Patent Application
20190064181
The invention relates to a method for monitoring the effectiveness of an anti-TNF-alpha-treatment, in particular an anti-TNF-alpha-treatment of an inflammatory bowel disease.
Inflammatory bowel disease (IBD) is characterized by chronic intestinal inflammation and dysfunction of the epithelial barrier. The disease initiating primary cause is unclear. Recent findings indicate that alternated interactions between the immune system, epithelial barrier function and microbiome play a crucial role for the development of IBD [1]. It is now well-accepted that inflammatory responses, most likely driven by the microbiome and defective barrier function, promote a vicious circle leading to chronic disease [12]. Since IBD is characterized by chronic inflammation, most therapies are directed against the inflammatory response. Tumor necrosis factor alpha (TNFα) is currently the key target of IBD therapy highlighting its pro-inflammatory properties. Anti-TNF-alpha therapy promotes mucosal healing, although the mechanism is currently unclear. It has been shown, that Interleukin 22 (IL-22) has both protective and pathogenic effects in mouse IBD models [4-8], suggesting that tight control of IL-22 activity is essential in order to maintain tissue protective functions and avoid detrimental effects. IL-22 promotes mucosal healing and is up-regulated in the intestine in patients with IBD [3, 13]. In mouse models it was however shown that IL-22 has also pro-inflammatory functions [7, 8]. Therefore, it is essential to tightly control IL-22 activity. This control is exerted by Inter-leukin-22 binding protein (IL-22BP), which has the same binding site for IL-22 as the membrane-bound Interleukin-22 receptor subunit 1 (IL-22R1). IL-22BP lacks a transmembrane and intracellular domain, however, and is therefore unable to induce signaling. IL-22BP specifically binds IL-22 and prevents binding of IL-22 to membrane-bound IL-22R1 [14-17]. IL-22 has a 20- to 1,000-fold higher binding affinity to IL-22BP compared to its binding to the membrane-bound IL-22R1 [3, 18]. Accordingly, it has previously been shown that endogenous IL-22BP controls IL-22, which uncontrolled can promote tumorigenesis in the intestine [10, 19, 20]. In line with these data, it was shown that transgenic overexpression of IL-22BP in the intestine causes increased colitis susceptibility in a mouse model, in which IL-22 has protective functions [8]. Taken together, these data suggest that a fine-tuned regulation of IL-22 and IL-22BP controls homeostasis in the intestine. Accordingly, it has been reported that IL-22 and IL-22BP exhibit an inverse expression pattern upon tissue damage in the intestine in mouse models: Under steady state conditions IL-22BP is expressed in the colon. It is down regulated locally during intestinal tissue damage and up regulated once again during colonic repair. In contrast, IL-22 expression has an inverse pattern, with maximal expression at the peak of colonic damage and low expression under homeostatic conditions [3, 10, 21]. This regulation of the IL-22-IL-22BP expression pattern is essential to control tissue repair in the intestine in murine models [6, 10].
To date, medical practitioners use clinical symptoms, endoscopic analysis, TNF-alpha antibody levels or the measurement of antibodies against TNF-alpha antibodies for monitoring the therapeutic success of an anti-TNF-alpha therapy. These methods, however, have only limited informative value. It is therefore an object of the invention to provide an improved method for monitoring the effectiveness of an anti-TNF-alpha-treatment of an inflammatory bowel disease.
For this purpose, the invention provides a method for monitoring the effectiveness of an anti-TNF-alpha-treatment in a subject, the method comprising determining the expression level of Interleukin-22 binding protein (IL-22BP) in a biological sample obtained from the subject.
It has surprisingly been found that the expression level of IL-22BP can serve as a prognostic biomarker in IBD. It has been found that the expression level of IL-22BP, in particular the expression level of T-cell derived IL-22BP in inflamed areas of the intestine, positively correlates with TNF alpha expression levels. Without wishing to be bound by theory, it is assumed that, in IBD, TNF alpha causes up-regulation of IL-22BP, which binds to IL-22 and thus prevents mucosal protecting/healing effects of IL-22. Anti-TNFα agents block TNFα, leading to down-regulation of IL-22BP, such that IL-22 is enabled to unfold its healing effects. Therefore the measurement of IL-22BP can serve as a direct biomarker for the effectiveness of anti-TNFalpha treatment.
