Patent Application
20220378878
This application claims the benefit AU Patent Application No 2019904873, filed 20 Dec. 2020, the entire contents of which are incorporated by reference herein.
THIS INVENTION relates to gastrointestinal diseases, disorders and conditions. More particularly, this invention relates to methods for the treatment and/or prevention of gastrointestinal diseases, disorders and conditions, such graft versus host disease (GVHD), inflammatory bowel disease (IBD) and autoimmune and therapy-related enteritis and/or colitis.
Allogeneic bone marrow or stem cell transplantation (hereinafter referred to as HSCT or BMT) offers curative therapy for patients with high-risk haematological malignancies. Despite advances in transplantation techniques, including advanced supportive care, reduced intensity conditioning, alternative donor sources and evolutions in GVHD prophylaxis; the overall benefit remains limited by transplant-related mortality and morbidity, reducing the number of patients who will realise cure with an acceptable quality of life. Graft-versus-host disease (GVHD) represents a major component of this morbidity and mortality. Prevention or treatment of GVHD has traditionally focused on T cell targeted immune suppression and is often accompanied by impairment in pathogen-specific immunity and graft-versus-leukemia (GVL) effects. Therapies that prevent GVHD whilst preserving leukemia and pathogen-specific immunity remain a clinical imperative.
The present invention broadly relates to methods of protecting and/or promoting proliferation of intestinal epithelium, including intestinal stem cells by administering an Interferon (IFN) λ ligand or an IFNλ receptor agonist to a subject in need thereof. In a particular form, the invention relates to methods of preventing or treating a gastrointestinal disease, disorder or condition, such as GVHD, inflammatory bowel disease and autoimmune and therapy-related enterocolitis.
In a first aspect, the invention provides a method of preventing or treating a gastrointestinal disease, disorder or condition in a subject, said method including the step of administering to the subject a therapeutically effective amount of an IFNλ receptor agonist to thereby prevent or treat the gastrointestinal disease, disorder or condition in the subject.
In one embodiment, the gastrointestinal disease, disorder or condition is at least partly characterized by gastrointestinal epithelial damage and/or loss.
Suitably, the gastrointestinal disease, disorder or condition is selected from the group consisting of an inflammatory intestinal disease, an infection, an autoimmune disease, radiation-induced intestinal damage, graft versus host disease (GVHD), and any combination thereof. In one embodiment, inflammatory intestinal disease comprises inflammatory bowel disease, ulcerative colitis and/or Crohn's disease.
In a specific embodiment, the gastrointestinal disease, disorder or condition is or comprises GVHD, such as acute GVHD. In this regard, GVHD in the subject is suitably characterised by or comprises gastrointestinal epithelial damage and/or loss, such as small intestinal and/or colonic epithelial damage and/or loss.
In certain embodiments, the subject has or is to be administered an immunosuppressive agent.
In some embodiments, the IFNλ receptor agonist is administered to the subject prior to, simultaneously with and/or subsequent to administration of a transplant. In this regard, the transplant is suitably a hematopoietic stem cell transplant, such as a bone marrow transplant or more particularly an allogeneic bone marrow transplant.
Suitably, the IFNλ receptor agonist does not modulate and/or preserves graft versus leukemia (GVL) and/or graft versus tumour (GVT) effects of the transplant.
In a second aspect, the invention provides a method of promoting survival and/or proliferation of an intestinal cell, said method including the step of contacting the intestinal cell with an IFNλ receptor agonist under conditions to promote survival and/or proliferation of the intestinal cell.
In this regard, the present method can be performed either in vitro or in vivo in a subject.
Suitably, the intestinal cell is or comprises an intestinal stem cell. More particularly, the intestinal stem cell may be positive for or expresses one or more of Lgr5, Ascl2 and Smoc2. In other embodiments, the intestinal stem cell is positive for or expresses one or more further surface markers denoting an intestinal stem cell.
In one embodiment, the IFNλ receptor agonist is administered prior to, simultaneously with and/or subsequent to administration of a cytotoxic agent to the subject. To this end, the cytotoxic agent suitably is or comprises a chemotherapeutic agent and/or a radiotherapy.
Suitably, the subject has or is suffering from a gastrointestinal disease, disorder or condition, such as an inflammatory intestinal disease, an infection, an autoimmune disease, immunotherapy-induced intestinal damage, an immune deficiency, a haematological malignancy, chemotherapy-induced intestinal damage, radiation-induced intestinal damage, GVHD, and any combination thereof.
In a third aspect, the invention provides a method of performing a transplant in a subject in need thereof, said method including the step of administering to the subject a therapeutically effective amount of an IFNλ receptor agonist prior to, simultaneously with and/or subsequent to administration of the transplant to the subject.
In certain embodiments, the transplant is or comprises a hematopoietic stem cell transplant, and more particularly is or comprises a bone marrow transplant.
In one embodiment, the present method includes the further step of administering the transplant to the subject.
In a fourth aspect, the invention relates to an IFNλ receptor agonist for use in:
(a) preventing or treating a gastrointestinal disease, disorder or condition;
(b) promoting survival and/or proliferation of an intestinal cell; and/or
(c) performing a transplant;
in a subject.
In a fifth aspect, the invention resides in use of an IFNλ receptor agonist in the manufacture of a medicament for:
(a) preventing or treating a gastrointestinal disease, disorder or condition;
(b) promoting survival and/or proliferation of an intestinal cell; and/or
(c) performing a transplant;
in a subject.
Referring to the aforementioned aspects, the IFNλ receptor agonist is suitably selected from the group consisting of IFN-λ1 (IL-29), IFN-λ2 (IL-28A), IFN-λ3 (IL-28B), IFN-λ4, including fragments, variants and derivatives thereof and any combination thereof. More particularly, the IFNλ receptor agonist suitably is or comprises recombinant IL-29. Even more particularly, the IFNλ receptor agonist suitably is or comprises pegylated recombinant IL-29. Further embodiments may relate to derivatives that include one or more modifications to target the IFNλ receptor agonist, and more particularly IL-29, to a specific tissue or tissue type (e.g., the gastrointestinal tract).
Unless the context requires otherwise, the terms “comprise”, “comprises” and “comprising”, or similar terms are intended to mean a non-exclusive inclusion, such that a recited list of elements or features does not include those stated or listed elements solely, but may include other elements or features that are not listed or stated.
The indefinite articles ‘a’ and ‘an’ are used here to refer to or encompass singular or plural elements or features and should not be taken as meaning or defining “one” or a “single” element or feature. For example, “a” cell includes one cell, one or more cells and a plurality of cells.
Quantification (n=9 per group, combined from 3 experiments). C) Quantification of donor T cells in spleen at day 4 post-BMT (n=8 per group, combined from 2 experiments). D) T cell expansion in colon at day 4 post-BMT (n=6, combined from 2 experiments). E) Proportion of donor T cells at day 4 post-BMT that are donor vs. host (n=8, combined from 2 experiments). F-G) Functional capacity of splenic DCs from naïve B6 WT or Ifnlr1−/− mice to stimulate BALB/c F) CD4+ or G) CD8+ T cells in a mixed lymphocyte reaction (n=3, Data from is from 1 of 2 representative experiments. H-I) BALB/c recipients were transplanted with WT or Ifnlr1−/− BM+T cells at day 0 and B6.TeaLuc T cells (reactive to BALB/c I-Ed derived TEa peptide expressed in donor B6 I-Ab) transferred at day 12 to assess donor-derived APC function in the GI tract as determined by bioluminescence of antigen-specific TEa T cells. H) Representative bioluminescence and I) Quantification (n=10, combined from 2 experiments). J) Proportions of donor T cells producing IFNγ in spleen at day 4 post-BMT (n=8, combined from 2 experiments). K) Dividing capacity of splenic BALB/c T cells transplanted into WT or Ifnlr1−/− recipients calculated at day 4 by CFSE dilution (n=14, combined from 3 experiments). L) Proportion of annexinV+7AAD− apoptotic donor T cells at day 4 as per K) (n=7, combined from 2 experiments). M) Number of NKp46+ cells in naïve recipient mice (WT, n=3; Ifnlr1−/−, n=6). N) B6 WT or Ifnlr1−/− recipients received αNK1.1 or IgG Isotype and were transplanted with BALB/c BM+T cells. IFNγ was determined in sera at day 4 post-BMT (n=9 per group, combined from 2 experiments). O-P) B6 CD45.2+ WT or Ifnlr1−/− recipients received αNK1.1 or IgG Isotype and were transplanted with CD45.1+ allogeneic BALB/c BM and CD45.1+CD45.2+ syngeneic B6 BM. O) Representative FACS plots from NK-depleted recipients showing syngeneic vs allogeneic cells in spleen 48 hours post BMT. P) Index of cytotoxicity as described in Methods (n=8, combined from 2 experiments). Q) Index of cytotoxicity in spleen of WT and Ifnlr1−/− recipient mice in addition to NKp46Cre.Ifnlr1fl.fl negative and positive recipients (n=12, combined from 2 experiments). Data are presented as mean±SEM. P values calculated using two tailed Mann-Whitney T test. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.
The present invention is at least partly predicated on the surprising discovery that signalling by way of the IFNλ receptor 1 (IFNLR1) can influence the progression of GVHD, particularly in the gastrointestinal tract. In this regard, the present invention is further at least partly predicated on the surprising discovery that agonists or ligands of the IFNLR1, such as IL-29, exert a protective and/or proliferative effect on intestinal stem cells, which can be utilised to preserve the gastrointestinal epithelial lining.
In a first aspect, the invention provides a method of preventing or treating a gastrointestinal disease, disorder or condition in a subject, said method including the step of administering to the subject a therapeutically effective amount of an IFNλ receptor agonist to thereby prevent or treat the gastrointestinal disease, disorder or condition in the subject.
