Methods for expanding or depleting t regulatory cells

Abstract

To provide methods for preparing compositions enriched in T regulatory cells (Tregs) and methods for treating immunological diseases using the compositions, and to provide methods for preparing compositions enriched in lymphocytes and depleted of Tregs and use of the compositions in treating proliferative diseases.SOLUTION: A method for preparing a composition enriched in CD4+CD25hi T regulatory cells (Tregs) is provided, comprising contacting in vitro a population of human cells including T lymphocytes obtained from a patient-derived human blood or bone marrow sample with a tumor necrotic factor receptor 2 (TNFR2) agonist or an NF-κB activator, thereby preparing the composition in which the Tregs are enriched, wherein the Tregs constitute at least 60% of the cells in the composition.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to methods for preparing compositions enriched in Tregs and methods for treating immunological diseases using these compositions. The present invention also relates to methods for preparing lymphocyte-enriched and Treg-depleted compositions and the use of these compositions in the treatment of proliferative diseases. [Background technology]

[0002] T regulatory cells (Tregs) are a small subset of T lymphocytes with diverse clinical applications in transplantation, allergy, asthma, infectious diseases, graft-versus-host disease (GVHD), and autoimmunity. Tregs are also involved in immune tolerance in conditions such as cancer. The use of Tregs in clinical applications has been challenging due to their rarity in blood and the difficulty of expanding them ex vivo into homogenous populations. Naturally occurring Tregs comprise only 1–5% of all CD4+ T cells in blood, and they remain largely quiescent until activated. Therefore, obtaining sufficient numbers of Tregs to study their role in basic biology and for clinical applications depends on the ability to expand Tregs ex vivo. Although more than a dozen protocols have been developed worldwide to expand Tregs ex vivo for reinfusion into patients, all of these protocols result in heterogeneous progeny consisting of a phenotypically and functionally mixed CD4+ T cell population. Heterogeneous CD4+ T cell populations pose risks because they can release proinflammatory cytokines and contain cells with diverse, sometimes antagonistic, functions. Because regulatory agencies consider heterogeneous CD4+ T cell populations impure and irreproducible, clinical trials have not progressed beyond Phase I. Therefore, an important research and clinical goal has been to find ways to selectively expand Tregs without stimulating the proliferation of other CD4+ T cell populations. A parallel goal in this field has been to find ways to selectively deplete Tregs and expand lymphocyte populations. Such lymphocyte populations would be useful for upregulating immune responses in the treatment of proliferative diseases such as cancer. Summary of the Invention

[0003] The present invention relates to CD4+CD25 hiThe present invention features a composition enriched for T regulatory cells (Tregs), wherein at least 60% (e.g., 70%, 80%, 90%, or 100%) of the cells in the composition are Tregs. Preferably, the composition contains a homogenous population of Tregs with desirable immunomodulatory properties, such as expression of the forkhead box P3 (FOXP3) protein. The composition also contains at least 5×10 6 (e.g., 5×10 7 , 5×10 8 , 5×10 9 , 5×10 10 , 5×10 11 , or 5 × 10 12 The Tregs in the composition can be characterized as positive for expression of one or more proteins selected from the group consisting of CTLA4, TNFR2, FOXP3, CD62L, Fas, HLA-DR, and CD45RO, and as low or negative for expression of one or more proteins selected from the group consisting of CD127, CCR5, CCR6, CCR7, CXCR3, IFN-γ, IL10, and ICOS.

[0004] The present invention also provides a method for treating CD4+CD25 hi The present invention features a method for preparing a composition enriched in Tregs, which generally involves contacting a population of human cells, including T lymphocytes (e.g., CD4+ cells, CD25+ cells, or CD4+CD25+ cells), in vitro with a tumor necrosis factor receptor 2 (TNFR2) agonist and / or an NF-κB activator (e.g., during one or more culture steps), thereby enriching the Treg population. hiThe method includes preparing a composition enriched in Tregs. The population of human cells can be obtained from a human blood sample or a human bone marrow sample from a patient. The population of human cells from the sample is or contains CD4+ cells, CD25+ cells, or CD4+CD25+ cells, and can be separated or enriched from the blood or bone marrow sample before contacting with a TNFR2 agonist and / or an NF-κB activator. The TNFR2 agonist and / or an NF-κB activator can be used to enrich the CD4+CD25+ cells present in the population of human cells according to the method. hi by promoting increased proliferation of Tregs and / or by increasing (e.g., by differentiation or activation) CD4+CD25+ T lymphocytes (e.g., CD4+ cells, CD25+ cells, or CD4+CD25+ cells) present within a population of human cells. hi By increasing the generation of Tregs, CD4+CD25 hi Promoting Enrichment of Tregs The above methods preferably result in a homogenous population of Tregs, e.g., at least 60% (e.g., 70%, 80%, 90%, or substantially 100%) of the cells in the composition are Tregs.

