and comprises a light chain variable region comprising a CDR3 domain as set forth in SEQ ID NO: 8, a CDR2 domain as set forth in SEQ ID NO: 7, and a CDR1 domain as set forth in SEQ ID NO: 6.

In another aspect, provided herein is a method of treating a human subject having an inflammatory bowel disease (IBD), said method comprising administering a humanized anti- 06407 antibody to the human subject, wherein the human subject having IBD has a decreased expression level before treatment of CLDN3, OCLN, and/or TJP1 relative to a reference level in a human subject without IBD, and/or wherein the human subject having IBD has a reversion after treatment in the expression level of CLDN3, OCLN, and/or TJP1 relative to a baseline expression level in the human subject having IBD, and wherein the humanized anti- 06407 antibody is an IgGl antibody and comprises a heavy chain variable region comprising a CDR3 domain as set forth in SEQ ID NO: 4, a CDR2 domain as set forth in SEQ ID NO: 3, and a CDR1 domain as set forth in SEQ ID NO: 2; and comprises a light chain variable region comprising a CDR3 domain as set forth in SEQ ID NO: 8, a CDR2 domain as set forth in SEQ ID NO: 7, and a CDR1 domain as set forth in SEQ ID NO: 6.

In another aspect, provided herein is a method of treating a human subject having an inflammatory bowel disease (IBD), said method comprising administering a humanized anti- a4p7 antibody to the human patient, wherein the human subject having IBD has an increased or decreased expression level before treatment of at least one effector marker selected from the group consisting of IGF2, PDGFB, IL6, NFKB2, MMP9, CLDN1, and CLDN2 relative to a reference level in a human subject without IBD, and/or wherein the human subject having IBD has a reversion after treatment in the expression level of the at least one effector marker selected from the group consisting of IGF2, PDGFB, IL6, NFKB2, MMP9, CLDN1, and CLDN2 relative to a baseline expression level in the human subject having IBD, and wherein the humanized anti-a4p7 antibody is an IgGl antibody and comprises a heavy chain variable region comprising a CDR3 domain as set forth in SEQ ID NO: 4, a CDR2 domain as set forth in SEQ ID NO: 3, and a CDR1 domain as set forth in SEQ ID NO: 2; and comprises a light chain variable region comprising a CDR3 domain as set forth in SEQ ID NO: 8, a CDR2 domain as set forth in SEQ ID NO: 7, and a CDR1 domain as set forth in SEQ ID NO: 6.

In another aspect, provided herein is a method of treating a human subject having an inflammatory bowel disease (IBD), said method comprising administering a humanized anti- a4p7 antibody and administering a pan-Janus kinase (JAK) inhibitor to the human subject, wherein the human subject having IBD has an increased or decreased expression level before treatment of at least one effector marker selected from the group consisting of WNT1, PPARG, IL22, TLR4, MICB, IL13, TGFBR2, EGF, C0L1A1, C0L1A2, and MSH2 relative to a reference level in a human subject without IBD, and/or wherein the human subject having IBD has a reversion after treatment in the expression level of the at least one effector marker selected from the group consisting of WNT1, PPARG, IL22, TLR4, MICB, IL13, TGFBR2, EGF, COL 1 Al, COL1 A2, and MSH2 relative to a baseline expression level in the human subject having IBD, and wherein the humanized anti-a4p7 antibody is an IgGl antibody and comprises a heavy chain variable region comprising a CDR3 domain as set forth in SEQ ID NO: 4, a CDR2 domain as set forth in SEQ ID NO: 3, and a CDR1 domain as set forth in SEQ ID NO: 2; and comprises a light chain variable region comprising a CDR3 domain as set forth in SEQ ID NO: 8, a CDR2 domain as set forth in SEQ ID NO: 7, and a CDR1 domain as set forth in SEQ ID NO: 6.

In another aspect, provided herein is a method of treating a human subject having an inflammatory bowel disease (IBD), said method comprising administering a humanized anti- 06407 antibody and administering a pan-Janus kinase (JAK) inhibitor to the human subject, wherein the human subject having IBD has an increased expression level before treatment of COL1 Al and/or COL1 A2 relative to a reference level in a human subject without IBD, and/or wherein the human subject having IBD has a reversion after treatment in the expression level of C0L1A1 and/or C0L1A2 relative to a reference level in a human subject without IBD, and wherein the humanized anti-a4p7 antibody is an IgGl antibody and comprises a heavy chain variable region comprising a CDR3 domain as set forth in SEQ ID NO: 4, a CDR2 domain as set forth in SEQ ID NO: 3, and a CDR1 domain as set forth in SEQ ID NO: 2; and comprises a light chain variable region comprising a CDR3 domain as set forth in SEQ ID NO: 8, a CDR2 domain as set forth in SEQ ID NO: 7, and a CDR1 domain as set forth in SEQ ID NO: 6.

In another aspect, provided herein is a method of treating a human subject having an inflammatory bowel disease (IBD), said method comprising administering a humanized anti- a4p7 antibody and administering a pan-Janus kinase (JAK) inhibitor to the human subject, wherein the human subject having IBD has an increased or decreased expression level before treatment of at least one effector marker selected from the group consisting of MSH2, PPARG, and EGF relative to a reference level in a human subject without IBD, and/or wherein the human subject having IBD has a reversion after treatment in the expression level of the at least one effector marker selected from the group consisting of MSH2, PPARG, and EGF relative to a baseline expression level in the human subject having IBD, and wherein the humanized anti-a4p7 antibody is an IgGl antibody and comprises a heavy chain variable region comprising a CDR3 domain as set forth in SEQ ID NO: 4, a CDR2 domain as set forth in SEQ ID NO: 3, and a CDR1 domain as set forth in SEQ ID NO: 2; and comprises a light chain variable region comprising a CDR3 domain as set forth in SEQ ID NO: 8, a CDR2 domain as set forth in SEQ ID NO: 7, and a CDR1 domain as set forth in SEQ ID NO: 6.

In another aspect, provided herein is a method of treating a human subject having an inflammatory bowel disease (IBD), said method comprising administering a humanized anti- 06407 antibody and administering a pan-Janus kinase (JAK) inhibitor to the human subject, wherein the human subject having IBD has an increased or decreased expression level before treatment of at least one effector marker selected from the group consisting of TLR4, MICB, IL22, and TGFBR2 relative to a reference level in a human subject without IBD, and/or wherein the human subject having IBD has a reversion after treatment in the expression level of the at least one effector marker selected from the group consisting of TLR4, MICB, IL22, and TGFBR2 relative to a baseline expression level in the human subject having IBD, and wherein the humanized anti-a4p7 antibody is an IgGl antibody and comprises a heavy chain variable region comprising a CDR3 domain as set forth in SEQ ID NO: 4, a CDR2 domain as set forth in SEQ ID NO: 3, and a CDR1 domain as set forth in SEQ ID NO: 2; and comprises a light chain variable region comprising a CDR3 domain as set forth in SEQ ID NO: 8, a CDR2 domain as set forth in SEQ ID NO: 7, and a CDR1 domain as set forth in SEQ ID NO: 6.

In one embodiment, the IBD is Crohn’s disease. In a further embodiment, the Crohn’s disease is moderately to severely active Crohn’s disease.

In one embodiment, the human subject has active inflammation on ileocolonoscopy.

In one embodiment, the human subject is biologic-naive.

In one embodiment, the human subject had a lack of an adequate response with, lost response to, or was intolerant to treatment with at least one of an immunomodulator, a tumor necrosis factor-alpha antagonist or combinations thereof.

In one embodiment, the human subject is intravenously administered a first dose of 300 mg of the humanized anti-a4p7 antibody at week 0, followed by a second dose of 300 mg of the humanized anti-a4p7 antibody at week 2. In a further embodiment, the method comprises intravenously administering a third dose of 300 mg of the humanized anti-a4p7 antibody at week 6, followed by a 300 mg dose of the humanized anti-a4p7 antibody every four or eight weeks thereafter.

In a further embodiment, the human subject is subcutaneously administered a dose of 108 mg of the humanized anti-a4p7 antibody at week 6, followed by a 108 mg dose of the humanized anti-a4p7 antibody every two weeks thereafter.

In a further embodiment, the method comprises intravenously administering a third dose of 300 mg of the humanized anti-a4p7 antibody at week 6, wherein the human patient is subcutaneously administered a dose of 108 mg of the humanized anti-a4p7 antibody at week 14, followed by a 108 mg dose of the humanized anti-a4p7 antibody every two weeks thereafter.

In one embodiment, the dose of 108 mg is self-administered.

In one embodiment, the humanized anti-a4p7 antibody and the pan-JAK inhibitor are administered to the human subject during an induction phase.

In one embodiment, the induction phase is followed by a maintenance phase comprising administration of the humanized anti-a4p7 antibody as a monotherapy. In a further embodiment, the maintenance phase begins when the human subject achieves clinical remission and/or an endoscopic response.

In one embodiment, the pan-JAK inhibitor is tofacitinib. In a further embodiment, a 10 mg dose of tofacitinib is administered orally twice daily.

In one embodiment, the expression level of the at least one effector marker is an mRNA expression level or protein expression level.

In one embodiment, the mRNA expression level is determined by in situ hybridization or RNA sequencing.

In one embodiment, the protein expression level is determined by flow cytometry, mass cytometry, or immunohistochemistry.

In one embodiment, the humanized anti-a4p7 antibody comprises a heavy chain variable domain comprising an amino acid sequence as set forth in SEQ ID NO: 1, and comprises a light chain variable domain comprising an amino acid sequence as set forth in SEQ ID NO: 5.

In one embodiment, the humanized anti-a4p7 antibody is vedolizumab.

Also contemplated in the invention are the dosing regimens and patient characteristics described in the Examples provided below. BRIEF DESCRIPTION OF DRAWINGS

FIG. 1A shows the Effect of VDZ+JAKi on CD pathophysiology: fSignal, the signal value of all response proteins (CD effectors) after stimulating the model (mean value). fSignal was slightly higher for VDZ than for JAKi.

FIG. IB and FIG. 1C show the Effect of VDZ+JAKi on CD pathophysiology: % reverted CD effectors by the drug over the disease pathophysiology, which measures if the causative effect of a protein effector characterized for CD is reverted in the treatment models.

FIG. 2 shows preliminary fSignal of VDZ or JAKi on CD and on each pathophysiological motive. Preliminary models of each drug individually were made to determine the effects of VDZ and JAKi on each identified CD motive and on whole CD physiopathology. None of the treatments have impact on motive M2 (defective innate immune response).

FIG. 3A-3D show simplified path representations of the mechanism of action of vedolizumab plus JAK inhibitors based on TPMS mechanism of action (MO A) predictions. Arrows show activation; T-bar lines show inhibition; dark arrow lines show complex or dual relationships; rhombus indicate drug targets; broken-lines indicate a node that contains more than one protein, all acting in the MOA together; full-filled circles indicate convergent effectors.

FIG. 4 shows simplified path representation of the mechanism of action of vedolizumab plus JAK inhibitors based on TPMS mechanism of action (MOA) predictions. Arrows show activation; T-bar lines show inhibition; dark lines show complex or dual relationships; rhombus indicate drug targets; broken-lines indicate a node that contains more than one protein, all acting in the MOA together.

FIG. 5 shows a heatmap of the expression of the identified JAKi and vedolizumab convergent effectors using VARSITY data at Week 0, Week 14, and Week 52. Shading indicates log-2 fold change in expression with higher values indicating enrichment in nonresponders and lower values indicating enrichment in responders.

FIG. 6 shows a heatmap of the expression of the identified complementary effectors reverted exclusively by vedolizumab using VARSITY data at Week 0, Week 14, and Week 52. Shading indicates log-2 fold change in expression with higher values indicating enrichment in non-responders and lower values indicating enrichment in responders.

FIG. 7 shows a heatmap of the expression of the identified complementary effectors reverted exclusively by JAKi using VARSITY data at Week 0, Week 14, and Week 52. Shading indicates log-2 fold change in expression with higher values indicating enrichment in nonresponders and lower values indicating enrichment in responders.

DETAILED DESCRIPTION OF THE INVENTION

I. Definitions

In order that the present invention may be more readily understood, certain terms are first defined. In addition, it should be noted that whenever a value or range of values of a parameter are recited, it is intended that values and ranges intermediate to the recited values are also part of this invention.

The cell surface molecule, “0.407 integrin,” or “a407” (used interchangeably throughout) is a heterodimer of an a4 chain (CD49D, ITGA4, OMIM 192975, human GenelD 3676) and a 07 chain (ITGB7, OMIM 147559; human GenelD 3695). Human a4- integrin and 07-integrin genes (GenBank (National Center for Biotechnology Information, Bethesda, Md.) RefSeq Accession numbers NM_000885 and NM_000889, respectively) are expressed by B and T lymphocytes, particularly memory CD4+ lymphocytes. Typical of many integrins, a407 can exist in either a resting or activated state. Ligands for a407 include vascular cell adhesion molecule (VCAM), fibronectin and mucosal addressin (MAdCAM (e.g., MAdCAM-1)).

As used herein, an antibody, or antigen-binding fragment thereof, that has “binding specificity for the a.407 complex” binds to 0.407, but not to a401 or aEB7. Vedolizumab is an example of an antibody that has binding specificity for the a407 complex.

As used herein, an “anti-a407 antibody” or “anti-a407 integrin antibody” refers to an antibody which specifically binds to human a407 integrin. In one embodiment, an anti-a407 antibody blocks or inhibits the binding of a407 integrin to one or more of its ligands. In one embodiment, an anti-a407 antibody binds to a.407, but not to a401 or aEB7. In one embodiment, an anti-a407 antibody is vedolizumab.

The term “antibody” broadly refers to an immunoglobulin molecule comprised of four polypeptide chains, two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds. Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region (CH). The heavy chain constant region is comprised of three domains, CHI, CH2 and CH3. Each light chain is comprised of a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region is comprised of one domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy -terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

As used herein, the term “antibody fragment” or “antigen-binding fragment” of an antibody refers to Fab, Fab’, F(ab)2, and Fv fragments, single chain antibodies, functional heavy chain antibodies (nanobodies), as well as any portion of an antibody having specificity toward at least one desired epitope, that competes with the intact antibody for specific binding (e.g., an isolated portion of a complementarity determining region having sufficient framework sequences so as to bind specifically to an epitope). Antigen binding fragments can be produced by recombinant techniques, or by enzymatic or chemical cleavage of an antibody.

As used herein, the term “humanized antibody” refers to an antibody that is derived from a non-human antibody (e.g., murine) that retains or substantially retains the antigen binding properties of the parent antibody but is less immunogenic in humans and contains minimal sequence derived from non-human immunoglobulins. Generally, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity, and capacity. In some instances, framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops (complementary determining regions) correspond to those of a non- human immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence. The humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones et al., Nature 321 :522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992).

The term “monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical and/or bind the same epitope, except for possible variants that may arise during production of the monoclonal antibody, such variants generally being present in minor amounts. In contrast to polyclonal antibody preparations that typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. The modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present invention may be made by the hybridoma method first described by Kohler et al., Nature, 256:495 (1975), or may be made by recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567). The “monoclonal antibodies” may also be isolated from phage antibody libraries using the techniques described in Clackson et al., Nature, 352:624- 628 (1991) and Marks et al., J. Mol. Biol., 222:581-597 (1991), for example.

As used herein, the term “recombinant antibody” refers to an antibody produced as the result of the transcription and translation of a gene carried on a recombinant expression vector. In one embodiment, the vector has been introduced into a host cell. Alternatively, a vector can be used in a cell free system.

As used herein, a “pan-JAK inhibitor” or “pan-Janus kinase (JAK) inhibitor” or “JAKi” refers to a therapeutic agent, e.g., a therapeutic compound, that inhibits the activity of at least two (e.g., at least three) of the Janus kinase family of enzymes (JAKI, JAK2, JAK3, and/or TYK2). An example of a pan-JAK inhibitor is tofacitinib (XELJANZ), which inhibits JAKI, JAK2, JAK3, and to a lesser extent TYK2 phosphorylation.

The term “effector” or “effector marker”, as used herein, refers generally to a molecule, e.g., mRNA (and a nucleotide sequence of such mRNA), a gene (and a nucleotide sequence of such gene), a peptide and a protein (and an amino acid sequence of such), the expression of which in or on a sample derived from a mammalian tissue or cell can be detected, for example, by standard methods in the art such as in situ hybridization.

The term "baseline" refers to a starting point used for a comparison. In a preferred embodiment, baseline is Day 1 of treatment.

The term "treatment" or "treating" means any treatment of a disease or disorder (e.g., IBD) in a human subject, including: preventing or protecting against the disease or disorder, that is, causing the clinical symptoms not to develop; inhibiting the disease or disorder, that is, arresting or suppressing the development of clinical symptoms; and/or relieving the disease or disorder that is, causing the regression of clinical symptoms. In one embodiment, treatment of IBD is achieved where a subject having IBD sees an improvement in symptoms as measured by an accepted IBD index (e.g., a clinical measure for Crohn’s disease) dosing regimen with an anti-a4p7 antibody and a pan-JAK inhibitor.

The term “combination therapy” refers to the use of two or more therapies, e.g., two or more agents, for the treatment of a disease or disorder. Use of the term "in combination" or “combination therapy” does not restrict the order in which therapeutic agents are administered to a subject having a disease. The term “combination therapy” is not intended to refer to a pharmaceutical composition comprising two active agents, e.g., an anti-a4p7 antibody and a pan-JAK inhibitor.

The terms "patient" and "subject" are used interchangeably herein. Preferably, a patient is a human patient.

As used herein, the term "about" is used synonymously with the term "approximately." Illustratively, the use of the term "about" indicates that values slightly outside the cited values, namely, plus or minus 5%.

As used herein, the term “expression” when used in connection with detecting the expression of an effector of the present disclosure, can refer to detecting transcription of the gene encoding a protein and/or detecting translation of the protein. To detect expression of an effector refers to the act of actively determining whether an effector is expressed or not. To quantitate expression refers to the act of determining the level of the given effector, e.g., ng/ml. Detecting and/or quantitating expression can include determining whether the effector expression is upregulated as compared to a known standard level, downregulated as compared to a known standard level, or substantially unchanged as compared to a known standard level. Therefore, the step of quantitating and/or detecting expression does not require that expression of the effector actually is upregulated or downregulated, but rather, can also include detecting no expression of the effector or detecting that the expression of the effector has not changed or is not different (i.e., detecting no significant expression of the effector or no significant change in expression of the effector as compared to a control).

The term “level” or “amount” of an effector, as used herein, refers to the measurable quantity of an effector, e.g., a transcript, a peptide, a protein (or polypeptide). The amount may be either (a) an absolute amount as measured in molecules, moles or weight per unit volume or cells or (b) a relative amount.

As used herein, the term “reference level” or “control level”, refers to an accepted or pre-determined level of an effector which is used to compare the effector level derived from the sample of a subject. The level of the effector may also be compared to a baseline level which is not accepted or pre-determined.