It is envisaged that the present method of treating the gastrointestinal disease, disorder or condition may be prophylactic, preventative or therapeutic and suitable for treatment of gastrointestinal diseases, disorders or conditions in mammals, particularly humans. As used herein, “treating” ,“treat” or “treatment” refers to a therapeutic intervention, course of action or protocol that at least ameliorates a symptom of the gastrointestinal disease, disorder or condition after the disease, disorder or condition and/or its symptoms have at least started to develop. As used herein, “preventing”, “prevent” or “prevention” refers to therapeutic intervention, course of action or protocol initiated prior to the onset of the gastrointestinal disease, disorder or condition and/or a symptom thereof so as to prevent, inhibit or delay or development or progression of the gastrointestinal disease, disorder or condition or the symptom.
The term “therapeutically effective amount” describes a quantity of a specified agent, such as of an IFNλ receptor agonist, sufficient to achieve a desired effect in a subject being treated with that agent. For example, this can be the amount of a composition comprising one or more IFNλ receptor agonists described herein, necessary to reduce, alleviate and/or prevent a gastrointestinal disease, disorder or condition, inclusive of gastrointestinal GVHD and recurrence thereof. In some embodiments, a “therapeutically effective amount” is sufficient to reduce or eliminate a symptom of a gastrointestinal disease, disorder or condition. In other embodiments, a “therapeutically effective amount” is an amount sufficient to achieve a desired biological effect, for example an amount that is effective to decrease the severity of or prevent a gastrointestinal disease, disorder or condition.
Ideally, a therapeutically effective amount of an agent is an amount sufficient to induce the desired result without causing a substantial cytotoxic effect in the subject. The effective amount of an agent useful for reducing, alleviating and/or preventing a gastrointestinal disease, disorder or condition will be dependent on the subject being treated, the type and severity of any associated disease, disorder and/or condition (e.g., the location of any associated disease), and the manner of administration of the therapeutic composition.
In one embodiment, the gastrointestinal disease, disorder or condition is at least partly characterized by gastrointestinal epithelial or epithelial cell damage and/or loss. To this end, it will be understood that gastrointestinal diseases, disorders and conditions can involve damage and/or at least partial loss of the gastrointestinal epithelial layer during initiation and/or progression thereof. Associated with such damage, subjects may be at risk of fibrosis and ulceration. The assessment and/or detection of gastrointestinal epithelial damage may be achieved by any method known in the art, such as endoscopy, colonoscopy, biopsy, histological analysis and faecal analysis.
The term “gastrointestinal epithelium” as used herein refers to epithelial tissues of the gastrointestinal tract, such as the oesophagus, the stomach, the small intestine (e.g., duodenum, jejunum, ileum) and/or the large intestine (e.g., caecum, colon). It may further include other structures involved in the gastrointestinal function of the body including the lower portion of the body, rectum and anus. To this end, the gastrointestinal disease, disorder or condition may affect the entire, or substantially the entire, gastrointestinal tract of a subject or alternatively may only affect one or more specific regions or portions thereof.
A “gastrointestinal epithelial cell” or “gut epithelial cell” as referred to herein refers to any epithelial cell on the surface of the gastrointestinal mucosa that faces the lumen of the gastrointestinal tract, including, for example, an epithelial cell of the stomach, an intestinal epithelial cell, a colonic epithelial cell, and the like.
The gastrointestinal or intestinal epithelium is a highly specialized tissue that lines the gastrointestinal tract and is critical to gut function, enabling both digestion and nutrient absorption, and provides both physical and chemical barriers to the gut microbiota. This gut barrier function also serves an important immunological role in preventing unwanted responses to microbes, but enables surveillance and appropriate immune control of infection. Intestinal epithelial cells (IECs) have a very high turnover rate (with replacement occurring every 4-5 days) and this continuous renewal is driven by long-lived intestinal stem cells (ISCs). This regenerative process is critical to maintenance of the intestinal epithelium, damage repair and preservation of gut-barrier integrity. Intestinal epithelium damage can occur in a range of settings including infection, chronic inflammation, autoimmunity as well as chemo/radiotherapy, and subsequent loss of barrier function can lead to further damage through feed-forward inflammatory responses. Damage to ISCs is particularly problematic due to their important regenerative role in forming intestinal epithelial cells.
Suitably, the gastrointestinal disease, disorder or condition is selected from the group consisting of an inflammatory intestinal disease, an infection, an autoimmune disease, such as autoimmune enterocolitis, radiation-induced intestinal damage, immunotherapy-induced intestinal damage (e.g., immunotherapy-related enterocolitis), an immune deficiency, a haematological malignancy, chemotherapy-induced intestinal damage (e.g., chemotherapy-related enterocolitis), graft versus host disease (GVHD), and any combination thereof.
As used interchangeably herein, the terms “inflammatory intestinal disease”, “inflammatory bowel diseases” or “IBD” includes art-recognized forms of a group of related inflammatory gastrointestinal conditions. Several major forms of IBD are known, such as Crohn's disease (regional bowel disease, inclusive of inactive and active forms) and ulcerative enterocolitis or colitis (inclusive of inactive and active forms). In addition, IBD encompasses irritable bowel syndrome, microscopic enterocolitis, lymphocytic-plasmocytic enteritis, coeliac disease, collagenous enterocolitis, lymphocytic enterocolitis and eosinophilic enterocolitis. Other less common forms of IBD include indeterminate enterocolitis, infectious enterocolitis (viral, bacterial or protozoan, e.g. amoebic enterocolitis) (e.g., Clostridium dificile enterocolitis), pseudomembranous enterocolitis (necrotizing enterocolitis), ischemic inflammatory bowel disease, Behcet's disease, sarcoidosis, scleroderma, IBD-associated dysplasia, dysplasia associated masses or lesions, and primary sclerosing cholangitis.
Accordingly, it will be appreciated that IBD represents chronic, inflammatory diseases of the gastrointestinal tract. IBD can be characterized by abdominal pain, diarrhoea (often bloody), a variable group of “extra-intestinal” manifestations (such as arthritis, uveitis, skin changes, etc.) and the accumulation of inflammatory cells within the small intestine and/or colon. Additional signs or symptoms of IBD include malabsorption of food, altered bowel motility, infection, fever, rectal bleeding, weight loss, signs of malnutrition, perianal disease, abdominal mass, and growth failure, as well as intestinal complications such as stricture, fistulas, toxic megacolon, perforation, and cancer, and including endoscopic findings, such as friability, aphthous and linear ulcers, cobblestone appearance, pseudopolyps and rectal involvement.
In a specific embodiment, the gastrointestinal disease, disorder or condition is or comprises GVHD. It will be appreciated that the term “graft-versus-host disease” or “GVHD” refers to a complication of allogeneic stem cell transplantation. In GVHD, donor hematopoietic stem cells recognize the transplant recipient as foreign and attack the patient's tissues and organs, which can impair the tissue or organ's function and/or cause it to fail. As used herein, GVHD includes, for example, acute GVHD and/or chronic GVHD. Acute and chronic GVHD are a major cause of morbidity and mortality among hematopoietic stem cell transplant recipients. Symptoms can include skin rash, intestinal problems similar to colitis or enterocolitis, and liver dysfunction. Accordingly, further non-limiting examples of GVHD include gastrointestinal GVHD, cutaneous GVHD and hepatic GVHD.
As used herein, the terms “gastrointestinal graft vs. host disease” and “GI-GVHD” refer to damage caused by donor immune cells to host tissue of the stomach and intestine, including the small intestine, large intestine and colon, which can cause loss of appetite, nausea, vomiting, or diarrhoea as part of GVHD, either acute or chronic. In severe cases, GI-GVHD can cause pain in the abdomen and bleeding in the stomach or intestines. To this end, GVHD in the subject is suitably characterised by or comprises colonic epithelial or epithelial cell damage and/or loss.
Accordingly, in certain embodiments, the method of the present aspect includes the further step of determining whether the subject has a gastrointestinal disease, disorder or condition, such as GVHD, or is at risk of developing a gastrointestinal disease, disorder or condition prior to administration of the IFNλ receptor agonist. The terms “determining”, “measuring”, “evaluating”, “assessing” and “assaying” are used interchangeably herein and may include any form of measurement known in the art.
The term “gastrointestinal infection” or “intestinal infection” as used herein refers to a viral, bacterial, fungal or parasitic infection in the gastrointestinal tract or intestine of said animal. Said infection may cause clinical or subclinical enteric disease in said animal.
The terms “autoimmune disease” or “autoimmune disorder” are used herein interchangeably and refer to any disease or disorder that occurs when the body's immune system erroneously attacks and destroys healthy body tissue, such as the gastrointestinal tract. According to one embodiment, the autoimmune disease is or comprises a gastrointestinal autoimmune disease or digestive autoimmune disease, such as ulcerative colitis, Crohn's disease, celiac disease, irritable bowel syndrome or autoimmune hepatitis. The terms “gastrointestinal autoimmune disease” and “digestive autoimmune disease” are used herein interchangeably and refer to autoimmune disease of a gastrointestinal tract of a subject.
It will be appreciated that radiation-induced damage to epithelial cells can lead to inflammation, epithelial cell apoptosis and death, and/or loss of epithelial cell (e.g., intestinal) barrier function. The mechanism by which radiation, e.g., abdominal radiation, causes such effects on cells (e.g., epithelial cells) is complex. Radiation has been shown to affect various components of the local intestinal response to injury and microbial invasion that includes rapid depletion of mucus, immune impairment, oxidant-mediated epithelial cell injury, and radiation-induced epithelial cell apoptosis. Exemplary symptoms include nausea, vomiting, diarrhoea, dehydration, electrolytic imbalance, loss of digestion ability, bleeding ulcers and sepsis.
It will be appreciated that the present aspect may include administration of one or more further treatments or therapeutic agents in addition to the IFNλ receptor agonist, as are known in the art.
In one particular embodiment, the subject has or is to be administered an immunosuppressive agent. In alternative embodiments, the subject has not or is not to be administered an immunosuppressive agent. In this regard, treatment of the subject with the IFNλ receptor agonist may preclude the need, at least partly, for further treatment with an immunosuppressive agent.