[0005] The TNFR2 agonist that can be used in the methods of the present invention can be an agent selected from the group consisting of antibodies (e.g., monoclonal anti-TNFR2 antibodies), peptides, small molecules, and proteins. Because TNFR2 signaling can proceed through the downstream NF-κB pathway, an NF-κB activator can be used to contact a population of human cells for the purpose of preparing a composition enriched for Tregs. The NF-κB activator can be selected from the group consisting of small molecules (e.g., betulinic acid, topoisomerase poison VP16, and doxorubicin), peptides, proteins, viruses, and small non-coding RNAs.

[0006] In addition to a TNFR2 agonist and / or an NF-κB activator, methods for preparing compositions enriched for Tregs can include contacting a population of human cells (e.g., CD4+ cells, CD25+ cells, or CD4+CD25+ cells) with one or more of interleukin-2 (IL2), rapamycin, anti-CD3 (e.g., an anti-CD3 antibody), and / or anti-CD28 (e.g., an anti-CD28 antibody). After in vitro expansion, the methods of the invention described above can be used to produce at least 5×10 6 (e.g., 5×10 6 , 5×10 7 , 5×10 8 , 5×10 9 , 5×10 10 , 5×10 11 , or 5 × 10 12 ) Tregs can be prepared, wherein at least 60% (eg, 70%, 80%, 90%, or substantially 100%) of the cells in the composition are Tregs.

[0007] The present invention also features a method for treating an immunological disease (e.g., allergy, asthma, autoimmune disease, GVHD, or transplant rejection) or an infectious disease (e.g., bacterial infection, viral infection, fungal infection, and / or parasitic infection) in a patient (e.g., a human patient) by administering to the patient any one or more of a Treg-enriched composition, a TNFR2 agonist (e.g., a monoclonal anti-TNFR2 antibody), and an NF-κB activator. For example, the method of treatment can include administering the Treg-enriched composition alone or in combination with an NF-κB activator. The Treg-enriched composition can be prepared by any method known in the art. One way to prepare the Treg-enriched composition is by using the method of the present invention described above. The TNFR2 agonist and NF-κB activator for use in the method of treating an immunological disease can be any one or more of those described above.

[0008] The allergies that can be treated by the methods of the present invention can be selected from the group consisting of food allergies, seasonal allergies, pet allergies, hives, hay fever, allergic conjunctivitis, poison ivy allergies, oak allergies, mold allergies, drug allergies, dust allergies, cosmetic allergies, and chemical allergies. Autoimmune diseases that can be treated by the methods of the present invention include type 1 diabetes, alopecia areata, ankylosing spondylitis, antiphospholipid syndrome, autoimmune Addison's disease, autoimmune hemolytic anemia, autoimmune hepatitis, Behcet's disease, bullous pemphigoid, cardiomyopathy, celiac sprue dermatitis, chronic fatigue immune deficiency syndrome (CFIDS), chronic inflammatory demyelinating polyneuropathy, Churg-Strauss syndrome, cicatricial pemphigoid, CREST syndrome, cold agglutinin disease, Crohn's disease, essential mixed cryoglobulinemia, fibromyalgia-fibromyositis, Graves' disease, Guillain-Barré syndrome, Hashimoto's thyroiditis, hypothyroidism, idiopathic pulmonary fibrosis, idiopathic thrombocytopenic purpura (ITP), and IgA. The disease may be selected from the group consisting of nephropathy, juvenile arthritis, lichen planus, systemic lupus erythematosus (Lupus), Meniere's disease, mixed connective tissue disease, multiple sclerosis, myasthenia gravis, pemphigus vulgaris, pernicious anemia, polyarteritis nodosa, polychondritis, polyglandular syndrome, polymyalgia rheumatica, polymyositis and dermatomyositis, primary agammaglobulinemia, primary biliary cirrhosis, psoriasis, Raynaud's phenomenon, Reiter's syndrome, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma, Sjogren's syndrome, stiff-man syndrome, Takayasu's arteritis, temporal arteritis / giant cell arteritis, ulcerative colitis, uveitis, vasculitis, vitiligo, and Wegener's granulomatosis.