The term “sample” or “biological sample” as used herein refers to cells or tissue obtained from a subject. The source of the tissue or cell sample may be solid tissue (as from a fresh, frozen and/or preserved organ or tissue sample or biopsy (e.g., mucosal biopsy) or aspirate); whole blood or any blood constituents (e.g., peripheral blood mononuclear cells (PBMCs)); or bodily fluids, such as serum, plasma, urine, feces, saliva, sweat or synovial fluid.

II. Combination Therapies

Disclosed herein is a treatment method comprising a combination therapy that decreases inflammation associated with IBD, e.g., Crohn’s disease, by blocking the trafficking of 01407 T cells to the lamina propria of the colon via an anti-a407 antibody, such as vedolizumab, and modulating the signaling of the JAK-dependent cytokines underlying the inflammatory burden and signs and symptoms of IBD using a pan-JAK inhibitor, such as tofacitinib.

Vedolizumab (ENTYVIO; Takeda) and tofacitinib (XELJANZ; Pfizer) are both effective for treating patients with IBD, particularly in patients who have had an inadequate response, lost response, or were intolerant to other treatments. Vedolizumab and tofacitinib have different mechanisms of action: vedolizumab is an antibody that binds to the human lymphocyte integrin a407, thus impairing the migration of gut homing lymphocytes to the gastrointestinal (GI) mucosa, whereas tofacitinib is a non-selective and reversible Janus kinase (JAK) inhibitor that modulates the signaling of the JAK-dependent cytokines associated with inflammation associated with IBD. Combining these two therapies may help overcome the efficacy ceiling for treating CD (Stalgis et al. 2021. Rational combination therapy to overcome the plateau of drug efficacy in inflammatory bowel disease. Gastroenterology, 161(2), 394-9). Provided herein are methods for treating a human patient having inflammatory bowel disease (IBD) using a combination therapy comprising an anti- oc407 antibody, e.g., vedolizumab, and a pan-JAK inhibitor, e.g., tofacitinib.

The combination therapy disclosed herein includes administering a pan-JAK inhibitor, such as tofacitinib, to the human patient in need thereof, e.g., patient having Crohn’s disease.

In some aspects, the therapeutic method has two phases, i.e., an induction phase and a maintenance phase. In one embodiment, a combination therapy as described herein is administered to a human patient having IBD in an induction phase. In the induction phase, an anti-a4p7 antibody and a pan-JAK inhibitor are administered in a way that quickly provides an effective amount of the antibody and pan-JAK inhibitor suitable for a certain purpose(s), such as inducing a clinical response and ameliorating inflammatory bowel disease symptoms,

A patient can be administered an induction phase treatment when first being treated by an anti-a4p7 antibody and/or a pan-JAK inhibitor, when being treated after a long absence from therapy, e.g., more than three months, more than four months, more than six months, more than nine months, more than one year, more than eighteen months or more than two years since anti-a4p7 antibody therapy or during maintenance phase of anti-a4 7 antibody therapy if there has been a return of inflammatory bowel disease symptoms, e.g., a relapse from remission of disease.

In the maintenance phase, the anti-a4p7 antibody is administered according to a dosing regimen such that treatment continues the response achieved by induction therapy with a stable level of anti-a4p7 antibody. A maintenance regimen can prevent return of symptoms or relapse of inflammatory bowel disease.

In a certain embodiment, a treatment method for treating IBD in a patient comprises an induction phase comprising a combination therapy (e.g., an anti-a4p7 antibody and a pan- JAK inhibitor) and a maintenance phase comprising a monotherapy (e.g., anti-a4p7 antibody in the absence of a pan-JAK inhibitor).

The disclosed combination therapy is effective for treating IBD, including Crohn’s disease or ulcerative colitis. In certain embodiments, the human patient receiving the therapeutic methods disclosed herein has Crohn’s disease, e.g., moderately to severely active Crohn’s disease. In other embodiments, the human patient has ulcerative colitis, e.g., moderately to severely active ulcerative colitis.

In a combination therapy, an anti-a4p7 antibody can be administered before, concurrently with, or after administration of a pan-JAK inhibitor to a subject having inflammatory bowel disease (IBD), e.g., Crohn’s disease. Similarly, the pan-JAK inhibitor can be administered before, concurrently with, or after administration of the anti-a4p7 antibody to a subject having inflammatory bowel disease (IBD), e.g., Crohn’s disease.

In one embodiment, the anti-a4p7 antibody (e.g., vedolizumab) is administered before the pan-JAK inhibitor, (e.g., tofacitinib), for treatment. In other embodiments, the anti-a4p7 antibody (e.g., vedolizumab) is administered after the pan-JAK inhibitor, (e.g., tofacitinib). In yet other embodiments, the anti-a407 antibody (e.g., vedolizumab) is administered concomitantly with the JAK inhibitor, (e.g., tofacitinib).

Further, an anti-a407 antibody (e.g., vedolizumab) and a pan-JAK inhibitor may be administered in a combination therapy whereby one agent is administered according to its own specific dosing regimen that overlaps in time, e.g., eight weeks, with the other agent.

The anti-a407 antibody (e.g., vedolizumab) may be administered according to a dosing regimen comprising administering a first dose of 300 mg of the anti-a407 antibody at week 0, followed by a second dose of 300 mg of the anti-a407 antibody at week 2. In some embodiments, the method further comprises administering a third dose of 300 mg of the anti- 0407 antibody at week 6. The dosing regimen of the anti-a407 antibody (e.g., vedolizumab) may further comprise intravenously administering 300 mg of the anti-a407 antibody to the human patient every eight weeks starting at 14 weeks. If needed, 300 mg of the anti-a407 antibody may be administered to the human patient every four weeks instead of every eight weeks during maintenance phase if the patient is not showing clinical improvement or experiences a return of inflammatory bowel disease symptoms, e.g., a relapse from remission of disease. A pan-JAK inhibitor is also administered according to the disclosure herein.

In other embodiments, the anti-a407 antibody (e.g., vedolizumab) is administered according to a dosing regimen comprising administering a first dose of 300 mg of the anti- 0407 antibody at week 0, followed by a second dose of 300 mg of the anti-a407 antibody at week 2, followed by third dose of 108 mg of the anti-a407 antibody at week 6, followed by a 108 mg dose every two weeks thereafter. If needed, 108 mg of the anti-a407 antibody may be administered to the human patient every week instead of every two weeks during maintenance phase if the patient is not showing clinical improvement or experiences a return of inflammatory bowel disease symptoms, e.g., a relapse from remission of disease. A pan- JAK inhibitor is also administered according to the disclosure herein.

In some embodiments, an anti-a407 (e.g., vedolizumab) is administered according to a dosing regimen comprising intravenously administered a first dose of 300 mg of the anti- 0.407 antibody at week 0, followed by a second dose of 300 mg of the anti-a407 antibody at week 2, further comprising intravenously administering a third dose of 300 mg of the anti- 0407 antibody at week 6 and subcutaneously administering a dose of 108 mg of the anti- 0407 antibody at week 14, followed by a 108 mg dose of the anti-a407 antibody every two weeks thereafter. If needed, 108 mg of the anti-a407 antibody may be administered to the human patient every week instead of every two weeks during maintenance phase if the patient is not showing clinical improvement or experiences a return of inflammatory bowel disease symptoms, e.g., a relapse from remission of disease.

The anti-a4p7 antibody may be administered to the human patient intravenously or subcutaneously. For example, the 300 mg dose of the anti-a4p7 antibody may be administered to the human patient intravenously, while the 108 mg dose of the anti-a4p7 antibody may be administered subcutaneously. In certain embodiments, the anti-a4p7 antibody may be self-administered subcutaneously by the human patient.

In one embodiment, the anti-a4p7 antibody is administered to the human patient having IBD, e.g., CD, according to a dosing regimen comprising intravenously administering a first dose of 300 mg of the anti-a4p7 antibody to the human patient at week 0, followed by a second dose of 300 mg of the anti-a4p7 antibody intravenously administered at week 2, followed by third dose of 300 mg of the anti-a4p7 antibody intravenously administered at week 6, followed by intravenously administering 300 mg of the anti-a4p7 antibody to the human patient every eight weeks starting at 14 weeks. A pan-JAK inhibitor is also administered according to the disclosure herein.

In another embodiment, the anti-a4p7 antibody is administered to the human patient having IBD, e.g., CD, according to a dosing regimen comprising intravenously administering a first dose of 300 mg of the anti-a4p7 antibody to the human patient at week 0, followed by a second dose of 300 mg of the anti-a4p7 antibody intravenously administered at week 2, followed by third dose of 108 mg of the anti-a4p7 antibody subcutaneously administered at week 6, followed by a 108 mg dose subcutaneously administered every two weeks thereafter. A pan-JAK inhibitor is also administered according to the disclosure herein.

In one embodiment, the pan-JAK inhibitor is tofacitinib and is administered orally twice daily. In certain embodiments, 10 mg of tofacitinib is administered to the human patient. In an embodiment, tofacitinib is administered orally twice daily for 8 weeks. In some embodiments, tofacitinib is administered for at least 8 weeks.

In some embodiments, the anti-a4p7 antibody is administered to a human patient having IBD as a first dose of 300 mg at week 0, followed by a second dose of 300 mg of the anti-a4p7 antibody at week 2, followed by third dose of 300 mg of the anti-a4p7 antibody at week 6. In another embodiment, the human patient is further administered a fourth dose of 300 mg of the anti-a4p7 antibody at week 10. The method may further comprise administering 300 mg of the humanized anti-a4p7 antibody to the human patient every eight weeks beginning 8 weeks after the third dose. In an alternative embodiment, the method described herein may comprise administering 300 mg of the anti-a4p7 antibody to the human patient every four weeks if the human patient is not showing clinical improvement, e.g., clinical remission of Crohn’s disease or ulcerative colitis. In other embodiments, the method comprises administering, e.g., intravenously, 300 mg of the anti-a4p7 antibody to the human patient every four weeks beginning 8 weeks after the third dose. In an embodiment, patients who achieve a CD Al reduction of >70 points from baseline at week 12 are administered 300 mg of the anti-a4p7 antibody to the human patient every eight weeks beginning 8 weeks after the third dose.

In one embodiment, 300 mg of the anti-a4p7 antibody (e.g., vedolizumab) is administered to the human patient every four weeks. In some embodiments, 300 mg of the anti-a4p7 antibody is administered to the human patient every four weeks, wherein the human patient does not achieve a CD Al reduction of >70 points from baseline at week 12 of treatment with an anti-a4p7 antibody or the CD Al score at the last visit, has a CD Al >150, and/or a CRP level >5 mg/L or fecal calprotectin level >250 pg/g.

In some embodiments in combination with dosing regimens described herein for the anti-a4p7 antibody, a pan-JAK inhibitor is administered orally (e.g., as oral capsules or tablets). In an embodiment, the pan-JAK inhibitor is administered twice daily. In some embodiments, the pan-JAK inhibitor is administered for at least 8 weeks. In an embodiment, 10 mg of the pan-JAK inhibitor is administered. In another embodiment, 10 mg of the pan- JAK inhibitor is administered twice daily.

In some embodiments, the human patient is administered a dosing regimen comprising a first dose of 300 mg of the anti-a4p7 antibody at week 0, followed by a second dose of 300 mg of the anti-a4p7 antibody, at week 2, followed by third dose of 300 mg of the anti-a4p7 antibody at week 6, and then a fourth and subsequent doses of 300 mg of the anti- a4p7 antibody every 8 weeks (8QW) in combination with 10 mg tofacitinib administered orally twice daily (i.e., 10 mg BID) for the first 8 weeks. In some embodiments, 10 mg tofacitinib is administered twice daily to a patient on an intensified vedolizumab maintenance dosing regimen. For example, in some embodiments, 300 mg of the anti-a4p7 antibody is administered to the human patient every four weeks with 10 mg tofacitinib administered twice daily. The combination therapy may be adjusted according to certain clinical achievements or failures seen in the human patient receiving the pan-JAK inhibitor and the anti-a407 antibody. For example, when a human patient having CD who has been administered the combination therapy, does not achieve a CD Al reduction of > 70 points from baseline by week 12, the human patient may be administered 300 mg of the anti-a407 antibody to the human patient every four weeks starting at 14 weeks, and administered 5 mg or 10 mg of tofacitinib orally twice daily for 12 weeks. If the human patient subsequently achieves a CD Al reduction of > 70 points from baseline by week 24, then the human patient may be administered 300 mg of the anti-a407 antibody every eight weeks beginning at week 26.

“CD Al” used herein refers to Crohn’s Disease Activity Index and can be used to determine clinical improvement of a patient having Crohn’s disease. CDAI-defined clinical remission is defined as CD Al <150 . CDAI-defined clinical response is defined as a > 100 point decrease from baseline in CD Al. Corticosteroid-free clinical remission is defined as CD Al <150 and not receiving corticosteroids at the assessment time point. CDAI-defined scores may be determined in comparison to a patient’s baseline score, which is obtained prior to treatment with a combination therapy described herein. A CD Al score includes 3 participant-reported items and 5 objective items. Scores are computed as a weighted sum of the items and range from 0 to 600 with higher scores indicating more disease activity. A score below 150 indicates remission; scores ranging from 150 to 219 indicate mildly active disease; scores ranging from 220 to 450 indicate moderately active disease; and scores above 450 indicate severe disease.

A human patient who is administered a therapy described herein comprising the anti- a407 antibody and the pan-JAK inhibitor may achieve certain clinical endpoints indicative of efficacy. For example, a human patient who is administered a combination therapy as disclosed herein may achieve a Crohn’s Disease Activity Index (CD Al) reduction of >70 points from baseline by week 12. In other embodiments, clinical remission is achieved.

In some embodiments, the method described herein may comprise administering 300 mg of the anti -0.407 antibody to the human patient every four weeks if the human patient is not showing clinical improvement, e.g., clinical remission of Crohn’s disease or ulcerative colitis. In some embodiments, the patient has Crohn’s disease and the clinical improvement is a CD Al reduction of >70 points from baseline at week 12 of treatment with an anti-a407 antibody or the CD Al score at the last visit, a CD Al >150, and/or a CRP level >5 mg/L or fecal calprotectin level >250 pg/g has not been achieved. In some embodiments, 300 mg of the anti-a4p7 antibody is administered to the human patient every four or eight weeks with oral tofacitinib twice daily, wherein the human patient does not achieve a CD Al reduction of >70 points from baseline at week 12 of treatment with an anti-a4p7 antibody, or the CD Al score at the last visit, have a CD Al >150, and/or a CRP level >5 mg/L or fecal calprotectin level >250 pg/g. In an embodiment, 300 mg of the anti-a4p7 antibody is administered to the human patient every four weeks with 10 mg oral tofacitinib twice daily. In some embodiments, the human patient is administered 10 mg tofacitinib orally for at least 8 weeks.

In some embodiments, patients who do not achieve a CD Al reduction of >70 points from baseline at week 12 of treatment with vedolizumab and tofacitinib are administered 12 additional weeks of combination therapy, comprising vedolizumab and tofacitinib.

In certain embodiments, administration of the anti-a4p7 antibody (e.g., vedolizumab) is continued after the pan-JAK inhibitor is no longer administered.

In one aspect, the invention provides a method of treating IBD in a subject comprising administering to a human subject an anti-a4p7 antibody (e.g., vedolizumab) and a pan-JAK inhibitor (e.g., tofacitinib) each in an amount effective to treat IBD. The human subject may be an adult (e.g., 18 years or older), an adolescent, or a child (juvenile or pediatric). The human subject may be a person 65 years or older. In certain embodiments, the human subject is a child who is less than 18 years old.

In certain embodiments, the combination therapy described herein results in a human subject having IBD achieving a clinical response as defined herein for Crohn’s disease or ulcerative colitis, e.g., as determined by a decrease from baseline in the Mayo score by greater than or equal to 30% and greater than or equal to 3 points and a decrease from baseline in the rectal bleeding subscore greater than or equal to 1 point or a rectal bleeding subscore of 0 or 1 by week 8 of treatment with the combination therapy.

Prior to treatment with a therapeutic method as disclosed herein, the human patient may be selected as having a certain characteristic or a combination of characteristics. For example, a human patient having Crohn’s disease may also have active inflammation on ileocolonoscopy. Thus, a human patient may be selected as having Crohn’s disease and active inflammation on ileocolonoscopy prior to administration of a combination therapy comprising an anti-a4p7 antibody and a pan-JAK inhibitor. In addition to or alternatively, a human patient did not have treatment for IBD with a biologic prior to treatment with a combination therapy disclosed herein, i.e., the patient is biologic-naive. In a separate embodiment, the human patient had an inadequate response or intolerance to one or more TNF-alpha inhibitors prior to treatment with a combination therapy disclosed herein.

In certain embodiments, the subject who is administered the method of the invention may have had a lack of an adequate response with, loss of response to, or was intolerant to treatment, e.g., initial treatment, with an immunomodulator, a TNF-alpha antagonist, or combinations thereof. In some embodiments, the subject who is administered the combination therapy of the invention may have had a lack of an adequate response or a lack of remission after initial treatment with an anti-a4p7 antibody (e.g., vedolizumab). In certain embodiments, the subject who is administered the combination therapy of the invention may have had a lack of an adequate response with, loss of response to, or was dependent on corticosteroid therapy. The patient may have previously received treatment with at least one corticosteroid (e.g., prednisone, prednisolone, budesonide, methylprednisolone, hydrocortisone) for the inflammatory bowel disease. An inadequate response to corticosteroids refers to signs and symptoms of persistently active disease despite a history of at least one 4-week induction regimen that included a dose equivalent to prednisone 30 mg daily orally for 2 weeks or intravenously for 1 week. A loss of response to corticosteroids refers to two failed attempts to taper corticosteroids to below a dose equivalent to prednisone 10 mg daily orally. Intolerance of corticosteroids includes a history of Cushing's syndrome, osteopenia/osteoporosis, hyperglycemia, insomnia and/or infection.

An immunomodulator may be, for example, oral azathioprine, 6-mercaptopurine, or methotrexate. An inadequate response to an immunomodulator refers to signs and symptoms of persistently active disease despite a history of at least one 8-week regimen of oral azathioprine, 6-mercaptopurine, or methotrexate. Intolerance of an immunomodulator includes, but is not limited to, nausea/vomiting, abdominal pain, pancreatitis, LFT abnormalities, lymphopenia. TPMT genetic mutation and/or infection.