The term “immunosuppressive agent” as used herein for adjunct therapy refers to substances that act to suppress or mask the immune system of the host into which a graft, such as haematopoietic stem cells, is being transplanted. This would include, for example, agents that suppress cytokine production, downregulate or suppress self-antigen expression, or mask the MHC antigens. Examples of such agents include non-steroidal anti-inflammatory drugs (NSAIDs); ganciclovir, tacrolimus, steroids such as corticosteroids, glucocorticoids or glucocorticoid analogues, e.g., prednisone, methylprednisolone, and dexamethasone, anti-inflammatory agents such as a cyclooxygenase inhibitor, a 5-lipoxygenase inhibitor, or a leukotriene receptor antagonist; purine antagonists such as azathioprine or mycophenolate mofetil (MMF); alkylating agents such as cyclophosphamide; bromocryptine; danazol; dapsone; glutaraldehyde (which masks the MHC antigens); anti-idiotypic antibodies for MHC antigens and MHC fragments; cyclosporin A; dihydrofolate reductase inhibitors such as methotrexate (oral or subcutaneous); anti-malarial agents such as chloroquine and hydroxychloroquine; sulfasalazine; leflunomide; cytokine or cytokine receptor antibodies including anti-interferon-alpha, -beta, or -gamma antibodies, anti-tumor necrosis factor (TNF)-alpha antibodies (infliximab (REMICADE®) or adalimumab), anti-TNF-alpha immunoadhesin (etanercept), anti-TNF-beta antibodies, anti-interleukin-2 (IL-2) antibodies and anti-IL-2 receptor antibodies, and anti-interleukin-6 (IL-6) receptor antibodies and antagonists (such as ACTEMRA™ (tocilizumab)); anti-LFA-1 antibodies, including anti-CD11a and anti-CD18 antibodies; anti-L3T4 antibodies; heterologous anti-lymphocyte globulin; pan-T antibodies, preferably anti-CD3 or anti-CD4/CD4a antibodies; streptokinase; transforming growth factor-beta (TGF-beta); streptodomase; chlorambucil; deoxyspergualin; rapamycin; and biologic agents that interfere with T cell helper signals.
In other embodiments, the IFNλ receptor agonist is administered to the subject prior to, simultaneously with and/or subsequent to administration of a transplant. Suitably, the transplant is a haematopoietic stem cell transplant.
The terms “haematopoietic stem cell transplant” and “haematopoietic cell transplant” are used interchangeably herein and refer to the transplantation of cells or stem cells derived at least partly from bone marrow, peripheral blood, placental blood and/or umbilical cord blood. Hematopoietic stem cells are stem cells that generate the red blood cells, white blood cells, and platelets that make up blood. Such a transplant may be autologous, involving cells collected from the subject in question, or allogeneic, utilizing cells from a donor other than the subject. It will be appreciated that a haematopoietic stem cell transplant may include the use of high dose chemotherapy or radiotherapy, including whole-body irradiation, to eliminate diseased cells, such as leukemic immune cells, prior to introduction of the transplanted haematopoietic stem cells. By way of example, patients with leukemia do not produce normal functioning blood cells and transplanting healthy haematopoietic stem cells from a donor with a histocompatibility antigen type (HLA) that is preferably similar to that of the patient with leukemia (and with or without chemotherapy or radiotherapy) into the leukemic patient can then produce blood cells that at least partly function normally.
In particular embodiments, the haematopoietic stem cell transplant can be an allogeneic bone marrow transplant, an umbilical cord blood transplant, a peripheral blood transplant, a placenta-derived blood transplant and/or a haematopoietic stem cell line such as conditionally immortalized haematopoietic stem cells. It will be appreciated that the transplant may be purified or at least partially purified prior to administration to the subject.
The skilled artisan will appreciate that haematopoietic stem cell transplants can benefit subjects with a cancerous disease or a noncancerous disease. Nonlimiting examples of such diseases include acute lymphoblastic leukemia, adrenoleukodystrophy, aplastic anaemia, bone marrow failure syndromes, chronic lymphoblastic leukemia, haemoglobinopathies, Hodgkin's lymphoma, immunodeficiencies, inborn errors of metabolism, multiple myeloma, myelodysplastic syndromes, neuroblastoma, non-Hodgkin's lymphoma, plasma cell disorders, POEMS syndrome and primary amyloidosis.
Suitably, the IFNλ receptor agonist does not modulate and/or preserves graft versus leukemia and/or graft versus tumour effects of the transplant. As used herein, the term “graft-versus-leukemia” or “GVL” or “graft-versus-tumour” or “GVT” refers to a beneficial therapeutic immune reaction of the transplanted immune cells, such as donor T lymphocytes, against the diseased bone marrow and/or residual cancer cells of the recipient subject.
In view of the above, in some embodiments, the IFNλ receptor agonist can be administered to the subject prior to, simultaneously with and/or subsequent to administration of a cytotoxic agent. In this regard, it is well established that gastrointestinal epithelial damage is a common side effect of many cytotoxic agents. Moreover, in the case of an allogeneic haematopoietic stem cell transplant, one or more cytotoxic agents, such as a chemotherapeutic agent and/or radiation can be administered to the subject to not only assist in removing any diseased or cancerous cells but also suppress the subject's immune system to allow the transplanted bone marrow to undergo a process called engraftment.
The term “cytotoxic agent” as used herein refers to a substance that inhibits or prevents a cellular function and/or causes cell death or destruction. Cytotoxic agents include, but are not limited to, radiotherapy, such as by way of radioactive isotopes (e.g., At211, I131, I125, Y90, Re186, Re188, Sm153, Bi212, P32, Pb212 and radioactive isotopes of Lu); chemotherapeutic agents; growth inhibitory agents; enzymes and fragments thereof such as nucleolytic enzymes; antibiotics; toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and/or variants thereof and other anticancer agents as are known in the art.
In one particular embodiment, the cytotoxic agent is or comprises a chemotherapeutic agent. As generally used herein, the term “chemotherapy” or “chemotherapeutic agent” broadly refers to a treatment or agent with a cytostatic or cytotoxic agent (i.e., a compound) to reduce or eliminate the growth or proliferation of undesirable cells, such as cancer cells. Accordingly, the terms can refer to a cytotoxic or cytostatic agent used to treat a proliferative disorder, for example cancer. The cytotoxic effect of the agent can be, but is not required to be, the result of one or more of nucleic acid intercalation or binding, DNA or RNA alkylation, inhibition of RNA or DNA synthesis, the inhibition of another nucleic acid-related activity (e.g., protein synthesis), or any other cytotoxic effect.
Exemplary chemotherapeutic agents include, but are not limited to, alkylating agents (e.g., nitrogen mustards such as chlorambucil, cyclophosphamide, isofamide, mechlorethamine, melphalan, and uracil mustard; aziridines such as thiotepa; methanesulphonate esters such as busulfan; nitroso ureas such as carmustine, lomustine, and streptozocin; platinum complexes such as cisplatin and carboplatin, oxaliplatin, nedaplatin, triplatin tetranitrate, phenanthriplatin, picoplatin, satraplatin and lipoplatin; bioreductive alkylators such as mitomycin, procarbazine, dacarbazine and altretamine); DNA strand-breakage agents (e.g., bleomycin); topoisomerase II inhibitors (e.g., amsacrine, dactinomycin, daunorubicin, idarubicin, mitoxantrone, doxorubicin, etoposide, and teniposide); DNA minor groove binding agents (e.g., plicamydin); antimetabolites (e.g., folate antagonists such as methotrexate and trimetrexate; pyrimidine antagonists such as fluorouracil (5-FU), fluorodeoxyuridine, CB3717, azacitidine, cytarabine, and floxuridine; purine antagonists such as mercaptopurine, 6-thioguanine, fludarabine, pentostatin; asparginase; and ribonucleotide reductase inhibitors such as hydroxyurea); tubulin interactive agents (e.g., vincristine, vinblastine, docetaxel and paclitaxel (Taxol)); hormonal agents (e.g., estrogens; conjugated estrogens; ethinyl estradiol; diethylstilbesterol; chlortrianisen; idenestrol; progestins such as hydroxyprogesterone caproate, medroxyprogesterone, and megestrol; and androgens such as testosterone, testosterone propionate, fluoxymesterone, and methyltestosterone); adrenal corticosteroids (e.g., prednisone, dexamethasone, methylprednisolone, and prednisolone); leutinizing hormone releasing agents or gonadotropin-releasing hormone antagonists (e.g., leuprolide acetate and goserelin acetate); and antihormonal antigens (e.g., tamoxifen, antiandrogen agents such as flutamide; and antiadrenal agents such as mitotane and aminoglutethimide).
As generally used herein, the terms “cancer”, “tumour”, “malignant” and “malignancy” refer to diseases or conditions, or to cells or tissues associated with the diseases or conditions, characterized by aberrant or abnormal cell proliferation, differentiation and/or migration often accompanied by an aberrant or abnormal molecular phenotype that includes one or more genetic mutations or other genetic changes associated with oncogenesis, expression of tumour markers, loss of tumour suppressor expression or activity and/or aberrant or abnormal cell surface marker expression.
Cancers may include any aggressive or potentially aggressive cancers, tumours or other malignancies such as listed in the NCI Cancer Index at http://www.cancer.gov/cancertopics/alphalist, including all major cancer forms such as sarcomas, carcinomas, lymphomas, leukemias and blastomas, although without limitation thereto. In particular embodiments, the subject has a cancer, such as a liquid cancer or a blood cell cancer inclusive of lymphoid cancers and myelomonocytic cancers, such as those hereinbefore described, that is potentially or at least partly amenable to treatment by administration of a transplant, such as a haematopoietic stem cell transplant.
As used herein, the term “IFNλ receptor agonist” refers to any substance, compound or molecule capable of activating an IFNλ receptor. It may activate the IFNλ receptor directly or indirectly. For clarity, the term “IFNλ receptor agonist” may also include an IFNλ receptor activator that activates the IFNλ receptor directly or indirectly. The IFNλ receptor comprises subunits IL10R2 (CRF2-4, UniProt Accession No: Q08334) and IFNλR1 (IL28RA, CRF2-12; Uniprot Accession No: Q8IU57). Activation of the IFNλ receptor triggers Janus kinase activation (Jak1 and Tyk2) and phosphorylation and activation of the transcription factors STAT1, STAT2 and STAT3, and interferon-regulated transcription factors IRFs. Upon phosphorylation, STATs translocate into the nucleus to induce hundreds of genes altogether termed IFN-stimulated genes or ISGs. Accordingly, an IFNλ receptor agonist is suitably capable of stimulating one or more of these downstream signalling events via the IFNλ receptor.