[0009] The above treatment method is at least 5 × 10 6 (e.g., 5×10 6 , 5×10 7 , 5×10 8 , 5×10 9 , 5×10 10 , 5×10 11 , or 5 × 10 12) Tregs. Tregs with desirable immunomodulatory properties include, for example, those that express FOXP3.

[0010] The present invention also features an isolated antibody or antigen-binding fragment thereof that selectively binds to a first epitope of TNFR2, the first epitope comprising positions 48-67 of SEQ ID NO:1. The antibody or antigen-binding fragment thereof has antagonistic activity against TNFR2 upon binding. The antibody or antigen-binding fragment thereof can further bind to a second epitope of TNFR2. The second epitope comprises position 135 of SEQ ID NO:1. The second epitope can comprise positions 135-147 of SEQ ID NO:1 (e.g., positions 130-149 of SEQ ID NO:1, positions 128-147 of SEQ ID NO:1, or positions 135-153 of SEQ ID NO:1). The antibody or antigen-binding fragment thereof can be a monoclonal antibody or antigen-binding fragment thereof, a polyclonal antibody or antigen-binding fragment thereof, a Fab, a humanized antibody or antigen-binding fragment thereof, a bispecific antibody or antigen-binding fragment thereof, a monovalent antibody or antigen-binding fragment thereof, a chimeric antibody or antigen-binding fragment thereof, a single-chain Fv molecule, a bispecific single-chain Fv ((scFv')2) molecule, a domain antibody, a diabody, a triabody, an affibody, a domain antibody, a SMIP, a nanobody, an Fv fragment, a Fab fragment, a F(ab')2 molecule, or a tandem scFv (taFv) fragment. The equilibrium dissociation constant ("K") for the binding of the antibody or antigen-binding fragment thereof to TNFR2 is D The equilibrium dissociation constant ("K") for the binding of the antibody or antigen-binding fragment thereof to TNFR2 can be less than about 50 nM (e.g., less than about 30 nM, less than about 20 nM, less than about 10 nM, less than about 5 nM, less than about 2 nM, less than about 1 nM, less than about 900 pM, less than about 800 pM, or less than about 700 pM). D ") can be in the range of about 10 pM to about 50 nM (e.g., about 20 pM to about 30 nM, about 50 pM to about 20 nM, about 100 pM to about 5 nM, about 150 pM to about 1 nM, or about 200 pM to about 800 pM).

[0011] The invention also features a lymphocyte-enriched and Treg-depleted composition, wherein less than 10% (e.g., less than 9%, less than 8%, less than 7%, less than 5%, or less than 2%, or substantially none) of the cells in the composition are Tregs. The composition can be prepared by any method known in the art.

[0012] The present invention further features a method for preparing a composition enriched in lymphocytes and depleted in Tregs. The method generally involves contacting a population of human cells, including Tregs, with a tumor necrosis factor receptor 2 (TNFR2) antagonist and / or an NF-κB inhibitor in vitro. The TNFR2 antagonist and / or NF-κB inhibitor is used to suppress Treg proliferation, thereby resulting in a composition substantially depleted of Tregs. The population of human cells can be obtained from a human blood sample or human bone marrow sample derived from a patient. The population of human cells can include, for example, CD4+ cells, CD25+ cells, or CD4+CD25+ cells, which can be separated or enriched from the blood or bone marrow sample before contacting with the TNFR2 antagonist and / or NF-κB inhibitor. The above methods can be used to prepare a lymphocyte-enriched composition (e.g., substantially 100% of the cells in the composition are lymphocytes), wherein less than 10% (e.g., less than 9%, less than 8%, less than 7%, less than 5%, or less than 2%, or substantially none) of the cells in the composition are Tregs.