In one aspect, the subject may have had a lack of an adequate response with, loss of response to, or was intolerant to treatment biologic therapy. In one aspect, the subject may have had a lack of an adequate response with, loss of response to, or was intolerant to treatment a TNF-alpha inhibitor, i.e., antagonist. A TNF-alpha antagonist is, for example, an agent that inhibits the biological activity of TNF-alpha, and preferably binds TNF-alpha, such as a monoclonal antibody, e.g., infliximab, adalimumab, certolizumab pegol, golimumab, or an Fc fusion protein such as ENBREL (etanercept). An inadequate response to a TNF-alpha antagonist refers to signs and symptoms of persistently active disease despite a history, for example, of at least one 4-week induction regimen of infliximab 5 mg/kg IV, 2 doses at least 2 weeks apart; one 80 mg subcutaneous dose of adalimumab, followed by one 40 mg dose at least two weeks apart; or 400 mg subcutaneously of certolizumab pegol, 2 doses at least 2 weeks apart. A loss of response to a TNF-alpha antagonist refers to recurrence of symptoms during maintenance dosing following prior clinical benefit. Intolerance of a TNF-alpha antagonist includes, but is not limited to infusion related reaction, demyelination, congestive heart failure, and/or infection.

In one embodiment, diseases which can be treated accordingly with the methods described herein include inflammatory bowel disease (IBD), such as ulcerative colitis, Crohn's disease, ileitis, Celiac disease, nontropical Sprue, enteropathy associated with seronegative arthropathies, microscopic or collagenous colitis, eosinophilic gastroenteritis, or pouchitis resulting after proctocolectomy, and ileoanal anastomosis. In one embodiment, the inflammatory bowel disease is Crohn's disease or ulcerative colitis. The ulcerative colitis may be moderate to severely active ulcerative colitis. Treatment may result in mucosal healing in patients suffering from moderate to severely active ulcerative colitis. Treatment may also result in a reduction, elimination, or reduction and elimination of corticosteroid use by the patient. Treatment may also result in a reduction, elimination, or reduction and elimination of immunomodulator use by the patient.

III. Anti-a4p7 Antibodies

The methods disclosed herein comprise administering an anti-a4p7 antibody and a pan-JAK inhibitor, e.g., tofacitinib, to a subject having an inflammatory disease such as IBD (e.g., Crohn’s disease or ulcerative colitis) for treatment.

An anti-a4p7 antibody that may be used in the methods disclosed herein, has certain characteristics. An anti-a4p7 antibody used herein can bind an a4p7 integrin, and can inhibit binding of the a4p7 integrin to one or more of its ligands (e.g. MAdCAM (e.g., MAdCAM- 1), VCAM-1, fibronectin), thereby inhibiting leukocyte infiltration of tissues (including, recruitment and/or accumulation of leukocytes in tissues). In another embodiment, an anti- a4p7 antibody used herein can bind a4p7 integrin, and can selectively inhibit binding of the a4p7 integrin to one or more of its ligands (e.g., MAdCAM (e.g., MAdCAM-1), VCAM-1, fibronectin), thereby inhibiting leukocyte infiltration of tissues (including recruitment and/or accumulation of leukocytes in tissues). Such anti-a4p7 antibodies can inhibit cellular adhesion of cells bearing an a4p7 integrin to vascular endothelial cells ire mucosal tissues, including gut-associated tissues, lymphoid organs or leukocytes (especially lymphocytes such as T or B cells) in vitro and/or in vivo. In yet another embodiment, the anti-a407 antibody used herein can inhibit the interaction of 06407 with MAdCAM (e.g., MAdCAM-1) and/or fibronectin. In another embodiment, the anti-a407 antibody used herein can inhibit the interaction of a407 with MAdCAM (e.g., MAdCAM-1) and/or fibronectin selectively, e.g., without inhibiting the interaction of a407 with VCAM.

Thus, an anti-a407 antibody (e.g., vedolizumab) used in the methods disclosed herein, can be used to modulate (e.g., inhibit (reduce or prevent)) binding function and/or leukocyte (e.g., lymphocyte, monocyte) infiltration function of a407 integrin. For example, humanized antibodies which inhibit the binding of a407 integrin to a ligand (i.e., one or more ligands) can be administered according to the method in the treatment of diseases associated with leukocyte (e.g., lymphocyte, monocyte) infiltration of tissues (including recruitment and/or accumulation of leukocytes in tissues), particularly of tissues which express the molecule MAdCAM (e.g., MAdCAM-1). Treatment methods using anti-a407 antibodies are described in publication nos. U.S. 2005/0095238, WO2012151248 and WO 2012/151247, each of which is incorporated herein by reference.

In one embodiment, the anti-a407 antibody is a humanized anti-a407 antibody that is an IgGl antibody; comprises a heavy chain variable region comprising a CDR3 domain as set forth in SEQ ID NO: 4, a CDR2 domain as set forth in SEQ ID NO: 3, and a CDR1 domain as set forth in SEQ ID NO: 2; and comprises a light chain variable region comprising a CDR3 domain as set forth in SEQ ID NO: 8, a CDR2 domain as set forth in SEQ ID NO: 7, and a CDR1 domain as set forth in SEQ ID NO: 6.

In certain embodiments, the anti-a407 antibody is a humanized anti-a407 antibody comprising a heavy chain variable domain comprising an amino acid sequence as set forth in SEQ ID NO: 1, and comprising a light chain variable domain comprising an amino acid sequence as set forth in SEQ ID NO: 5. In certain embodiments, the anti-a407 antibody is vedolizumab.

In particular, the methods disclosed herein include administration of the anti-a.407 antibody vedolizumab, or antibodies having antigen binding regions of vedolizumab. Vedolizumab is also known by its trade name ENTYVIO® (Takeda Pharmaceuticals, Inc.). Vedolizumab is a humanized antibody that comprises mutated human IgGl framework regions and antigen-binding CDRs from the murine antibody Act-1 (which is described in US Patent No. 7,147,851, incorporated by reference herein). Vedolizumab specifically binds to the a4p7 integrin and blocks the interaction of a4p7 integrin with mucosal addressin cell adhesion molecule- 1 (MAdCAM-1) and fibronectin and inhibits the migration of memory T-lymphocytes across the endothelium into inflamed gastrointestinal parenchymal tissue. Vedolizumab does not bind to or inhibit function of the a4pi and aEp7 integrins and does not antagonize the interaction of a4 integrins with vascular cell adhesion molecule-1 (VCAM-1).

The heavy chain variable region of vedolizumab is provided herein as SEQ ID NO: 1, and the light chain variable region of vedolizumab is provided herein as SEQ ID NO: 5. Vedolizumab comprises a heavy chain variable region comprising a CDR1 of SEQ ID NO: 2, a CDR2 of SEQ ID NO: 3, and a CDR3 of SEQ ID NO: 4. Vedolizumab comprises a light chain variable region comprising a CDR1 of SEQ ID NO: 6, a CDR2 of SEQ ID NO: 7 and CDR3 of SEQ ID NO: 8. Vedolizumab and the sequences of vedolizumab are also described in U.S. Patent Publication No. 2014/0341885 and U.S. Patent Publication No. 2014/0377251, the entire contents of each which are expressly incorporated herein by reference in their entireties. Alpha4beta7 antibodies and their corresponding amino acid sequences are described in U.S. Patent No. 10143752, which is incorporated by reference herein.

Antibodies useful as anti-a4p7 antibodies suitable in the methods and uses described herein can also be identified using techniques known in the art, such as hybridoma production. Hybridomas can be prepared using, e.g., a murine system. Protocols for immunization and subsequent isolation of splenocytes for fusion are known in the art. Fusion partners and procedures for hybridoma generation are also known. In making a desired antibody, a target protein (antigen) of choice (whole protein or fragments thereof) is isolated and/or purified. Immunization of animals can be performed by any method known in the art. See, e.g., Harlow and Lane, Antibodies: A Laboratory Manual, New York: Cold Spring Harbor Press, 1990. Methods for immunizing animals such as mice, rats, sheep, goats, pigs, cattle and horses are well known in the art. See, e.g., Harlow and Lane, supra, and U.S. Pat. No. 5,994,619. A desired antigen may be administered with an adjuvant to stimulate the immune response. Adjuvants known in the art include complete or incomplete Freund's adjuvant, RIBI (muramyl dipeptides) or ISCOM (immunostimulating complexes). After immunization of an animal with a desired antigen, antibody-producing immortalized cell lines are prepared from cells isolated from the immunized animal. After immunization, the animal is sacrificed and lymph node and/or splenic B cells are immortalized by methods known in the art (e.g., oncogene transfer, oncogenic virus transduction, exposure to carcinogenic or mutating compounds, fusion with an immortalized cell, e.g., a myeloma cell, and inactivating a tumor suppressor gene. See, e.g., Harlow and Lane, supra. Hybridomas can be selected, cloned and further screened for desirable characteristics, including robust growth, high antibody production and desirable antibody characteristics.

Methods for high throughput screening of antibody, or antibody fragment, libraries for molecules capable of binding a target protein (antigen) can be used to identify and affinity mature antibodies useful for the methods of the present disclosure. Such methods include in vitro display techniques known in the art, such as phage display, bacterial display, yeast display, mammalian cell display, ribosome display, mRNA display, and cDNA display, among others. The use of phage display to isolate ligands that bind biologically relevant molecules has been reviewed, for example, in Felici et al., Biotechnol. Annual Rev. 1 : 149- 183, 1995; Katz, Annual Rev. Biophys. Biomol. Struct. 26:27-45, 1997; and Hoogenboom et al., Immunotechnology 4: 1-20, 1998, the disclosures of each of which are incorporated herein by reference as they pertain to in vitro display techniques. Randomized combinatorial peptide libraries have been constructed to select for polypeptides that bind cell surface antigens as described in Kay, Perspect. Drug Discovery Des. 2:251-268, 1995 and Kay et al., Mol. Divers. 1 : 139-140, 1996, the disclosures of each of which are incorporated herein by reference as they pertain to the discovery of antigen-binding molecules. Proteins, such as multimeric proteins, have been successfully phage-displayed as functional molecules (see, for example, EP 0349578; EP 4527839; and EP 0589877, as well as Chiswell and McCafferty, Trends Biotechnol. 10:80-84 1992, the disclosures of each of which are incorporated herein by reference as they pertain to the use of in vitro display techniques for the discovery of antigen-binding molecules). In addition, functional antibody fragments, such as Fab and scFv fragments, have been expressed in in vitro display formats (see, for example, McCafferty et al., Nature 348:552- 554, 1990; Barbas et al., Proc. Natl. Acad. Sci. USA 88:7978-7982, 1991; and Clackson et al., Nature 352:624-628, 1991, the disclosures of each of which are incorporated herein by reference as they pertain to in vitro display platforms for the discovery of antigen-binding molecules). These techniques, among others, can be used to identify and improve the affinity of antibodies that bind to a target antigen.

In addition to in vitro display techniques, computational modeling techniques can be used to design and identify antibodies, or antibody fragments, in silico that bind a target antigen. For example, using computational modeling techniques, one of skill in the art can screen libraries of antibodies, or antibody fragments, in silico for molecules capable of binding specific epitopes, such as extracellular epitopes of the target antigen. IV. JAK Inhibitors

Provided herein is a combination therapy comprising an anti-a4p7 antibody (e.g., vedolizumab) and a pan-Janus kinase (JAK) inhibitor, for the treatment of an IBD (e.g., Crohn’s disease) in a human patient in need thereof.

In certain embodiments, a pan-JAK inhibitor, which can be used in the combination therapy disclosed herein, is tofacitinib. Tofacitinib is also known as XELJANZ.

Tofacitinib is a potent inhibitor of the JAK family enzymes that consists of intracellular tyrosine kinases JAK 1, 2, and 3 and the related kinase tyrosine kinase 2 (TYK2). Upon binding of cytokines to their cell surface receptors, JAKs phosphorylate and activate Signal Transducers and Activators of Transcription (STATs) a family of DNA- binding proteins that regulates the expression of genes relevant to immune and inflammatory cellular responses. This includes the synthesis of inflammatory proteins, primarily members of the interleukin (IL) family, including IL-2, -4, -6, -7, -9, -12, -15, -21, -23, and -27. Among these, IL-6, IL-12, and IL-23 are important drivers of disease activity in inflammatory bowel disease (IBD). Tofacitinib inhibits JAK1, JAK2, JAK3, and to a lesser extent TYK2, phosphorylation, thus preventing STAT activation, thereby restricting intracellular growth factor and cytokine-mediated signals to be transduced by the JAK-STAT pathway and downregulating a number of inflammatory mediators.

Other exemplary pan-JAK inhibitors suitable for use in the combination therapy disclosed herein include, but are not limited to, baricitinib, cerdulatinib, delgocitinib, gusati cinib, izencitinib, momelotinib, and peficitinib.

In certain embodiments, a pan-JAK inhibitor, which can be used in the combination therapy disclosed herein, is baricitinib. Baricitinib inhibits Jak 1 and Jak 2 and additionally has moderate activity against TYK2 and minimal activity against JAK3 (Harrington, Robert et al. “JAK Inhibitors in Rheumatoid Arthritis: An Evidence-Based Review on the Emerging Clinical Data.” Journal of inflammation research vol. 13 519-531. 14 Sep. 2020). Baricitinib is also known as OLUMIANT®. In some embodiments in combination with dosing regimens described herein for the anti-a4p7 antibody, baricitinib is orally administered at 2 mg once daily or 4 mg once daily to a subject.

In certain embodiments, a pan-JAK inhibitor, which can be used in the combination therapy disclosed herein, is cerdulatinib. Cerdulatinib is an investigational oral, dual spleen tyrosine kinase (Syk) and janus kinase (JAK) 1/2/3 inhibitor. In some embodiments in combination with dosing regimens described herein for the anti-a407 antibody, cerdulatinib is orally administered at 30, 25, 20, or 15 milligrams twice daily to a subject.

In certain embodiments, a pan-JAK inhibitor, which can be used in the combination therapy disclosed herein, is delgocitinib. Delgocitinib is also known as CORECTIM®, and inhibits Jak 1, Jak 2, Jak 3, and Tyk2 (Shih, Pei- Yun, Chia-Jung Li, and Su-Boon Yong. "Emerging trends in clinical research on Janus kinase inhibitors for atopic dermatitis treatment." International Immunopharmacology 124 (2023): 111029). Topical delgocitinib is currently approved in Japan for treating atopic dermatitis.

In certain embodiments, a pan-JAK inhibitor, which can be used in the combination therapy disclosed herein, is gusaticinib. Gusaticinib is an oral spleen tyrosine kinase/Janus Kinase (JAK 1, 2, 3, and TYK2) inhibitor (Jimenez, Pablo A., et al. "Oral spleen tyrosine kinase/Janus Kinase inhibitor gusacitinib for the treatment of chronic hand eczema: Results of a randomized phase 2 study." Journal of the American Academy of Dermatology (2023).). In some embodiments in combination with dosing regimens described herein for the anti- 06407 antibody, gusaticinib is orally administered at 80 mg once daily to a subject (e.g., for 12 weeks).

In certain embodiments, a pan-JAK inhibitor, which can be used in the combination therapy disclosed herein, is momelotinib (Desai, Jigar, et al. "Optimisation of momelotinib with improved potency and efficacy as pan-JAK inhibitor." Bioorganic & Medicinal Chemistry Letters 66 (2022): 128728). Momelotinib is also known as OJJAARA®. In some embodiments in combination with dosing regimens described herein for the anti -06.407 antibody, momelotinib is orally administered at 100 mg, 150 mg, or 200 mg once daily to a subject (e.g., for 24 weeks, 48 weeks or longer).

In certain embodiments, a pan-JAK inhibitor, which can be used in the combination therapy disclosed herein, is peficitinib (Kaneko, Yuko. "Efficacy and safety of peficitinib in rheumatoid arthritis." Modern rheumatology 30.5 (2020): 773-778.; Markham, Anthony, and Susan J. Keam. "Peficitinib: first global approval." Drugs 79.8 (2019): 887-891.). Peficitinib is also known as SMYRAF®. Peficitinib is a Janus kinase (JAK)l, JAK2, JAK3 and tyrosine kinase (Tyk)2 (pan-JAK) inhibitor currently approved in Japan for the treatment of rheumatoid arthritis. In some embodiments in combination with dosing regimens described herein for the anti-a407 antibody, peficitinib is orally administered at 100 mg or 150 mg once daily to a subject. In certain embodiments, a pan-JAK inhibitor, which can be used in the combination therapy disclosed herein, is izencitinib. In some embodiments in combination with dosing regimens described herein for the anti-a4p7 antibody, izencitinib is orally administered at 80 mg or 200 mg once daily to a subject (Schreiber, S., et al. "P375 Izencitinib induction treatment in patients with moderately-to-severely-active Crohn’s Disease: A phase 2 doubleblind, randomized, placebo-controlled study." Journal of Crohn's and Colitis 17.Supplement_l (2023): i505-i507).

V. Effectors

The present disclosure is based, at least in part, on the discovery that certain molecular effectors (e.g., mRNA and/or protein) are associated with the combination of vedolizumab with pan-JAK inhibitors. In particular, the effectors described herein are mRNA and/or protein molecular effectors.

The level of one or more mRNA effectors in a biological sample may be determined by any suitable method. Any reliable method for measuring the level or amount of mRNA in a sample may be used. Generally, mRNA can be detected and quantified from a sample (including fractions thereof), such as samples of isolated RNA by various methods known for mRNA, including, for example, amplification-based methods (e.g., Polymerase Chain Reaction (PCR), Real-Time Polymerase Chain Reaction (RT-PCR), Quantitative Polymerase Chain Reaction (qPCR), rolling circle amplification, etc.), hybridization-based methods (e.g., hybridization arrays (e.g., microarrays), NanoString analysis, Northern Blot analysis, branched DNA (bDNA) signal amplification, in situ hybridization, etc.), and sequencingbased methods (e.g., transcriptome sequencing via next-generation sequencing methods, for example, using a MGI2000 sequencer or Illumina or lonTorrent platforms). In some embodiments, RNA is converted to DNA (cDNA) prior to analysis. cDNA can be generated by reverse transcription of isolated mRNA using conventional techniques. Other exemplary techniques include ribonuclease protection assay (RPA) and mass spectroscopy. In one embodiment, the level of the mRNA in a sample is determined using quantitative PCR (qPCR) or a Northern blot.

In certain embodiments, the protein product of one or more effectors in a biological sample may be determined by any suitable method. In certain embodiments, it may be possible to assay for the expression of one or more effectors at the protein level using a detection reagent that detects the protein product encoded by the mRNA of the effector(s). In some embodiments of the present teachings, an antibody binding assay is used to detect a protein effector; e.g., a sample from the subject is contacted with an antibody reagent that binds the effector analyte, a reaction product (or complex) comprising the antibody reagent and analyte is generated, and the presence (or absence) or amount of the complex is determined. The antibody reagent useful in detecting effector analytes can be monoclonal, polyclonal, chimeric, recombinant, or a fragment of the foregoing, and the step of detecting the reaction product can be carried out with any suitable immunoassay. For example, if an antibody reagent is available that binds specifically to the effector protein product to be detected, and not to other proteins, then such an antibody reagent can be used to detect the expression of the effector of interest in a cellular sample from the subject, or a preparation derived from the cellular sample, using standard antibody -based techniques known in the art, such as flow cytometry (e.g., Fluorescence-Activated Cell Sorting (FACS); multi-color flow cytometry), mass cytometry (CyTOF), immunohistochemistry, oligonucleotide sequencing (e.g., CITE-Seq), ELISA and the like.

