Use of IL-27 antagonists for the treatment of EBV-driven B-lymphoproliferative disorders

Abstract

We found that upon EBV infection, IL-27 is produced by infected B lymphocytes and that the IL27RA-IL-27 interaction is required for the in vitro maintenance and expansion of EBV-transformed B cells, which may explain the favorable outcome of EBV viral disease in IL27RA-deficient patients. Additionally, we identified neutralizing anti-IL27 autoantibodies in individuals who developed sporadic infectious mononucleosis (thus potentially phenocopying IL27RA deficiency). Collectively, these results demonstrate a critical role for the IL27-IL27RA axis in immunity to EBV, but also demonstrate EBV's hijacking of this defense to promote the expansion of infected cells. Therefore, IL27-IL27RA may represent a novel therapeutic target for abrogating EBV-driven B lymphoproliferative disorders.

Description

[Technical Field]

[0001] The present invention is in the field of medicine, particularly oncology. [Background technology]

[0002] Epstein-Barr virus (EBV) is a gamma-herpesvirus that infects a large proportion of humans and has a pronounced tropism for B lymphocytes. Importantly, EBV is known to be one of the most potent inducers of innately uncontrolled B cell proliferation and lymphoma formation. Rare genetic disorders specifically predispose to the miscontrol of EBV infection, leading to lymphoproliferative disorders (LPDs), such as virus-associated hemophagocytic syndrome, Hodgkin's lymphoma, and non-Hodgkin's lymphoma. Selective vulnerability to EBV has been reported in association with inherited mutations that impair T cell immunity to EBV (Tangye, SG & Latour, S. Primary immunodeficiencies reveal the molecular requirements for effective host defense against EBV infection. Blood 135, 644-655, doi:10.1182 / blood.2019000928 (2020)). Investigations into these primary immunodeficiencies have revealed crucial pathways involved in the T cell response to EBV-infected B lymphocytes, and more generally in T cell function. In healthy individuals, the efficiency of the immune response to EBV is in fact dependent on the expression of specific CD8 T cells that eliminate EBV-infected B cells. + This is primarily due to the large expansion of cytotoxic T cells. For example, CTPS1, SH2D1A, and MAGT1 deficiency results in a significant increase in CD8 T cells against EBV-infected B lymphocytes. + T cell responses include cell-mediated cytotoxicity and / or specific cytotoxic CD8 +T cell proliferation is impaired as a result of any defect. IL-27 is a two-chain cytokine consisting of the EB13 and IL-27p28 subunits belonging to the IL-12 family, and signals through its heterodimeric receptor consisting of the gp130 and IL-27 receptor alpha (WSX-1) subunits. Several lines of evidence have shown that IL-27 has potent antitumor activity associated with the induction of tumor-specific Th1 and cytotoxic T lymphocyte (CTL) responses. However, the role of IL-27 in EBV infection susceptibility and progression to lymphoma has not been investigated. Summary of the Invention

[0003] The present invention is defined by the claims. In particular, the present invention relates to the use of IL-27 antagonists for the treatment of EBV-driven B lymphoproliferative disorders.

[0004] Detailed Description of the Invention Epstein-Barr virus infection can cause severe B-cell lymphoproliferative disorders in immunocompromised individuals 1,2 In immunocompetent individuals, primary infection is often asymptomatic or leads to infectious mononucleosis, a self-limiting lymphoproliferative disorder. Selective vulnerability to EBV has been reported in association with inherited mutations that impair T-cell immunity to EBV. 3 Herein, we report biallelic loss-of-function mutations in IL-27RA in humans that cause acute and severe primary EBV infection but have a favorable spontaneous outcome. IL-27RA encodes the α subunit of the receptor for IL-27. 4,5 In the absence of IL27RA, STAT1 and STAT3 phosphorylation in response to IL-27 is abolished in patient T cells. IL-27 exerts a synergistic effect on TCR-dependent proliferation of T cells. 6This is lost in patient cells, leading to a reduced expansion of potent anti-EBV effector cytotoxic CD8+ cells. We found that upon EBV infection, IL-27 is produced by infected B lymphocytes, and that the IL27RA-IL-27 interaction is required for the in vitro maintenance and expansion of EBV-transformed B cells, which may explain the favorable outcome of EBV viral disease in IL27RA-deficient patients. Additionally, we identified neutralizing anti-IL27 autoantibodies in individuals who developed sporadic infectious mononucleosis (thus potentially phenocopying IL27RA deficiency). Collectively, these results demonstrate a critical role for the IL27-IL27RA axis in immunity to EBV, but also demonstrate EBV's hijacking of this defense to promote the expansion of infected cells. Therefore, IL27-IL27RA may represent a novel therapeutic target to abrogate EBV-driven B lymphoproliferative disorders. DETAILED DESCRIPTION OF THE INVENTION

[0005] A first object of the present invention relates to a method for treating an EBV-driven B-lymphoproliferative disorder in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of an IL-27 antagonist.

[0006] As used herein, the term "B-lymphoproliferative disorder" includes any type of leukemia or lymphoma of B cells. The term "B-cell lymphoma" refers to cancer that arises in cells of the lymphatic system from B cells. B cells are white blood cells that develop in the bone marrow and produce antibodies. They are also known as B lymphocytes. B-cell malignancies include non-Hodgkin's lymphoma, Burkitt's lymphoma, small lymphocytic lymphoma, primary effusion lymphoma, diffuse large B-cell lymphoma, splenic marginal zone lymphoma, MALT (mucosa-associated lymphoid tissue) lymphoma, hairy cell leukemia, chronic lymphocytic leukemia, B-cell prolymphocytic leukemia, B-cell lymphomas (e.g., Hodgkin's disease, B-cell non-Hodgkin's lymphoma (NHL) and various forms of related lymphomas (e.g., Waldenstrom's macroglobulinemia (also called lymphoplasmacytic lymphoma or immunocytoma) or central nervous system lymphoma), leukemias (e.g., acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL; also called B-cell chronic lymphocytic leukemia, BCLL), hairy cell leukemia, and chronic lymphocytic leukemia (CLL). leukemia), and myeloma (e.g., multiple myeloma). Additional B-cell malignancies include, but are not limited to, small lymphocytic lymphoma, B-cell prolymphocytic leukemia, lymphoplasmacytic lymphoma, splenic marginal zone lymphoma, plasma cell myeloma, isolated plasmacytoma of bone, extraskeletal plasmacytoma, extranodal marginal zone B-cell lymphoma of mucosa-associated lymphoid tissue (MALT), nodal marginal zone B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, diffuse large B-cell lymphoma, mediastinal (thymic) large B-cell lymphoma, intravascular large B-cell lymphoma, primary effusion lymphoma, Burkitt's lymphoma / leukemia, gray zone lymphoma, B-cell hyperplasia of unspecified grade, lymphomatoid granulomatosis, and post-transplant lymphoproliferative disorder.

[0007] As used herein, the term "EBV-driven B lymphoproliferative disease" refers to a lymphoproliferative disease caused by the Epstein-Barr virus (EBV).

[0008] As used herein, the term "treatment" or "treating" refers to both prophylactic or preventative treatment and curative or disease-modifying treatment, including treatment of patients at risk of or suspected of having a disease, as well as patients who are ill or have been diagnosed with a disease or medical condition, including the suppression of clinical recurrence. Treatment can be administered to a subject with a medical disorder or who may ultimately suffer from a disorder to prevent, cure, delay, reduce the severity of, or ameliorate one or more symptoms of a disorder or relapsing disorder, or to extend the subject's survival beyond that expected in the absence of such treatment. "Therapeutic regimen" refers to a pattern of disease treatment, e.g., a pattern of medication used during therapy. Therapeutic regimens can include induction regimens and maintenance regimens. The phrase "induction regimen" or "induction period" refers to a therapeutic regimen (or portion of a therapeutic regimen) used in the initial treatment of a disease. The general goal of an induction regimen is to provide high levels of drug to the patient during the initial period of the treatment regimen. The induction regimen may employ (in part or in whole) a "loading regimen," which may involve administering a higher dose of drug than the physician would employ during the maintenance regimen, administering the drug more frequently than the physician would administer the drug during the maintenance regimen, or both. The phrase "maintenance regimen" or "maintenance period" refers to a treatment regimen (or a portion of a treatment regimen) used to maintain a patient during disease treatment, for example, to keep a subject in remission for an extended period of time (months or years). A maintenance regimen may employ continuous therapy (e.g., administering a drug at regular intervals, such as weekly, monthly, or yearly) or intermittent therapy (e.g., intermittent treatment, intermittent treatment, treatment upon relapse, or treatment upon achievement of certain predetermined criteria (e.g., disease symptoms, etc.)).

[0009] As used herein, the term "IL-27" has its general meaning in the art and refers to a heterodimeric cytokine comprising the subunits p28 and EBI3.

[0010] As used herein, the term "p28" refers to interleukin-27 subunit alpha. The term is also known as IL-27-A; IL27-A, or interleukin-30. An exemplary amino acid sequence for p28 is represented by SEQ ID NO:1.

[0011] [ka]

[0012] As used herein, the term "EBI3" refers to interleukin-27 subunit beta. The term is also known as IL-27 subunit beta; IL-27B, or Epstein-Barr virus-inducible gene 3 protein (EBV-inducible gene 3 protein). An exemplary amino acid sequence for EBI3 is represented by SEQ ID NO:2.

[0013] [ka]

[0014] As used herein, the term "IL-27 receptor" or "IL-27R" refers to a heterodimeric receptor comprising the IL-27 receptor, the alpha subunit, and gp130.