[0013] The TNFR2 antagonist that can be used in the above method for preparing a lymphocyte-enriched and Treg-depleted composition can be an agent selected from the group consisting of an antibody (e.g., a monoclonal anti-TNFR2 antibody), a peptide, a small molecule, and a protein. The NF-κB inhibitor that can be used in the above method can be an agent selected from the group consisting of a small molecule, a peptide (e.g., a cell-permeable inhibitory peptide), a protein, a virus, and a small non-coding RNA. For example, the NF-κB inhibitor can be a small molecule selected from the group consisting of 2-(1,8-naphthyridin-2-yl)-phenol, 5-aminosalicylic acid, BAY 11-7082, BAY 11-7085, CAPE (caffeic acid phenethyl ester), diethyl maleate, ethyl 3,4-dihydroxycinnamate, helenalin, gliotoxin, and NF-κB activation inhibitor II. JSH-23, NF-κB activation inhibitor III, glucocorticoid receptor modulator, CpdA, PPM-18, pyrrolidine dithiocarbamic acid ammonium salt, (R)-MG-132, rocaglamide, sodium salicylate, QNZ, MG-132 [Z-Leu-Leu-Leu-CHO], astaxanthin, (E)-2-fluoro-4'-methoxystilbene, CHS-828, disulfiram, olmesartan, triptolide, withaferin, celastrol, tanshinone IIA, Ro 106-9920, cardamonin, BAY 11-7821, PSI, HU 211, ML130, PR 39, honokiol, CDI 2858522, andrographolide, and dithiocarbamates.

[0014] The TNFR2 antagonist can be a TNFR2 antagonist antibody that binds to a first epitope of TNFR2, the first epitope comprising positions 48-67 of SEQ ID NO:1. The antibody or antigen-binding fragment thereof can bind to a second epitope of TNFR2, the second epitope comprising position 135 of SEQ ID NO:1 (e.g., positions 135-147 of SEQ ID NO:1). The antibody or antigen-binding fragment thereof can be a monoclonal antibody or antigen-binding fragment thereof, a polyclonal antibody or antigen-binding fragment thereof, a Fab, a humanized antibody or antigen-binding fragment thereof, a bispecific antibody or antigen-binding fragment thereof, a monovalent antibody or antigen-binding fragment thereof, a chimeric antibody or antigen-binding fragment thereof, a single-chain Fv molecule, a bispecific single-chain Fv ((scFv')2) molecule, a domain antibody, a diabody, a triabody, an affibody, a domain antibody, a SMIP, a nanobody, an Fv fragment, a Fab fragment, a F(ab')2 molecule, or a tandem scFv (taFv) fragment. The equilibrium dissociation constant ("K") of the binding of the antibody or antigen-binding fragment thereof to TNFR2 D The equilibrium dissociation constant ("K") for the binding of the antibody or antigen-binding fragment thereof to TNFR2 can be less than about 50 nM (e.g., less than about 30 nM, less than about 20 nM, less than about 10 nM, less than about 5 nM, less than about 2 nM, less than about 1 nM, less than about 900 pM, less than about 800 pM, or less than about 700 pM). D ") can be in the range of about 10 pM to about 50 nM (e.g., about 20 pM to about 30 nM, about 50 pM to about 20 nM, about 100 pM to about 5 nM, about 150 pM to about 1 nM, or about 200 pM to about 800 pM).

[0015] The present invention features a method of treating a proliferative disease (e.g., cancer or solid tumor) in a patient (e.g., a human patient) by administering to the patient one or more of a lymphocyte-enriched and Treg-depleted composition, a TNFR2 antagonist (e.g., a monoclonal anti-TNFR2 antibody), or an NF-κB inhibitor. For example, the method of treating a proliferative disease can include administering a lymphocyte-enriched (and Treg-depleted) composition alone or in combination with an NF-κB inhibitor. The lymphocyte-enriched composition can be prepared by any method known in the art. Preferably, the lymphocyte-enriched composition can be prepared by the method of the present invention as described above. The TNFR2 antagonist and NF-κB inhibitor for use in the method of treating a proliferative disease can be any one or more of those described above.

[0016] The present invention features a method for treating an infectious disease (e.g., a bacterial infection, a viral infection, a fungal infection, or a parasitic infection) in a patient by administering to the patient a lymphocyte-enriched and Treg-depleted composition, a TNFR2 antagonist (e.g., a monoclonal anti-TNFR2 antibody), or an NF-κB inhibitor. For example, the method for treating an infectious disease can include administering a lymphocyte-enriched (and Treg-depleted) composition alone or in combination with an NF-κB inhibitor. The lymphocyte-enriched composition can be prepared by any method known in the art. Preferably, the lymphocyte-enriched composition can be prepared by the method of the present invention as described above. The TNFR2 antagonist and NF-κB inhibitor for use in the method for treating an infectious disease can be any one or more of those described above.