The term “anti-TNF-alpha-treatment” as used herein means any treatment aimed at directly or indirectly inhibiting the action of tumor necrosis factor alpha (TNFα). Anti-TNFα treatment involves, for example, the use of TNF-alpha inhibitors, i.e. agents directed against TNFα. TNFα inhibitors include, but are not limited to (monoclonal) antibodies binding TNFα. Examples of such antibodies are Infliximab, adalimumab, certolizumab pegol and golimumab. Other examples of TNF-alpha inhibitors are receptor proteins like etanercept, or substances like thalidomide and its derivatives, and xanthine derivatives.
The term “inflammatory bowel disease”, IBD, relates to a group of chronic inflammatory conditions of the gastrointestinal (GI) tract, in particular the large intestine (colon) and the small intestine. IBD primarily includes ulcerative colitis (UC) and Crohn's disease (CD). Both conditions usually involve severe diarrhea, pain, fatigue, vomiting and weight loss. Ulcerative colitis mainly causes long-lasting inflammation and sores (ulcers) in the innermost lining of the large intestine (colon) and rectum. Crohn's disease mainly causes inflammation of the lining of the digestive tract, but can also affect the entire thickness of the bowel wall. It most commonly affects the end of the small bowel (the ileum) and the beginning of the colon. It may, however, affect any part of the GI tract, from the mouth to the anus. In Crohn's disease, the inflammation of the intestine can be “patchy”, i.e. it may leave uninflamed areas in between patches of diseased intestine. Although ulcerative colitis and Crohn's disease account for the most cases of IBD, other conditions are also encompassed by the term. Such other conditions are collagenous colitis, lymphocytic colitis, diversion colitis, Behcet's disease, and indeterminate colitis.
The term “subject” as used herein preferably refers to a vertebrate, further preferred to a mammal, and most preferred to a human.
The term “expression level of Interleukin-22 binding protein” means the quantity of Interleukin-22 binding protein produced in a defined quantity of a biological sample, e.g. a cell suspension or a tissue sample. The expression level may be measured by real-time quantitative reverse transcriptase PCR (qRT-PCR) or on a protein level using ELISA, Western blotting, immune histochemistry, immune fluorescense or flow cytometry. Relative expression levels can e.g. be calculated by methods known in the art (see e.g. [25]).
The term “Interleukin-22 binding protein” (IL-22BP), also known as “cytokine receptor family (CRF) 2-10”, “CRF2-X”, and “IL22RA2”, relates to a soluble IL-22 glycoprotein belonging to the type II cytokine receptor family. IL-22BP lacks a trans-membrane and intracellular domain and specifically binds to IL-22 but not other IL-10 family members, and prevents the binding of IL-22 to membrane bound IL-22R1. As regards human IL-22BP the term in particular relates to its isoform 2 (see e.g. [9]). The term “T-cell derived IL-22BP” relates to IL-22BP produced by T-cells, in particular CD4+CD3+CD11− T-cells.
The term “biological sample” or “biological specimen” as used herein relates to any sample of biological origin or of biological material, e.g. a sample of body fluid, e.g. blood or saliva, feces, a cell sample or tissue sample. The term “biopsy sample” or “biopsy specimen” relates to tissue removed from the body.
The method of the invention may be used to monitor the effectiveness of any anti-TNF-alpha-treatment in a subject subjected to such a treatment. Conditions treated with an anti-TNF-alpha-treatment comprise e.g. psoriasis, rheumatoid arthritis or inflammatory bowel disease (IBD). It is, however, preferred that the condition for which the effectiveness of an anti-TNF-alpha-treatment is monitored, is an inflammatory bowel disease.
For monitoring the effectiveness of any anti-TNF-alpha-treatment it may be sufficient to determine the expression level of Interleukin-22 binding protein (IL-22BP) at any given time during the treatment. The expression level thus determined may, e.g. be compared with accepted standard expression levels for subjects under effective anti-TNF-alpha-treatment or with average expression levels in healthy subjects or subjects in a disease remission state or a state of minimal disease activity. It may thus be sufficient to examine only one sample, without the need for taking any subsequent or previous samples.
In a preferred embodiment of the method of the invention the method comprises determining the expression level of Interleukin-22 binding protein (IL-22BP) in a first biological sample obtained from the subject at a first date and in a second biological sample obtained from the subject at a later second date, and comparing the IL-22BP expression levels at the first and second date. In this embodiment of the method of the invention the expression levels of Interleukin-22 binding protein in a first and a later second biological sample are compared in order to evaluate the effectiveness of an anti-TNF-alpha treatment in a subject, e.g. a subject having IBD. A reduction of the IL-22BP expression level indicates that the treatment is efficient, whereas an essentially unchanged or higher IL-22BP expression level indicates that the treatment is inefficient.