It will be readily understood by the ordinarily skilled artisan that essentially any technical means established in the art for detecting effectors, at either the nucleic acid or protein level, can be adapted to detection of the effectors discussed herein and applied in the methods of the current invention.

As described in the Examples below, in silico models revealed that the combined vedolizumab plus JAKi therapy could modulate a high array of effectors altered in CD, more than any of the therapies alone, by enhancing the effects of the individual drugs. Thus, in one embodiment, the disclosure relates to the use of differential expression of mRNA and/or protein levels in a biological sample from an IBD patient relative to a reference level in a patient without the disease to provide rationale for employing the combination of vedolizumab with pan-JAK inhibitors. In some embodiments, an effector is increased or decreased in the human patient with IBD relative to a reference level in individuals without the disease, and administration of the humanized anti-a4p7 antibody and/or pan-JAK inhibitor to the human patient results in a reversion of the expression level of the at least one effector to said reference level.

The effectors that can be used in the compositions and methods disclosed herein include nucleic acid sequences (e.g., mRNA transcripts) that encode the following proteins, alone or in combination: CCR9: CCR9 (also known as CC-CKR-9, CDwl99, GPR-9-6, GPR28, C-C motif chemokine receptor 9) encodes a G protein-coupled receptor that is expressed on several types of immune cells, including dendritic cells (DCs), CD4+ T cells, and B cells. CCR9 is also known to drive the migration of immune cells to gradients of its cognate ligand CCL25 (TECK), which is produced by gut and thymic epithelial cells (Pathak M, Lal G. The Regulatory Function of CCR9+ Dendritic Cells in Inflammation and Autoimmunity. Front Immunol. 2020 Oct 2; 11 :536326. doi: 10.3389/fimmu.2020.536326). The amino acid sequence of CCR9 is SEQ ID NO: 9 and can be found under UniProt Accession No. P51686.

In one embodiment, the methods disclosed herein include determining a level of expression of a gene (mRNA) encoding SEQ ID NO: 9 and/or the protein level thereof.

FASLG: FASLG (also known as FasL, ALPS1B, APT1LG1, APTL, CD178, CD95-L, CD95L, FASL, TNFSF6, TNLG1 A, Fas ligand) encodes a type-II transmembrane protein that is expressed on cytotoxic T lymphocytes and natural killer cells and has been found to induce apoptosis in target cells through the death receptor Fas/Apol/CD95. It has been reported that certain tumors may escape FasL-dependent immune-cytotoxic attack by expressing a decoy receptor that blocks FasL (Pitti RM, et al. Genomic amplification of a decoy receptor for Fas ligand in lung and colon cancer. Nature. 1998 Dec 17;396(6712):699- 703). The amino acid sequence of FASLG is SEQ ID NO: 10 and can be found under UniProt Accession No. P48023.

In one embodiment, the methods disclosed herein include determining a level of expression of a gene (mRNA) encoding SEQ ID NO: 10 and/or the protein level thereof.

IFNG: IFNG (also known as IFN-y, IFG, IFI, interferon, gamma, interferon gamma, IMD69) encodes a dimerized soluble cytokine associated antiproliferative, pro-apoptotic and antitumor mechanisms and is considered as a major effector of immunity (Castro F, et al. Interferon-Gamma at the Crossroads of Tumor Immune Surveillance or Evasion. Front Immunol. 2018 May 4;9:847. doi: 10.3389/fimmu.2018.00847). The amino acid sequence of IFNG is SEQ ID NO: 11 and can be found under UniProt Accession No. P01579.

In one embodiment, the methods disclosed herein include determining a level of expression of a gene (mRNA) encoding SEQ ID NO: 11 and/or the protein level thereof.

MAPK1 : MAPK1 (also known as ERK, ERK-2, ERK2, ERT1, MAPK2, P42MAPK, PRKM1, PRKM2, p38, p40, p41, p41mapk, p42-MAPK, mitogen-activated protein kinase 1, NS13) encodes an enzyme that has both independent functions of phosphorylating histones as a kinase and directly binding the promoter regions of genes to regulate gene expression as a transcription factor (Wang Y, et al. MAPK1 promotes the metastasis and invasion of gastric cancer as a bidirectional transcription factor. BMC Cancer. 2023 Oct 10;23(l):959. doi: 10.1186/S12885-023-11480-3). The amino acid sequence ofMAPKl is SEQ ID NO: 12 and can be found under UniProt Accession No. P28482.

In one embodiment, the methods disclosed herein include determining a level of expression of a gene (mRNA) encoding SEQ ID NO: 12 and/or the protein level thereof.

MAPK3: MAPK3 (also known as ERK-1, ERK1, ERT2, HS44KDAP, HUMKER1A, P44ERK1, P44MAPK, PRKM3, p44-ERKl, p44-MAPK, mitogen-activated protein kinase 3) encodes a member of the mitogen-activated protein kinase family that has been shown to have an important role in the induction of T-cell anergy (Bendix I, et al. MAPK3 deficiency drives autoimmunity via DC arming. Eur J Immunol. 2010 May;40(5): 1486-95. doi: 10.1002/eji.200939930). The amino acid sequence of MAPK3 is SEQ ID NO: 13 and can be found under UniProt Accession No. P27361.

In one embodiment, the methods disclosed herein include determining a level of expression of a gene (mRNA) encoding SEQ ID NO: 13 and/or the protein level thereof.

MMP1 : MMP1 (also known as Matrix Metallopeptidase 1, Interstitial Collagenase, fibroblast collagenase, CLG, MMP-1, Matrix Metalloproteinase- 1) encodes a zinc-dependent endopeptidase that has been found to be a potential regulator of tumor progression and dedifferentiation in Papillary thyroid cancer (PTC) (Zhou J, et al. MMP1 acts as a potential regulator of tumor progression and dedifferentiation in papillary thyroid cancer. Front Oncol. 2022 Nov 21;12: 1030590. doi: 10.3389/fonc.2022.1030590). The amino acid sequence of MMP1 is SEQ ID NO: 14 and can be found under UniProt Accession No. P03956.

In one embodiment, the methods disclosed herein include determining a level of expression of a gene (mRNA) encoding SEQ ID NO: 14 and/or the protein level thereof.

NFKB 1 : NFKB1 (also known as Nuclear factor NF-kappa-B pl 05 subunit, EBP-1, KBF1, NF-kB 1, NF-kappa-B, NF-kappaB, NFKB-pl05, NFKB-p50, NFkappaB, pl 05, p50, CVID12, nuclear factor kappa B subunit 1, NF-kappa-B 1, NF-kB, NF-kappabeta) encodes one of the five subunits of NF-KB, widely implicated in carcinogenesis, in some cases driving cancer progression and in others acting as a tumour-suppressor (Concetti J, Wilson CL. NFKB1 and Cancer: Friend or Foe? Cells. 2018 Sep 7;7(9):133. doi: 10.3390/cells7090133). The amino acid sequence of NFKB1 is SEQ ID NO: 15 and can be found under UniProt Accession No. P19838.

In one embodiment, the methods disclosed herein include determining a level of expression of a gene (mRNA) encoding SEQ ID NO: 15 and/or the protein level thereof.

PLA2G1B: PLA2G1B (also known as PLA2, PLA2A, PPLA2, phospholipase A2 group IB) encodes an enzyme that catalyzes the release of fatty acids from glycero-3- phosphocholines and has been found to catalyze phospholipid hydrolysis in the intestinal lumen (Hui DY. Intestinal phospholipid and lysophospholipid metabolism in cardiometabolic disease. Curr Opin Lipidol. 2016 Oct;27(5):507-12. doi: 10.1097/MOL.0000000000000334). The amino acid sequence of PLA2G1B is SEQ ID NO: 16 and can be found under UniProt Accession No. P04054.

In one embodiment, the methods disclosed herein include determining a level of expression of a gene (mRNA) encoding SEQ ID NO: 16 and/or the protein level thereof.

TCF4: TCF4 (also known as E2-2, ITF-2, ITF2, PTHS, SEF-2, SEF2, SEF2-1, SEF2- 1A, SEF2-1B, SEF2-1D, TCF-4, bHLHbl9, FECD3, transcription factor 4, CDG2T, immunoglobulin transcription factor 2) encodes a transcription factor that binds to the immunoglobulin enhancer mu-E5/kappa-E2 motif. The TCF4-CCL2-CCR2 axis has been found to have an essential role in colorectal cancer liver metastasis by enhancing tumor- associated macrophage (TAMs) recruitment and M2 polarization (Tu W, et al. TCF4 enhances hepatic metastasis of colorectal cancer by regulating tumor-associated macrophage via CCL2/CCR2 signaling. Cell Death Dis. 2021 Sep 27;12(10):882). The amino acid sequence of TCF4 is SEQ ID NO: 17 and can be found under UniProt Accession No. P15884.

In one embodiment, the methods disclosed herein include determining a level of expression of a gene (mRNA) encoding SEQ ID NO: 17 and/or the protein level thereof.

CDH1 : CDH1 (also known as CADH1, Arc-1, CD324, CDHE, ECAD, LCAM, UVO, cadherin 1, BCDS1, E-cadherin, uvomorulin) encodes a calcium-dependent cell-cell adhesion glycoprotein composed of five extracellular cadherin repeats, a transmembrane region, and a highly conserved cytoplasmic tail. Germline mutations of CDH1 have been found to be associated with the development of multiple cancers (Corso G. Pleiotropic cancer manifestations of germline CDH1 mutations: Risks and management. J Surg Oncol. 2022 Jun; 125(8): 1326-1331. doi: 10.1002/jso.26847. Epub 2022 Mar 12). The amino acid sequence of CDH1 is SEQ ID NO: 18 and can be found under UniProt Accession No. P55287.

In one embodiment, the methods disclosed herein include determining a level of expression of a gene (mRNA) encoding SEQ ID NO: 18 and/or the protein level thereof.

NFKB2: NFKB2 (also known as Nuclear factor NF-kappa-B pl 00 subunit, C VID 10, H2TF1, LYT-10, LYT10, NF-kB2, pl 05, p52, pl 00, p49/pl00, nuclear factor kappa B subunit 2) encodes an inhibitor of KB (IKB) protein that is partially degraded to produce the NF-KB2/p52 (p52) transcription factor, and heterozygous NFKB2 mutations are thought to be a cause of immunodeficiency and autoimmunity (Wirasinha RC, et al. Nfkb2 variants reveal a plOO-degradation threshold that defines autoimmune susceptibility. J Exp Med. 2021 Feb l;218(2):e20200476). The amino acid sequence of NFKB2 is SEQ ID NO: 19 and can be found under UniProt Accession No. Q00653.

In one embodiment, the methods disclosed herein include determining a level of expression of a gene (mRNA) encoding SEQ ID NO: 19 and/or the protein level thereof.

CLDN 1 : CLDN1 (also known as CLD1, ILVASC, SEMP1, claudin 1) encodes a member of the tight junction protein family. It has been found that targeting CLDN1 reverted inflammation-induced hepatocyte profibrogenic signaling and cell fate and suppressed the myofibroblast differentiation of hepatic stellate cells (Roehlen N., et al. A monoclonal antibody targeting nonjunctional claudin- 1 inhibits fibrosis in patient-derived models by modulating cell plasticity. Sci Transl Med. 2022 Dec 21;14(676):eabj4221). The amino acid sequence of CLDN1 is SEQ ID NO: 20 and can be found under UniProt Accession No. 095832.

In one embodiment, the methods disclosed herein include determining a level of expression of a gene (mRNA) encoding SEQ ID NO: 20 and/or the protein level thereof.

CLDN2: CLDN2 (also known as Claudin-2, SP82, OAZON) encodes a pore-forming tight junction protein associated with inflammatory bowel disease. It has been shown that starvation-induced macroautophagy/autophagy enhances the tight junction barrier by degrading pore-forming CLDN2 (Ganapathy AS, et al. AP2M1 mediates autophagy-induced CLDN2 (claudin 2) degradation through endocytosis and interaction with LC3 and reduces intestinal epithelial tight junction permeability. Autophagy. 2022 Sep;18(9):2086-2103). The amino acid sequence of CLDN2 is SEQ ID NO: 21 and can be found under UniProt Accession No. P57739.

In one embodiment, the methods disclosed herein include determining a level of expression of a gene (mRNA) encoding SEQ ID NO: 21 and/or the protein level thereof.

CLDN3: CLDN3 (also known as C7orfl, CPE-R2, CPETR2, HRVP1, RVP1, claudin 3) encodes an integral tight junction membrane protein, which has been found to play a critical role in maintaining the tight junction’s barrier function. CLDN3 has been suggested to be an epigenetically silenced metastasis suppressor gene in hepatocellular carcinoma (HCC) (Jiang L, et al. CLDN3 inhibits cancer aggressiveness via Wnt-EMT signaling and is a potential prognostic biomarker for hepatocellular carcinoma. Oncotarget. 2014 Sep 15;5(17):7663-76. doi: 10.18632/oncotarget.2288). The amino acid sequence of CLDN3 is SEQ ID NO: 22 and can be found under UniProt Accession No. 015551.

In one embodiment, the methods disclosed herein include determining a level of expression of a gene (mRNA) encoding SEQ ID NO: 22 and/or the protein level thereof.

OCLN: OCLN (also known as BLCPMG, PPP1R115, occludin, PTORCH1) encodes a protein that is an important component of the tight junction complex, providing apical intercellular connections between adjacent cells in endothelial and epithelial tissue (Jenkinson EM, et al. Comprehensive molecular screening strategy of OCLN in band-like calcification with simplified gyration and polymicrogyria. Clin Genet. 2018 Feb;93(2):228-234). The amino acid sequence of OCLN is SEQ ID NO: 23 and can be found under UniProt Accession No. QI 6625.

In one embodiment, the methods disclosed herein include determining a level of expression of a gene (mRNA) encoding SEQ ID NO: 23 and/or the protein level thereof.

TJP1 : TJP1 (also known as Zonula occludens-1, ZO-1, tight junction protein-1) encodes a peripheral membrane protein that belongs to the family of zonula occludens proteins, which are tight junction-associated proteins. TJP1 has been shown to function as a mediator of mTOR to modulate the hepatic circadian clock (Liu Y, et al. The tight junction protein TJP1 regulates the feeding-modulated hepatic circadian clock. Nat Commun. 2020 Jan 30; 11(1):589). The amino acid sequence of TJP1 is SEQ ID NO: 24 and can be found under UniProt Accession No. Q07157. In one embodiment, the methods disclosed herein include determining a level of expression of a gene (mRNA) encoding SEQ ID NO: 24 and/or the protein level thereof.

ITGA4: ITGA4 (also known as CD49D, IA4, integrin subunit alpha 4) encodes an integrin alpha subunit that makes up half of a4pi lymphocyte homing receptor. ITGA4 is thought to play an essential role in mediating both cell-cell and cell-matrix interactions in Chronic lymphocytic leukemia (CLL)-involved tissues eventually delivering prosurvival signals and protecting CLL cells from drug-induced damages and has been found to be a prognostic biomarker of CLL (Tissino E, et al. CD49d promotes disease progression in chronic lymphocytic leukemia: new insights from CD49d bimodal expression. Blood. 2020 Apr 9; 135(15): 1244-1254. doi: 10.1182/blood.2019003179). The amino acid sequence of ITGA4 is SEQ ID NO: 25 and can be found under UniProt Accession No. P13612.

In one embodiment, the methods disclosed herein include determining a level of expression of a gene (mRNA) encoding SEQ ID NO: 25 and/or the protein level thereof.

ITGB7: ITGB7 (also known as Integrin Subunit Beta 7, Gut Homing Receptor Beta Subunit) encodes an integrin protein that with ITGA4 can form the heterodimeric integrin receptor a4p7, or form aEp7 when paired with ITGAE (CD103) (Byron A, et al. Anti- integrin monoclonal antibodies. J Cell Sci. 2009 Nov 15;122(Pt 22):4009-l l. doi: 10.1242/jcs.056770). The amino acid sequence of ITGB7 is SEQ ID NO: 26 and can be found under UniProt Accession No. P26010.

In one embodiment, the methods disclosed herein include determining a level of expression of a gene (mRNA) encoding SEQ ID NO: 26 and/or the protein level thereof.

PDGFB: PDGFB (also known as IBGC5, PDGF-2, PDGF2, SIS, SSV, c-sis, platelet derived growth factor subunit B) encodes a pro-angiogenic factor that acts as a transcriptional target of super enhancer-driven KLF6 and can activate the mTORCl signaling pathway in clear cell renal cell carcinoma (Abuhamad AY, et al. Cancer Cell-Derived PDGFB Stimulates mTORCl Activation in Renal Carcinoma. Int J Mol Sci. 2023 Mar 29;24(7):6447). The amino acid sequence of PDGFB is SEQ ID NO: 27 and can be found under UniProt Accession No. P01127.

In one embodiment, the methods disclosed herein include determining a level of expression of a gene (mRNA) encoding SEQ ID NO: 27 and/or the protein level thereof. IL5 : IL5 (also known as Interleukin 5, IL-5, TRF, Eosinophil Differentiation Factor, T-Cell Replacing Factor, EDF, B-Cell Differentiation Factor I) encodes a type-1 cytokine that stimulates B cell growth and increases immunoglobulin secretion. IL5 is thought to be central to the initiation and sustenance of eosinophilic airway inflammation (Mukherjee M, et al. Anti-IL5 therapy for asthma and beyond. World Allergy Organ J. 2014 Dec 4;7(1):32). The amino acid sequence of IL5 is SEQ ID NO: 28 and can be found under UniProt Accession No. P05113.

In one embodiment, the methods disclosed herein include determining a level of expression of a gene (mRNA) encoding SEQ ID NO: 28 and/or the protein level thereof.

IL6: IL6 (also known as BSF2, HGF, HSF, IFNB2, IL-6, BSF-2, CDF, IFN-beta-2, interleukin 6) encodes an interleukin that acts as both a pro-inflammatory cytokine and an anti-inflammatory myokine. IL-6 blockade has been proposed as a therapeutic strategy for acute systemic and chronic inflammatory diseases (Tanaka T, et al. Interleukin (IL-6) Immunotherapy. Cold Spring Harb Perspect Biol. 2018 Aug l;10(8):a028456). The amino acid sequence of IL6 is SEQ ID NO: 29 and can be found under UniProt Accession No. P05231.

In one embodiment, the methods disclosed herein include determining a level of expression of a gene (mRNA) encoding SEQ ID NO: 29 and/or the protein level thereof.