It is envisaged that the IFNλ receptor agonist or activator can be any as are known in the art. For example, the agonist/activator of IFNλ receptor may be a small molecule, an antibody or an antibody fragment, a synthekine, a peptide, a polynucleotide expressing IFNλ, an IFNλ ligand or molecule or a fragment, variant or derivative thereof. An activator of an IFNλ receptor may also be an agent that triggers an increase of endogenous IFNλ in a subject after administration of the agent to the subject.
Lambda IFNs (IFNλs), type III IFNs or IL-28/29 constitute one of the most recent additions to the interferon family (Lazear et al., 2015). They consist of four members in humans (IFNλ1/IL-29, Uniprot Accession No. Q8IU54; IFNλ2/IL-28A, Uniprot Accession No. Q8IZJ0; IFNλ3/IL-28B, Uniprot Accession No. Q8IZI9; IFNλ4, Uniprot Accession No. K9M1U5) and although a broad range of pathogens are capable of inducing IFNλs, the cellular sources of IFNλs are relatively limited and include epithelial cells, especially at mucosal interfaces, conventional and plasmacytoid dendritic cells, and monocytes.
The IFNλ receptor agonist may further encompass fragments, variants, derivatives and functional equivalents of IFNλ1/IL-29, IFNλ2/IL-28A, IFNλ3/IL-28B and IFNλ4.
By the term “variants”, substantially similar amino acid sequences of wild-type IFNλ ligands or molecules are intended. The IFNλ polypeptides in accordance with the present invention may be altered in various ways including amino acid substitutions, deletions, truncations and insertions. They may also undergo posttranslational modification. Novel proteins having properties of interest may be created by combining elements and fragments of IFNλ proteins or their receptors, as well as with other proteins. This may lead to greater stability or greater bioavailability in the GI tract, for example. Methods for such manipulations are generally known in the art. The IFNλ proteins encompass naturally occurring proteins as well as variant, truncated and modified forms or derivatives thereof.
A “fragment” is a segment, domain, portion or region of a protein, such as an IFNλ ligand or cytokine described herein, which constitutes less than 100% of the amino acid sequence of the protein. In general, fragments may comprise up to 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 200, 250, 300, 350, 400, 450, 550 or up to about 600 amino acids of an amino acid sequence. Suitably, the fragment contains or comprises a portion of an IFNλ ligand capable of binding and/or activating an IFNλ receptor.
As used herein, “derivative” proteins have been altered, for example by conjugation or complexing with other chemical moieties, by post-translational modification (e.g. phosphorylation, acetylation and the like), modification of glycosylation (e.g. adding, removing or altering glycosylation) and/or inclusion of additional amino acid sequences as would be understood in the art. In particular embodiments, the IFNλ receptor agonist comprises an IFNλ derivative that has been modified to extend the serum half-life thereof, such as by the addition of one or more PEG groups or albumin binding domains (ABD). In some embodiments, this may lead to greater stability or greater delivery and bioavailability in the GI tract, for example.
Additional amino acid sequences may include fusion partner amino acid sequences which create a fusion protein. By way of example, fusion partner amino acid sequences may assist in detection and/or purification of the isolated fusion protein. Non-limiting examples include metal-binding (e.g. polyhistidine) fusion partners, maltose binding protein (MBP), Protein A, glutathione S-transferase (GST), fluorescent protein sequences (e.g. GFP), epitope tags such as myc, FLAG and haemagglutinin tags. For example, the IFNλ receptor agonist may comprise an IFNλ ligand that is pegylated (e.g., pegylated recombinant IL29), in particular monopegylated, and/or conjugated with a polyalkyl oxide moiety. Such fragments, variants and derivatives will suitably continue to possess the desired IFNλ activity (i.e., functional variants, fragments or derivatives). By way of example, exemplary IL-29 variants and fragments are provided in US20110243888, which is incorporated by reference herein.
Other derivatives contemplated by the invention include, but are not limited to, modification to side chains, incorporation of unnatural amino acids and/or their derivatives during peptide, or protein synthesis and the use of crosslinkers and other methods which impose conformational constraints on the immunogenic proteins, fragments and variants of the invention.
In some embodiments, IFNλ receptor agonist variants or derivatives may include the IFNλ receptor agonist being in a more stabilised form or in a prodrug form, preferably suited for oral delivery or otherwise for delivery to the GI tract, for example. In some embodiments, IFNλ receptor agonist variants or derivatives may be capable of generating an orally bioavailable cytokine targeted to the GI tract. In some embodiments, the IFNλ receptor agonist variants or derivatives may be suitable for oral administration. In some embodiments, IFNλ receptor agonist variants or derivatives may include the IFNλ receptor agonist being in a prodrug form, for providing greater bioavailability of the IFNλ receptor agonist in the GI tract, for example.
Suitably, the IFNλ receptor agonist is administered to the subject as a pharmaceutical composition comprising a pharmaceutically-acceptable carrier, diluent or excipient.
By “administering” or “administration” is meant the introduction of an IFNλ receptor agonist described herein into an animal subject by a particular chosen route.
By “pharmaceutically-acceptable carrier, diluent or excipient” is meant a solid or liquid filler, diluent or encapsulating substance that may be safely used in systemic administration. Depending upon the particular route of administration, a variety of carriers, well known in the art may be used. These carriers may be selected from a group including sugars, starches, cellulose and its derivatives, malt, gelatine, talc, calcium sulfate, liposomes and other lipid-based carriers, vegetable oils, synthetic oils, polyols, alginic acid, phosphate buffered solutions, emulsifiers, isotonic saline and salts such as mineral acid salts including hydrochlorides, bromides and sulfates, organic acids such as acetates, propionates and malonates and pyrogen-free water.
A useful reference describing pharmaceutically acceptable carriers, diluents and excipients is Remington's Pharmaceutical Sciences (Mack Publishing Co. N.J. USA, 1991), which is incorporated herein by reference.
Any safe route of administration may be employed for providing a patient with the composition of the invention. For example, oral, rectal, parenteral, sublingual, buccal, intravenous, intra-articular, intra-muscular, intra-dermal, subcutaneous, inhalational, intraocular, intraperitoneal, intracerebroventricular, transdermal and the like may be employed. Intra-muscular and subcutaneous injection is appropriate, for example, for administration of immunotherapeutic compositions, proteinaceous vaccines and nucleic acid vaccines.
Dosage forms include tablets, dispersions, suspensions, injections, solutions, syrups, troches, capsules, suppositories, aerosols, transdermal patches and the like. These dosage forms may also include injecting or implanting controlled releasing devices designed specifically for this purpose or other forms of implants modified to act additionally in this fashion. Controlled release of the therapeutic agent may be effected by coating the same, for example, with hydrophobic polymers including acrylic resins, waxes, higher aliphatic alcohols, polylactic and polyglycolic acids and certain cellulose derivatives such as hydroxypropylmethyl cellulose. In addition, the controlled release may be effected by using other polymer matrices, liposomes and/or microspheres.
Compositions of the present invention suitable for oral or parenteral administration may be presented as discrete units such as capsules, sachets or tablets each containing a pre-determined amount of one or more therapeutic agents of the invention, as a powder or granules or as a solution or a suspension in an aqueous liquid, a non-aqueous liquid, an oil-in-water emulsion or a water-in-oil liquid emulsion. Such compositions may be prepared by any of the methods of pharmacy but all methods include the step of bringing into association one or more agents as described above with the carrier which constitutes one or more necessary ingredients. In general, the compositions are prepared by uniformly and intimately admixing the agents of the invention with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product into the desired presentation.
The above compositions may be administered in a manner compatible with the dosage formulation, and in such amount as is pharmaceutically-effective. The dose administered to a patient, in the context of the present invention, should be sufficient to effect a beneficial response in a patient over an appropriate period of time. The quantity of agent(s) to be administered may depend on the subject to be treated inclusive of the age, sex, weight and general health condition thereof, factors that will depend on the judgement of the practitioner.
In a further aspect, the invention provides a method of promoting survival and/or proliferation of an intestinal cell, said method including the step of contacting the intestinal cell with an IFNλ receptor agonist under conditions to promote survival and/or proliferation of the intestinal cell.
Exemplary intestinal cells include intestinal stem cells, enterocytes, goblet cells, enteroendocrine cells, Paneth cells, microfold or M cells, cup cells, Tuft cells Suitably, the intestinal cell is or comprises an intestinal stem cell (ISC). As used herein, the term “intestinal stem cell” refers to a multipotent stem cell, such as those intestinal cells that express or are positive for one or more of Lgr5, ALCAM, ASCL2, BMI1, DCLK1, EPHB2, HOPX, Igfbp4, Itgb1, Lrig1, mTert, Musashi-1, OLFM4, PHLDA1, Prom1, PW1, Smoc2, Sox9 and TNFRSF19.
It is envisaged that the present method can be performed in vitro. In this regard, the present method may be utilised to generate intestinal cells or organoids, such as intestinal stem cells or organoids thereof, that may be subsequently administered to a subject as a prophylactic or therapeutic agent for a gastrointestinal disease, disorder or condition. In other embodiments, the intestinal cells, tissues and/or organoids described herein may be useful for toxicity screening or for in vitro drug safety testing. By way of example, some drugs used for therapeutic interventions can cause unexpected toxicity in intestinal cells or tissue, often leading to significant morbidity and disability.
In alternative embodiments, the method of the present aspect can be performed in vivo. To this end, the present method relates to the promotion of survival and/or proliferation of an intestinal cell in a subject, which includes the step of administering to the subject a therapeutically effective amount of an IFNλ receptor agonist to thereby promote survival and/or proliferation of the intestinal cell therein. To this end, the IFNλ receptor agonist may be administered to a subject to promote preservation of the gastrointestinal epithelial layer and/or promote proliferation and/or regeneration thereof.