[0015] As used herein, the term "IL-27RA" has its common meaning in the art and refers to interleukin-27 receptor subunit alpha. The term is also known as IL-27R subunit alpha; IL-27R-alpha; WSX-1, cytokine receptor-like 1, type I T-cell cytokine receptor (TCCR), or ZcytoR1. An exemplary amino acid sequence for IL-27RA is represented by SEQ ID NO:3. In particular, the extracellular domain of IL-27RA spans from amino acid residue 33 to amino acid residue 516 in SEQ ID NO:3.

[0016] [ka]

[0017] As used herein, the term "gp130" has its common meaning in the art and refers to interleukin-6 receptor subunit beta. The term is also known as IL-6 receptor subunit beta; IL-6R subunit beta; IL-6R-beta; IL-6RB, CDw130, interleukin-6 signal transducer, membrane glycoprotein 130, oncostatin-M receptor subunit alpha, or CD130. An exemplary amino acid sequence for gp130 is represented by SEQ ID NO:4. The extracellular domain of gp130 spans from amino acid residue 23 to amino acid residue 619 in SEQ ID NO:4.

[0018] [ka]

[0019] As used herein, the term "IL-27 activity" or "biological activity of IL-27" includes any biological effect of IL-27. In some embodiments, IL-27 activity includes the ability of IL-27 to interact with or bind to a substrate or receptor. In some embodiments, the biological activity of IL-27 is the ability of IL-27 to stimulate the JAK / STAT pathway, primarily STAT1 and STAT3 phosphorylation. In some embodiments, the biological activity of IL-27 includes any biological activity that results from IL-27-mediated signaling, particularly in EBV-infected cells such as B cells.

[0020] As used herein, the term "antagonist" is used in the broadest sense and includes any molecule that partially or fully inhibits or neutralizes the biological activity of a polypeptide such as IL-27, or partially or fully inhibits the transcription or translation of a nucleic acid encoding the polypeptide. Exemplary antagonist molecules include, but are not limited to, antagonist antibodies, polypeptide fragments, oligopeptides, organic molecules (including small molecules), and antisense nucleic acids.

[0021] As used herein, the term "IL-27 antagonist" refers to a molecule that interacts with at least one factor selected from IL-27 heterodimer, p28, EBI3, IL-27 receptor (IL-27R) heterodimer, IL-27RA, and gp130, and inhibits IL-27-mediated signaling.

[0022] Exemplary IL-27 antagonists include antibodies that bind to IL-27 heterodimers, antibodies that bind to p28, antibodies that bind to EBI3, antibodies that bind to IL-27R heterodimers, antibodies that bind to IL-27RA, the IL-27RA extracellular domain (ECD), and IL-27RA ECD fusion molecules. In some embodiments, the IL-27 antagonist is an antibody that binds to IL-27 heterodimers. In some embodiments, an IL-27 antibody that binds to an IL-27 heterodimer binds to the p28 subunit of IL-27 but does not bind to the EBI3 subunit of IL-27. In some embodiments, an IL-27 antibody that binds to p28 but not EBI3 blocks binding of IL-27 heterodimers to IL-27R. In some embodiments, the IL-27 antagonist blocks binding of IL-27 to IL-27R.

[0023] As used herein, the term "antibody" has its common meaning in the art and refers to an immunoglobulin molecule that recognizes and specifically binds to a target, such as a protein, polypeptide, peptide, carbohydrate, polynucleotide, lipid, or a combination of the foregoing, through at least one antigen recognition site within the variable region of the immunoglobulin molecule. As used herein, the term "antibody" encompasses intact polyclonal antibodies, intact monoclonal antibodies, chimeric antibodies, humanized antibodies, human antibodies, fusion proteins containing an antibody, and any other modified immunoglobulin molecule so long as the antibody exhibits the desired biological activity. Antibodies can be of any of the five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, or their subclasses (isotypes) (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), based on the identity of their heavy chain constant domains, designated alpha, delta, epsilon, gamma, and mu, respectively. The light chain contains two domains: a variable domain (VL) and a constant domain (CL). Heavy chains contain three (α, δ, γ) to five (μ, ε) domains, a variable domain (VH), and three to four constant domains (CH1, CH2, CH3, and CH4, collectively referred to as CH). The variable regions of both the light (VL) and heavy (VH) chains determine antigen binding recognition and specificity. The constant region domains of the light (CL) and heavy (CH) chains confer important biological properties, such as antibody chain association, secretion, transplacental transport, complement fixation, and Fc receptor (FcR) binding. An Fv fragment is the N-terminal portion of an immunoglobulin Fab fragment and consists of one light chain and one heavy chain variable region. Antibody specificity resides in the structural complementarity between the antibody-combining site and an antigenic determinant. The antibody-combining site is composed of residues primarily from hypervariable or complementarity-determining regions (CDRs). In some cases, residues from non-hypervariable or framework regions (FR) may participate in the antibody binding site or influence the overall domain structure and hence the binding site. CDRs refer to amino acid sequences which together define the binding affinity and specificity of the natural Fv region of a native immunoglobulin binding site.The light and heavy chains of an immunoglobulin each have three CDRs designated L-CDR1, L-CDR2, L-CDR3, and H-CDR1, H-CDR2, H-CDR3, respectively. Thus, an antigen-binding site typically contains six CDRs, including a set of CDRs from each of the heavy and light chain V regions. Framework regions (FRs) refer to the amino acid sequences intervening between the CDRs. Residues in antibody variable domains are conventionally numbered according to a system devised by Kabat et al. This system is set forth in Kabat et al., 1987, in Sequences of Proteins of Immunological Interest, US Department of Health and Human Services, NIH, USA (hereinafter "Kabat et al."). This numbering system is used herein. Kabat residue designations do not necessarily correspond directly to the linear numbering of amino acid residues in SEQ ID NO: sequences. The actual linear amino acid sequence may contain fewer or additional amino acids than in the strict Kabat numbering, corresponding to shortening of, or insertion into, structural components of the basic variable domain structure, whether framework or complementarity-determining region (CDR). The correct Kabat numbering of residues can be determined for a given antibody by aligning homologous residues within the antibody sequence with the "standard" Kabat numbering sequence. The CDRs of the heavy chain variable domain are located at residues 31-35B (H-CDR1), residues 50-65 (H-CDR2), and residues 95-102 (H-CDR3) according to the Kabat numbering system. The CDRs of the light chain variable domain are located at residues 24-34 (L-CDR1), residues 50-56 (L-CDR2), and residues 89-97 (L-CDR3) according to the Kabat numbering system.

[0024] In some embodiments, the IL-27 antagonist is an IL-27R antibody.

[0025] As used herein, the term "IL-27 antibody" or "antibody that binds IL-27" refers to an antibody that binds to the IL-27 heterodimer.

[0026] In some embodiments, antibodies that bind to IL-27 inhibit IL-27-mediated signaling. IL-27 antibodies include antibodies that bind to the IL-27 heterodimer but do not bind to either p28 or EBI3 alone, antibodies that bind to p28 (alone and / or complexed with EBI3), and antibodies that bind to EBI3 (alone and / or complexed with p28). In some embodiments, the antibodies bind to p28 but not to EBI3. In some embodiments, the antibodies bind to EBI3 but not to p28. In some embodiments, the IL-27 antibodies block binding of IL-27 to IL-27R. In one embodiment, the extent of binding of an anti-IL-27 antibody to an unrelated, non-IL-27 protein is less than about 10% of the binding of the antibody to IL-27, as measured, for example, by radioimmunoassay (RIA). In some embodiments, an antibody that binds to IL-27 has a IL-27 activity of ≦1 μM, ≦100 nM, ≦10 nM, ≦1 nM, ≦0.1 nM, ≦0.01 nM, or ≦0.001 nM (e.g., 10 -8 M or less, e.g. 10 -8 M~10 -13 M, e.g. 10 -9 M~10 -13 In some embodiments, the anti-IL-27 antibody binds to an epitope of IL-27 that is conserved among IL-27 from different species.

[0027] In some embodiments, the IL-27 antagonist is a p28 antibody.

[0028] As used herein, the term "p28 antibody" or "antibody that binds to p28" refers to an IL-27 antibody that binds to an epitope located in the extracellular domain of p28.

[0029] In some embodiments, an antibody that binds to p28 inhibits IL-27-mediated signaling. The p28 antibody may bind to p28 alone, to p28 when it is complexed with EBI3, or both. In some embodiments, the p28 antibody binds to p28 in the IL-27 heterodimer but not to EBI3. In some embodiments, the p28 antibody blocks the association of p28 with EBI3. In some embodiments, the p28 antibody blocks the binding of IL-27 to IL-27R, as defined above.

[0030] In some embodiments, the p28 antibody is SRF388, a fully human IgG1 blocking antibody against IL-27. Upon administration, the p28 monoclonal antibody SRF388 targets and binds to p28 and inhibits the interaction of IL-27 with IL-27 receptor subunit alpha (IL-27RA). This prevents activation of IL-27RA and blocks IL-27-mediated signal transduction. This reduces signal transducer and activator of transcription 1 (STAT1) phosphorylation.

[0031] In some embodiments, the IL-27 antibody is an EBI3 antibody.

[0032] As used herein, the term "EBI3 antibody" or "antibody that binds to EBI3" refers to an IL-27 antibody that binds to EBI3.

[0033] In some embodiments, antibodies that bind to EBI3 inhibit IL-27-mediated signaling. The EBI3 antibody may bind to EBI3 alone, to EBI3 when it is complexed with p28, or both. In some embodiments, the EBI3 antibody blocks the association of EBI3 with p28. In some embodiments, the EBI3 antibody blocks the binding of IL-27 to IL-27R, as defined above.

[0034] In some embodiments, the IL-27 antagonist is an IL-27R antibody.

[0035] As used herein, the term "IL-27R antibody" or "antibody that binds to IL-27R" refers to an antibody that binds to the IL-27R heterodimer.