[0017] The invention features methods of treating an infectious disease in a patient by administering to the patient an effective amount of an antibody or antigen-binding fragment thereof described herein. The invention also features methods of treating a proliferative disease (e.g., cancer) in a patient by administering to the patient an effective amount of an antibody or antigen-binding fragment thereof described herein.

[0018] Cancers that can be treated according to the methods of the invention (e.g., by administering any one or more of a lymphocyte-enriched (and Treg-depleted) composition, a TNFR2 antagonist, and / or an NF-κB inhibitor) can be selected from the group consisting of acute lymphoblastic leukemia, acute lymphocytic leukemia, acute myeloid leukemia, adrenocortical carcinoma, AIDS-related lymphoma, AIDS-related malignancies, anal cancer, astrocytoma, cholangiocarcinoma, bladder cancer; bone cancer, osteosarcoma / malignant fibrous histiocytoma, brainstem glioma, visual pathway and hypothalamic glioma, breast cancer, bronchial adenoma / carcinoid. , chronic lymphocytic leukemia, chronic myeloid leukemia, chronic myeloproliferative disorders, clear cell sarcoma of tendon sheath, colon cancer, colorectal cancer, cutaneous T-cell lymphoma, endometrial cancer, epithelial carcinoma, esophageal cancer, Ewing's tumor, extracranial germ cell tumor, extragonadal germ cell tumor, extrahepatic bile duct cancer, eye cancer, intraocular melanoma, retinoblastoma, gallbladder cancer, gastric cancer, hairy cell leukemia, head and neck cancer, hepatocellular (liver) carcinoma, Hodgkin's lymphoma, hypopharyngeal cancer, Kaposi's sarcoma, kidney cancer, laryngeal cancer, pituitary cancer, plasma cell neoplasm / multiple myeloma, pleuropulmonary blastoma, skin cancer, small cell lung cancer, small intestine cancer, sarcoma of the soft tissue, squamous cell cervical carcinoma, testicular cancer, thyroid cancer, urethral cancer, uterine sarcoma, and vaginal cancer. Solid tumors that can be treated with the methods of the present invention include solid tumors of the brain, lung, breast, lymphatic system, gastrointestinal tract, genitourinary tract, pharynx, prostate, or ovaries.

[0019] definition The term "about" is used herein to mean a value that is ±10% of the stated value. As used herein, the term "antibody" includes whole antibodies or immunoglobulins, as well as antigen-binding fragments or single chains thereof. Antibodies, as used herein, can be mammalian (e.g., human or murine), humanized, chimeric, recombinant, synthetically produced, or naturally isolated. In most mammals, including humans, whole antibodies have at least two heavy (H) chains and two light (L) chains connected by disulfide bonds. Each heavy chain contains a heavy chain variable region (referred to herein as V H The heavy chain constant region consists of three domains, C H 1. C H 2 and C H 3 and C H 1 and C H Each light chain consists of a light chain variable region (referred to herein as V L The light chain constant region consists of one domain, C L V H and V L The regions can be further subdivided into regions of hypervariability (called complementarity determining regions (CDRs)) interspersed with more conserved regions (called framework regions (FRs)). H and V LEach antibody is composed of three CDRs and four FRs arranged in the following order from amino to carboxy terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant regions of the antibody can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. The antibodies of the present invention include all known forms of antibodies and other protein scaffolds with antibody-like properties. For example, the antibody can be a monoclonal antibody, a polyclonal antibody, a human antibody, a humanized antibody, a bispecific antibody, a monovalent antibody, a chimeric antibody, or a protein scaffold with antibody-like properties, such as fibronectin or ankyrin repeats. The antibody can have any of the following isotypes: IgG (e.g., IgG1, IgG2, IgG3, and IgG4), IgM, IgA (e.g., IgA1, IgA2, and IgAsec), IgD, or IgE.