IGF 2: IGF 2 (also known as Cl lorf43, GRDF, IGF-II, PP9974, insulin like growth factor 2, SRS3) encodes a growth factor that shares structural similarities to insulin. It is thought to play an important role in human growth regulation, metabolism, and tumor susceptibility (Selenou C, et al. IGF2: Development, Genetic and Epigenetic Abnormalities. Cells. 2022 Jun 10; 11(12): 1886). The amino acid sequence of IGF2 is SEQ ID NO: 30 and can be found under UniProt Accession No. P01344.

In one embodiment, the methods disclosed herein include determining a level of expression of a gene (mRNA) encoding SEQ ID NO: 30 and/or the protein level thereof.

MMP9: MMP9 (also known as CLG4B, GELB, MANDP2, MMP-9, 92 kDa type IV collagenase, 92 kDa gelatinase, gelatinase B, matrix metallopeptidase 9) encodes matrixin, a class of enzymes that belong to the zinc-metalloproteinases family involved in the degradation of the extracellular matrix. Matrix metalloproteinase 9 (MMP9) is highly expressed in gastric cancer (Fu CK, et al. The Association of MMP9 Promoter Rs3918242 Genotype With Gastric Cancer. Anticancer Res. 2021 Jul;41(7):3309-3315). The amino acid sequence of MMP9 is SEQ ID NO: 31 and can be found under UniProt Accession No. P14780.

In one embodiment, the methods disclosed herein include determining a level of expression of a gene (mRNA) encoding SEQ ID NO: 31 and/or the protein level thereof.

WNT1 : WNT1 (also known as Proto-oncogene Wnt-1, Proto-oncogene Int-1 homolog, BMND16, INTI, 0115, Wnt family member 1) encodes a ligand of Wnt/p-catenin signaling known to promote pro-angiogenesis and reduce myocardial infarction. Wntl is also involved in various cancers, genetic type XV osteogenesis imperfecta, osteoporosis, and neurological diseases (Peng C, et al. Comprehensive bioinformatic analysis of Wntl and Wntl -associated diseases. Intractable Rare Dis Res. 2020 Feb;9(l): 14-22). The amino acid sequence of WNT1 is SEQ ID NO: 32 and can be found under UniProt Accession No. P04628.

In one embodiment, the methods disclosed herein include determining a level of expression of a gene (mRNA) encoding SEQ ID NO: 32 and/or the protein level thereof.

PPARG: PPARG (also known as CIMT1, GLM1, NR1C3, PPARG1, PPARG2, PPARgamma, peroxisome proliferator activated receptor gamma, PPAR-y, PPARG, PPARG5, glitazone reverse insulin resistance receptor, NR1C3) encodes a type II nuclear receptor that functions as a transcription factor. PPARG has been shown to play an important role in urothelial cells for mitochondrial biogenesis, cellular differentiation and regulation of inflammation in response to urinary tract infection (UTI) (Liu C, et al. Pparg promotes differentiation and regulates mitochondrial gene expression in bladder epithelial cells. Nat Commun. 2019 Oct 9;10(l):4589). The amino acid sequence of PPARG is SEQ ID NO: 33 and can be found under UniProt Accession No. P37231.

In one embodiment, the methods disclosed herein include determining a level of expression of a gene (mRNA) encoding SEQ ID NO: 33 and/or the protein level thereof.

IL22: IL22 (also known as Interleukin-22, IL-21, IL-22, IL-D110, IL-TIF, ILTIF, TIFIL-23, TIFa, zcytol8, interleukin 22) encodes an a-helical cytokine. IL22 has been proposed as a therapeutic target in diseases of the intestine, including inflammatory bowel disease, GvHD, and cancer (Keir M, et al. The role of IL-22 in intestinal health and disease. J Exp Med. 2020 Feb 13;217(3):e20192195). The amino acid sequence of IL22 is SEQ ID NO: 34 and can be found under UniProt Accession No. Q9GZX6. In one embodiment, the methods disclosed herein include determining a level of expression of a gene (mRNA) encoding SEQ ID NO: 34 and/or the protein level thereof.

TLR4: TLR4 (also known as ARMDIO, CD284, TLR-4, TOLL, toll like receptor 4) encodes a transmembrane protein whose activation leads to intracellular signaling pathway NF-KB and inflammatory cytokine production. Prolonged activation of TLR4 is thought to be linked with several hereditary human diseases, neurodegeneration and also with autoimmune diseases and cancer (Ciesielska, A., Matyjek, M., & Kwiatkowska, K. (2021). TLR4 and CD14 trafficking and its influence on LPS-induced pro-inflammatory signaling. Cellular and molecular life sciences : CMLS, 78(4), 1233-126). The amino acid sequence of TLR4 is SEQ ID NO: 35 and can be found under UniProt Accession No. 000206.

In one embodiment, the methods disclosed herein include determining a level of expression of a gene (mRNA) encoding SEQ ID NO: 35 and/or the protein level thereof.

MICB: MICB (also known as PERBI 1.2, MHC class I polypeptide-related sequence B) encodes a protein expressed by many human cancers as a result of cellular stress, and can tag cells for elimination by cytotoxic lymphocytes through NKG2D receptor activation (Ferrari de Andrade L, et al. Antibody -mediated inhibition of MICA and MICB shedding promotes NK cell-driven tumor immunity. Science. 2018 Mar 30;359(6383): 1537-1542). The amino acid sequence of MICB is SEQ ID NO: 36 and can be found under UniProt Accession No. Q29980.

In one embodiment, the methods disclosed herein include determining a level of expression of a gene (mRNA) encoding SEQ ID NO: 36 and/or the protein level thereof.

IL13: IL13 (also known as IL-13, P600, interleukin 13) encodes an immunoregulatory cytokine produced primarily by activated Th2 cells. It has been found that IL-4 or IL- 13 alone is not sufficient, but IL-4 or IL-13 together with apoptotic cells induced the tissue repair program in macrophages (Bosurgi L, et al. Macrophage function in tissue repair and remodeling requires IL-4 or IL-13 with apoptotic cells. Science. 2017 Jun 9;356(6342): 1072- 1076). The amino acid sequence of IL13 is SEQ ID NO: 37 and can be found under UniProt Accession No. P35225.

In one embodiment, the methods disclosed herein include determining a level of expression of a gene (mRNA) encoding SEQ ID NO: 37 and/or the protein level thereof. TGFBR2: TGFBR2 (also known as AAT3, FAA3, LDS1B, LDS2, LDS2B, MFS2, RIIC, TAAD2, TGFR-2, TGFbeta-RII, transforming growth factor beta receptor 2, TBR-ii, TBRII) encodes a member of the serine/threonine protein kinase family and the TGFB receptor subfamily that plays a role in regulating the transcription of a subset of genes related to cell proliferation. TGFBR2 has a synergistic interaction with mismatch repair (MMR) in inflammation-associated colon tumorigenesis (Tosti E, et al. Loss of MMR and TGFBR2 Increases the Susceptibility to Microbiota-Dependent Inflammation-Associated Colon Cancer. Cell Mol Gastroenterol Hepatol. 2022;14(3):693-717). The amino acid sequence of TGFBR2 is SEQ ID NO: 38 and can be found under UniProt Accession No. P37173.

In one embodiment, the methods disclosed herein include determining a level of expression of a gene (mRNA) encoding SEQ ID NO: 38 and/or the protein level thereof.

EGF : EGF (also known as H0MG4, URG, epidermal growth factor, epithelial growth factor) encodes a 6-kDa protein that stimulates cell growth and differentiation by binding to its receptor, EGFR. The presence of EGF in human gastric cancer is thought to indicate a higher malignant potential (Tokunaga A, et al. Clinical significance of epidermal growth factor (EGF), EGF receptor, and c-erbB-2 in human gastric cancer. Cancer. 1995 Mar 15;75(6 Suppl): 1418-25). The amino acid sequence of EGF is SEQ ID NO: 39 and can be found under UniProt Accession No. P01133.

In one embodiment, the methods disclosed herein include determining a level of expression of a gene (mRNA) encoding SEQ ID NO: 39 and/or the protein level thereof.

C0L1A1 : COL1A1 (also known as EDSC, Oil, 012, 013, 014, collagen type I alpha 1, collagen type I alpha 1 chain, EDSARTH1, CAFYD, alpha-1 type I collagen) encodes a member of the collagen family that is involved in epithelial-mesenchymal transition, which is closely linked to malignant tumorigenesis (Li X, et al. C0L1 Al : A novel oncogenic gene and therapeutic target in malignancies. Pathol Res Pract. 2022 Aug;236: 154013). The amino acid sequence of COL1 Al is SEQ ID NO: 40 and can be found under UniProt Accession No. P02452.

In one embodiment, the methods disclosed herein include determining a level of expression of a gene (mRNA) encoding SEQ ID NO: 40 and/or the protein level thereof.

COL1A2: COL1A2 (also known as Collagen alpha-2(I) chain, 014, collagen type I alpha 2, collagen type I alpha 2 chain, EDSCV, EDSARTH2) encodes one of the chains for type I collagen, the fibrillar collagen found in most connective tissues. COL1A2 has been found to mediate the pro- and anti-migratory effects of T-box transcription factor 3 (TBX3) in chondrosarcoma and fibrosarcoma cells respectively (Omar R, et al. C0L1A2 is a TBX3 target that mediates its impact on fibrosarcoma and chondrosarcoma cell migration. Cancer Lett. 2019 Sep 10;459:227-239). The amino acid sequence of COL1A2 is SEQ ID NO: 41 and can be found under UniProt Accession No. P08123.

In one embodiment, the methods disclosed herein include determining a level of expression of a gene (mRNA) encoding SEQ ID NO: 41 and/or the protein level thereof.

MSH2: MSH2 (also known as DNA mismatch repair protein Msh2, MutS homolog 2, COCAI, FCC1, HNPCC, HNPCC1, LCFS2, hMMRCS2, MSH-2) encodes a tumor suppressor gene which forms a heterodimer with MSH6 to make the human MutSa mismatch repair complex. MSH2 has been found to be mutated or rearranged in Lynch syndrome (LS), which is characterized by a high risk of tumor development, including colorectal cancer (Liccardo R, et al. MSH2 Overexpression Due to an Unclassified Variant in 3 '-Untranslated Region in a Patient with Colon Cancer. Biomedicines. 2020 Jun 19;8(6): 167). The amino acid sequence of MSH2 is SEQ ID NO: 42 and can be found under UniProt Accession No. P43246.

In one embodiment, the methods disclosed herein include determining a level of expression of a gene (mRNA) encoding SEQ ID NO: 42 and/or the protein level thereof.

In certain embodiments, methods disclosed herein include effectors that are selected from any one of (or a combination of, or combinations of) CCR9, FASLG, IFNG, MAPK1, MAPK3, MMP1, NFKB1, PLA2G1B, and TCF4, CDH1, NFKB2, CLDN1, CLDN2, CLDN3, OCLN, TJP1, ITGA4, ITGB7, PDGFB, IL5, IL6, IGF2, MMP9, WNT1, PPARG, IL22, TLR4, MICB, IL13, TGFBR2, EGF, COL1A1, COL1A2, and/or MSH2.

In some embodiments, a humanized anti-a4p7 antibody and pan-JAK inhibitor is administered to a human subject having IBD and an increased expression level of at least one effector marker selected from the group consisting of CCR9, FASLG, IFNG, MAPK1, MAPK3, MMP1, NFKB1, PLA2G1B, and TCF4 relative to a reference level in a human subject without IBD. In certain embodiments, administration of a humanized anti-a4p7 antibody and pan-JAK inhibitor results in a decreased expression level of at least one effector marker selected from the group consisting of CCR9, FASLG, IFNG, MAPK1, MAPK3, MMP1, NFKB1, PLA2G1B, and TCF4. In some embodiments, a humanized anti-a4p7 antibody and pan-JAK inhibitor is administered to a human subject having IBD and an increased expression level before treatment of at least one effector marker selected from the group consisting of CCR9, FASLG, IFNG, MAPK1, MAPK3, MMP1, NFKB1, PLA2G1B, and TCF4 relative to a reference level in a human subject without IBD. In a further embodiment, the human subject having IBD has a decreased expression level after treatment of the at least one effector marker selected from the group consisting of CCR9, FASLG, IFNG, MAPK1, MAPK3, MMP1, NFKB1, PLA2G1B, and TCF4 relative to a baseline expression level in the human subject having IBD. The IBD can be Crohn’s disease or ulcerative colitis. In a further embodiment, the Crohn’s disease is moderately to severely active Crohn’s disease. In a further embodiment, the ulcerative colitis is moderately to severely active ulcerative colitis.

In some embodiments, a humanized anti-a4p7 antibody and pan-JAK inhibitor is administered to a human subject having IBD and a decreased expression level of CDH1 relative to a reference level in a human subject without IBD. In certain embodiments, administration of a humanized anti-a4p7 antibody and pan-JAK inhibitor results in an increased expression level of CDH1.

In some embodiments, a humanized anti-a4p7 antibody and pan-JAK inhibitor is administered to a human subject having IBD and a decreased expression level before treatment, of CDH1 relative to a reference level in a human subject without IBD. In a further embodiment, the human subject having IBD has an increased expression level after treatment of CDH1 relative to a baseline expression level in the human subject having IBD. The IBD can be Crohn’s disease or ulcerative colitis. In a further embodiment, the Crohn’s disease is moderately to severely active Crohn’s disease. In a further embodiment, the ulcerative colitis is moderately to severely active ulcerative colitis.

In some embodiments, a humanized anti-a4p7 antibody and pan-JAK inhibitor is administered to a human subject having IBD and an increased expression level before treatment, of MAPK1 and/or MAPK3 relative to a reference level in a human subject without IBD. In a further embodiment, the human subject having IBD has a decreased expression level after treatment of MAPK1 and/or MAPK3 relative to a baseline expression level in the human subject having IBD. The IBD can be Crohn’s disease or ulcerative colitis. In a further embodiment, the Crohn’s disease is moderately to severely active Crohn’s disease. In a further embodiment, the ulcerative colitis is moderately to severely active ulcerative colitis. In some embodiments, a humanized anti-a4p7 antibody and pan-JAK inhibitor is administered to a human subject having IBD and an increased expression level before treatment of at least one effector marker selected from the group consisting of TCF4, NFKB1, MMP1, and IFNG relative to a reference level in a human subject without IBD. In a further embodiment, the human subject having IBD has a decreased expression level after treatment of the at least one effector marker selected from the group consisting of TCF4, NFKB1, MMP1, and IFNG relative to a baseline expression level in the human subject having IBD. The IBD can be Crohn’s disease or ulcerative colitis. In a further embodiment, the Crohn’s disease is moderately to severely active Crohn’s disease. In a further embodiment, the ulcerative colitis is moderately to severely active ulcerative colitis.

In some embodiments, a humanized anti-a4p7 antibody is administered to a human subject having IBD and an increased or decreased expression level of at least one effector marker selected from the group consisting of NFKB2, CLDN1, CLDN2, CLDN3, OCLN, TJP1, ITGA4, ITGB7, PDGFB, IL5, IL6, IGF2, and MMP9 relative to a reference level in a human subject without IBD. In certain embodiments, administration of a humanized anti- a4p7 antibody results in a reversion in the expression level of the at least one effector marker selected from the group consisting of NFKB2, CLDN1, CLDN2, CLDN3, OCLN, TJP1, ITGA4, ITGB7, PDGFB, IL5, IL6, IGF2, and MMP9 relative to a baseline expression level in the human subject having IBD.

In some embodiments, a humanized anti-a4p7 antibody is administered to a human subject having IBD and an increased or decreased expression level before treatment, of at least one effector marker selected from the group consisting of NFKB2, CLDN1, CLDN2, CLDN3, OCLN, TJP1, ITGA4, ITGB7, PDGFB, IL5, IL6, IGF2, and MMP9 relative to a reference level in a human subject without IBD. In a further embodiment, the human subject having IBD has a reversion after treatment in the expression level of the at least one effector marker selected from the group consisting of NFKB2, CLDN1, CLDN2, CLDN3, OCLN, TJP1, ITGA4, ITGB7, PDGFB, IL5, IL6, IGF2, and MMP9 relative to a baseline expression level in the human subject having IBD. The IBD can be Crohn’s disease or ulcerative colitis. In a further embodiment, the Crohn’s disease is moderately to severely active Crohn’s disease. In a further embodiment, the ulcerative colitis is moderately to severely active ulcerative colitis.

In some embodiments, a humanized anti-a4p7 antibody is administered to a human subject having IBD and a decreased expression level before treatment of CLDN3, OCLN, and/or TJP1 relative to a reference level in a human subject without IBD. In a further embodiment, the human subject having IBD has a reversion after treatment in the expression level of CLDN3, OCLN, and/or TJP1 relative to a baseline expression level in the human subject having IBD. The IBD can be Crohn’s disease or ulcerative colitis. In a further embodiment, the Crohn’s disease is moderately to severely active Crohn’s disease. In a further embodiment, the ulcerative colitis is moderately to severely active ulcerative colitis.

In some embodiments, a humanized anti-a4p7 antibody is administered to a human subject having IBD and an increased or decreased expression level before treatment of at least one effector marker selected from the group consisting of IGF2, PDGFB, IL6, NFKB2, MMP9, CLDN1, and CLDN2 relative to a reference level in a human subject without IBD. In a further embodiment, the human subject having IBD has a reversion after treatment in the expression level of the at least one effector marker selected from the group consisting of IGF2, PDGFB, IL6, NFKB2, MMP9, CLDN1, and CLDN2 relative to a baseline expression level in the human subject having IBD. The IBD can be Crohn’s disease or ulcerative colitis. In a further embodiment, the Crohn’s disease is moderately to severely active Crohn’s disease. In a further embodiment, the ulcerative colitis is moderately to severely active ulcerative colitis.

In some embodiments, a pan-Janus kinase (JAK) inhibitor is administered to a human subject having IBD and an increased or decreased expression level of at least one effector marker selected from the group consisting of WNT1, PPARG, IL22, TLR4, MICB, IL13, TGFBR2, EGF, COL 1 Al, COL1 A2, and MSH2 relative to a reference level in a human subject without IBD. In certain embodiments, administration of a pan-Janus kinase (JAK) inhibitor results in a reversion in the expression level of the at least one effector marker selected from the group consisting of WNT1, PPARG, IL22, TLR4, MICB, IL13, TGFBR2, EGF, COL1 Al, COL1 A2, and MSH2 relative to a baseline expression level in the human subject having IBD.

In some embodiments, a humanized anti-a4p7 antibody is administered to a human subject having IBD and an increased or decreased expression level before treatment of at least one effector marker selected from the group consisting of WNT1, PPARG, IL22, TLR4, MICB, IL13, TGFBR2, EGF, COL1 Al, COL1 A2, and MSH2 relative to a reference level in a human subject without IBD. In a further embodiment, the human subject having IBD has a reversion after treatment in the expression level of the at least one effector marker selected from the group consisting of WNT1, PPARG, IL22, TLR4, MICB, IL13, TGFBR2, EGF, C0L1 Al, COL1 A2, and MSH2 relative to a baseline expression level in the human subject having IBD. The IBD can be Crohn’s disease or ulcerative colitis. In a further embodiment, the Crohn’s disease is moderately to severely active Crohn’s disease. In a further embodiment, the ulcerative colitis is moderately to severely active ulcerative colitis.