In a preferred embodiment of the method of the invention the first biological sample is obtained at a date before the treatment and the second biological sample is obtained at a date during or after the treatment, or the first biological sample is obtained at a date during the treatment and the second biological sample is obtained at a later date during the treatment or at a date after the treatment. The method of the invention can thus be used to monitor the effectiveness of an anti-TNF-alpha-treatment over time.
In a further preferred embodiment of the method of the invention the biological sample is a biopsy specimen. In case of the condition being treated with an anti-TNF-alpha-treatment being inflammatory bowel disease (IBD), the biopsy specimen is taken from the intestine of the subject, preferably a biopsy specimen taken from an inflamed area of the large intestine or small intestine of the subject.
In a further preferred embodiment of the method of the invention the expression level of T-cell derived IL-22BP is determined. In this embodiment of the invention, cells from the biological specimen, e.g. a biopsy specimen from an inflamed area of an affected organ or tissue, e.g. the intestine, are sorted with methods known in the art, and the IL-22BP expression levels of (CD4−) T-cells, in particular CD4+CD3+CD11− T-cells, are determined and compared.
In the following, the invention is described in more detail by way of examples and the attached figures for illustration purposes only.
Il22−/− and Il22bp−/− mice are described elsewhere [10, 8. Rag1−/−, Tnfr1−/−, and Tnfr2−/− mice were obtained from the Jackson Laboratory. Age- and sex-matched knock out mice and wild type littermates (in case of Il22−/−, Il22bp−/−) or co-housed in house breed C57/BL6 wild-type mice (in case of Tnfr1−/− and Tnfr2−/− mice) between 8 to 14 weeks of age were used for all experiments. All animals were cared for in accordance with the Institutional Animal Care and Use Committee of Yale University or the institutional review board ‘Behörde für Soziales, Familie, Gesundheit and Verbraucherschutz’ (Hamburg, Germany).
Lymphocytes from spleen and lymph nodes were collected from 8 to 12 weeks old wild-type mice and indicated knock out strains. CD4+ T cells were enriched using MACS (Miltenyi Biotec GmbH) following the manufacturer's instructions and further sorted to collect CD25−CD45RBhi cells using a FACS Aria II. 4×105 CD4+CD25−CD45RBhi cells were injected intraperitoneally into Rag1−/−, Il22bp−/−Rag1−/− or Il22−/−Rag1−/− mice as indicated. Colitis development was measured by changes in weight, endoscopic and histological findings. At indicated time points the mice were sacrificed and lymph nodes and colon were isolated for further analysis.
Infection with Citrobacter rodentium 6 weeks old Rag1−/− mice were repopulated with 4×106 CD4+ T cells from either Il22bp−/− or wild-type mice. 5 weeks upon transfer mice were orally gavaged with C. rodentium (109 CFU/200 μl). On day 8 post-infection the mice were sacrificed and the colon and caecum was isolated for analysis.
Colonoscopy was performed in a blinded fashion for colitis scoring using the Coloview system (Karl Storz, Germany) as previously described [27]. In brief: Colitis scoring was based on granularity of mucosal surface, stool consistency, vascular pattern, translucency of the colon and the visibility of fibrin (0-3 points for each). For histological colitis scoring colons were evaluated and were assigned scores by investigators blinded to experimental manipulation. Each section was evaluated by a semiquantitative criterion based method (score 0-5 for edema, inflammation, hyperplasia, crypt loss, and ulceration) essentially as described before [28].
Small intestinal samples were taken from the ileum part of the small intestine from patients undergoing laparoscopic gastric bypass surgery due to morbid obesity. Paired endoscopic biopsy specimens were obtained from the colon (ascending colon, transverse colon, descending colon, sigma/rectum) and terminal ileum from patients with IBD or suspicion of intestinal disease. An additional paired biopsy was taken from the largest foci of macroscopic inflammation. One of the paired biopsies was used for histological assessments and the other one for RNA extraction or cell isolation. Colon biopsies of diverticulitis were obtained from patients undergoing surgical resection due to recurrent diverticulitis. Disease severity was based on Mayo score for UC (remission: 0-2, mild: 3-5, moderate: 6-10, severe 11-12 points) and Harvey-Bradshaw index for CD (remission: 0-4, mild: 5-7, moderate: 8-16, severe >16 points). For comparison of IBD patients we assigned scores for disease severity (remission: 0, mild: 1, moderate: 2, severe 3 points). Human studies were approved by the local ethical committee (Ethik-Kommission der Ärztekammer Hamburg PV4444).