In one embodiment, the IFNλ receptor agonist is administered prior to, simultaneously with and/or subsequent to administration of a cytotoxic agent, such as those hereinbefore described, to the subject. To this end, the cytotoxic agent suitably is or comprises a chemotherapeutic agent and/or a radiotherapy.
Suitably, the subject has or is suffering from a gastrointestinal disease, disorder or condition, such as inflammatory intestinal disease, autoimmune disease, immunotherapy-induced intestinal damage, an immune deficiency, a haematological malignancy, chemotherapy-induced intestinal damage, radiation-induced intestinal damage, GVHD, and any combination thereof.
Suitably, the IFNλ receptor agonist is selected from the group consisting of IFN-λ1 (IL-29), IFN-λ2 (IL-28A), IFN-λ3 (IL-28B), IFN-λ4, including variants and derivatives thereof and any combination thereof. More particularly, the IFNλ receptor agonist suitably is or comprises recombinant IL-29, such as pegylated recombinant IL-29.
In another aspect, the invention provides a method of performing a transplant in a subject in need thereof, said method including the step of administering to the subject a therapeutically effective amount of an IFNλ receptor agonist prior to, simultaneously with and/or subsequent to administration of the transplant to the subject.
In certain embodiments, the transplant is or comprises a hematopoietic stem cell transplant, such as those described herein. In one particular embodiment, the transplant is or comprises a bone marrow transplant.
In one embodiment, the present method includes the further step of administering the transplant to the subject.
Suitably, the IFNλ receptor agonist is that described herein, such as IFN-λ1 (IL-29), IFN-λ2 (IL-28A), IFN-λ3 (IL-28B), IFN-λ4, including fragments, variants and derivatives thereof and any combination thereof. More particularly, the IFNλ receptor agonist suitably is or comprises recombinant IL-29, such as pegylated recombinant IL-29.
Suitably, the IFNλ receptor agonist and/or the transplant are administered to a subject as a pharmaceutical composition comprising a pharmaceutically-acceptable carrier, diluent or excipient. In this regard, any dosage form and route of administration, such as those provided herein, may be employed for providing a subject with the composition of the invention.
In a further aspect, the invention relates to an IFNλ receptor agonist for use in:
(a) preventing or treating a gastrointestinal disease, disorder or condition;
(b) promoting survival and/or proliferation of an intestinal cell; and/or
(c) performing a transplant;
in a subject.
In a related aspect, the invention resides in use of an IFNλ receptor agonist in the manufacture of a medicament for:
(a) preventing or treating a gastrointestinal disease, disorder or condition;
(b) promoting survival and/or proliferation of an intestinal cell; and/or
(c) performing a transplant;
in a subject.
For the two aforementioned aspects, the IFNλ receptor agonist is suitably selected from the group consisting of IFN-λ1 (IL-29), IFN-λ2 (IL-28A), IFN-λ3 (IL-28B), IFN-λ4, including fragments, variants and derivatives thereof and any combination thereof. More particularly, the IFNλ receptor agonist suitably is or comprises recombinant IL-29, such as pegylated recombinant IL-29.
Suitably, the gastrointestinal disease, disorder or condition of the two aforementioned aspects is that hereinbefore described. In one embodiment, the gastrointestinal disease, disorder or condition is selected from the group consisting of an inflammatory intestinal disease, an infection, an autoimmune disease, immunotherapy-induced intestinal damage, an immune deficiency, a haematological malignancy, chemotherapy-induced intestinal damage, radiation-induced intestinal damage, GVHD, and any combination thereof.
Suitably, the intestinal cell of the two aforementioned aspects is or comprises an intestinal stem cell, such as that described herein.
Suitably, the transplant of the two aforementioned aspects is that hereinbefore described and more particularly is or comprises a haematopoietic stem cell transplant.
With respect to the aforementioned aspects, the term “subject” includes but is not limited to mammals inclusive of humans, performance animals (such as horses, camels, greyhounds), livestock (such as cows, sheep, horses) and companion animals (such as cats and dogs). In one particular embodiment, the subject is a human.
So that preferred embodiments of the invention may be fully understood and put into practical effect, reference is made to the following non-limiting examples.
Despite recent recognition of the important and unique immunology of the gastrointestinal (GI) mucosal barrier, the factors controlling the interface between the luminal microbiome and local immune cell populations in driving lethal acute GVHD (aGVHD) remain poorly defined (Jenq et al., 2012; Simms-Waldrip et al., 2017; Zeiser et al., 2016). The manipulation of targets preferentially active at this interface constitutes a rational therapeutic strategy that avoids the detriment of broad immune suppression. Type II interferons (i.e. IFNγ) are known to be directly pathogenic to the GI tract (Burman et al., 2007). In contrast, previous work by Robb (Robb et al., 2011) and Fischer (Fischer et al., 2017) identified a protective role for type I IFNs in the GI tract, although their relative importance on the initiation of MHC class II-dependent immunity by hematopoietic antigen presenting cell (APC) versus direct effects on the GI tract remain unclear. Recently, type III IFNs (lambda interferon (IFNλ)) (Kotenko et al., 2002; Sheppard et al., 2002) are increasingly recognized as the dominant IFN subtype driving innate immune responses at mucosal barriers (Baldridge et al., 2015; Galani et al., 2017; Lin et al., 2016) that can be disrupted by GVHD. We thus sought to define the role for type III, or IFNλ in the setting of transplantation. The IFNλ receptor is a heterodimer containing a specific IFNλ receptor chain (Ifnlr1) and the IL-10RB subunit (Kotenko et al., 2002). Genes for lambda IFNs and Ifnlr1 appear to be evolutionarily conserved, with humans possessing genes for 4 ligands IFNL1 (IL-29), IFNL2 (IL-28A), IFNL3 (IL-28B) and IFNL4. Mice express IFNL2 and 3, whilst IFNL1 and IFNL4 exist as pseudogenes (Hamming et al., 2013; Lasfar, 2006; Prokunina-Olsson et al., 2013). IFNλ shares downstream signalling pathways with type I IFNs and receptor ligation triggers phosphorylation of STAT 1, 2, 3 and 5 (Dumoutier et al., 2003; Kotenko et al., 2002). However, in contrast to the ubiquitous expression of type I IFN receptors, Ifnlr1 is preferentially expressed in mucosal epithelia tissues, especially in the GI and respiratory tracts (Galani et al., 2017; Lin et al., 2016; Sommereyns et al., 2008). Compartmentalization of IFNλ cytokine effects within the GI tract, by virtue of the restricted expression of the cognate receptor, offers the potential to impact GI tract homeostasis with limited effects on systemic GVL and immunity.
The importance of lambda IFNs in mediating innate viral defence was initially demonstrated in hepatitis C infection (Ge et al., 2009), where increased rates of spontaneous viral remission and improved responses to therapy were shown in patients with polymorphisms in IFNλ gene loci (Tanaka et al., 2009). IFNλ-dependent defence from gastro-tropic viruses such as norovirus and rotavirus is apparent (Baldridge et al., 2015; Lin et al., 2016; Rocha-Pereira et al., 2018). Homeostatic and protective paracrine functions were recently described (Galani et al., 2017) and demonstrate an anti-inflammatory viral response in respiratory epithelia. While anti-inflammatory effects in other cells including neutrophils have also been demonstrated (Blazek et al., 2015; Broggi et al., 2017), the function of IFNλ in immunopathology at mucosal surfaces unrelated to viral infections is largely unexplored. In the present Example, we document that IFNλ acts as an intestinal stem cell cytoprotectant and pre-BMT recipient treatment with IFNλ preserves intestinal stem cell function and mucosal integrity thereby attenuating GVHD. Together these data support the use of IFNλ as a strategy to prevent aGVHD within the GI tract. Since peg -rIL-29 is currently in late phase III clinical trials as an adjunctive therapy to treatment for hepatitis C (Muir et al., 2014; Muir et al., 2010), a rational strategy for rapid translation to the clinic can be envisioned.
Female adult C57BL/6 (B6.WT, H-2b, CD45.2+), B6D2F1 (H-2b/d, CD45.2+) and BALB/c (H-2d, CD45.2+) mice (Mus musculus) of greater than 6 weeks of age were purchased from Animal Resources Centre, Canning Vale, Western Australia, Australia. Female adult B6.Ifnlr1−/−, B6.Ifnar1−/−, BALB/cLuc, BALB/c CD45.1, B6.TEaLuc, B6.Lgr5-EGFP-IREScreERT2, Ifnlr1−/−, NKp46Cre, Ifnlr1fl/fl, Lgr5-EGFP-IREScreERT2.Ifnlr1fl/fl and B6.IL-22−/− of greater than 6 weeks of age were bred in-house at QIMR Berghofer Medical Research Institute in a pathogen-free animal facility. B6.Inflr1−/−.Ifnar1−/− were derived from B6.Ifnlr1−/− and B6.Infar1−/− crosses. All procedures were approved by the QIMR Berghofer Medical Research Institute animal ethics committee. Mice were housed in groups of up to 6 animals per cage for experiments other than co-housing, where cages of up to 20 mice were maintained. Separately housed and co-housed strains remained in these housing arrangements post-transplant.
Animals were housed in sterile micro-isolator cages and received acidified autoclaved water and were supplied with standard chow. Water was supplemented with enrofloxacin for the first two weeks post transplantation. No enrofloxacin was supplied in experiments where fecal microbial 16S sequencing was performed. Littermates of the same gender were randomly assigned to experimental groups. Lgr5-EGFP-IREScreERT2 mice were genotyped using the following primers in standard PCR conditions (15020 mutant reverse—CTGAACTTGTGGCCGTTTAC (SEQ ID NO:1), 26840 wild type reverse—GTCTGGTCAGAATGCCCTTG (SEQ ID NO:2), 8060 Common—CTGCTCTCTGCTCCCAGTCT (SEQ ID NO:3)). Tamoxifen (1 mg/day) was administered via the intraperitoneal route for 5 days, 2 weeks before transplant to induce tamoxifen-dependent Cre recombinase under the control of the Lgr5 promotor as indicated. Ifnlr1fl/fl mice were genotyped using the following primers in standard PCR conditions forward: TCT GAC ATC CGC TCA GCA CCA A (SEQ ID NO:4), reverse: GGG CCC GCC CAA ATA TAA ACC (SEQ ID NO:5). Sample sizes based on estimates from initial and previously published results to ensure appropriate power.