[0020] As used herein, the term "antigen-binding fragment" refers to one or more fragments of an antibody that retain the ability to specifically bind to a particular antigen (e.g., CD21 receptor). The antigen-binding function of an antibody is performed by a fragment of a full-length antibody. An antibody fragment can be a Fab, Fab'2, scFv, SMIP, diabody, triabody, affibody, nanobody, aptamer, or domain antibody. Examples of binding fragments encompassed by the term "antigen-binding fragment" of an antibody include, but are not limited to, the following: (i) V L , V H , C L , and C H (ii) a monovalent fragment consisting of one Fab fragment; (iii) a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region, the F(ab')2 fragment; H and C H (iv) a single-arm V of an antibody; Land V H (v) Fv fragment consisting of V domains; H and V L (vi) V domain-containing dAb; H (vii) dAb fragments consisting of domains (Ward et al., Nature 341:544-546, 1989); H or V L (viii) an isolated complementarity determining region (CDR); and (ix) a combination of two or more isolated CDRs, optionally joined by a synthetic linker. Additionally, the two domains of an Fv fragment, V L and V H Although the V and VD are encoded by separate genes, they can be joined by a synthetic linker using recombinant methods. L and V H This allows the antibody fragments to be produced as a single protein chain in which the regions pair to form monovalent molecules (known as single-chain Fvs (scFvs)) (see, e.g., Bird et al., Science 242: 423-426, 1988; and Huston et al., Proc. Natl. Acad. Sci. USA 85: 5879-5883, 1988). These antibody fragments are obtained using conventional techniques known to those skilled in the art, and the fragments are screened for utility in the same manner as intact antibodies. Antigen-binding fragments can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact immunoglobulins.

[0021] The term "chimeric antibody" refers to an immunoglobulin or antibody whose variable regions derive from a first species and whose constant regions derive from a second species. Chimeric antibodies can be constructed, for example, by genetic engineering, from immunoglobulin gene segments belonging to different species (e.g., from mouse and human).

[0022] The term "human antibody," as used herein, is intended to include antibodies or fragments thereof having variable regions in which both the framework and CDR regions are derived from human germline immunoglobulin sequences, e.g., as described in Kabat et al. (Sequences of proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242, 1991). Furthermore, if the antibody contains a constant region, the constant region also is derived from human germline immunoglobulin sequences. Human antibodies may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, the term "human antibody," as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences (i.e., humanized antibodies or antibody fragments).

[0023] The term "humanized antibody" refers to any antibody or antibody fragment comprising at least one immunoglobulin domain having a variable region comprising a variable framework region substantially derived from a human immunoglobulin or antibody and a complementarity determining region (e.g., at least one CDR) substantially derived from a non-human immunoglobulin or antibody.

[0024] As used herein, the term "TNF-α mutein" refers to a polypeptide having an amino acid sequence that differs from that of TNF-α by one or more amino acids while retaining the ability to activate or inhibit TNFR2. For example, a TNF-α mutein can have an amino acid sequence that has more than 90% but less than 100% sequence identity to the amino acid sequence of a reference polypeptide (TNF-α).

[0025] As used herein, the terms "substantially 100%" or "substantially homogeneous" with respect to the Treg-enriched compositions of the invention mean that at least 90%, 95%, 96%, 97%, 98%, 99%, or more (e.g., all) of the cells in the composition are Tregs. As used herein, the term "treat" means to stabilize or alleviate adverse symptoms associated with a disease; to reduce the severity of disease symptoms; to slow the rate of disease progression; to inhibit or stabilize the progression of a disease state; or to alter a metric associated with a disease state in a desired manner. [Brief explanation of the drawings]