[0036] In some embodiments, antibodies that bind to IL-27R inhibit IL-27-mediated signaling. IL-27R antibodies include antibodies that bind to the IL-27R heterodimer but do not bind to either IL-27RA or gp130 alone, and antibodies that bind to IL-27RA (alone and / or complexed with gp130) and antibodies that bind to gp130 (alone and / or complexed with IL-27RA). In some embodiments, IL-27R antibodies block the binding of IL-27 to IL-27R, as defined above.

[0037] In some embodiments, the IL-27 antagonist is an IL-27RA antibody.

[0038] As used herein, the terms "IL-27RA antibody" or "antibody that binds IL-27RA" refer to an IL-27R antibody (defined below) that binds to IL-27RA. In some embodiments, an antibody that binds IL-27RA inhibits IL-27-mediated signaling. An IL-27RA antibody may bind to IL-27RA alone, to IL-27RA when it is complexed with gp130, or both. In some embodiments, an IL-27RA antibody blocks the association of IL-27RA with gp130. In some embodiments, an IL-27RA antibody blocks the binding of IL-27 to IL-27RA, as defined above.

[0039] In some embodiments, the antibodies of the invention are human, humanized, or chimeric antibodies.

[0040] In some embodiments, the IL-27 antagonist is an IL-27RA ECD fusion molecule.

[0041] As used herein, the term "IL-27RA ECD fusion molecule" refers to a molecule comprising an IL-27RA extracellular domain (ECD) and one or more "fusion partners." In some embodiments, the IL-27RA ECD and fusion partner are covalently linked ("fused"). When the fusion partner is also a polypeptide (a "fusion partner polypeptide"), the IL-27RA ECD and fusion partner polypeptide can be part of a contiguous amino acid sequence, and the fusion partner polypeptide can be linked to either the N-terminus or C-terminus of the IL-27RA ECD. In such cases, the IL-27RA ECD and fusion partner polypeptide can be translated as a single polypeptide from a coding sequence encoding both the IL-27RA ECD and the fusion partner polypeptide (an "IL-27RA ECD fusion protein"). In some embodiments, the IL-27RA ECD and fusion partner are covalently linked through other means, such as chemical linkages other than peptide bonds. Many known methods of covalently linking polypeptides to other molecules (e.g., fusion partners) can be used. In other embodiments, the IL-27RA ECD and fusion partner may be fused through a "linker" composed of at least one amino acid or chemical moiety. In some embodiments, the fusion partner polypeptide is an immunoglobulin constant domain (Fc region), forming an immunoadhesin. Immunoadhesins can possess many of the beneficial chemical and biological properties of human antibodies. Because immunoadhesins can be constructed from human protein sequences with the desired specificity linked to appropriate human immunoglobulin hinge and constant domain (Fc) sequences, the binding specificity of interest can be achieved using entirely human components. The immunoglobulin sequence is typically, but not necessarily, an immunoglobulin constant domain. The immunoglobulin portion in the chimeras of the present invention may be obtained from IgG1, IgG2, IgG3, or IgG4 subtypes, IgA, IgE, IgD, or IgM, but typically IgG1 or IgG3.In some embodiments, the functional equivalent of PD-1 or NRP-1 and the immunoglobulin sequence portion of the immunoadhesin are linked by a minimal linker.

[0042] As used herein, the term "therapeutically effective amount" refers to a sufficient amount of an IL-27 antagonist to treat EB-driven B-lymphoproliferative disorders. However, it is understood that the total daily dosage of the agent will be determined by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular subject will depend on a variety of factors, including the disorder being treated and the severity of the disorder; the activity of the specific compound employed; the specific composition employed; the subject's age, weight, general health, sex, and diet; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of treatment; drugs used in combination with or concomitantly with the specific agent; and similar factors well known in the medical field. For example, it is well within the skill of the art to start dosing the compound at a level lower than that required to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved. However, the daily dosage of the agent can vary over a wide range, from 0.01 to 1,000 mg per adult per day. Preferably, the compositions contain 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 250, and 500 mg of the active ingredient for symptomatic administration to the subject to be treated. Medicaments typically contain from about 0.01 mg to about 500 mg of the active ingredient, preferably from 1 mg to about 100 mg. An effective amount of the drug is usually supplied at a dosage level of from 0.0002 mg / kg to about 20 mg / kg of body weight per day, more preferably from about 0.001 mg / kg to 7 mg / kg of body weight per day.

[0043] Typically, the IL-27 antagonist of the present invention is combined with a pharmaceutically acceptable excipient and, optionally, a sustained-release matrix, such as a biodegradable polymer, to form a pharmaceutical composition. "Pharmaceutically" or "pharmaceutically acceptable" refers to molecular entities and compositions that do not produce adverse, allergic, or other untoward reactions when administered to mammals, particularly humans, as appropriate. A pharmaceutically acceptable carrier or excipient refers to any type of non-toxic solid, semi-solid, or liquid filler, diluent, encapsulating material, or formulation aid. Typically, pharmaceutical compositions contain a pharmaceutically acceptable vehicle for injectable formulations. These may be, in particular, isotonic, sterile saline solutions (such as monosodium or disodium phosphate, sodium, potassium, calcium, or magnesium chloride, or mixtures of such salts), or dried, especially lyophilized, compositions that, optionally, upon addition of sterile water or saline, allow for the constitution of an injectable solution. Pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations containing sesame oil, peanut oil, or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. Sterile injectable solutions are prepared by incorporating the active ingredient in the required amount in an appropriate solvent, with some of the other ingredients listed above, as needed, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterile active ingredients into a sterile vehicle containing a basic dispersion medium and the required other ingredients from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred method of preparation is vacuum drying and freeze-drying techniques, which yield a powder of the active ingredient and any additional desired ingredients from a previously sterile-filtered solution thereof.

[0044] The present invention is further illustrated by the following figures and examples, which, however, should not be construed as limiting the scope of the present invention in any way. [Brief explanation of the drawings]

[0045] [Figure 1a] EBV infection induces IL-27 production by B cells, which is essential for their maintenance. a, Curve of the percentage of viable LCLs cultured for 14 days. LCLs from three different control, P1.1, P1.2, and P2 patients, and four STAT1 LOF patients at different days of culture. Values ​​are normalized to the cell number on day 0. Data from FACS analysis of DAPI staining and cell counts of three independent experiments. [Figure 1b] b, Curves of the percentage of viable LCLs as in (a) for three control LCLs cultured in the presence or absence of blocking anti-27 or anti-IL2 antibodies. Values ​​from three independent experiments. (a) Median ± sd, two-tailed Mann-Whitney test; (b) Median ± sd, paired t-test. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. [Figure 2a] Inhibition of proliferation and IL27RA signaling in LCLs by anti-IL-27 antibodies. a, Curves of the percentage of viable LCLs cultured for 14 days for three different controls (control LCLs, black) in the presence of blocking anti-IL-27 (red) or anti-IL2 (gray) antibodies. For controls #1 and #2, curves of two independent experiments. [Figure 2b] b, Immunoblot for phospho-STAT1, STAT1, phospho-STAT3, STAT3, and actin expression in LCLs stimulated with IL-27 (+) or unstimulated (-) with two controls (control 1 and control 2) in the presence or absence of 2 μg.ml-1 (+) or 20 μg.ml-1 (++) of anti-IL27 blocking antibody. Data from one representative experiment out of two. [Example]

[0046] method Human Subjects and Samples, and Study Approval. The patients and relatives studied here resided and were followed in France. Written informed consent was obtained from the donors, patients, and their families. The study and protocol conformed to the 1975 Declaration of Helsinki, local regulations, and ethical guidelines from the Île-de-France II Ethics Review Committee (Comite de Protection des Personnes de l'Ile de France II) and the French Advisory Committee for Data Handling in Medical Research. Experiments using samples from human subjects were conducted in France and Australia in accordance with local regulations and with approval from the corresponding institutional IRBs. Healthy controls were individuals without symptomatic EBV infection. Plasma and serum samples from patients and controls were frozen at -80°C immediately after collection.

[0047] Patient Case Reports P1.1 was born to consanguineous Caucasian parents. Her past medical history was unremarkable except for two benign episodes of bronchiolitis at age 13 months and a brief hospitalization for chickenpox skin superinfection. At age 20 months, she was hospitalized for a 7-day persistent fever with sore throat. Physical examination revealed hepatosplenomegaly (HSM) (3 cm and 5 cm from the costal margin, respectively). Blood counts showed marked hyperleucocytosis (up to 62 G / L) with lymphocytosis (up to 45 G / L) and excess basophils, mild anemia, and a normal platelet count. LDH was elevated, while bilirubin was normal. Triglycerides were elevated, but ferritin and fibrinogen were within normal limits. EBV serology was compatible with primary EBV infection (anti-VCA IgM and IgG positive, anti-EBNA IgG negative), and EBV PCR was positive at 3.56–4.33 log copies / mL. Peripheral immunophenotyping demonstrated a rapid CD8+ lymphocytosis with an activation profile. A blood smear revealed rare cytophagocytosis, excluding malignancy. 15 days later, due to persistent symptoms (spiking fever, increased HSM) and significant biological abnormalities reproducing incomplete hemophagocytosis (3 / 8 positive criteria), steroids (methylprednisolone 2 mg / kg) were administered. The partial response and the development of excessive B-cell lymphocytosis prompted the initiation of rituximab (four injections, 375 mg / m² per week), an anti-CD20 monoclonal antibody, which resulted in complete clinical and biological normalization. Due to transient B-cell depletion, immunoglobulin replacement therapy was initiated for 12 months. Three years later, at age 5.5 years, partial B-cell deficiency was diagnosed due to the absence of antitetanus antibodies despite complete vaccination and persistent memory B-cell lymphopenia. Due to recurrent upper respiratory tract infections, weekly subcutaneous immunoglobulin replacement therapy was restarted. At last follow-up at age 8 years, the patient was asymptomatic and well. Her immunophenotype, including memory B cells, was within normal limits. Circulating EBV viral load was low.