Donor mice were euthanized and bone marrow isolated from pelvis, femur and tibia as required and T cell depleted using complement where indicated. T cells were isolated from spleens and purified using magnetic separation. For all transplants 5×106 T cells were introduced simultaneously with 10×106 unmanipulated bone marrow cells for GVHD inducing grafts, and bone marrow only for T cell depleted grafts. Grafts were introduced via tail vein injection 24 hours after lethal irradiation given in two divided doses; 1000 cGy for B6 recipients, 1100 cGy for B6D2F1 recipients and 950 cGy for bone marrow chimeras. GVHD was assessed clinically using established scoring systems (Cooke et al., 1996) and mice with clinical scores>6 were sacrificed in accordance with institutional guidelines. For GVL experiments 1×106 viable BCR-ABL nup98hoxA9 or MLL-AF9 transformed leukemia cells were introduced simultaneously with the graft.
For chimeric transplantation, WT or Ifnlr1−/− recipients were transplanted with bone marrow only from Ifnlr1−/− or WT donors in a syngeneic transplant establishing hematopoietic, non-hematopoietic cells, or both as unable to signal through Ifnlr1. WT to WT and Ifnlr1−/− to Ifnlr1−/− controls were included. Secondary MHC disparate transplantation with BALB/c bone marrow and T cells was then performed to evaluate post-transplant outcomes. Where necessary congenically marked BALB/c strains (CD45.1 and CD45.2) were used to allow identification of donor derived cells in mixed chimeras, or where the source of differing grafts was desired, including in in vivo cytotoxicity experiments. For NK cell depletion; anti-NK1.1 antibody derived from PK136 (ATCC® HB-191™) or IgG control was given at the dose of 1 mg on day −1 and at 0.5 mg on days +3 and +6 via the intraperitoneal route. PEG-rIL-29 treatment; 5 μg per day was delivered in 200 μL PBS via the intraperitoneal route either three days before harvest of GI tissues, or on days −2, −1 and 0 of transplantation.
Samples were collected and fixed in 10% formalin, and transferred to 70% ethanol prior to paraffin embedding. 5 μm samples were cut and stained with Hematoxylin and Eosin to allow blinded semi-quantitative GVHD scoring using a published system (Hill et al., 1997) by an experienced anatomical pathologist. Paneth cell numbers were quantitated after immunohistochemical staining with horseradish peroxidase and anti-lysozyme antibody with enumeration performed electronically using Aperio ImageScope software (Version 12.3.2.8013) and the cytoplasmic staining algorithm. Ki67 was quantitated after immunohistochemical staining with horseradish peroxidase and enumeration performed using Aperio ImageScope (Version 12.3.2.8013) IHC nuclear algorithm. Three well sectioned areas devoid of processing artefact were chosen per slide and average % of positive cells per slide depicted.
Tissues were fixed with 4% paraformaldehyde, then placed in 30% sucrose prior to being frozen in Tissue-Tek OCT compound (Sakura Finetek). Sections (7 μm thickness) were treated with Background Sniper (Biocare Medical) and 2% BSA for 30 min and then stained with anti-GFP (Abcam ab290) and Ki-67 (DakoM7248) for 120 minutes at room temperature in the dark. After washing, sections were counterstained with DAPI for 5 min and cover-slipped with Vector Vectashield Hard Set mounting media. Images were taken with ×10 non-oil lens using a Zeiss 780-NLO Point Scanning Confocal Microscope with Zen software (Zen software).
Seven days post-transplantation, mice were fasted of food and water for 4 hours prior to oral gavage with 8 mg of FITC labelled Dextran (MW 4 kDa, Sigma-Aldrich) in 200 μL of PBS. Peripheral blood was collected 4 hours later and serum separated. FITC-Dextran concentration in serum was determined using a Synergy H4 Fluorometer (Biotek) at excitation 485 nm and emission 535 nm.
Serum IL-6, IL-17A, TNF and IFNγ were measured via murine Flex Array™ sets (BD Biosciences Pharmingen, San Diego, Calif., USA) according to manufacturer's instructions. Samples were acquired on a BD LSR Fortessa and analyzed using FCAP Array™ Software (BD Biosciences).
Serum IL-28A/B and SI and colon IL28-A/B were measured using R&D Systems Mouse IL-28A/B (IFN-lambda 2/3) DuoSet ELISA on samples obtained from either serum or mucosal intestinal homogenate as per Invitrogen. Briefly the mucosa was scraped from the underlying muscle layer with a glass slide. The cells of the mucosa were lysed with Tris EDTA (10 mM Tris-HCl, and 1 mM EDTA, pH 7.4) containing 0.05% sodium azide, 1% Tween-80, 2 mM PMSF, and 1 microgram per milliliter of each of the following protease inhibitors: aprotinin, leupeptin, and pepstatin A. The mucosa was homogenized (20 s). The homogenate was centrifuged (11,000×g, 10 minutes at 4° C.). The supernatant was collected. The supernatant was filtered (4.5 micron filter).
RT-qPCR was performed on RNA isolated from tissues isolated from naïve mice and mice post-transplant. Tissues were frozen in 500 μL Trizol and then mechanically homogenated. RNA was then extracted using QIAGEN RNeasy micro kit, was converted to cDNA and PCR performed using Taqman reagents. For Ifnlr1 Taqman Gene Expression Assay SM Mm00558035_m1 was used and for housekeeping SM Mm03024075_m1 (Hprt). For Reg3b, Reg3g, LysP and GAPDH primers were used with Sybr Green Supermix using standard PCR conditions on an ABI ViiA7 PCR machine. The primers used were; Reg3b F ACTCCCTGAAGAATATACCCT (SEQ ID NO:6) R GCTATTGAGCACAGARACGAG (SEQ ID NO:7), Reg3g F ATGCTTCCCCGTATAACCATCA (SEQ ID NO:8) R GGCCATATCTGCATCATACCAG (SEQ ID NO:9), Lysp F ATGGCTACCGTCGRGRCAAG (SEQ ID NO:10) R CGGTCTCCACGGTTGTAGTT (SEQ ID NO:11) and Gapdh F GACATGCCGCCTGGAGAAAC (SEQ ID NO:12) R AGCCCAGGATGCCCTTAGT (SEQ ID NO:13), all from Sigma Aldrich.
Expansion of luciferase expressing T cells was quantitated through measurement of luciferin-luciferase signal intensity using the Xenogen imaging system (Xenogen IVIS 100; Caliper Life Sciences, CA., USA). Fur on the ventral surfaces was shaved and mice were injected with 500 μg of luciferin subcutaneously and imaged 5 minutes later under continuous isoflurane-based anesthesia. After total body imaging mice were again injected with luciferin and then euthanized and single organs were isolated and imaged. For assessment of donor T cell expansion after transplant, BALB/cLuc T cells were given at time of BMT. For assessment of APC function, expansion of TEaLuc T cells in response to selected antigen presenting cells was performed at day 15 post-transplantation, with T cells given on day 12 post-transplant.
Single cell organ suspensions were prepared and all FACS enumeration was performed on a BD LSR Fortessa flow cytometer (BD Biosciences) unless otherwise specified. Post transplantation samples were incubated with 50 μL 24.G2 antibody for 5 minutes at room temperature to block Fc receptors. Samples and antibodies were incubated for 15 minutes at 4° C. in 50 μL of PBS containing 2% FBS. Proliferation assays were performed on T cells labelled with CFSE prior to transplantation. Cells were stained in 0.001 mM CFSE at 37° C. for 10 minutes, then washed and re-infused during BMT. Apoptosis assays were performed using BD Pharmingen Annexin V Apoptosis detection kit. Intracellular cytokine expression was measured after stimulation with PMA (5 μg/mL) and ionomycin (50 μg/mL) (Sigma-Aldrich) for 4 hours at 37° C. with Brefeldin A (BioLegend) included in the last 3 hours of culture. Cells were surfaced labelled, fixed and permeabilized (BD Biosciences—Cytofix/Cytoperm Kit) and stained with cytokine specific antibodies. Cell sorting was performed using a FACSAria II or III. Data were processed using FlowJo version 10 (FlowJo LLC). Detail of antibodies used is included in the Key Resources Table.
For isolation and separation of cells from GI tract prior to FACS staining and/or sort separation the MACS Miltenyi Biotec Lamina Propria Dissociation Kit was used as per the manufacturer's instructions.
BALB/c T cells were isolated from spleen and purified by magnetic bead selection. WT or Ifnlr1−/− DC were isolated from spleen via density gradient and further purified by magnetic bead selection. Peritoneal macrophages were obtained by lavage. DC and macrophages were irradiated with 2100 cGy. Serial dilutions (20,000, 10,000, 5,000 and 0) of stimulator DC APC or 100,000 or 0 macrophage APC were plated with either CD4+ or CD8+ T cells at 200,000 cells per well in triplicate. After 4 days of culture, 100 μL per well of 1:1000 3H-thymidine was added. 18 hours later proportionate inclusion of 3H-thymidine was measured scintigraphically.