Isolation of Hematopoietic Cells from Murine and Human Intestine
Hematopoietic cells were isolated from freshly obtained human colon biopsies, human small intestine or murine colon. From human small intestine samples the mucosa was isolated. After removal of the Peyer's patches and the adventitial fat the murine colon was cut longitudinally. Prepared samples were washed with PBS. For isolation of intraepithelial lymphocytes (IEL) the intestinal tissue was incubated in HBSS containing 1 mM dithioerythritol (DTE) followed by a dissociation step using 1.3 mM EDTA for 20 min at 37° C. respectively. To isolate lamina propria lymphocytes (LPL) the tissue was further cut in small pieces and minced with a scalpel. The remaining tissue was incubated for 45 min at 37° C. on a shaking incubator in HBSS (with Ca2+ and Mg2+) with Collagenase (1 mg/ml) and DNase I (10 U/ml) and supernatant was collected. Leukocytes were further enriched by Percoll gradient centrifugation (GE Healthcare). If not stated otherwise, IEL and LPL were collected and pooled.
Immunohistochemistry was performed on 5 μm formalin-fixed and paraffin-embedded sections of human colonic biopsies. Slides were deparaffinized and exposed to heat-induced antigen retrieval for 5 minutes in an autoclave at 121° C. in pH 7.8 wash buffer (Dako, Glostrup, Denmark) and primary antibody specific for IL-22BP (dilution 1:450; R&D Systems) was applied.
Bound antibody was then visualized using the EnVision Kit (Dako). All sections were counter-stained with hematoxylin. IL-22BP+ cells were counted in a blinded fashion from at least 4 areas of a given histological section and divided by the total tissue surface obtained from the 4 areas. For indicated analysis histological slides were stained with Haematoxylin and Eosin.
Total RNA was extracted from tissue and cells of the small intestine, colon and lymph nodes using Trizol® Reagent (Invitrogen). The High capacity cDNA synthesis Kit (Applied Biosystems) was used for synthesis of cDNA. Primers and probes were purchased from Applied Biosystems. Human primers and probes including reference: IL-22 (Hs01574154_m1), IL-22RA1 (Hs00222035_m1), IL-22RA2 (Hs00364814_m1), IL-17A (Hs00174383_m1), IL-23 (Hs00900828_g1), IL-18 (Hs01038788_m1), IFN-γ (Hs00989291_m1), TNF (Hs01113624_g1), and HPRT1 (Hs02800695_m1), IL-6 (HS00189606_m1), IL-5 (Hs01548712_g1), TNFR1 (Hs01042313_m1) and TNFR2 (Hs00961749_m1), CD4 (Hs01058407_m1), CD11c (Hs00174217_m1), FOXP3 (Hs01085834_m1), GATA-3 (Hs00231122_m1), TBX21 (Hs00203436_m1), RORC (Hs01076122_m1).
Mouse primers and probes including reference: HPRT (Mm01545399_m1), IL-22 (Mm00444241_m1), IL-22RA2 (Mm01192969_m1).
Real-time PCR was performed using the Kapa Probe Fast qPCR Master Mix (Kapa Biosystems) on the StepOne Plus system (Applied Biosystems). For both human and mouse, relative expression was normalized to HPRT and calculated using the 2-ΔΔCt method.
Western blot analyses were undertaken as previously described [29]. Total protein extracts (50m) were separated by electrophoresis and transferred onto nitrocellulose membranes. Blots were incubated with IL-22BP antibody (MAB1087, R&D Systems) at 1:1000, polyclonal rabbit anti-mouse HRP (Dako) at 1:2000 and reprobed with mouse anti-actin (Santa Cruz). Recombinant IL-22BP was purchased from Sino Biological.
Human antibodies anti-CD45, anti-CD4, anti-CD3, anti-CD11c, anti- Siglec-8, and mouse antibodies anti-CD45.2, anti-CD45.1, anti-MHCII, anti-CD11c, anti-CD3, anti-CD4, anti-CD44, anti-CD62L, anti-CD25, anti-Foxp3, anti-IFNγ, anti- IL-17A, and anti-CD45RB were purchased from Biolegend. Anti-human CD3 and anti-human CD4 were purchased from BD Biosciences. Anti-human IL-22BP antibody (clone 87554) and IgG2B isotype control were purchased from R&D Systems. To identify dead cells, 7-AAD staining (Biolegend) was performed. For extracellular staining isolated hematopoietic cells of the small intestine, the colon and the lymph nodes were incubated for 20 min at 4° C. Cells were sorted on a FACS Aria II or acquired on a LSRII Fortessa flow cytometer (BD), respectively. Data were analyzed with FlowJo software (Treestar).