[0048] The eldest son, P1.2, was hospitalized at 8 months of age with severe mononucleosis compatible with primary infection (positive anti-VCA IgM and IgG antibodies, but negative anti-EBNA IgG), fever, HSM, lymphadenopathy, leukocytosis (40 g / L), and mildly elevated liver enzymes (AST 4N, ALT 7N, and GGT 13N) and LDH (4N). He spontaneously improved within 2 weeks without treatment. He presented with benign bronchiolitis, one episode of pneumonia in infancy, and several episodes of upper respiratory tract infections during childhood. At last follow-up at age 18 years, he is healthy and well. His immunophenotype shows mild CD4 and CD8 lymphopenia. Humoral immunity is normal (normal immunoglobulin dosage, positive postvaccine response, normal memory B cell count). EBV viral load is negative. Two children in the family died in infancy (neonatal anoxia in a premature infant and a 4-month-old boy from dysplastic syndrome and hypertrophic cardiomyopathy).

[0049] P2 was admitted at age 17 years for severe mononucleosis with hepatitis and HLH consistent with primary infection (EBV blood load 5.56 log copies / mL, positive anti-VCA IgM and IgG antibodies, but negative anti-EBNA IgG). She had no family or medical history. She presented with fever, sore throat, liver failure, hepatic stenosis, lymphadenopathy, bicytopenia with anemia (7.7 g / dL), leukocytosis (40 g / L), elevated liver enzymes (AST 3.5N, ALT 5.2N, GGT 22N, and PAL 966 UI / L), and elevated LDH (4 N). Triglycerides (7.25 g / L) and ferritin (1086 ng / mL) were elevated, and fibrinogen (1.32 g / L) was decreased. Myelography showed accumulation of macrophages with rare hemophagocytic lesions. Lymphocyte immunophenotyping revealed a high proportion of activated HLA-DR1. + CD8 + Hyperlymphocytosis (11808 / mm3) with T cells (70%) 3) She was treated with corticosteroid therapy with methylprednisolone, which led to rapid improvement within a few days. After discontinuing corticosteroid therapy, she had a relapse 1 month later characterized by hepatocellular lysis with extensive infiltration of T lymphocytes. One and a half years later, the blood EBV load had significantly decreased to 2.95 log copies / ml. However, serology remained abnormal with no detectable IgG anti-EBNA antibodies. Two and a half years later, at final follow-up, she was well.

[0050] Exome Sequencing and Analysis Exome capture and analysis were performed as previously described. 31,32 The IL27RA variation (15:38803380 G / T) identified in the patient, a homozygous substitution c.286C>T (p.G96X), has a 4.10 nucleotide sequence in the Genome Aggregation Database (gnomad.broadinstitute.org). -6 It was reported in frequency but not in the homozygous state.

[0051] Sanger sequencing Genomic DNA from peripheral blood cells of the patient, her parents, and her siblings was isolated according to standard methods. Variants were identified by genomic DNA amplification using oligonucleotide primers flanking exon 3 of the IL27RA gene. PCR products were amplified using High-Fidelity Platinum Taq DNA Polymerase (Invitrogen) according to the manufacturer's recommendations, purified using a QIAquick gel extraction kit (Qiagen), and sequenced using an ABI PRISM BigDye Terminator Cycle Sequencing Ready Reaction Kit (PerkinElmer) according to the manufacturer's recommendations. All collected sequences were analyzed using 4peaks software (version 1.8; A. Griekspoor and T. Groothuis, http: / / nucleobytes.com / index.php / 4peaks).

[0052] cell culture Peripheral blood mononuclear cells (PBMCs) collected from patients and healthy donors were isolated from blood samples by Ficoll-Paque density gradient (Lymphoprep, Proteogenix) using standard procedures. T-cell blasts were expanded by incubating PBMCs with phytohemagglutinin (PHA) (2.5 μg / ml, Sigma-Aldrich) for 72 hours in Panserin 401 (Pan Biotech) supplemented with 5% human AB serum (BioWest), penicillin (100 U / ml), and streptomycin (100 μg / ml). After 3 days, dead cells were removed by Ficoll-Paque density gradient, and blasts were maintained in culture with IL-2 (100 UI / ml). T-cell blasts were phenotyped for CD3, CD4, CD8, CD25, CD27, CD45RA, and CD57 expression before being tested in various assays. The phenotype of T cell blasts from healthy donors and patients was comparable in expression of these various markers. EBV-transformed LCLs were obtained according to previously described procedures (Izawa et al., 2017; Martin et al., 2014). Patient and control LCLs were cultured in RPMI 1640 (Life Technologies) supplemented with 10% heat-inactivated fetal bovine serum (Gibco), penicillin (100 U / ml), and streptomycin (100 μg / ml).

[0053] EBV-transformed LCL and EBV-specific T cell lines EBV-transformed LCLs were obtained according to previously described procedures (Izawa et al., 2017; Martin et al., 2014). Patient and control LCLs were cultured in RPMI 1640 (Life Technologies) supplemented with 10% heat-inactivated fetal bovine serum (Gibco), penicillin (100 U / ml), and streptomycin (100 μg / ml). EBV-specific T cell lines were obtained from patient and control healthy donors using PBMCs cocultured with 45 Gy-irradiated autologous LCLs at a PBMC / LCL ratio of 40:1. After 8–10 days, surviving cells were stimulated with 45 Gy-irradiated autologous LCLs at a PBMC / LCL ratio of 4:1. Cells were restimulated weekly with 45 Gy-irradiated autologous LCLs in the presence of IL-2 (40 U / ml).

[0054] EBV-specific T cell detection HLA phenotyping of the patient showed that he was a carrier of HLA-A*2, so HLA-A*2 reagents could be used to assess EBV-specific T cells. EBV-specific CD8 T cells from PBMCs of the patient and healthy control carriers of HLA-A*2 were also detected. + T cells were detected using a mixture of unlabeled EBV HLA-A2:01 Pro5 pentamers (Proimmune) mixed with R-PE Pro5 Fluorotag in addition to BV785-anti-CD3, APC-anti-CD19, BV510-anti-CD4, and BV650-anti-CD8 antibodies according to the manufacturer's instructions. The EBV HLA-A2:01 Pro5 pentamer mixture contains four different pentamers presenting peptides derived from the EBV BMLF-1, LMP-1, and LMP-2 proteins: GLCTLVAML (residues 259–267 from BMLF-1), FLYALALLL (residues 356–364 from LMP-2), CLGGLLTMV (residues 426–434 from LMP-2), or YLLEMLWRL (residues 125–133 from LMP-1).

[0055] Flow cytometry Cell staining and flow cytometry-based phenotypic analysis of PBMCs and cells was performed according to standard flow cytometry methods. Fluorescein isothiocyanate (FITC), R-phycoerythrin (PE), phycoerythrin-cyanine 5 (PE-Cy5), phycoerythrin-cyanine 5.5 (PE-Cy5.5), phycoerythrin-cyanine 7 (PE-Cy7), peridinin-chlorophyll (PerCP), peridinin-chlorophyll-cyanine 5.5 (PerCP-Cy5.5), allophycocyanin (APC), allophycocyanin-cyanine 7 (APC-Cy7), allophycocyanin-Vio7 (APC-Vio7), alexa-700, Brilliant Violet 421 (BV421), Brilliant Violet 510 (BV510), Brilliant Violet 605 (BV605), Brilliant Violet 711 (BV711), Brilliant Violet 650 (BV650), or Brilliant Violet The following monoclonal antibodies conjugated to 785 (BV785) were used: anti-CD3 (UCHT1), anti-CD4 (OKT4), anti-CD8 (RPA-T8), anti-CD11c (3.9), anti-CD14 (M5E2), anti-CD16 (3G8), anti-CD19 (HIB19), anti-CD25 (BC96), anti-CD27 (LG.3A10), anti-CD28 (CD28.2), anti-CD31 (WM59), anti-CD45RA (HI100), anti-CD45RO (UCHL1), anti-CD56 (HCD56), anti-CD57 (HNK-1), anti-CD70 (113-16), and anti-CD137 (4B4-1). Antibodies against CD161 (HP-3G10), CD183 (G025H7), CD185 (J252D4), CD196 (G034E3), CD197 (G043H7), and CD279 (EH12.EH7), CD303 (BDCA-2), IgM (G20-127), IgD (IA6-2), CD355 (29A1.4), TCRαβ (IP26), TCRγδ (B1), IgM (MHM-88), IgD (IA6-2), and HLA-Dr (LN3) were all purchased from BioLegend. Anti-IL-27RA (FAB14791P) was purchased from R&D.

[0056] iNKT cells were detected by staining with anti-Vα24-Jα18 (6B11-BioLegend) and anti-Vβ11 (C21-Beckman Coulter). MAIT cells were detected by staining with anti-Vα7.2 (3C10-BioLegend) and anti-CD161 (HP-3G10-BioLegend) or by using 5-OP-RU-loaded MR1 tetramers (NIH Tetramer Core Facility, Atlanta, GA).

[0057] For intracellular staining for phosphorylated STAT1 and STAT3 proteins, cells were treated with human recombinant IL-27 protein (Preprotech) (50 ng.mL -1 The cells were stimulated with 1000kJ / mL phospho-STAT1 (pY701; clone 14 / P-STAT-1) and 1000kJ / mL phospho-STAT3 (pY705; clone 4 / P-STAT-3) for 15–20 min, then fixed and permeabilized using a Phosflow kit (BD Biosciences) according to the manufacturer's instructions. The following antibodies were used: anti-phospho-STAT1 (pY701; clone 14 / P-STAT-1) and anti-phospho-STAT3 (pY705; clone 4 / P-STAT-3), both from BD Biosciences.