DNA was extracted from 50-100 mg of fecal material using an initial bead beating step followed by extraction using the Maxwell 16 Research Instrument (Promega, USA) according to the manufacturer's protocol with the Maxwell 16 Tissue DNA Kit (Promega, USA). DNA concentration was measured using a Qubit assay (Life Technologies, USA) and was adjusted to a concentration of 5 ng/μl. The 16S rRNA gene encompassing the V6 to V8 regions was targeted using the 803F (5′-AAACTYAAAKGAATTGRCGG-3′ (SEQ ID NO:14)) and 1392R (5′-ACGGGCGGTGWGTRC-3′ (SEQ ID NO:15)) primers (Kunin et al., 2010) modified to contain Illumina specific adapter sequence (803F :5′ TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGTTAGAKACCCBNGTAGTC 3′ (SEQ ID NO:16) and 1392wR:5′GTCTCGTGGGCTCGGGTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG ACGGGCGGTGWGTRC3′ (SEQ ID NO:17)). Preparation of the 16S library was performed as described, using the workflow outlined by Illumina (#15044223 Rev. B). In the first stage, PCR products of ˜466 bp were amplified according to the specified workflow with an alteration in polymerase used to substitute Q5 Hot Start High-Fidelity 2X Master Mix (New England Biolabs, USA) in standard PCR conditions. Resulting PCR amplicons were purified using Agencourt AMPure XP beads (Beckman Coulter, USA). Purified DNA was indexed with unique 8 bp barcodes using the Illumina Nextera XT 384 sample Index Kit A-D (#FC-131-1002, Illumina, USA) in standard PCR conditions with Q5 Hot Start High-Fidelity 2X Master Mix. Indexed amplicons were pooled together in equimolar concentrations and sequenced on the MiSeq Sequencing System (Illumina, USA) using paired end sequencing with V3 300 bp chemistry at the Australian Centre for Ecogenomics according to manufacturer's protocol. Heat map includes OTUs identified as significantly different (p<0.001) between separately housed WT and Ifnlr1−/− at week 4 where OTU relative abundance exceeds 2% in at least one sample. Each column includes scaled read counts for one mouse. Read counts normalized using metagenomeSeq.
Organoids were grown from crypt preparations harvested from small intestine or colon of naïve WT or Ifnlr1−/− mice around 6-8 weeks of age. Crypt preparations were isolated following a recognized method (Miyoshi and Stappenbeck, 2013) with growth media derived from L-WRN (ATCC® CRL-3276™) cell line (ATCC). 50 viable crypts per well were plated in triplicate and numbers of growing, viable GI organoids were assessed at day 5 of culture. Four representative fields per well were counted at 10× Obj using the Evos FL inverted microscope and the average number from 4 fields of each of 3 replicate cultures for an individual crypt donor is reported. Images were recorded to allow quantitation of maximal cross-sectional area using Image J (Wayne Rasband, NIH). Organoids were also grown from FACS sorted single cell preparations of Lgr5+ cells obtained by a modification of the crypt isolation method used previously in order to generate a single cell suspension. Fifty Lgr5+ cells were plated per well in triplicate. Numbers of growing organoids from the entire well were counted to allow assessment of growth efficiency. Organoids were imaged and surface area calculated as above.
Single cells were isolated per the MACS Miltenyi Biotec Lamina Propria dissociation kit. Cells were stained with 7AAD, CD45.2 and EpCAM and sorted per GFP expression into Lgr5+ and Lgr5− populations. Sorting was performed on a BD FACSAria III cell sorter. TRIzol was added to single cell suspensions and cryopreserved at −80° C. RNA was subsequently extracted after a second chloroform extraction step using QIAGEN RNeasy Micro Kit. RNA libraries were prepared using the NEBnext Ultra RNA Library Prep Kit for Illumina (New England Biolabs), assessed for size and quantified using the 2100 Bioanalyzer (Agilent Technologies) and Qubit fluorometer (Thermofischer Scientific). Libraries were sequenced using high output single-end 75 cycle sequencing kits (version 2) on the Illumina Nextseq 550 platform. Sequence reads in each fastq file were trimmed for adapter sequences using Cutadapt (Martin, 2011b) (version 1.11) and aligned using STAR (Dobin et al., 2013) (version 2.5.2a) to the mm19 assembly with the gene, transcript, and exon features of Ensembl (release 75) gene model. Expression was estimated using RSEM (Li and Dewey, 2011) (version 1.2.30) and was used as input to assess differential gene expression between groups.
Survival curves were plotted using Kaplan-Meier estimates and compared by log-rank analysis. The parametric nonparametric Mann-Whitney U test (two-sided) was used for comparison between two groups and 1 way ANOVA with Tukey's multiple comparison test was used for comparison between two or more groups for statistical analysis of data. Data are presented as mean±standard error of the mean (SEM). Calculations were performed using Prism 7 for windows software (GraphPad).
Sequence analysis 16S microbial sequencing. Reads were cleaned of adapter sequences using Cutadapt (Martin, 2011a) and trimmed using Trimmomatic (Bolger et al., 2014) employing a sliding window of 4 bases with an average base quality above 15, followed by hard-trimming to 250 bases with exclusion of reads less than this length. Remaining forward reads were processed following the QIIME2 workflow (https://qiime2.org) (Caporaso et al., 2010) using DADA2 (Callahan et al., 2016) to de-noise sequences. Taxonomy assignment was performed on amplicon sequence variants using BLAST (Altschul et al., 1990) against the SILVA (Quast et al., 2013) reference database version 132. Differential abundance analysis was performed on raw read counts using DESeq2 (Love et al., 2014). Counts were normalized prior to principal component analysis (PCA) and heat map visualization using cumulative sum scaling implemented within metagenomeSeq (Paulson et al., 2013). PCA was performed using the rda function within the vegan R package (Oksanen). Heat maps were generated using pheatmap (Kolde).
Intestinal cell differential gene expression was determined using the edgeR package (Robinson et al., 2010) within R v3.3.4 and significance defined as p<0.05 after Benjamini-Hochberg false discovery rate correction. Pathway analysis was performed by single sample gene set variation analysis via the GSVA package (Hanzelmann et al., 2013) using KEGG, BioCarta, Reactome and Gene Ontology (GO) pathway databases and significance defined as p<0.05 and only gene sets between 25-500 genes considered. Heat maps were generated using heatmap.2 function in gplots 3.0.1 R package. Gene set enrichment analysis (GSEA) was performed using GSEA 3.0 (Mootha et al., 2003; Subramanian et al., 2005). Canonical Pathway enrichment analysis for differentially expressed genes (log 2 Fold-change>|0.58| and adj. p-value<0.05) across PEG-IL29-treated Lgr5+ and Lgr5− samples relative to genotype-matched PBS-treated samples was done using Ingenuity Pathway Analysis (IPA) (Kramer et al., 2014). IPA function enrichment was calculated using a right-tailed Fisher exact test with a threshold of significance set at P value of 0.05. Inferences in the significant activation state (z-score>|2|) of canonical pathways, upstream regulatory transcriptional regulators, cytokines and kinases were done using IPA. Positive z-scores reflect a predicted activation state, while negative z-scores reflect the inhibition of upstream regulatory activity.
Both open source and commercially available software was used in this study as described in context within the methods. RNAseq data from Lgr5− and Lgr5+ cells derived from PBS treated and PEG-rIL-29 treated mice has been deposited under the NCBI GEO profile accession number: GEO:pending.
We utilized B6.Ifnlr1−/− mice (henceforth referred to as Ifnlr1−/−) that lack expression of the unique IFNλ receptor heterodimeric subunit Ifnlr1, rendering them unable to respond to IFNλ. These mice do not display any obvious developmental abnormalities in GI function including weight and histological appearance (
To define whether the protective role of Ifnlr1 signaling is within cells that initiate GVHD (i.e. hematopoietic) or within GVHD targets (non-hematopoietic tissue), we generated bone marrow chimeras lacking Ifnlr1 in hematopoietic and/or non-hematopoietic tissues as previously described (Burman et al., 2007; Robb et al., 2011) (
We next sought to determine the IFNλ-responsive hematopoietic cell that attenuates aGVHD and the inflammatory cytokine phenotype observed. Initial characterization of immune cell lineages revealed no differences in either myeloid or lymphoid subset frequencies and absolute numbers in Ifnlr1−/− mice (data not shown). Interrogation of the donor T cell response after BMT revealed enhanced expansion of CD4+ and CD8+ cells in Ifnlr1−/− relative to WT recipients. This was evident by measurement of total T cell expansion via quantification of donor T cell-derived bioluminescence in vivo (
To determine whether APC function was enhanced by IFNλ, we assessed T cell responses to WT or Ifnlr1−/− dendritic cells (DC) or macrophages in mixed lymphocyte cultures. No difference was seen between CD4+ and CD8+ T cell expansion when stimulated by WT or Ifnlr1−/− APC (
Assessment of T cell function demonstrated no increase in the proportion of donor CD4+ or CD8+ T cells that were making IFNγ (
Given the importance of IFNλ in mucosal immunity and the increasing recognition of the GI tract microbiome in determining transplant outcomes (Teshima et al., 2016; Varelias et al., 2017; Zeiser et al., 2016), we evaluated the microbiota in WT vs. Ifnlr1−/− mice by 16S ribosomal sequencing. Principal component analysis of the microbiota in the two trains was significantly different at baseline but coalesced after 4 weeks of co-housing (
Considering alteration in microbial defenses as a factor determining post-transplant outcomes in Ifnlr1−/− mice, we also evaluated Paneth cells. Paneth cells synthesize antimicrobial peptides, have been implicated in the maintenance of the GI stem cell niche (Sato et al., 2011) and are reduced in number during aGVHD (Eriguchi et al., 2012; Levine et al., 2013). We observed equivalent numbers of Paneth cells after BMT in WT and Ifnlr1−/− recipients, suggesting they are unlikely to be involved in the protective effect of IFNλ within the GI tract (
Given the protective role for Ifnlr1 signaling in prevention of early acute GI GVHD seen in our transplantation models, we sought to test the effect of IFNλ on GI epithelial growth. An Ifnlr1 ligand, PEGylated recombinant IL-29 (PEG-rIL-29), has been developed and tested in phase I-III clinical trials in humans as adjunctive therapy to antiviral agents for the treatment of Hepatitis C virus (Muir et al., 2014; Muir et al., 2010; Nelson et al., 2017). It is known to be cross-reactive with the murine Ifnlr1 (Souza-Fonseca-Guimaraes et al., 2015) and has an estimated in vivo half-life of 50-80 hours (Muir et al., 2010). Initial ability of PEG-rIL-29 to support the GI stem cell compartment was assessed through generation of colon organoids from WT mice receiving either PEG-rIL-29 or PBS for three days prior to crypt harvest. Increased numbers of colon organoids were generated in the primary growth phase from mice receiving treatment with PEG-rIL-29 compared to those receiving PBS (
To test the capacity of IFNλ to prevent GVHD in vivo, recipient mice were treated with PEG-rIL-29 from day −2 to day 0 prior to transplantation. We tested both the BALB/c→B6 and B6→B6D2F1 systems, the latter to confirm results in the absence of T cell-mediated graft rejection. In both models, recipients receiving PEG-rIL-29 had prolonged survival (
Given the observed proliferative effects in intestinal stem cells seen in our in vitro experiments, we examined the proliferation of epithelia within the GI tract after BMT. A significantly greater proportion of both colon and small intestinal epithelial cells, inclusive of the crypt base site of stem cell localization, were Ki67 positive, consistent with the ability of pre-transplant PEG-rIL-29 administration to drive epithelial proliferation after BMT (
Finally, we sought to exclude detrimental effects of PEG-rIL-29 treatment on GVL. PEG-rIL-29 treatment of allograft recipients of GFP expressing BCR-ABL nup98HoxA9 AML did not influence leukemia growth or death due to leukemia relative to controls receiving PBS (
Top 25 differentially expressed genes in ISC treated in vivo with PEG-rIL-29. RNA sequencing from sort purified single colonic stem cells (LGR5+) derived from either rIL-29 or PBS treated mice (n=5 mice per group). Log FC=the log 2 transformed fold change obtained from edgeR analyses. FDR (false discovery rate)=the Benjamini-Hochberg (FDR) adjusted P value obtained from edgeR analyses.