In some embodiments, a humanized anti-a4p7 antibody is administered to a human subject having IBD and an increased expression level before treatment of C0L1A1 and/or COL1 A2 relative to a reference level in a human subject without IBD. In a further embodiment, the human subject having IBD has a reversion after treatment in the expression level of COL1 Al and/or COL1 A2 relative to a reference level in a human subject without IBD. The IBD can be Crohn’s disease or ulcerative colitis. In a further embodiment, the Crohn’s disease is moderately to severely active Crohn’s disease. In a further embodiment, the ulcerative colitis is moderately to severely active ulcerative colitis.

In some embodiments, a humanized anti-a4p7 antibody is administered to a human subject having IBD and an increased or decreased expression level before treatment of at least one effector marker selected from the group consisting of MSH2, PPARG, and EGF relative to a reference level in a human subject without IBD. In a further embodiment, the human subject having IBD has a reversion after treatment in the expression level of the at least one effector marker selected from the group consisting of MSH2, PPARG, and EGF relative to a baseline expression level in the human subject having IBD. The IBD can be Crohn’s disease or ulcerative colitis. In a further embodiment, the Crohn’s disease is moderately to severely active Crohn’s disease. In a further embodiment, the ulcerative colitis is moderately to severely active ulcerative colitis.

In some embodiments, a humanized anti-a4p7 antibody is administered to a human subject having IBD and an increased or decreased expression level before treatment of at least one effector marker selected from the group consisting of TLR4, MICB, IL22, and TGFBR2 relative to a reference level in a human subject without IBD. In a further embodiment, the human subject having IBD has a reversion after treatment in the expression level of the at least one effector marker selected from the group consisting of TLR4, MICB, IL22, and TGFBR2 relative to a baseline expression level in the human subject having IBD. The IBD can be Crohn’s disease or ulcerative colitis. In a further embodiment, the Crohn’s disease is moderately to severely active Crohn’s disease. In a further embodiment, the ulcerative colitis is moderately to severely active ulcerative colitis. The following examples exemplify improved methods and compositions described herein. The following examples are offered for illustrative purposes only, and are not intended to limit the scope of the present invention in any way.

EXAMPLES

EXAMPLE 1. Unveiling the molecular mechanisms of the combination of vedolizumab with JAK inhibitors in Crohn’s Disease through a systems biology and artificial intelligence-based approach

Background

Clinical evidence supports the combination therapy of vedolizumab (a gut-selective, anti -lymphocyte trafficking drug inhibiting a4p7-integrin) with JAK 1, 2, 3 inhibitors (JAKi) for Inflammatory Bowel Disease. However, the mechanistic explanation for the improved outcomes in Ulcerative Colitis patients treated with vedolizumab plus JAKi remains unknown and even more in Crohn's Disease (CD).

The incidence and prevalence of IBD have shown a significant increase globally over recent decades, now affecting up to 1 in 200 individuals in Western countries. In Spain, a recent a population-based nationwide study yielded an overall incidence of 7.5 cases/100,000 person-years for CD. Understanding the etiology of CD necessitates recognizing its complex, multifactorial nature. The interplay among the three main factors - genetic susceptibility, gut immune response, and microbiota dysbiosis is influenced by environmental triggers, ultimately contributing to disease development. In this setting, the development of CD is proposed to occur in three stages: (1) penetration of bacteria and antigenic material into the bowel wall, facilitated by environmental factors and disruption of the intestinal barrier; (2) altered acute inflammatory response resulting from defective secretion of pro-inflammatory cytokines by macrophages and impaired clearance of foreign material; and (3) chronic inflammation and adaptive autoimmune response mediated by Thl and Thl7 cells, triggered by the previous stages.

Therapeutic strategies for CD have evolved over time, with both non-biological and biological therapies being employed. However, in certain instances, these approaches have shown limited efficacy, loss of response, and adverse events. Certain advanced treatments report clinical remission rates at 1 year up to a maximum of 50%, suggesting that a therapeutic ceiling has been reached using single agents. The efficacy plateau observed with advanced monotherapies underscores the growing interest in innovative treatment modalities, such as combination therapies. Taken together, clinical and preclinical data suggest that combination approach might potentially break through an efficacy ceiling, although data from controlled studies are still scarce.

In this study, an unbiased in silico systems biology and artificial intelligence approach was used to gain a holistic view of the mechanisms that may underpin the potential benefits of the mentioned combination therapy in the treatment of CD.

• Although more evidence is needed, literature suggests that combination therapy with vedolizumab (VDZ) and Janus Kinase (JAK) 1,2,3 inhibitors (JAKi) may be useful for Inflammatory Bowel Disease (Llano et al. Crohns Colitis 360. 2021 Jun 9;3(3) and Kolar et al. Inflammatory Bowel Diseases, Volume 29, Issue Supplement !, February 2023, Page S78).

• The mechanism of action (MoA) for the improved outcomes in Ulcerative Colitis patients after VDZ+JAKi remains unknown, and even more in Crohn’s Disease (CD).

• The combination of VDZ, a lymphocyte trafficking inhibitor, with JAKi, distinguishes itself from other treatment combinations by addressing distinct stages and immune response locations of CD. Specifically, this combination targets both the initial, milder phase characterized by inflammatory conditions as well as the more advanced phase marked by complications, thus potentially offering benefits to CD patients.

• This study used an in silico systems biology and artificial intelligence approach to gain a holistic view of the MoA underpinning the benefits of the combination therapy in the treatment of CD.

Methods

The drugs under study - vedolizumab and JAKi (tofacitinib, upadacitinib, and filgotinib) - and CD were characterized at protein level by compiling data from an extensive literature search. Through it, human protein interaction networks focused on CD protein effectors were generated and used to construct mathematical models using Therapeutic Performance Mapping System (TPMS) technology (Anaxomics Biotech, Barcelona, Spain; Jorba et al. PLoS One. 2020;15(2):e0228926). Sampling-based methods were employed to assess the impact on CD of the combined therapy vedolizumab plus JAKi and to describe underlying molecular mechanisms. Data compilation

Molecular characterization of CD was performed through a structured review of the literature. This study identified 4 pathophysiological processes (motives) and 148 nonduplicated proteins (effectors) involved in CD. Condition effectors are defined as protein/gene candidates whose activity (or lack thereof) is functionally associated with the development of the disease.

• Ml : Intestinal barrier disruption (50 effectors)

• M2: Reduced innate immune response (24 effectors)

• M3: Chronic inflammation & Thl/Thl7 adaptive immune response (45 effectors)

• M4: Tissue remodeling (59 effectors)

Innate immune response dysfunction, represented by M2, was not taken into account for modelling since it occurs before administering VDZ+JAKi. Nevertheless, molecular players involved in innate immunity are included in other motives of the disease.

Mathematical models generation

Computer simulation of CD pathophysiology was then performed by generating mathematical models for (1) CD patients treated with VDZ, (2) CD patients treated with JAKi, and (3) CD patients treated with VDZ+JAKi.

Mathematical models analysis

The impact of the therapies over CD was the quantified through Therapeutic Performance Mapping System (TPMS) technology:

• fSignal: signal value of all response proteins (CD effectors) after stimulating the model (mean value).

• % effectors reverted by the drug over the disease pathophysiology. It measures if the causative effect of a protein effector characterized for CD is reverted in the treatment models.

TPMS mathematical models were built over the Human Protein Network, as previously described (Jorba et al. PLoS One 2020;15(2):e0228926; and Segu-Verges et al. Arthritis Res Ther 2021;23(l): 126). This protein-protein interaction human network incorporates the available relationships between proteins from a regularly updated in-house database drawn from public sources.

In order to transform this network map into mathematical models capable of both reproducing existing knowledge and predicting new data, a collection of known input (drugs)-output (clinical conditions) physiological relationships was used as training data, or “training set”, to train the models. This training set was constructed using a compendium of biological and clinical databases through text mining techniques and manual review of the information to obtain biological and pharmacological input-output relationships. Every mathematical model created must satisfy this training set, as previously detailed (Jorba et al. PLoS One 2020;15(2):e0228926; Segu-Verges et al. Arthritis Res Ther 2021;23(l): 126; and Segu-Verges et al. PLoS One 2023;18(2):e0280677).

TPMS technology was used to simulate in silico clinical responses of CD pathophysiology, exploring all plausible relationships between an input or stimulus (drug targets) and an output or response (CD motives). This type of analysis elucidates the molecular mechanisms by which drug-pathophysiology relationships function and how the treatment can modulate the pathophysiology. Specifically, three models were developed: one for VDZ treatment, one for JAK inhibitors treatment, and one for the combined treatment. The latter included integrin a4 and integrin P7 as VDZ targets, and JAKI, JAK2, JAK3, and TYK2 as JAKi targets. Due to limitations in the modelling system, MADCAM1 was added as a pseudotarget of VDZ to reduce the distance from the stimulus to the response, thereby enabling an adequate reproduction of the impact of integrin modulation on the pathophysiology of CD.

Sampling methods were used to assess the impact of VDZ, JAKi, and VDZ plus JAKi on CD-simulated patients through mathematical models. The resulting models, comprising a set of solutions, were able to describe all possible mechanistic relationships, or MoAs, between an input and an output.

“Predicted protein activity” was defined as the value between 1 and -1 (indicating activation and inhibition, respectively) that each protein in the MoA subnetwork achieves. To elucidate the impact of the drugs on each pathophysiological motive featuring the condition, the Full T Signal (fSignal) and the percentage of effectors reverted by the drug over the disease pathophysiology was analyzed, as previously described in detail (Jorba et al. PLoS One 2020;15(2):e0228926; Segu-Verges et al. PLoS One 2023;18(2):e0280677). The fSignal retrieves the signal values of all response proteins (CD effectors) after stimulating the model, that is, propagating a signal through the drugs’ targets over the network, and computes the mean value. This measure indicates how much signal generated by the stimulus (drug) reaches the response set (disease) as a mean. The percentage of reversed effectors measures the ability of each treatment to reverse the protein activity status in these pathological mechanisms, according to the molecular characterization.

“Reversed effectors” are defined as the proteins that are activated/inactivated by the drug in the opposite activation state than in the CD molecular characterization, with at least |0.11 protein activity. The percentage of reversed proteins in each motive was calculated to gain a quantitative point of view of the stimulus’s impact. These proteins were considered to evaluate the contribution of each drug to the combination efficacy. However, it is not appropriate to interpret these values as a quantitative measure of efficacy. Only model solutions complying with the training set biological restrictions, with a cross-validated accuracy greater than 90% (percentage of compliance of all drug-clinical condition relationships included in the training set), were considered as valid.

Accuracy of the final MoA models was calculated as the mean of the accuracies of all considered solutions. Once the individual mechanisms for each drug were identified, they were jointly evaluated to identify the potential of the combination in the treatment of CD. The percentage of reversed effectors measures the ability of each treatment to reverse protein activity status in the pathological mechanisms, according to the molecular characterization.

Results

Mechanistic model ofVDZ plus JAKi on CD through TPMS

A comprehensive review of the literature identified four hallmark processes, so-called motives, which are as follows: 1) intestinal barrier disruption (motive 1, Ml); 2) altered innate immune response (motive 2, M2); 3) chronic inflammation and predominant Thl/Thl7 adaptive immune response (motive 3, M3); and 4) tissue remodelling (motive 4, M4). The molecular and functional characterization of these motives yielded 50, 24, 45, and 59 proteins (effectors) associated with Ml, M2, M3, and M4, respectively. The total number of unique effector proteins was 148.

Defective innate immunity (M2) represents an early pathophysiological event that triggers chronic inflammatory responses, eventually leading to CD establishment. However, VDZ and JAKi are approved for the treatment of moderate to severe CD, when the disease is fully established, not during the time-period when M2 occurs. In addition, a preliminary evaluation of the models for each drug showed that no signal reached M2 effectors, having no clear impact on this motive (see FIG. 2). Considering these factors, M2 was not included in the model, as it realistically would not be relevant when patients are administered the combination of VDZ plus JAKi. Nevertheless, exclusion of M2 does not imply the complete exclusion of the innate immune response from the modelling analysis, as molecular players involved in innate immunity is also represented in our modelling in motives Ml, M3, and M4. More specifically, 14 out of the 50 effectors found in Ml, 21 out of the 45 effectors in M3, and 17 out of the 59 effectors in M4 involved in the innate immune response were identified.

Effects of the combination therapy in the treatment of CD

The predicted MoAs for each drug were jointly evaluated, aiming to elucidate the likely pharmacological mechanisms evoked by the combination therapy on CD. The impact of each treatment on the specific biological processes (motives) involved in this disease was assessed through two complementary parameters: 1) the percentage of altered effectors reversed by the drug, and 2) the intensity of the response measured using fSignal. These parameters provided an idea of the scope and the strength of the treatment’s impact on a specific biological process.

As shown in FIG. 1A and FIG. IB, at single treatment level, the fSignal was slightly higher for VDZ than for JAKi; however, both drugs modulated a similar number of protein effectors on disease pathophysiology. More specifically, VDZ was able to revert more protein effectors in Ml and M3, while JAKi were able to revert a higher percentage of proteins effectors in M4. Therefore, the combination of VDZ plus JAKi, unlike other combined treatments, would act at different stages of the disease, thus offering potential greater benefits to CD patients.

The combination of vedolizumab and JAKi induced a high degree of reversion of the effectors altered in CD (54.8%) than any of the therapies individually. Specifically, the combined therapy reversed the activity of more effectors than the individual treatments in Ml and M4 and equaled the number of effectors reversed by VDZ in M3. FIG. 1C shows that both VDZ and JAKi converged in the modulation of 45 proteins, which represents 36% of the total CD effectors. Moreover, each drug provided complementary mechanisms for the improvement of CD pathophysiology, since 10% (13 proteins) of the reversed effectors were due to the exclusive effects of VDZ, while 9% (11 proteins) were reverted by the exclusive effects of JAKi.

Combined vedolizumab plus JAKi therapy mainly modulated M3, where both drugs mostly converge although by distinct mechanisms, thus providing additional individual benefits. The combination therapy reverted a great percentage of M3 effectors, for example, CCR9 and FASLG which were increased in CD, were reverted (or downregulated). However, the complementary mechanisms of these drugs are evident across all motifs, with a pronounced impact observed on Ml (intestinal barrier disruption) and M4 (tissue remodeling). In Ml, vedolizumab is the one that would provide more complementary mechanisms apart from those that already converge in both drugs. Regarding M4, it seems that the main contribution comes from complementarity pathways of the different mechanisms of action of each individual drug. This differential targeting of Ml and M4, along with the convergence on M3, potentially amplifies therapeutic coverage and implies synergistic and additive effects between VDZ and JAKi.

• fSignal was slightly higher for VDZ than for JAKi (FIG. 1A); ; however, both drugs modulated a similar number of protein effectors on disease pathophysiology.

• VDZ was able to revert more protein effectors in Ml and M3, while JAKi were able to revert a higher percentage of proteins effectors in M4

• VDZ+JAKi had a higher fSignal than VDZ and JAKi alone (except in M3) (FIG. 1A).

• The combined therapy reversed more CD effectors than the drugs alone in all motives, except in M3 where the combination reverted more effectors than JAKi but the same as VDZ alone (FIG. IB and FIG. 1C)

• However, complementary mechanisms provided by each individual drug are observed in all the motives but mainly M4 (tissue remodeling) and Ml (intestinal barrier disruption) (FIG. IB and FIG. 1C).

Contribution of each drug to the combined therapy

The contribution of each drug to the overall MoAs was evaluated focusing on complementary mechanisms and convergent effectors. Complementary mechanisms describe the pathways characteristic of the individual drugs that are maintained in the combination treatment.

Results are summarized in Table 1, Table 2, Table 4A, Table 4B, and Table 4C.

Specifically, VDZ exclusively reverted 13 CD effectors: 6 from Ml (NFKB2, CLDN1, CLDN2, CLDN3, OCLN and TJP1), 3 from M3 (ITGA4 and ITGB7 (drug targets) and NFKB2), and 6 from M4 (PDGFB, IL5, IL6, IGF2, MMP9 and NFKB2). Likewise, JAKi exclusively reverted 11 CD effectors: 3 from Ml (WNT1, PPARG and IL22), 3 from M3 (TLR4, MICB and IL22), and 7 from M4 (IL13, TGFBR2, EGF, C0L1A1, C0L1A2, MSH2 and WNT1). Thus, the combination therapy provides complementary pathways from each drug that promote mainly the reversion of Ml and M4 motives. Table 1: Complementary mechanisms provided by the individual drugs to the combined treatment. Table 2: Complementary mechanisms

COMPLEMENTARY MECHANISMS: describe the pathways through which the individual drugs in the combination contribute to the overall mechanism of action.

Convergent effectors are defined as CD protein effectors reversed by both VDZ and JAKi, and predicted to be greatly reversed when both drugs are combined. 10 proteins were identified as convergent effectors (TCF4, IFNG, FASLG, CCR9, MMP1, CDH1, NFKB1, PLA2G1B, MAPK3 and MAPK1). These effectors are almost equally involved in the three evaluated motives of CD: 5 effectors from intestinal barrier disruption (Ml), 4 from chronic inflammation and Thl/Thl7 adaptive immune response (M3), and 4 from tissue remodelling (M4). Results are shown in Table 3 and summarized in Table 4A, Table 4B, and Table 4C.

Table 3: Convergent mechanisms from the individual drugs in the combined treatment.

CONVERGENT EFFECTORS: CD protein effectors that are individually reversed by VDZ and JAKi but predicted to be greatly reverted when combined.

Table 4A: Summary of identified effectors from Motive 1 (Ml) - Intestinal Barrier Disruption (molecules primarily involved in maintaining or disrupting the structural integrity and function of the intestinal barrier). Table 4B: Summary of identified effectors from Motive 3 (M3) - Chronic Inflammation and Thl/Thl7 Adaptive Immune Response (molecules are associated with immune cell signaling and inflammatory pathways)

Table 4C: Summary of identified effectors from Motive 4 (M4) - Tissue Remodeling (molecules implicated in fibrosis, extracellular matrix remodeling, and structural chanes in the intestinal tissue). Mechanism of action (Mo A) of the combined therapy on CD

The combined therapy seems to affect several processes implicated in motives Ml, M3, and M4. Specifically, in Ml, VDZ appears to mitigate intestinal barrier dysfunction by inhibiting TNF-a (inhibition of tight junction (TJ) dysfunction), while JAKi would act by decreasing metalloprotease activity, and both VDZ plus JAKi, when used together, by activation of CADH1 (restoration of TJ) and reduction of IFNG expression. Interestingly, VDZ might have an additive effect on JAKi’s mechanism of action reducing the production of the pro-inflammatory agents TNF-a, IFNG, IL 17, and IL6, which normally promote the activation of their associated JAKs. Moreover, the damage to the intestinal architecture caused by local inflammation might be reversed by VDZ’s inhibition of TNF-a and NF-kB pathway, as well as by the inhibition of IFNG by both drugs.