[0058] All data were collected on an LSR-Fortessa cytometer (BD Biosciences) and analyzed using FlowJo version 10.8.0 software (Tree Star).

[0059] Cytokine Assays PBMCs were incubated with monoclonal antibodies against CD8, CD4, CD45RA, CCR7, CD127, and CD25. First, Treg cells (CD4 + CD45RA - CD25 hi CD127 lo ) and then CD4 + or CD8 + CD45RA + CCR7 + or CD45RA - CXCR5- CCR7 + / - Naive and memory CD4 by sorting cells + and CD8 + T cells were isolated. Naive and memory CD4 + or CD8 + T cells were then cultured in 96-well round-bottom (30–40 × 10) plates using T cell activation and expansion beads (coated with mAbs against CD2 / CD3 / CD28; Miltenyi Biotech) alone (Th0) / +IL-12 (Th1) / +IL-27 for CD4 T cells, or beads alone (Th0) / +IL-2 / +IL-27 for CD8 T cells. 3 After 5 days, supernatants were harvested and production of IL-4, IL-5, IL-9, IL-10, IL-13, IL-17A, IL-17F, and IFN-γ was determined by cytometric bead array (Becton Dickinson); IL-22 secretion was measured by ELISA (eBioscience). For cytokine expression, activated CD4 + CD4+ and CD8+ T cells were restimulated with PMA (100 ng / ml) / ionomycin (750 ng / ml) for 6 hours, followed by the addition of brefeldin A (10 μg / ml) 2 hours later. Cells were then harvested, stained for extracellular markers, and fixed and permeabilized using a Cytofix / Cytoperm kit according to the manufacturer's instructions. Cells were then stained with specific antibodies to quantify the intracellular levels of IL-4, IL-9, IL-13, IL-10, IL-17A, IL-17F, IL-22, IL-21, IFN-γ or IL-2, TNF-α, IFN-γ, granzyme A, granzyme B, and perforin in CD4+ and CD8+ T cells, respectively.

[0060] We assessed IL-27 secretion using an ELISA kit (R&D Systems, #DY2526) according to the manufacturer's instructions. We also performed intracellular staining to determine IL-27 expression in LCLs and various PBMC subpopulations. Cells were incubated overnight with monensin and brefeldin A (Golgistop and Golgiplug, BD Biosciences) 2 hours after stimulation. Cells were then fixed / permeabilized using Cytofix / Cytoperm (BD Biosciences) according to the manufacturer's instructions and labeled with an anti-IL-27 antibody and the corresponding isotype antibody (R&D Systems, clone 307426) kit. All data were collected on an LSR-Fortessa cytometer (BD Biosciences) and analyzed using FlowJo version 10.8.0 software (Tree Star).

[0061] Proliferation assay T-cell blasts or PBMCs were cultured at 50 ng.ml in complete medium alone or with increasing doses of coated anti-CD3 antibody (clone OKT3 functional grade, eBiosciences) or anti-CD3+CD28 coated beads (Invitrogen). -1 The cells were cultured for 3 and 6 days, respectively, with or without the addition of human recombinant IL-27 (PeproTech) at a concentration of 0.1%. Cell proliferation was monitored by labeling the cells with violet dye (Violet Proliferation Dye 450, BD Biosciences) before stimulation. EBV-transformed LCLs were washed three times in PBS and starved overnight in RPMI medium supplemented with 0.1% fetal bovine serum. The LCLs were then labeled with violet dye and cultured in complete medium for 3 and 5 days. After 3, 5, or 6 days of culture, the cells were harvested, and violet dye dilution was assessed by flow cytometry. Proliferation and expansion indices were calculated using Flowjo software as the total number of divisions / cells entering division (for proliferation) and the total number of cells / cells at the start of culture (for expansion).

[0062] Cytotoxicity Assay EBV-transformed LCLs from patients and healthy donors contained a caspase-3 cleavage sequence (DEVD). 33 LCLs were infected with lentivirus containing the pMSCV-CFP-DEVD-YFP construct (a gift from P. Bousso, Pasteur Institute), which allows expression of CFP and YFP proteins linked by GFP. Three days after infection, infected cells were sorted using GFP reporter protein expression. Infected LCLs were then cocultured with autologous or HLA-matched EBV-specific CTLs at various effector / target ratios for 3 hours. LCL cell death was quantified by FRET using flow cytometry analysis.

[0063] Immunoblotting T cell blasts were stimulated with human recombinant IL-27 protein (R&D) or anti-CD3 / 28 beads for various times. The following antibodies were used for immunoblotting: anti-phospho-STAT1 (anti-phospho-Y701, clone #D4A7), anti-phospho-STAT3 (anti-phospho-Y705, clone #D3A7), and anti-actin (clone #D3A7D18C11), all purchased from Cell Signaling Technology; anti-KU80 (clone #C48E7); and anti-IL-27RA (clone #191106) from R&D Systems. The membranes were then washed and incubated with anti-mouse or anti-rabbit HRP-conjugated antibodies from Cell Signaling Technology. Pierce ECL Western blotting substrate was used for detection.

[0064] Plasmid constructs, CRISPR-Cas9 genome editing, and infection Full-length IL27RA cDNA was obtained from blasts by RT-PCR. The cDNA was verified by sequencing and inserted into a bicistronic lentiviral expression vector (pLVX-EF1α-IRES-mCherry vector, Clontech) encoding mCherry as a reporter. Viral particles for infection were obtained by co-expression of the lentiviral vector containing IL-27RA with a third-generation lentiviral plasmid containing the Gag-Pol, Rev, and G proteins of vesicular stomatitis virus (VSVG) in HEK 293T cells using calcium phosphate. Viral supernatants were collected every 12 hours for two consecutive days, starting 48 hours after transfection, and viral particles were concentrated by ultracentrifugation at 49,000 g for 1.5 hours at 12°C. Control and patient T-cell blasts were transfected with 10 8 TU ml -1 Cells were infected with viral particles at a minimum titer of 10 (MOI). 48 hours after infection, cells were maintained in IL-2 for culture bis-STAT phosphorylation or proliferation assays. For IL-27 gene knockdown using CRISP-Cas9, the SpCas9(BB)-2A-eGFP(PX458) plasmid (plasmid no. 48138; Adgene) was used for genome editing. All single-guide RNAs (sgRNAs) were designed according to previously reported procedures. 31 The synthesized oligonucleotide pair was annealed, phosphorylated, ligated to the linearized PX458 plasmid, and transferred to Stabl3 bacteria (Thermo Fischer Scientific). The efficiency of the sgRNA was tested in HEK-293T cells. EBV-transformed LCLs from healthy donors, either transduced with a lentiviral vector containing IL-27RA or not, were transfected twice with the PX458 plasmid by electroporation using a Nepa21 electroporator (Nepagene, Japan), selected for eGFP expression, and transfected with human recombinant IL-27 (100 ng / ml). -1) in culture. IL-27 expression and production were analyzed by intracellular flow cytometry and ELISA in the culture supernatant of LCLs, respectively. Clones lacking IL-27 expression were sequenced for IL27 by Sanger sequencing using specific primers to determine the mutations introduced by the CRISPR Cas9 genome. To monitor the survival of CRISPR IL27 or empty LCLs, IL-27 supplementation was stopped, and cell death analysis was assessed by flow cytometry using Annexin V and DAPI labeling. All constructs were verified by Sanger sequencing using the BigDye™ Terminator v3.1 Cycle Sequencing Kit (Life Technologies) and a 3500xL Genetic Analyzer (Applied Biosystems) according to the manufacturer's instructions. Sequence analysis was performed using DNADynamo (BlueTractor Software).

[0065] Anti-IL-27 autoantibody detection ELISA was performed as previously described 24,26Briefly, 96-well ELISA plates (Maxisorp; Thermo Fisher Scientific) were coated with 1 μg / ml rIL-27 (PeproTech) by overnight incubation at 4°C. Plates were then washed (PBS-Tween 0.05%), blocked by incubation with the same buffer supplemented with 5% nonfat dry milk, washed, and incubated with serum or plasma samples (1 / 50 diluted) from patients and controls for 2 hours at room temperature. Plates were thoroughly washed. Fc-specific HRP-conjugated IgG fraction of polyclonal goat antisera against human IgG, IgA, and IgM (Nordic Immunology Laboratories) was added to a final concentration of 2 μg / ml. Plates were incubated for 1 hour at room temperature and washed. Substrate was added, and optical density was measured (Victor X4™; Perkin Elmer). The presence of anti-IL-27 autoantibodies was then confirmed by Western blot according to the following method. Five hundred nanograms of human recombinant IL-27 (PeproTech) was separated by SDS-PAGE (10% acrylamide) under reducing conditions and transferred to a PVDF membrane (Millipore). The membrane was blocked by incubation with PBS supplemented with 5% BSA and 0.05% Tween 20, washed, and incubated overnight at 4°C with patient or control plasma samples diluted 1 / 500 in PBS with 5% BSA and 0.01% Tween 20. The membrane was then washed three times and incubated for 1 hour at room temperature with HRP-conjugated anti-human IgG, IgA, and IgM secondary antibodies (GAHu / Ig(Fc) / PO, Nordic MUbio) used at the final concentration. The membrane was washed three times and Pierce ECL Western blotting substrate was used for detection.

[0066] Neutralization Assay The blocking activity of anti-IL-27 autoantibodies was assessed by measuring the activity of IL-27 (50 ng mL ) in the presence of 10% healthy control or patient plasma / serum. -1 ) or IL-27 + anti-CD3 (OKT3; 0.1 μg / mL -1Neutralization was determined by assessing STAT1 phosphorylation and T cell proliferation (see procedures below) in healthy control cells after stimulation with IL-27. An anti-IL-27 commercial antibody (Ultra-Leaf™ purified anti-human IL-27p28 Biolegend, clone MM27-7B) was used at 2 μg.ml as a positive neutralization control. -1 was used at a concentration.