Top 25 differentially expressed genes in intestinal epithelial cells treated in vivo with PEG-rIL-29. RNA sequencing from sort purified single colonic epithelial cells (LGR5−) derived from either rIL-29 or PBS treated mice (n=5 mice per group). Log FC=the log 2 transformed fold change obtained from edgeR analyses. FDR (false discovery rate)=the Benjamini-Hochberg (FDR) adjusted P value obtained from edgeR analyses.
Here we demonstrate that IFNλ plays a dual role in preventing acute GI GVHD following murine allotransplantation through both hematopoietic and non-hematopoietic effects in recipient tissues. In the hematopoietic compartment we observed significantly impaired NK cell function in the absence of Ifnlr1 signaling, resulting in uncontrolled donor T cell expansion and subsequent recipient tissue damage. In concert with this, we found IFNλ responses in the non-hematopoietic compartment are vital for the maintenance of intestinal epithelial barrier function and regeneration after transplant. Critically, we found these protective effects can be successfully exploited therapeutically, such that PEG-rIL-29 administration prior to BMT enhanced intestinal stem cell function after transplant and attenuated aGVHD in the GI tract.
IFNλ is a key effector cytokine of mucosal immunity, with most data to date showing a unique and non-redundant role in immune defense at mucosal barriers (Baldridge et al., 2015; Ferguson et al., 2019; Lin et al., 2016; Mordstein et al., 2010; Pott et al., 2011; Selvakumar et al., 2017). This is contextually important in understanding the role we have demonstrated for IFNλ in the pathophysiology of aGVHD, an immunopathology with a predilection for damage at sites including the GI tract. Whilst responses to IFNλ are described in immune cell populations including NK cells, DC and neutrophils (Blazek et al., 2015; Broggi et al., 2017; Koltsida et al., 2011; Souza-Fonseca-Guimaraes et al., 2015; Zanoni et al., 2017), it is clear that IFNλ receptors are predominantly expressed on tissues of epithelial origin (Pott and Stockinger, 2017). This contributes to the tissue-specific activity of type III IFNs in relation to innate anti-viral effects (Baldridge et al., 2015; Galani et al., 2017; Hernandez et al., 2015; Lin et al., 2016; Pott et al., 2011; Rocha-Pereira et al., 2018). Thus, GI tract and respiratory tissues appear to mediate direct IFNλ-dependent anti-viral responses first, followed by the induction of additional innate immunological responses at high levels of viral replication or in response to alternate TLR ligand binding and preservation of GI barrier function (Odendall et al., 2017b; Ramos et al., 2019). IFNλ also induces a specific transcriptional profile after receptor ligand binding (Forero et al., 2019). In our model, we noted IFNλ-dependent recipient NK responses controlled the rate of engraftment and systemic inflammatory cytokine generation. Enhanced engraftment of donor T cells compounds conditioning-induced local tissue damage in the GI tract, linking the hematopoietic and non-hematopoietic effects of IFNλ in aGVHD, similar to that in models of viral infection (Ye et al., 2019). Our data support a model where IFNλ is important locally at epithelial sites, and in influencing systemic immune responses specifically through NK and GI stem cell compartments. These dual roles inform the role IFNλ has in specific anti-viral immunity, for example hepatitis C virus, where it is recognized to increase both the rate of spontaneous viral remission and viral clearance in the context of anti-viral treatment for HCV (Aka et al., 2014; Akkarathamrongsin et al., 2014; Ge et al., 2009; Marcello et al., 2006; Robek et al., 2005).
Our data support a specific protective effect within GI epithelia independent of immune-driven systemic inflammation. In the absence of robust and effective immunological measures to separate GVHD and GVL, protecting key tissue targets has been suggested as an effective means by which to attain this goal (Wu and Reddy, 2017). IFNλ treatment was able to directly improve proliferation and regenerative capacity of Lgr5+ stem cells. The GI tract however is increasingly recognized as a crossroads of immunity with the balance of local pathogen control exerted by resident immune cell populations, and immune responses within mucosal cell populations also influenced by the luminal microbial contents and the immunomodulatory products they produce (Jenq et al., 2012; Taur et al., 2014). As an immunopathology, GVHD (and transplantation) causes disruption to the integrity of the epithelial barrier, and preservation of this barrier is critical to the prevention of GVHD lethality (Koyama et al., 2015; Koyama and Hill, 2016). IFNλ appears to be beneficial in providing protection in this setting through multiple mechanisms in our models. Lack of Ifnlr1 does not result in pathological dysbiosis, nor apparent reduction in innate antimicrobial defenses mediated by Paneth cells. Ifnlr 1 mediates maintenance of GI barrier integrity and RNAseq suggests potential for direct immune-shielding in addition to the proliferative effects seen functionally in the epithelial compartment. In-vivo PEG-rIL-29 treatment of mice resulted in the regulation by Lgr5+ stem cells of pathways of antigen presentation and T cell mediated immunity (
Our findings are in contrast with a recent study that did not identify significant IFNλ-mediated effects on mortality after BMT (Fischer et al., 2019). Unfortunately, the absence of specific data from this prior study surrounding clinical signs of GVHD, tissue pathology, as well as a range of other methodologies reported here, make these findings difficult to compare. Importantly, there were key differences between the studies with regard to transplant model systems, therapeutic products and treatment strategies that influence GVHD kinetics, GVHD severity and treatment exposure, which are highly likely account for this disparity.
The ability to shield the GI tract from GVHD damage has been a holy grail in transplantation for some time (Hill and Ferrara, 2000) but has yet to come to clinical fruition. IL-22 secretion from innate lymphoid cells has recently been shown to protect the Lgr5+ intestinal stem cell compartment from aGVHD (Hanash et al., 2012; Lindemans et al., 2015) including through secretion of antimicrobial peptides (Ferrara et al., 2011; Zhao et al., 2018). However, IL-22 has also been shown to mediate cutaneous GVHD (Gartlan et al., 2018) and so treatment with this cytokine may have undesired effects. In contrast, treatment with IFNλ does not appear to cause pathology within the hematopoietic compartment, specifically there does not appear to be effects on antigen presenting cells or alterations to T cell function that are likely to impair GVL or pathogen-specific immunity. In contrast, improving NK cell responses could arguably improve this component of the GVL response, particularly in patients receiving T cell depleted grafts where this pathway is maximally active and conditioning intensity is often very high, causing significant GI tract toxicity (Bunting et al., 2017).
Manipulation of cytokine signaling has a generated significant interest as a method to interfere with immune-mediated pathologies, where GVHD represents an extreme example (Hanash et al., 2012; Kennedy et al., 2014; Kleist et al., 2011; Koreth et al., 2011). To date cytoprotection within the GI tract has been explored in relation to a number of cytokines including IL-11 and keratinocyte growth factor (Hill et al., 1998; Krijanov ski et al., 1999; Panoskaltsis-Mortari et al., 1998; Teshima et al., 1999). The latter has shown clinical activity in relation to limiting the severity of mucositis in chemoradiotherapy-conditioned allotransplant patients but this has not translated to the attenuation of aGVHD (Blazar et al., 2006; Hill and Ferrara, 2000). More recently, IL-22 has shown to be capable of preserving Paneth cell numbers and the stem cell niche to attenuate aGVHD in the GI tract (Hanash et al., 2012; Lindemans et al., 2015). Importantly, the effects mediated by IFNλ are independent of IL-22. Most recently, recipient-derived IL-17A has been shown to be important in preventing dysbiosis and maintaining intestinal barrier function after BMT, an effect at least partially mediated by MALT cells (Varelias et al., 2018; Varelias et al., 2017). Thus a number of cytokines in the IFN and IL-17/IL-22 family appear important and clinically tractable to enhance intestinal barrier function (Ferguson et al., 2019; Odendall et al., 2017a). The availability of clinical grade IFNλ with an established safety profile makes our findings rapidly translatable and testable in the clinic.
There is clear potential for the use of IFNλ outside of BMT, particularly in other immunopathologies that cause specific damage to GI epithelia such as inflammatory bowel disease (Pott and Stockinger, 2017; Vlachiotis and Andreakos, 2019). Regeneration of epithelial components in ulcerative colitis would rationally limit morbidity and the link to disease severity and luminal microbial components has also been made in Crohn's disease (Gevers et al., 2014; Manichanh et al., 2006). Reduction of morbidity associated with therapeutic radiation of fields encompassing the GI tract is an additional logical application for the effects seen in our experiments. Similarly, specific GI tract protection from damage as a consequence of intensive chemotherapy regimens, such as those during induction therapy for acute myeloid leukemia is also testable. Our data provides a sound basis for the therapeutic exploitation of IFNλ mediated protection of the GI epithelia in allogeneic BMT that avoids off target effects on leukemia and pathogen-specific immunity.
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| Number | Date | Country | Kind |
|---|---|---|---|
| 2019904873 | Dec 2019 | AU | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/AU2020/051389 | 12/18/2020 | WO |