The combined treatment may impact M3 through restriction of T cell gut-homing, diminution of Thl cell activity in CD-affected tissue, and blockage of Thl7 response. VDZ- mediated reduction in TNF-a expression might decrease NFKB1 activity and therefore FASLG expression. Regarding the diminution of Thl cell activity in CD-affected tissue, JAKi’s effect in combination with VDZ-mediated reduction in TNF-a expression in the lamina propria could in turn further decrease Thl activity in CD. The predicted MoA suggests that Thl7 response might be blocked through the reduction of IL6, IFNG, and IL17 intestinal production by VDZ, resulting in reduced migration of effector T lymphocytes to the inflamed gut.

Finally, regarding M4, the combination therapy might reduce intestinal fibrosis by blocking extracellular matrix remodelling. This effect is achieved through the reduction of collagen synthesis (by VDZ through decrease of TNFA (TNF-alpha), IL17 and IL6 expression) and the inhibition of MMPs’ extracellular matrix (ECM) remodelling function (by JAKi via NF-KB p65 nuclear translocation inhibition and TCF4 expression reduction). Moreover, VDZ plus JAKi therapy might promote improvement in CD patients by decreasing stricture formation through the modulation of TNFA, IL 17, IL6, NF-kB, and MMPs expression.

The main identified pathways that seem to be predominant in the improvement of these characteristic processes of CD are simplified and represented in FIGS. 3A-3D and FIG. 4

I. Reduction of the intestinal barrier dysfunction

TNFA modulates the transcription of tight-junction proteins and induces apoptosis of enterocytes leading to altered permeability, while E-cadherin (CADH1), which is downregulated in CD, is essential to maintain cell-cell adhesion and epithelial integrity expression. The cytokine interferon y (IFNG), similar to TNFA, enhances paracellular permeability. Other important effectors that disrupt the intestinal barrier in CD are metalloproteinases (MMP1, MMP3 and MMP13). Vedolizumab seems to reduce intestinal barrier dysfunction by inhibition of TNFA (inhibition of TJ dysfunction) (FIG. 3A), JAKi by decreasing metalloprotease activity (FIG. 3C), and both, VDZ+JAKi, by activation of CADH1 (restoration of TJ) and reduction of IFNG expression (FIG. 3A and FIG. 3B). VDZ might have an additive effect on JAKi’s mechanism of action due to its reducing effect in the production of the pro-inflammatory mediators TNFA, IFNG, IL 17 and IL6, which normally promote activation of their associated JAKs (FIG. 3A).

II. Inhibition of the inflammatory damage in the intestinal structure

IFNG and TNFA are central mediators of intestinal inflammation in CD. Together, NF-kB strongly influences the course of mucosal inflammation promoting the expression of various pro-inflammatory genes. Thus, the damage in the intestinal structure caused by local inflammation might be reverted by the inhibition of TNFA and NF-kB (FIG. 3 A) by VDZ and by the inhibition of IFNG by both drugs (FIGS. 3A-3D).

Ill Restriction of T cell gut-homing

Theoretically, VDZ-provoked blocking of integrin a4p7 binding to MADCAM1 prevents T cells recruitment to the intestine, leading to chronic inflammation improvement. Very likely this might occur via the reduction of TNFA, IFNG, IL6 and IL17 in the gut. Moreover, as MADCAM1 is able to upregulate CCR9 under costimulatory conditions, its blockage by VDZ diminishes CCR9 expression in T cells surface reducing even more T cells recruitment (FIG. 3A). Endothelial tumour necrosis factor ligand superfamily member 6 (FASLG) expression also has a role in the access of lymphocytes to the intestinal mucosa; it is overexpressed in CD. FIG. 3A shows how VDZ-mediated TNFA reduced expression might decrease NFKB1 activity and therefore FASLG expression.

IV. Diminution of Thl cell activity in CD affected tissue

Crohn’s disease is characterized by an IL12-driven T-helper 1 (Thl)-cell mediated immune response. Biologically active IL 12 comes from IL12A and IL12B genes and promotes IFNG production. IL12 also enables Thl response self-maintenance through intracellular JAKI -activated TBX21 protein. IL23 and TNFA can also support Thl cell differentiation. According to the predicted mechanism of action (FIG. 3D) JAKi could reduce Thl cell activity by inhibiting IL12A and IL12B, leading to reduced IFNG. Additionally, JAKi treatment could affect Thl activity through the decrease of TBX21 activity. Thus, JAKi’s effect in combination with VDZ-mediated TNFA reduced expression in the lamina propria (FIG. 3) could enhance the diminution of Thl activity in CD. V. Blockage of Thl 7 response

T helper type 17 (Thl7) cells also play a pivotal role in Crohn’s disease. Thl7 differentiation occurs under the stimulation of JAK/STAT pathway mainly by IL6 and IL23. Thl7 immune response is mainly driven by IL17, but also by IFNG. Hence, the predicted MoA suggests that Thl7 response might be blocked through the reduction of IL6, IFNG and IL 17 intestinal production by VDZ due to a lesser migration of effector T lymphocytes to the inflamed gut (FIG. 3A).

VI. Reduction of intestinal fibrosis through the blockage of extracellular matrix remodeling

When extracellular matrix (ECM)/collagen production is increased and degradation is surpassed, intestinal fibrosis occurs in CD patients. This is mainly mediated by matrix metalloproteinases (MMPs) and metalloproteinase inhibitors (TIMPs). The predicted mechanism of action suggests that VDZ might decrease TNFA, IL 17 and IL6 expression what leads to a reduce collagen synthesis (FIG. 3 A); while JAKi, probably via NF-KB p65 nuclear translocation inhibition and TCF4 expression reduction, might be able to inhibit MMPs expression, including MMP9, which is the predominant upregulated remodeling protease in inflamed intestinal tissue (FIG. 3B and FIG. 3C). Thus, the combinatory therapy might reduce intestinal fibrosis through the reduction of collagen synthesis and MMPs’ ECM remodeling function.

VII. Diminution of stricture formation

ECM remodeling and collagen accumulation together with NF-kB -promoted mesenchymal cell contraction might conclude in stricture formation. Thus, VDZ plus JAKi therapy might promote CD patients’ improvement by decreasing stricture formation through TNFA, IL17, IL6, NF-kB and MMPs expression modulation.

Mathematical modelling corroboration through bioflags analysis

Genes or proteins altered by the effects of the drug on its targets and/or off-targets, at the downstream level, were referred to as “bioflags”. The bioflags described in the literature for VDZ’s and JAKi’s actions on CD were evaluated in the single and combined treatment generated models to corroborate the simulated drug effects. Results of bioflag analysis are summarized in Table 5A and Table 5B. From the 21 bioflags described for VDZ and included in both mathematical models (single drug and combined), 20 were optimally modulated to promote a healthy status in CD (Table 5A). Likewise, from the 14 bioflags described for JAKi and included in both mathematical models, 11 were correctly modulated in CD (Table 5B). Only bioflags from the characterization of the drugs with higher evidence of modulation, other than just high-throughput data, were evaluated in our analysis. Overall, the models accurately represented the effects of VDZ and JAKi treatment observed and reported in patients according to scientific literature.

Table 5A: Corroboration of the VDZ bioflags’ activation status after treatment with individual and combined treatment.

Table 5B: Corroboration of the JAKi bioflags’ activation status after treatment with individual and combined treatment.

Conclusion

CD presents a formidable challenge in the field of gastroenterology, characterized by its chronic, inflammatory, and potentially life-threatening course. Despite significant advancements, the etiology of CD remains elusive, its incidence is increasing worldwide, and therapeutic options often fall short in providing long-term remission and relief for afflicted patients.

The constructed in silico models revealed that the combined vedolizumab plus JAKi therapy could modulate a high array of effectors altered in CD, more than any of the therapies alone, by enhancing the effects of the individual drugs. Furthermore, both drugs converge in the modulation of several effectors through different pathways, which could enhance their action and reinforce the beneficial effects observed in IBD patients treated with this combined therapy. The results report the superior effect of vedolizumab in the early stages of the disease (6 over 3 effectors), which reinforces the rationale of an earlier use of vedolizumab to slow down the evolutionary process of the disease, according to purely immunological criteria. Moreover, the combined treatment distinguishes itself from others, by addressing distinct stages and immune sites on CD. Specifically, this combination targets both the initial, milder phase characterized by inflammatory conditions as well as the more advanced phase marked by complications, thus potentially offering potential benefits to CD patients.

By leveraging the complementary and convergent effects of both drugs, the combined therapy emerged as a compelling therapeutic strategy, since it holds the potential to achieve more comprehensive and sustained disease control in CD patients, particularly those with refractory or severe disease phenotypes. While VDZ acts by blocking immune cell trafficking to the intestinal submucosa, JAKi act locally at the inflammatory site.

As an example, the model predicted that VDZ+JAKi could more effectively revert E- cadherin (CDH1), which is required for the integrity the intestinal epithelial lining. Therefore, the combination of VDZ plus JAKi, unlike other combined treatments, would act at two different stages of the disease, thus offering potential greater benefits to CD patients.

Indeed, the combination of vedolizumab plus JAKi allows modulation of greater proportion of CD effectors (54.8%) than either drug alone, potentially offering enhanced therapeutic benefits for patients. Both drugs overlap in the modulation of several effectors (36%), which could probably enhance their combined therapeutic effects and increase efficacy, as observed for UC patients. Additionally, each drug provides complementary mechanisms that improve CD pathophysiology. Thus, 10% of the reversed effectors are given by the effects of VDZ and 9% by JAKi. The combination mainly affects M3, where both drugs show higher coincidence, although mechanistically they are different, thus each providing complementary benefits. The combination can also impact on Ml and M4. Although the mechanisms of convergence are less than in M3, in M4 there is a greater contribution or individual complementarity of each drug. Regarding Ml, vedolizumab is the one that would provide more complementary mechanisms apart from those that already converge in both drugs.

By modulating a significantly higher percentage (54.8%) of effectors compared to individual drug treatments, combination therapy of VDZ and JAKi holds the promise of delivering greater therapeutic benefits to patients. Intriguingly, the convergence of effectors modulated by both VDZ and JAKi, accounting for 36% of the total. This overlap suggests a potential synergistic enhancement of their respective effects when used in tandem, mirroring the observed efficacy in UC patients who have undergone combination therapy (Kolar et al. Inflammatory Bowel Diseases 2023;29(Supplement_l):S78). Additionally, this drug combination might be beneficial for non-responder patients to current treatments for CD.

EXAMPLE 2. VARSITY Mucosa Transcriptomics: Analysis of VDZ & JAK markers for IBD

A large-scale transcriptomics analysis of tissue biopsies derived from moderate-to- severe UC patients treated with vedolizumab (VDZ) or adalimumab (ADA) as part of the VARSITY trial (Sands et al. N Engl J Med. 2019 Sep 26;38I(I3): 1215-1226) was performed. The aim of this study was to identify biomarkers of response to VDZ and ADA treatment. Bulk RNA sequencing of 954 biopsies collected using the flexible sigmoidoscopy from pre- treatment/baseline, week 14 and week 52. The dataset comprised complete longitudinal tissue sampling of n=151 non-responders, n=117 responders based on clinical remission at week 52. Methods

Study design and recruitment

VARSITY is a phase 3b, randomized, double-blind, double-dummy trial conducted between 2015 and 2019 across 34 different countries (Sands et al. N Engl J Med. 2019 Sep 26;381(13): 1215-1226). The primary objective was to evaluate and compare VDZ effectiveness and safety profiles versus ADA in individuals diagnosed with moderately to severely active ulcerative colitis. Participants were required to have a total Mayo score between 6 and 12 with a minimum subscore of 2 on the endoscopic component, and colonic involvement extending at least 15 cm. Individuals who had not previously used an anti-TNF treatment and who had shown inadequate response or loss of response to standard treatments were eligible to participate, with a limit placed on the enrollment of those who had ceased due to non-safety -related reasons. Participants were randomized centrally, with stratification based on prior TNF inhibitor use and concurrent oral corticosteroid use. Those in the vedolizumab group received 300 mg intravenous infusions on day 1 and at weeks 2, 6, 14, 22, 30, 38, and 46, along with placebo subcutaneous injections on day 1 (four injections), week 2 (two injections), and every two weeks until week 50. The adalimumab group received 40 mg subcutaneous injections on days 1 and 2 (either four injections on day 1 or two injections per day for two days), two injections at week 2, and 40 mg every two weeks until week 50, with placebo intravenous infusions on the same schedule as the vedolizumab group. During week 14, patients were evaluated whether response or not defined as a reduction in the partial Mayo score [stool frequency, rectal bleeding, and physician’s global assessment] of >2 points and of >25% from baseline, with an accompanying decrease in rectal bleeding subscore of >1 point or absolute rectal bleeding sub score of <1 point; and safety (as assessed by the incidence of adverse events). The primary outcome was achieving clinical remission at week 52, defined by a total Mayo score of <2 with no subscore exceeding 1. Secondary outcomes included endoscopic improvement, indicated by a Mayo endoscopic subscore of <1, and histologic remission defined by Robarts Histopathology Index score <3. Individuals who were defined as responder/non-responder at both week 14 and week 52 by the primary outcome were defined as complete responder (CR)/complete non-responder(NCR).

Bioinformatics preprocessing on RNA sample

In the VARSITY study, RNA-Seq quality control was performed and summarized with Omicsoft platform (Hu et al. Bioinformatics. 2012 Jul 15;28(14): 1933-4). 3’ adapters and low-quality reads were trimmed, and then aligned to the hg38 human reference genome, the latest version available at the time, using the Omicsoft Sequence Aligner (OSA) RNA-seq (Hu et al. Bioinformatics. 2012 Jul 15;28(14): 1933-4), and gene expression was quantified in the Omicsoft platform. Samples exhibiting poor overall quality or low alignment rates were excluded from further analysis. After quality control, genes with low expression (mean count < 2), low variation (standard deviation of count < 0.1), and those located on sex chromosomes were removed from downstream analyses. Following preprocessing and filtering, a total of 16,301 genes were retained for the VARSITY analyses.

Differential gene expression (DGE) analysis

Differential expression (DE) analysis of clinical remission responsiveness at week 52 in biopsy tissue samples from VARSITY subjects was performed using a multivariable negative binomial regression framework implemented in the DESeq26 package in R 4.2.1. Multiple testing correction was applied using the Benjamini -Hochberg false discovery rate (FDR) method. This multivariable analysis was conducted separately for VDZ and ADA at baseline, week 14, and week 52, with adjustments for age, gender, race, sampling country, and prior TNF-alpha status. Significant up-regulated differentially expressed genes (DEGs) were defined as those with an FDR p-value < 0.05 and a positive log2 fold change, indicating overexpression in non-responders, while significant down-regulated DEGs were defined as those with an FDR p-value < 0.05 and a negative log2 fold change. Similar analysis were later conducted for the complete R/NR, histologic remission and endoscopic improvement. Subsequently, gene set enrichment analysis? was performed on each set of results using the MsigDB Hallmarks databases, with genes pre-ranked by fold change. Enriched gene sets were considered significant if the FDR p-value was < 0.05.

Results

VARSITY longitudinal clinical study overview

In the VARSITY clinical study, a total of 771 patients were enrolled, with 385 patients randomized to receive vedolizumab (VDZ) treatment and 386 patients assigned to the adalimumab (ADA) group. For the tissue biopsy transcriptomic analysis, a total of 400 samples were included, comprising 214 samples from VDZ-treated patients and 186 samples from ADA-treated patients. At baseline, biopsy tissue samples were collected from 330 individuals for RNA-seq. At week 14, samples were collected from 316 patients, and at week 52, 254 samples were collected. The age of patients in the ADA group (mean = 40.79, sd = 13.3) was not significantly different from those in the VDZ group (mean = 39.5, sd = 13.7). In the VDZ group, 64% were male, compared to 54.8% in the ADA group, though this difference was not statistically significant. The majority of participants in both treatment groups were white (-89%). The BMI of patients in the VDZ group (mean = 24.3, sd = 4.8) was significantly lower than that in the ADA group (mean 25.5, sd = 5.5).

Based on clinical remission at the week 52 endpoint, 76 (47.2%) patients in the VDZ group were classified as responders, compared to 47 (39.2%) in the ADA group. When using the complete definition of response, a significantly higher proportion of patients in the VDZ group (38.9%) were responders compared to those in the ADA group (25%, p < 0.01).

Comparison of gene expression across patients revealed treatment response signatures

To uncover the molecular and cellular mechanisms influencing therapeutic outcomes, differential gene expression (DGE) analysis in VDZ and ADA treated patients was conducted at the three timepoints, focusing on clinical remission at Week 52. Differential gene expression (DGE) analysis on baseline samples demonstrated limited discriminative power when comparing non-responders and responders, resulting in a relatively small number of significant differentially expressed genes (DEGs) defined by an FDR p-value < 0.05.

Analysis of convergent protein effectors in Table 3

The convergent protein effectors identified in the in silico systems biology and artificial intelligence approach described in Table 1 were analyzed using VARSITY transcriptomics data from tissue biopsies derived from moderate-to-severe UC patients treated with VDZ or ADA. In particular, expression of CCR9, CDH1, FASLG, IFNG, MAPK1, MAPK3, MMP1, NFKB1, PLA2G1B, and TCF4 were analyzed at Week 0, Week 14, and Week 52 in VDZ and ADA treated patients from the VARSITY trial (see FIG. 5). These CD protein effectors were individually reversed by VDZ and JAKi but predicted to be greatly reverted when combined based on the in silico analysis described in Example 1. FIG. 5 indicates that MAPK3, CDH1, and MAPK1 are markers that are enriched in vedolizumab responders and are addressed by vedolizumab treatment. FIG. 5 further indicates that TCF4, NFKB1, MMP1, and IFNG are markers that are enriched in vedolizumab non-responders and may benefit from combination therapy. Analysis of complementary protein effectors exclusively reverted by VDZ in Table 2

The complementary protein effectors identified in the in silico systems biology and artificial intelligence approach described in Table 2 to be exclusively reverted by VDZ were analyzed using VARSITY transcriptomics data from tissue biopsies derived from moderate- to-severe UC patients treated with VDZ or ADA. In particular, expression of TJP 1, OCLN, CLDN3, IGF2, PDGFB, IL6, NFKB2, MMP9, CLDN1, IL5, ITGA4, and ITGB7 were analyzed at Week 0, Week 14, and Week 52 in VDZ and ADA treated patients from the VARSITY trial (see FIG. 6). These CD protein effectors were individually reversed by VDZ based on the in silico analysis described in Example 1.