For serum cytokine quantification LEGENDplexTM human T helper cell cytokine panel was used. Experimental procedures have been performed according to the manufacturer's instructions.
Statistical analysis was performed with GraphPad Prism® Software (GraphPad Software, San Diego, Calif., USA). For comparison of groups, the non-parametric two-sided Mann-Whitney test was used. Bonferroni correction was used to counteract the problem in case of multiple comparisons. For time-dependent weight-loss data a repeated-measures ANOVA to assess the significance of the main effects and an experimental group-time interaction was used. The significance level a was set to 0.05. The Pearson correlation was used for correlative analyses. The significance level was set to 0.05.
IL-22 and IL-22BP expression in intestinal biopsy specimens from patients with ulcerative colitis (UC) or Crohn's disease (CD) (table 1) who either were in remission (UC, n=13; CD, n=12) or had active disease based on clinical, endoscopic, and histological findings (UC, n=18; CD, n=21) was analyzed.
In patients with active IBD, we analyzed samples from both healthy (uninflamed) and diseased (inflamed) areas in the colon and terminal ileum, based on endoscopic and histological findings, which were further validated by analyzing expression of the genes for the inflammatory markers IL-22, IL-17A, and interferon-γ (IFN-γ) (
In order to identify the cellular source of IL-22BP in the intestine, different cell populations from human intestine were sorted. In line with previous publications [10, 22, 9, 26], it was confirmed that dendritic cells (DCs) and eosinophils express high levels of IL-22BP (
Murine CD4+ T cells in the lymph nodes also expressed Il22bp (
T cell-specific up-regulation of IL-22BP in active IBD suggests a pathogenic role in intestinal inflammation. On the basis of these findings, the relevance of DC-versus T cell-derived IL-22BP in colitis was examined. To that end, the murine CD45RBhigh transfer colitis model was used, in which IL-22 mediates a protective function (10, 22). To discriminate between CD4+ T cell-derived and innate immune cell-derived IL-22BP, Il22bp−/− and Il22bp+/+ CD4+CD25−CD45RBhigh T cells were transferred into Rag1−/− and Rag1−/−Il22bp−/− mice. Transfer of wild-type T cells into Rag1−/− and Rag1−/−Il22bp−/− mice caused equally severe disease, characterized by weight loss and endoscopic and histological scores of colitis (
In a next step the question was addressed whether the effect of IL-22BP is due to the free activity of IL-22 or to indirect mechanisms. Therefore, the transfer colitis experiment was performed in an IL-22-deficient environment. Il22−/− and Il22−/−Il 22bp−/− CD4+CD25−CD45RBhigh T cells were transferred into Rag1−/−Il22−/− mice. In this setting, both Il22−/− and Il22−/−Il22bp−/− CD4+CD25−CD45RBhigh T cells caused equally severe colitis (
Anti-TNF-α therapy is the most effective treatment of IBD at present. It was therefore tested whether the efficacy of this therapy is linked to IL-22BP. A positive correlation between IL22BP and TNF-α expression in the intestine of IBD patients with active disease was found (P=0.004, correlation coefficient r=0.36;
CD4+ T cells and DCs were isolated from intestinal biopsy specimens obtained from IBD patients who were receiving anti-TNF-α therapy (adalimumab or infliximab) or other immune-modulating treatment in order to test whether anti-TNF-α treatment influences the expression of IL22BP. IL22BP expression was markedly reduced in CD4+ T cells of IBD patients who were responsive to anti-TNF-α treatment, compared with those of patients on other medications (
This effect did not seem to be due to differences in disease activity [disease activity index (mean±SEM) for anti-TNF-α, 0.6±0.25; for other treatment, 0.3±0.13; P=0.29] and appeared to be specific to CD4+ T cells, because DCs did not show a significant down-regulation of IL22BP in IBD patients treated with anti-TNF-α (
Next it was tested whether anti-TNF-α therapy is simply inversely correlated with IL-22BP expression or whether the effect of this treatment is dependent on IL-22BP regulation, and thus on the protective effect of IL-22. In case of the latter, one would expect that this therapy would not work in an Il22-deficient environment. To test this, wild-type and Il22−/− CD4+CD25−CD45RBhigh T cells were transferred into Rag1−/− and Rag1−/−Il22−/− mice, respectively, and the mice were treated with anti-TNF-α upon colitis development. Anti-TNF-α treatment was not effective in the Il22-deficient environment (
| Number | Date | Country | Kind |
|---|---|---|---|
| 10 2016 103 356.1 | Feb 2016 | DE | national |
| 92982 | Feb 2016 | LU | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2017/054218 | 2/23/2017 | WO | 00 |