[0026] [Figure 1A] Figure 1A is a series of graphs showing that in a small double-blind, placebo-controlled study of human subjects, BCG treatment induces TNF-α (top left graph) versus placebo (right graph), and shortly thereafter, Tregs appear in treated subjects (bottom left graph). [Figure 1B] Figure 1B is a series of graphs showing that TNF-α alone does not induce FOXP3 in culture in freshly isolated CD4+ cells from fresh human blood (left graph), but when incubated with IL-2, it induces FOXP3 to higher levels compared to IL-2 alone (right graph). Data are from 14 subjects (left panel) and 10 subjects (right panel). [Figure 1C] Figure 1C is a series of representative flow cytometry histograms confirming greater intracellular induction of FOXP3 in CD4+CD25hi Tregs after co-incubation with TNF-α and IL-2 than with IL-2 alone. Numbers in flow diagrams are %. [*P<0.05 or **P<0.01 by paired t-test]. [Figure 2A] FIG. 2A is a series of graphs showing that TNFR2 is preferentially expressed on CD4+CD25hi T cells. [Figure 2B]FIG. 2B is a graph showing that one TNFR2 antibody acted as an agonist to induce FOXP3, while the other TNFR2 antibody acted as an antagonist to suppress FOXP3+ expression. [Figure 2C] Figure 2C is a graph showing that incubation of purified CD4+ cells with IL-2, TNFR2 agonists, and antagonists in a signaling pathway assay results in differences in the relative downstream expression of mRNA, particularly for the signaling proteins TRAF2, TRAF3, and the inhibitor of apoptosis cIAP2, which are preferentially induced by TNFR2 agonism. Data shown are means ± SEM from four subjects. [Figure 2D] Figure 2D is a series of graphs showing that TNFR2 agonists elicited a greater percent increase in proliferation in samples from six subjects, as measured by flow cytometry (left panel) and carboxyfluorescein diacetate succinimidyl ester (CFSE) assays (right panel). Representative results from a typical experiment are presented by CFSE assays (right panel). Numbers within the bars represent the percent of cells that entered division. TNFR2 antagonists suppressed CD4+ proliferation (left panel) and inhibited proliferation as measured by CFSE dilution (right panel). *P<0.05 or ***P<0.01 by paired t-test. [Figure 3A] Figure 3A is a schematic diagram showing the protocol for purifying CD4+CD25hi cells from fresh blood-derived CD4+ cells and expanding them for 16 days by incubating them in 96-well round-bottom plates (2 x 104 cells / well) with anti-CD3 antibody, anti-CD28 antibody, human IL-2, and rapamycin. [Figure 3B] Figure 3B is a series of graphs showing representative CD25 and FOXP3 flow diagrams of CD4+ cells before versus after CD25hi purification and expansion, indicating the purity of the population. [Figure 3C]Figure 3C is a graph showing the number of purified Treg cells by treatment group, revealing that the TNFR2 agonist induced greater proliferation than any other group. *P<0.05, **P<0.01 by paired t-test. The TNFR2 antagonist suppressed proliferation compared to no treatment. Data in Figure 3C are from samples from 10 subjects. [Figure 4A] FIG. 4A is a series of graphs showing that all treatment groups were highly positive for Treg markers such as CD25, FOXP3, CTLA4, TNFR2, CD62L, and Fas, and negative for CD127. [Figure 4B] Figure 4B is a series of graphs showing that Tregs treated with TNFR2 agonists nearly uniformly express HLA-DR and CD45RO and nearly uniformly lack markers such as ICOS, CXCR3, CCR5, CCR6, CCR7, and CXCR3. [Figure 4C] Figure 4C is a series of representative flow diagrams showing that Tregs treated with TNFR2 agonists have greater homogeneity of Treg markers than other groups (*P<0.05, **P<0.01 by t test). [Figure 5A] Figure 5A is a series of graphs showing that, in a representative case, Tregs treated with TNFR2 agonists exerted more potent, dose-dependent suppression of CD8+ cell numbers than the other groups at all dilutions or suppression ratios (left panel, third column). With a suppression index of 2:1 (CD8+ responders vs. Tregs), TNFR2 agonist suppression of CD8+ cells was greater than no treatment and TNFR2 antagonist treatment (right panel). Data in Figure 5A (right panel) are from samples from five subjects, while data in Figure 5B (bottom panel) are from eight subjects. [Figure 5B] FIG. 5B is a series of graphs showing that Tregs treated with TNFR2 agonists produce a lower percentage of IFNγ+ cells. [Figure 5C]FIG. 5C is a graph showing lower numbers of T-bet+ cells after 24 hours of stimulation with PMA and ionomycin. [Figure 6] Figure 6 is a schematic diagram outlining the results of studies using TNFR2 agonists versus antagonists. After purification and expansion, TNFR2 agonists are superior to TNFR2 antagonists in expanding and generating phenotypically more homogeneous Tregs (CD4+ CD25hi FOXP3+ CTLA4+ TNFR2+ CD45RO+ CD62L+ CD127-, HLA-DRhi CCR5- CCR7- CXCR3- ICOS-) with higher suppressive capacity against CD8+ cells and lower cytokine production. [Figure 7] Figure 7 is a graph showing the induction of FOXP3 expression by different TNFR agonist and antagonist antibodies. Screening of anti-TNFR1 and anti-TNFR2 mAbs reveals that not all of the tested antibodies induce or suppress FOXP3+ expression. [Figure 8] Figure 8 is a graph showing the percentage of CD25+FOXP3- cells after overnight incubation with IL-2 in the presence and absence of TNFR2 agonists or antagonists. A significant percentage increase was observed when treatment groups were incubated in the presence of TNF or TNFR2 agonists (**; P<0.001). Data are from samples from 10 subjects. [Figure 9A] Figure 9A is a schematic diagram showing the expansion protocol. After 16 days of expansion, Treg Expander beads were removed and allowed to rest overnight for cell counting. [Figure 9B] Figure 9B is a graph showing the magnitude of proliferation by each treatment group (*; p<0.05 by paired t-test). The data in Figure 9B are from samples from 10 subjects. [Figure 10A] FIG. 10A is a series of graphs showing that all cells were Fas positive. [Figure 10B]Figure 10B is a series of graphs showing that several surface markers showed diverse expression patterns in similar cell growth methods compared to cells grown with rapamycin (*; p<0.05, **; p<0.01, as determined by paired t-test). Data are from six subjects. [Figure 11A] FIG. 11A is a graph showing the phenotype of freshly isolated Tregs before expansion (N=3, samples from 3 subjects). [Figure 11B] FIG. 11B is a series of graphs showing a representative flow diagram of Treg markers before expansion. [Figure 12] Figure 12 is a series of graphs showing the density of cell surface markers as measured by mean fluorescence intensity (MFI). The MFI of Tregs shows a clear difference between TNFR2 agonist- and TNFR2 antagonist-proliferated cells (*p<0.05, **p<0.01, as determined by paired t-test). [Figure 13A] Figure 13A is a graph showing that the suppressive capacity of expanded CD4+CD25+ cells was determined by CFSE dilution of CD8+ T responder cells. A flow cytometry diagram of a typical result and a summary of the suppression index calculated based on a 2:1 responder:Treg ratio from four independent experiments are also shown in Figure 13A. [Figure 13B] Figure 13B is a series of graphs showing that CD4+CD25+ cells expanded by TNFR2 agonists exhibited significantly enhanced suppressive capacity (N=5). These cells also exhibited the lowest cytokine production capacity (IFN, IL-10, and TNF after 24 hours of stimulation with PMA and ionomycin (*; p<0.05, **; p<0.01 by paired t-test)). DETAILED DESCRIPTION OF THE INVENTION