[0067] LCL proliferation monitoring assay. A flow cytometry-based method that can allow cell counting has been developed for the assessment of cell proliferation. LCL cells were seeded at 125,000 cells / well in 96-well plates or 1 million cells / mL in T25. Cells in wells were counted every 2 days by flow cytometry using fluorescent beads (Precision Count Beads™, Biolegend catalog: 424902) according to the supplier's instructions. Where indicated, cells were counted every 2 days (3 times a week) at 2 and 3 μg.ml, respectively. -1 The cells were cultured with antagonist blocking antibodies against IL-27 (Ultra-Leaf™ purified anti-human IL27p28 Biolegend, clone MM27-7B) or anti-IL-2 (Ultra-Leaf™ purified anti-human IL2 Biolegend, clone MQ1-17H12).

[0068] IL27RA intracellular and surface staining. T cell blasts were stimulated with anti-CD3 / CD28-activated Dynabeads (Life Technologies) for 3 days. Surface IL-27RA was stained with purified anti-IL27RA antibody (R&D Systems, clone #191106) followed by Alexa Flour 488-conjugated secondary antibody (Invitrogen). Cells were then fixed and permeabilized using a FOXP3 staining kit (eBioscience) and incubated with PE-conjugated anti-IL-27RA antibody (R&D Systems, clone #191106). Stained cells were analyzed using a BD LSRII-Fortessa (BD Biosciences), and data were processed using FlowJo software (Tree Star).

[0069] Immunofluorescence T cell blasts were stimulated with anti-CD3 / CD28-activated Dynabeads (Life Technologies) for 3 days. Surface IL-27RA was stained with anti-IL27RA antibody (R&D Systems, clone #191106) followed by Alexa Flour 488-conjugated secondary antibody (Invitrogen). Cells were then fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, and incubated with anti-IL-27RA antibody (R&D Systems, clone #191106) followed by Alexa Flour 546-conjugated secondary antibody (Invitrogen). Cell nuclei were stained with 49-6-diamidino-2-phenylindole. Images were acquired with a Leica SP8 STED confocal microscope (Leica Microsystems).

[0070] statistical analysis P values ​​were calculated using the unpaired or paired Student's t-test or Mann-Whitney test with a two-tailed distribution. P values ​​less than 0.05 were considered significant.

[0071] result: Identification of biallelic mutations in IL27RA causing loss of function in three patients with severe primary EBV infection Infectious mononucleosis (IM) is a benign condition caused by primary EBV infection that occurs primarily in adolescents and adults. We studied three children (P1.1, P1.2, and P2) from two families with severe primary Epstein-Barr virus (EBV) infection. In Family 1, P1.1 and P1.2 developed unusual early-onset IM symptoms at 20 and 8 months of age, respectively, whereas in Family 2, P2's primary infection occurred at 17 years of age (data not shown). Severe EBV infection in these patients was characterized by persistent fever, hepatitis, splenomegaly, and signs of HLH, requiring relapse-related hospitalization and treatment with anti-CD20 (P1.1) or corticosteroids (P2). In addition, the patients had a history of ear, nose, and throat infections (P2), bronchiolitis (P1.2), and severe VZV infection (P1.1) during their first year of life. In P1.1, high blood levels of EBV (up to 1 × 10 5 The high EBV count (EBV copies / mL) persisted for more than 12 months without any clinical symptoms. Thereafter, no clinical signs were observed in P1.1, P1.2, and P2 (6, 17, and 3 years after IM, respectively). Immunophenotyping of P1.1, P1.2, and P2 PBMCs revealed memory effector CD8 + The numbers and percentages of various leukocyte subpopulations were within normal limits, with the exception of a decreased fraction of CD4+ T cells (data not shown). The number of memory CD27+ B cells was also decreased in P1.1 and P2. Ig levels were normal in patients, with the exception of IgA, which was low in P1.1. T-cell proliferation in response to PHA and anti-CD3 antibodies was normal for P1.1, P1.2, and P2. Based on these characteristics, a genetic defect leading to immunodeficiency associated with vulnerability to EBV infection was suspected in these patients.

[0072] To identify potential genetic defects contributing to the disease in the three patients, we performed whole-exome sequencing (WES) on P1.2 and P2. Gene variants identified by WES were filtered based on their combined annotation-dependent depletion / mutation significance cutoff (CADD / MSC) scores and their allele frequencies in Gnomad and our institutional exome databases. Significant genetic variants in the IL27RA gene were detected in both P1.2 and P2, consisting of a homozygous premature stop codon (g.19:14150387C>T (rs375317876), c.286C>T; p.Gln96X) in P1.2 and two heterozygous compound variants in P2, including a missense mutation (g.19:14160060C>G (rs201107107), c.1336C>G, p.Arg446Gly) and an essential acceptor splice mutation (g.19:14159791A>C (rs778365769), c.1142-2A>C). All three variations are located in exons encoding the extracellular domain of the protein encoded by IL27RA, are predicted to have a high impact using CADD / MSC scoring (MSC of 3.3 for IL27RA), and have a low allele frequency (AF) in gnomAD and in our own database (p.Gln96X, CADD: 33, gnomAD AF: 7.96.10). -6 ;p.Arg446Gly, CADD:8.7, gnomAD AF:5.84.10 -4 ;c.1142-2A>C, CADD:19.1, gnomAD AF:7.96.10 -6). Segregation of the IL27RA variant was examined in members of both families by Sanger sequencing (data not shown). In Family 1, both parents were heterozygous for g.19:14150387C>T, and the affected sibling (P1.2) was homozygous, as expected. In Family 2, the mother and father were heterozygous carriers of the c.1336C>G and c.1142-2A>C variants, respectively. These data were consistent with autosomal recessive inheritance of the disease. The p.Gln96X premature stop codon is predicted to remove most of the protein, including the extracellular, transmembrane, and intracellular domains (data not shown). A splice mutation was found to cause aberrant splicing characterized by the use of an alternative acceptor site within exon 9, leading to a 45-nucleotide in-frame deletion resulting in a shorter protein (p.Gln381_Ala395del) lacking 15 amino acids (data not shown).

[0073] IL-27RA encodes the α subunit of the IL-27 receptor, which, together with gp130, forms a heterodimeric receptor belonging to type 1 group 2 of the cytokine receptor family. 5,7 The only known ligand for this receptor is the IL-27 cytokine. IL-27RA is expressed predominantly in lymphoid tissues, specifically in the thymus, spleen, lymph nodes, and peripheral blood leukocytes. 7,8IL27RA protein expression was assessed in patient T cells (data not shown). To determine the impact of the identified genetic variations in the patient, IL27RA expression was examined in T cells. IL27RA was found to be upregulated in activated control T cells in response to CD3 / CD28 stimulation, whereas no or very weak IL27RA expression was detected on the surface of activated T cells from P1.1, P1.2, and P2 (data not shown). IL27RA expression was also absent in T cell extracts from P1.1 and P1.2, as shown by Western blot, in contrast to detectable amounts of IL27RA in control cell extracts (data not shown). In contrast, in P2, IL27RA protein was evident, but most of the detected protein had a significantly lower molecular weight (data not shown). This lower molecular weight may be explained by abnormal post-translational modifications, such as truncations and / or aberrant glycosylation, resulting from the two mutations, p.Arg446Gly and p.Gln381_Ala395del (data not shown). Notably, although the IL27RA mutant protein was not expressed on the surface of P2 cells, it was detectable intracellularly by flow cytometry and immunohistochemistry, indicating IL27RA accumulation in the cytoplasm. T cells from P2's parents showed intracellular IL27RA staining and reduced IL27RA surface staining, consistent with their heterozygous status and indicating the deleterious effects of the two mutations (data not shown).

[0074] IL-27 is known to activate the JAK / STAT pathway, primarily STAT1 and STAT3, when bound to the gp130-IL27RA heterodimer. This activation is mediated by the gp130 cytoplasmic domain, which contains the binding sites for JAK1 / 2. 4,5,8Therefore, the ability of IL-27 to activate IL27RA signaling was examined in patient T cell blasts by flow cytometry and Western blotting. STAT1 and STAT3 were rapidly phosphorylated in lysates of control T cells in response to IL-27, starting at 2 min, and STAT1 and STAT3 phosphorylation persisted at 30 min (data not shown). Phospho-STAT1 and STAT3 proteins were also detectable by intracellular staining at 15 min of stimulation with IL-27 (data not shown). In contrast, STAT1 and STAT3 phosphorylation was undetectable or weakly detectable in IL-27-stimulated T cell blasts from P1.1 and P2, respectively (data not shown). Thus, these data demonstrate that the genetic variants in IL27RA identified in the three patients are deleterious and behave as loss-of-function variants.

[0075] Effects of IL27RA deficiency on T cell proliferation and effector differentiation We next assessed the functional consequences of IL27RA deficiency. Initial studies have shown that IL-27 can promote Th1 differentiation in mice. 9~12 Accordingly, Th1-associated cytokine production in P1.1- and P1.2-derived T cells was found to be impaired (data not shown), while Th1 numbers were within the normal range (data not shown). IL-27 is also known to promote T cell proliferation of naive CD4+ and CD8+ T cells. 13~15 More recently, it was found that memory-like CD8+ T cell expansion during persistent viral infection in mice is IL-27 dependent. 6 Thus, we demonstrate robust expansion of EBV-specific T cells, a key step in the immune control of EBV infection. 2 We hypothesized that the proliferation of T cells may be IL-27 dependent. To assess this possibility, we analyzed T cell proliferation upon stimulation with anti-CD3 antibody in the presence or absence of IL-27 (data not shown). The addition of IL-27 alone did not induce any proliferation of control T cells, whereas the addition of low doses of anti-CD3 antibody (0.001–0.1 μg mL -1) provided a synergistic effect on proliferation induced by the highest concentration of anti-CD3 antibody (10 μg mL -1 ) and was inhibited by the addition of a blocking anti-IL-27 antibody (data not shown). Importantly, this synergistic effect of IL-27 was restricted to naive T cells (data not shown). Conversely, the addition of IL-27 had no effect on CD3-induced proliferation of T cells from patients (P1.1 and P2) (data not shown). Furthermore, as expected from the critical role of STAT1 in IL-27RA signaling and function, T cells from patients with STAT1 deficiency did not proliferate in response to IL-27 (data not shown).