FIG. 6 indicates that TJP1, OCLN, and CLDN3 are markers that are enriched in vedolizumab responders and are addressed by vedolizumab treatment. FIG. 6 further indicates that IGF2, PDGFB, IL6, NFKB2, MMP9, CLDN1, and CLDN2 are markers that are enriched in vedolizumab non-responders and may benefit from combination therapy. Analysis of complementary protein effectors exclusively reverted by JAKi in Table 2

The complementary protein effectors identified in the in silico systems biology and artificial intelligence approach described in Table 2 to be exclusively reverted by JAKi were analyzed using VARSITY transcriptomics data from tissue biopsies derived from moderate- to-severe UC patients treated with VDZ or ADA. In particular, expression of WNT1, TLR4, MICB, IL22, TGFBR2, COL 1 Al, COL1A2, MSH2, PPARG, and EGF were analyzed at Week 0, Week 14, and Week 52 in VDZ and ADA treated patients from the VARSITY trial (see FIG. 7). These CD protein effectors were individually reversed by JAKi based on the in silico analysis described in Example 1.

FIG. 7 indicates that MSH2, PPARG, and EGF are markers that may be associated with an additive or synergistic effect following combination therapy. FIG. 7 further indicates that TLR4, MICB, IL22, TGFBR2, COL1 A2, and COL1 Al are markers that are enriched in vedolizumab non-responders and may benefit from combination therapy.

SEQUENCE TABLE

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims. The contents of all references, patents and published patent applications cited throughout this application are incorporated herein by reference.

Claims

CLAIMS What is claimed:

1. A method of treating a human subject having an inflammatory bowel disease (IBD), said method comprising administering a humanized anti-a4p7 antibody and administering a pan-Janus kinase (JAK) inhibitor to the human subject, wherein the human subject having IBD has an increased expression level before treatment, of at least one effector marker selected from the group consisting of CCR9, FASLG, IFNG, MAPK1, MAPK3, MMP1, NFKB1, PLA2G1B, and TCF4 relative to a reference level in a human subject without IBD, and/or wherein the human subject having IBD has a decreased expression level after treatment, of the at least one effector marker selected from the group consisting of CCR9, FASLG, IFNG, MAPK1, MAPK3, MMP1, NFKB1, PLA2G1B, and TCF4 relative to a baseline expression level in the human subject having IBD, and wherein the humanized anti-a4p7 antibody is an IgGl antibody and comprises a heavy chain variable region comprising a CDR3 domain as set forth in SEQ ID NO: 4, a CDR2 domain as set forth in SEQ ID NO: 3, and a CDR1 domain as set forth in SEQ ID NO: 2; and comprises a light chain variable region comprising a CDR3 domain as set forth in SEQ ID NO: 8, a CDR2 domain as set forth in SEQ ID NO: 7, and a CDR1 domain as set forth in SEQ ID NO: 6.

2. A method of treating a human subject having an inflammatory bowel disease (IBD), said method comprising administering a humanized anti-a4p7 antibody and administering a pan-Janus kinase (JAK) inhibitor to the human subject, wherein the human subject having IBD has a decreased expression level before treatment, of CDH1 relative to a reference level in a human subject without IBD, and/or wherein the human subject having IBD has an increased expression level after treatment, of CDH1 relative to a baseline expression level in the human subject having IBD, and wherein the humanized anti-a4p7 antibody is an IgGl antibody and comprises a heavy chain variable region comprising a CDR3 domain as set forth in SEQ ID NO: 4, a CDR2 domain as set forth in SEQ ID NO: 3, and a CDR1 domain as set forth in SEQ ID NO: 2; and comprises a light chain variable region comprising a CDR3 domain as set forth in SEQ ID NO: 8, a CDR2 domain as set forth in SEQ ID NO: 7, and a CDR1 domain as set forth in SEQ ID NO: 6.

3. A method of treating a human subject having an inflammatory bowel disease (IBD), said method comprising administering a humanized anti-a4p7 antibody and administering a pan-Janus kinase (JAK) inhibitor to the human subject, wherein the human subject having IBD has an increased expression level before treatment, of MAPK1 and/or MAPK3 relative to a reference level in a human subject without IBD, and/or wherein the human subject having IBD has a decreased expression level after treatment, of MAPK1 and/or MAPK3 relative to a baseline expression level in the human subject having IBD, and wherein the humanized anti-a4p7 antibody is an IgGl antibody and comprises a heavy chain variable region comprising a CDR3 domain as set forth in SEQ ID NO: 4, a CDR2 domain as set forth in SEQ ID NO: 3, and a CDR1 domain as set forth in SEQ ID NO: 2; and comprises a light chain variable region comprising a CDR3 domain as set forth in SEQ ID NO: 8, a CDR2 domain as set forth in SEQ ID NO: 7, and a CDR1 domain as set forth in SEQ ID NO: 6.

4. A method of treating a human subject having an inflammatory bowel disease (IBD), said method comprising administering a humanized anti-a4p7 antibody and administering a pan-Janus kinase (JAK) inhibitor to the human subject, wherein the human subject having IBD has an increased expression level before treatment of at least one effector marker selected from the group consisting of TCF4, NFKB1, MMP1, and IFNG relative to a reference level in a human subject without IBD, and/or wherein the human subject having IBD has a decreased expression level after treatment of the at least one effector marker selected from the group consisting of TCF4, NFKB1, MMP1, and IFNG relative to a baseline expression level in the human subject having IBD, and wherein the humanized anti-a4p7 antibody is an IgGl antibody and comprises a heavy chain variable region comprising a CDR3 domain as set forth in SEQ ID NO: 4, a CDR2 domain as set forth in SEQ ID NO: 3, and a CDR1 domain as set forth in SEQ ID NO: 2; and comprises a light chain variable region comprising a CDR3 domain as set forth in SEQ ID NO: 8, a CDR2 domain as set forth in SEQ ID NO: 7, and a CDR1 domain as set forth in SEQ ID NO: 6.

5. The method of any one of claims 1-4, wherein the IBD is Crohn’s disease.

6. The method of claim 5, wherein the Crohn’s disease is moderately to severely active Crohn’s disease.

7. The method of any one of the preceding claims, wherein the human subject has active inflammation on ileocolonoscopy.

8. The method of any one of the preceding claims, wherein the human subject is biologic-naive.

9. The method of any one of claims 1-7, wherein the human subject had a lack of an adequate response with, lost response to, or was intolerant to treatment with at least one of an immunomodulator, a tumor necrosis factor-alpha antagonist or combinations thereof.

10. The method of any one of the preceding claims, wherein the human subject is intravenously administered a first dose of 300 mg of the humanized anti -01407 antibody at week 0, followed by a second dose of 300 mg of the humanized anti-a407 antibody at week 2.

11. The method of claim 10, further comprising intravenously administering a third dose of 300 mg of the humanized anti -0.407 antibody at week 6, followed by a 300 mg dose of the humanized anti-a407 antibody every four or eight weeks thereafter.

12. The method of claim 10, wherein the human subject is subcutaneously administered a dose of 108 mg of the humanized anti-a407 antibody at week 6, followed by a 108 mg dose of the humanized anti-a407 antibody every two weeks thereafter.

13. The method of claim 10, further comprising intravenously administering a third dose of 300 mg of the humanized anti-a4p7 antibody at week 6, wherein the human patient is subcutaneously administered a dose of 108 mg of the humanized anti-a4p7 antibody at week 14, followed by a 108 mg dose of the humanized anti-a4p7 antibody every two weeks thereafter.

14. The method of claim 12 or 13, wherein the dose of 108 mg is self-administered.

15. The method of any one of claims 1-9, wherein the humanized anti-a4p7 antibody and the pan-JAK inhibitor are administered to the human subject during an induction phase.

16. The method of claim 15, wherein the induction phase is followed by a maintenance phase comprising administration of the humanized anti-a4p7 antibody as a monotherapy.

17. The method of claim 16, wherein the maintenance phase begins when the human subject achieves clinical remission and/or an endoscopic response.

18. The method of any one of the preceding claims, wherein the pan-JAK inhibitor is tofacitinib.

19. The method of claim 18, wherein a 10 mg dose of tofacitinib is administered orally twice daily.

20. A method of treating a human subject having an inflammatory bowel disease (IBD), said method comprising administering a humanized anti-a4p7 antibody to the human patient, wherein the human subject having IBD has an increased or decreased expression level before treatment, of at least one effector marker selected from the group consisting of NFKB2, CLDN1, CLDN2, CLDN3, OCLN, TJP1, ITGA4, ITGB7, PDGFB, IL5, IL6, IGF2, and MMP9 relative to a reference level in a human subject without IBD, and/or wherein the human subject having IBD has a reversion after treatment, in the expression level of the at least one effector marker selected from the group consisting of NFKB2, CLDN1, CLDN2, CLDN3, OCLN, TJP1, ITGA4, ITGB7, PDGFB, IL5, IL6, IGF2, and MMP9 relative to a baseline expression level in the human subject having IBD, and wherein the humanized anti-a4p7 antibody is an IgGl antibody and comprises a heavy chain variable region comprising a CDR3 domain as set forth in SEQ ID NO: 4, a CDR2 domain as set forth in SEQ ID NO: 3, and a CDR1 domain as set forth in SEQ ID NO: 2; and comprises a light chain variable region comprising a CDR3 domain as set forth in SEQ ID NO: 8, a CDR2 domain as set forth in SEQ ID NO: 7, and a CDR1 domain as set forth in SEQ ID NO: 6.

21. A method of treating a human subject having an inflammatory bowel disease (IBD), said method comprising administering a humanized anti-a4p7 antibody to the human subject, wherein the human subject having IBD has a decreased expression level before treatment of CLDN3, OCLN, and/or TJP1 relative to a reference level in a human subject without IBD, and/or wherein the human subject having IBD has a reversion after treatment in the expression level of CLDN3, OCLN, and/or TJP1 relative to a baseline expression level in the human subject having IBD, and wherein the humanized anti-a4p7 antibody is an IgGl antibody and comprises a heavy chain variable region comprising a CDR3 domain as set forth in SEQ ID NO: 4, a CDR2 domain as set forth in SEQ ID NO: 3, and a CDR1 domain as set forth in SEQ ID NO: 2; and comprises a light chain variable region comprising a CDR3 domain as set forth in SEQ ID NO: 8, a CDR2 domain as set forth in SEQ ID NO: 7, and a CDR1 domain as set forth in SEQ ID NO: 6.

22. A method of treating a human subject having an inflammatory bowel disease (IBD), said method comprising administering a humanized anti-a4p7 antibody to the human patient, wherein the human subject having IBD has an increased or decreased expression level before treatment of at least one effector marker selected from the group consisting of IGF2, PDGFB, IL6, NFKB2, MMP9, CLDN1, and CLDN2 relative to a reference level in a human subject without IBD, and/or wherein the human subject having IBD has a reversion after treatment in the expression level of the at least one effector marker selected from the group consisting of IGF2, PDGFB, IL6, NFKB2, MMP9, CLDN1, and CLDN2 relative to a baseline expression level in the human subject having IBD, and wherein the humanized anti-a4p7 antibody is an IgGl antibody and comprises a heavy chain variable region comprising a CDR3 domain as set forth in SEQ ID NO: 4, a CDR2 domain as set forth in SEQ ID NO: 3, and a CDR1 domain as set forth in SEQ ID NO: 2; and comprises a light chain variable region comprising a CDR3 domain as set forth in SEQ ID NO: 8, a CDR2 domain as set forth in SEQ ID NO: 7, and a CDR1 domain as set forth in SEQ ID NO: 6.

23. A method of treating a human subject having an inflammatory bowel disease (IBD), said method comprising administering a humanized anti-a4p7 antibody and administering a pan-Janus kinase (JAK) inhibitor to the human subject, wherein the human subject having IBD has an increased or decreased expression level before treatment of at least one effector marker selected from the group consisting of WNT1, PPARG, IL22, TLR4, MICB, IL13, TGFBR2, EGF, COL1 Al, COL1 A2, and MSH2 relative to a reference level in a human subject without IBD, and/or wherein the human subject having IBD has a reversion after treatment in the expression level of the at least one effector marker selected from the group consisting of WNT1, PPARG, IL22, TLR4, MICB, IL13, TGFBR2, EGF, C0L1A1, C0L1A2, and MSH2 relative to a baseline expression level in the human subject having IBD, and wherein the humanized anti-a4p7 antibody is an IgGl antibody and comprises a heavy chain variable region comprising a CDR3 domain as set forth in SEQ ID NO: 4, a CDR2 domain as set forth in SEQ ID NO: 3, and a CDR1 domain as set forth in SEQ ID NO: 2; and comprises a light chain variable region comprising a CDR3 domain as set forth in SEQ ID NO: 8, a CDR2 domain as set forth in SEQ ID NO: 7, and a CDR1 domain as set forth in SEQ ID NO: 6.

24. A method of treating a human subject having an inflammatory bowel disease (IBD), said method comprising administering a humanized anti-a4p7 antibody and administering a pan-Janus kinase (JAK) inhibitor to the human subject, wherein the human subject having IBD has an increased expression level before treatment of COL1 Al and/or COL1 A2 relative to a reference level in a human subject without IBD, and/or wherein the human subject having IBD has a reversion after treatment in the expression level of C0L1A1 and/or C0L1A2 relative to a reference level in a human subject without IBD, and wherein the humanized anti-a4p7 antibody is an IgGl antibody and comprises a heavy chain variable region comprising a CDR3 domain as set forth in SEQ ID NO: 4, a CDR2 domain as set forth in SEQ ID NO: 3, and a CDR1 domain as set forth in SEQ ID NO: 2; and comprises a light chain variable region comprising a CDR3 domain as set forth in SEQ ID NO: 8, a CDR2 domain as set forth in SEQ ID NO: 7, and a CDR1 domain as set forth in SEQ ID NO: 6.

25. A method of treating a human subject having an inflammatory bowel disease (IBD), said method comprising administering a humanized anti-a4p7 antibody and administering a pan-Janus kinase (JAK) inhibitor to the human subject, wherein the human subject having IBD has an increased or decreased expression level before treatment, of at least one effector marker selected from the group consisting of MSH2, PPARG, and EGF relative to a reference level in a human subject without IBD, and/or wherein the human subject having IBD has a reversion after treatment in the expression level of the at least one effector marker selected from the group consisting of MSH2, PPARG, and EGF relative to a baseline expression level in the human subject having IBD, and wherein the humanized anti-a4p7 antibody is an IgGl antibody and comprises a heavy chain variable region comprising a CDR3 domain as set forth in SEQ ID NO: 4, a CDR2 domain as set forth in SEQ ID NO: 3, and a CDR1 domain as set forth in SEQ ID NO: 2; and comprises a light chain variable region comprising a CDR3 domain as set forth in SEQ ID NO: 8, a CDR2 domain as set forth in SEQ ID NO: 7, and a CDR1 domain as set forth in SEQ ID NO: 6.

26. A method of treating a human subject having an inflammatory bowel disease (IBD), said method comprising administering a humanized anti-a4p7 antibody and administering a pan-Janus kinase (JAK) inhibitor to the human subject, wherein the human subject having IBD has an increased or decreased expression level before treatment, of at least one effector marker selected from the group consisting of TLR4, MICB, IL22, and TGFBR2 relative to a reference level in a human subject without IBD, and/or wherein the human subject having IBD has a reversion after treatment in the expression level of the at least one effector marker selected from the group consisting of TLR4, MICB, IL22, and TGFBR2 relative to a baseline expression level in the human subject having IBD, and wherein the humanized anti-a4p7 antibody is an IgGl antibody and comprises a heavy chain variable region comprising a CDR3 domain as set forth in SEQ ID NO: 4, a CDR2 domain as set forth in SEQ ID NO: 3, and a CDR1 domain as set forth in SEQ ID NO: 2; and comprises a light chain variable region comprising a CDR3 domain as set forth in SEQ ID NO: 8, a CDR2 domain as set forth in SEQ ID NO: 7, and a CDR1 domain as set forth in SEQ ID NO: 6.

27. The method of any one of claims 20-26, wherein the IBD is Crohn’s disease.

28. The method of claim 27, wherein the Crohn’s disease is moderately to severely active Crohn’s disease.

29. The method of any one of claims 20-28, wherein the human subject has active inflammation on ileocolonoscopy.

30. The method of any one of claims 20-29, wherein the human subject is biologic-naive.

31. The method of any one of claims 20-29, wherein the human subject had a lack of an adequate response with, lost response to, or was intolerant to treatment with at least one of an immunomodulator, a tumor necrosis factor-alpha antagonist or combinations thereof.

32. The method of any one of claims 20-31, wherein the human subject is intravenously administered a first dose of 300 mg of the humanized anti-a407 antibody at week 0, followed by a second dose of 300 mg of the humanized anti -01407 antibody at week 2.

33. The method of claim 32, further comprising intravenously administering a third dose of 300 mg of the humanized anti-a407 antibody at week 6, followed by a 300 mg dose of the humanized anti-a407 antibody every four or eight weeks thereafter.

34. The method of claim 32, wherein the human subject is subcutaneously administered a dose of 108 mg of the humanized anti-a407 antibody at week 6, followed by a 108 mg dose of the humanized anti-a407 antibody every two weeks thereafter.

35. The method of claim 32, further comprising intravenously administering a third dose of 300 mg of the humanized anti-a407 antibody at week 6, wherein the human patient is subcutaneously administered a dose of 108 mg of the anti-a407 antibody at week 14, followed by a 108 mg dose of the humanized anti-a407 antibody every two weeks thereafter.

36. The method of claim 34 or 35, wherein the dose of 108 mg is self-administered.

37. The method of any one of claims 20-31, wherein the humanized anti-a407 antibody and the pan-JAK inhibitor are administered to the human subject during an induction phase.

38. The method of claim 37, wherein the induction phase is followed by a maintenance phase comprising administration of the humanized anti-a407 antibody as a monotherapy.

39. The method of claim 38, wherein the maintenance phase begins when the human subject achieves clinical remission and/or an endoscopic response.

40. The method of any one of claims 20-39, wherein the pan-JAK inhibitor is tofacitinib.

41. The method of claim 40, wherein a 10 mg dose of tofacitinib is administered orally twice daily.

42. The method of any one of the previous claims, wherein the expression level of the at least one effector marker is an mRNA expression level or protein expression level.

43. The method of claim 42, wherein the mRNA expression level is determined by in situ hybridization or RNA sequencing.

44. The method of claim 42, wherein the protein expression level is determined by flow cytometry, mass cytometry, or immunohistochemistry.

45. The method of any one of the preceding claims, wherein the humanized anti-a4p7 antibody comprises a heavy chain variable domain comprising an amino acid sequence as set forth in SEQ ID NO: 1, and comprises a light chain variable domain comprising an amino acid sequence as set forth in SEQ ID NO: 5.

46. The method of any one of the preceding claims, where the humanized anti-a4p7 antibody is vedolizumab.