[0076] To formally demonstrate that IL27RA deficiency directly interferes with IL-27 function in T cells, P1.1 T cells were transduced with a lentiviral vector (pLVX) containing cDNA encoding wild-type IL27RA (pLVX-IL27RA) to restore IL27RA expression (data not shown). The pLVX plasmid also contains an mCherry reporter gene, allowing for tracking of transduced cells. Transduction of P1.1 T cells with pLVX-IL27RA, but not empty pLVX, restored IL27RA expression as assessed by Western blot (data not shown). When IL27RA expression was restored in P1.1 T cells, cell lysates restored detectable amounts of phosphorylated STAT1 and STAT3 in response to IL-27 stimulation. Phosphorylated STAT1 and STAT3 were significantly increased by pLVX-IL27RA-transduced mCherry. + It was also detected by intracellular staining in patient T cells, but mCherry - In untransduced T cells, phosphorylated STAT1 and STAT3 were not detected, whereas in patient T cells transduced with empty pLVX, no phosphorylated STAT1 or STAT3 was found (data not shown). IL-27 synergistically enhanced CD3-dependent proliferation, and mCherry was upregulated to levels comparable to those observed in control T cells. + Recovery in patient T cells, but mCherry -This was not restored in patient T cells (data not shown). No such effect was observed in cells transduced with empty pLVX. Notably, overexpression of IL27RA in control T cells resulted in enhanced IL-27-induced phosphorylation of STAT1 and STAT3, but this was not associated with increased proliferation. Collectively, these data indicate that IL-27 has the potency to sustain T cell proliferation, whereas this synergistic effect is lost in patients with IL27RA deficiency.

[0077] Therefore, this mechanism may be applicable to the expansion and differentiation of (naive) EBV-specific T cells. To address this question, we first screened PBMCs from P1.1 and P1.2, both of which express class I MHC HLA-A02* molecules, for the presence of EBV-specific T cells using HLA-A2 pentamers containing EBV peptides. EBV-specific T cells were detected in PBMCs from both P1.1 and P1.2 (data not shown), thus seemingly suggesting that IL-27 is not required for the induction / expansion and survival of EBV-specific T cells. However, in contrast to EBV-specific T cells from HLA-A2-positive healthy individuals, EBV-specific T cells from P1.1 and P1.2 did not expand when cocultured with irradiated autologous EBV-transformed B cells, also known as lymphoblastoid cell lines (LCLs) (data not shown). Furthermore, unlike control EBV-specific T cells, EBV-specific T cells from P1.1 and P1.2 expressed elevated levels of exhaustion and activation markers, such as CD25, CD137, CD40L, PD1-L, KGRL1, CD57, LAG3, and 2B4, after 9 days of coculture with LCLs (data not shown). Analysis of blood samples from P1.1 during acute / severe infectious mononucleosis (SIM) revealed EBV-specific T cells with a similar exhaustion and activation phenotype, confirming the data from in vitro expansion experiments. When tested, EBV-specific T cells from P1.1 exhibited a reduced ability to kill autologous or HLA-A2-matched LCLs compared with control EBV-specific T cells (data not shown). Because P2 did not express class I MHC HLA-A02* molecules, only EBV-specific pentameric HLA-A2 was available, and we were therefore unable to analyze EBV-specific T cells in P2. However, in vitro expansion of CD8+ T cells, which should contain most of the EBV-specific T cells, was analyzed after coculture of P2-derived PBMCs with autologous LCLs. Expanded CD8+ T cells from P2, in contrast to expanded CD8+ cells from controls, exhibited increased levels of activation and exhaustion markers, as did expanded EBV-specific T cells from P1.1 and P1.2 (data not shown).

[0078] Notably, P1.1, P1.2, and P2 LCLs expressed HLA-A2, CD137L, and CD70 molecules at levels similar to those found in control LCLs (data not shown), whereas EBV-specific T cells (day 0) from P1.1, P1.2, and P2 cells displayed CD137 and CD27 expression levels comparable to those of control cells (data not shown). These data exclude interference between IL-27 and the CD27-CD70 and CD137-CD137L pathways, which are known to be important for the efficient expansion of EBV-specific T cells. 1,3 Taken together, these data indicate that in the absence of IL27RA, proliferation and differentiation of EBV-specific T cells into potent effector T lymphocytes are impaired, associated with an abnormal activation- and exhaustion-like phenotype of EBV-specific T cells. Thus, the absence of IL-27 signaling may result in unresolved activation and accelerated exhaustion of T cells and less efficient control of EBV-infected B cells.

[0079] IL-27 is produced by B cells during EBV infection IL-27 is a heterodimeric cytokine composed of IL-27p28 (encoded by IL27) and EBV-inducible gene 3 (encoded by EBI3). IL-27 is produced primarily by antigen-presenting cells (APCs), i.e., macrophages, dendritic cells, and B cells. 16~18 Interestingly, most of these observations were made in mice in the context of persistent viral infection, and B cell-derived IL-27 in particular appeared to be important for controlling persistent LCMV infection. 17 EBI3 was first identified in EBV-transformed B cell lines or LCLs. 19 , which was found to be expressed in EBV-positive lymphoma. 19~21Therefore, we hypothesized that B cells might be one major source of IL-27 during EBV infection. This was also supported by the IL27RA-dependent proliferation of EBV-specific T cells when cocultured with LCLs (data not shown). We initially found that IL-27 expression was induced in 20–30% of B cells when stimulated with PMA plus ionomycin, along with IL-2 expression in a small proportion of B cells (data not shown). Next, we infected B cells with GFP-tagged EBV and analyzed their IL-27 production. IL-27 expression was upregulated in GFP-positive, EBV-infected B cells, whereas it was not upregulated in GFP-negative B cells (data not shown). Furthermore, IL-27 accumulated in the supernatant of PBMCs upon EBV infection, as well as in the supernatant of LCLs derived from long-term cultures of EBV-infected PBMCs (data not shown). Taken together, these results indicate that B cells are IL-27 producers when infected and immortalized by EBV, and this direct production of IL-27 by EBV-infected B cells may therefore be involved in initiating and supporting the expansion of EBV-specific T cells and their differentiation into potent effector T cells.

[0080] IL-27 is required for B cell proliferation in EBV-transformed B cells During these experiments, we noticed that EBV-transformed B cell lines (LCLs) derived from the PBMCs of three IL27RA-deficient patients proliferated slowly and expanded less efficiently than control LCLs (Fig. 1a). These observations may suggest an unexpected additional role for IL-27 in promoting LCL proliferation through an autocrine loop. Indeed, control LCLs were found to express IL27RA, whereas IL27RA expression was undetectable on the surface of LCLs derived from P1.1, P1.2, and P.2 (data not shown). Notably, upon incubation with IL-27, STAT1 and STAT3 phosphorylation was detectable in control LCLs, indicating that IL27RA is functional in LCLs. In contrast, these phosphorylations were absent in IL-27-incubated IL27RA-deficient LCLs derived from P1.1, P1.2, and P2, as well as in LCLs derived from STAT1-deficient patients (data not shown). STAT1-deficient LCLs also failed to proliferate (Fig. 1a). The reduced growth of IL27RA-deficient LCLs (and STAT1 LOF LCLs) was associated with significantly reduced proliferation indices compared with control LCLs (data not shown). The reduced proliferation associated with defective STAT1 phosphorylation in IL27RA-deficient LCLs was corrected to levels comparable to those of control LCLs when IL27RA expression was restored by transduction with the expression vector pLVX-IL27RA. No correction was observed in cells transduced with the empty pLVX vector (data not shown). Notably, IL27RA- and STAT1-deficient LCLs excluded a defect in IL-27 production that could be responsible for their inability to produce IL-27 and proliferate like control LCLs (data not shown). These data demonstrate that the reduced proliferation and STAT1 phosphorylation of IL27RA-deficient LCLs are directly caused by IL27RA deficiency. Furthermore, the addition of blocking antibodies against IL-27, but not against IL-2, significantly reduced cell proliferation of control LCLs (Fig. 1b and Fig. 2a) and abolished IL-27-induced STAT1 and STAT3 phosphorylation (Fig. 2b), supporting a role for autocrine IL-27 production in LCL expansion.To further support this possibility, CRISPR-Cas9 genome editing in control LCLs, which selectively inactivated IL-27 by targeting exon 1 or exon 2 of IL-27, was performed using DAPI in culture. + This resulted in a rapid accumulation of dead cells, whereas genome editing with an empty CRISPR-Cas9 vector did not affect cell viability (data not shown). As expected, the addition of exogenous IL-27 prevented LCL cell death induced by IL-27 inactivation. However, cells did not proliferate under these conditions. This may be explained by the reduced expression of IL27RA in the absence of IL-27; IL-27, when bound to IL27RA, may be important for stabilizing IL27RA at the cell surface and / or activating IL27RA gene expression. This speculation was supported by the acquisition of control LCLs overexpressing ectopic IL27RA (after transduction with pLVX-IL27RA), in which IL27 was inactivated by CRISPR-Cas9, resulting in reduced IL-27 production (data not shown). In contrast to control LCLs in which IL27 was inactivated, control LCLs overexpressing IL27RA (IL27 inactivated) proliferated and expanded when exogenous IL-27 was added (data not shown). Importantly, deprivation of exogenous IL-27 rapidly induced cell death in these cells. No such effect was observed in cells transduced with an empty CRISPR-Cas9 vector. Thus, these results indicate that autocrine IL-27 production is required for the proliferation of EBV-transformed B cells. Importantly, this indicates that severe infectious mononucleosis in all three patients was not observed in patients with impaired immune T-cell responses to EBV (e.g., those on immunosuppressive treatment or suffering from inherited immunodeficiencies associated with increased susceptibility to EBV). 1,3This may explain why these patients did not further develop B-cell lymphoproliferation or lymphoma, as observed in EBV-infected B cells. Nevertheless, three patients developed severe infectious mononucleosis, suggesting that in vivo, expansion of infected B cells during primary infection does not depend on IL-27. The early EBV latency program activated during primary infection may be sufficient to sustain B-cell proliferation independently of IL-27. Further studies are needed to elucidate the precise role of IL-27 in the expansion and maintenance of EBV-infected B cells.

[0081] Identification of neutralizing anti-IL-27 autoantibodies in patients with EBV viral disease Neutralizing anti-cytokine autoantibodies have been identified in several infectious and immunological conditions that cause immune deficiency and / or immune dysregulation. 22 Some of these anti-cytokine autoantibodies are IL-17A / F 23,24 and IL-6 25,26It has been shown to phenocopy genetically determined immune deficiencies, such as deficiencies in IL-27RA. Therefore, we hypothesized that anti-IL-27 autoantibodies could phenocopy IL27RA deficiency and explain the sporadic acute EBV-driven infectious mononucleosis observed in adolescents and adults. We first set up an ELISA to detect anti-IL-27 autoantibodies and screened sera from patients with autoimmune and / or inflammatory diseases, including autoimmune polyendocrinopathy candidiasis ectodermal dystrophy (APECED) syndrome and AIRE deficiency, which is known to cause STAT1 gain-of-function (GOF) mutations (data not shown). High titers of autoantibodies that reacted positively with recombinant IL-27 in ELISA were found in most patients with STAT1 GOF, correlating with the presence of detectable serum levels of IL-27 in these patients (data not shown). In contrast, titers in patients with APECED titers were very low or undetectable, not significantly different from those detected in healthy controls. We next investigated the risk of EBV+ B-cell lymphoma, acute infectious mononucleosis (IM), and chronic active EBV infection, a severe viral disease characterized by persistent infection of T and / or NK cells. 27,28We tested sera from patients with a variety of EBV-associated conditions, including STAT1 GOF patients. Surprisingly, as in patients with IM and CAEBV, significantly higher titers of anti-IL-27 autoantibodies were detected in association with detectable levels of plasma IL-27. In patients with EBV+ lymphoma, most of which included Hodgkin lymphoma, the titers of anti-IL-27 autoantibodies were low, while the levels of IL-27 were comparable to those detected in patients with IM and CAEBV. Consistent with the ELISA data, sera from patients with infectious mononucleosis and STAT1 GOF mutations were able to detect recombinant IL-27 (data not shown), whereas sera from healthy control donors (data not shown) did not detect recombinant IL-27. Thus, these results are consistent with the presence of IL-27-specific autoantibodies in patients with infectious mononucleosis and STAT1 GOF mutations. Importantly, these anti-IL-27 autoantibodies (from both patients with IM and STAT1 GOF) exerted specific neutralizing activity, as they were able to inhibit phospho-STAT1 induction (data not shown) and IL-27-induced synergistic CD3-dependent proliferation in T cells (data not shown), whereas control sera did not. Collectively, these data demonstrate the presence of neutralizing anti-IL-27 autoantibodies in patients with IM, CAEBV, and STAT1 GOF.

[0082] Consideration: We report a novel primary immunodeficiency characterized by high susceptibility to herpesviruses, mostly EBV, and demonstrate the importance of IL-27 responses in anti-EBV immunity. The role of IL-27 in antiviral immunity in humans has been suspected for some time. Herein, we show that IL-27 is required for the expansion of naive EBV-specific T cells and their differentiation into potent effector T cells. IL-27, like other costimulatory pathways, namely SLAM, CD27, and CD137, is required for the expansion of naive EBV-specific T cells and their differentiation into potent effector T cells. 1, can be considered to provide an additional costimulatory signal that contributes to the necessary large amplification of the T cell immune response against infected B cells. Indeed, vigorous expansion of EBV-specific T cells is required to efficiently control and eliminate EBV-infected cells. Of note, EBV-specific T cells can account for up to 40% of circulating T cells in healthy individuals during primary EBV infection. 29,30 The crucial protective role of IL-27 in immune control against EBV is also supported by the presence of anti-IL-27 autoantibodies that interfere with IL-27 in individuals presenting with acute sporadic severe infectious mononucleosis, potentially phenocopying patients with IL27RA deficiency. Interestingly, patients with CAEBV also exhibit high titers of IL-27 autoantibodies. In contrast to infectious mononucleosis, CAEBV disease is characterized by EBV-infected T and / or T cells associated with high circulating EBV loads that persist for years. The pathophysiological mechanisms of CAEBV remain largely unknown. Anti-IL-27 autoantibodies may play a role in the persistence of EBV infection in individuals with CAEBV. As has been shown for other anti-cytokine autoantibodies, it may be hypothesized that anti-IL-27 autoantibodies are present prior to EBV infection in individuals whose infection with EBV leads to infectious mononucleosis. 22Patients with immune deficiencies caused by STAT1 GOF also exhibited high levels of anti-IL-27 autoantibodies, although these patients were not particularly susceptible to EBV infection. However, these patients have impaired effector T cell function, which may compensate for the defective IL-27 response. Interestingly, detectable levels of IL-27 (potentially elevated) were detected in the sera of patients with sporadic IM or STAT1 GOF and correlated with high titers of anti-IL-27 autoantibodies. These high levels of IL-27 may precede autoantibodies, trigger their development, or / and represent a compensatory mechanism to circumvent the blocking effect of anti-IL-27 autoantibodies. Finally, the observation that IL-27 is involved in the proliferation of EBV-transformed B cells may explain the self-limiting phenotype of EBV infection in IL27RA-deficient patients, suggesting that in vivo EBV "captures" the IL-27 pathway for its own benefit by providing a selective advantage to infected B cells. Therefore, IL-27 may be considered a novel therapeutic target for inhibiting the proliferation of EBV+ B cells in immunocompromised individuals or in patients with EBV+ lymphoma. Importantly, this hypothesis is further supported by the fact that patients with EBV+ lymphoma have fewer or undetectable autoantibodies to IL-27 (in contrast to patients with sporadic EBV+ IM), while exhibiting detectable serum levels of IL-27 (similar to those of patients with sporadic EBV+ IM). In conclusion, this report points to a dual role for IL-27 as a costimulatory molecule for T cell proliferation and differentiation into effector cells required for the control of EBV infection, and as a proliferation and / or survival signal for EBV-infected B cells, observations that merit further investigation to assess their mechanisms.

[0083] References: Throughout this application, various references describe the state of the art to which this invention pertains, the disclosures of which are incorporated herein by reference into the present disclosure.

[0084] [Table 1] TIFF2025539147000006.tif218169TIFF2025539147000007.tif150169

Claims

1. A method of treating an EBV-driven B-lymphoproliferative disorder in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of an IL-27 antagonist.

2. B-lymphoproliferative disorders include non-Hodgkin's lymphoma, Burkitt's lymphoma, small lymphocytic lymphoma, primary effusion lymphoma, diffuse large B-cell lymphoma, splenic marginal zone lymphoma, MALT (mucosa-associated lymphoid tissue) lymphoma, hairy cell leukemia, chronic lymphocytic leukemia, B-cell prolymphocytic leukemia, B-cell lymphomas (e.g., Hodgkin's disease, B-cell non-Hodgkin's lymphoma (NHL) and various forms of related lymphomas (e.g., Waldenstrom's macroglobulinemia (also called lymphoplasmacytic lymphoma or immunocytoma) or central nervous system lymphoma), leukemias (e.g., acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL; also called B-cell chronic lymphocytic leukemia, BCLL)), hairy cell leukemia, 2. The method of claim 1, wherein the tumor is selected from the group consisting of myeloma, myeloma (e.g., multiple myeloma), small lymphocytic lymphoma, B-cell prolymphocytic leukemia, lymphoplasmacytic lymphoma, splenic marginal zone lymphoma, plasma cell myeloma, isolated plasmacytoma of bone, extraskeletal plasmacytoma, extranodal marginal zone B-cell lymphoma of mucosa-associated lymphoid tissue (MALT), nodal marginal zone B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, diffuse large B-cell lymphoma, mediastinal (thymic) large B-cell lymphoma, intravascular large B-cell lymphoma, primary effusion lymphoma, Burkitt's lymphoma / leukemia, gray zone lymphoma, B-cell hyperplasia of unspecified grade, lymphomatoid granulomatosis, and post-transplant lymphoproliferative disorder.

3. 2. The method of claim 1, wherein the EBV-driven B-lymphoproliferative disorder is a B-cell lymphoma.

4. 4. The method of any one of claims 1 to 3, wherein the IL-27 antagonist is selected from the group consisting of an antibody that binds to an IL-27 heterodimer, an antibody that binds to p28, an antibody that binds to EBI3, an antibody that binds to an IL-27R heterodimer, an antibody that binds to IL-27RA, an IL-27RA extracellular domain (ECD), and an IL-27RA ECD fusion molecule.

5. The method of claim 4, wherein the IL-27 antagonist is an antibody that binds to p28 but does not bind to EBI3.

6. The method of claim 4, wherein the IL-27 antagonist is an antibody that binds to EBI3 but does not bind to p28.

7. The method of claim 4, wherein the IL-27 antagonist is an IL-27RA antibody.

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