[0027] The present invention relates to Treg (e.g., CD4+, CD25 hiThe invention features methods for preparing compositions enriched in Tregs (e.g., Treg-enriched compositions), such as those prepared by the methods described below. The invention also features methods for treating immunological and infectious diseases using Treg-enriched compositions, such as those prepared by the methods described below. The invention also features methods for preparing lymphocyte-enriched (and Treg-depleted) compositions, as well as methods for treating proliferative diseases using the compositions.

[0028] Tregs and TNFR2 T regulatory cells (Tregs) are a small subset of T lymphocytes with diverse clinical applications in transplantation, allergy, asthma, infectious diseases, GVHD, and autoimmunity. Tregs can be used in patients in need to suppress aberrant immune responses. Tregs are also known to be involved in immune tolerance in conditions such as cancer. Naturally occurring Tregs comprise only 1–5% of all CD4+ T cells in the blood and remain mostly resting until activated. In humans, Tregs are composed of CD4+ T cells and highly expressed interleukin-2 (IL-2) receptor α chain CD25. hi Tregs are defined by their co-expression with TNFR1 and TNFR2. Tregs are also characterized by inducible levels of the intracellular transcription factor FOXP3, and FOXP3 expression can be used to identify Tregs. TNF-α has two receptors, TNFR1 and TNFR2, which each regulate distinct signaling pathways. Unlike TNFR1, which has ubiquitous cellular expression, TNFR2 is expressed in a more restricted manner, primarily restricted to subpopulations of T cells (especially Tregs), endothelial cells, and neurons. Studies in primates suggest that TNFR2-specific ligands may have minimal systemic toxicity due to the limited cellular distribution of TNFR2. Naturally occurring Tregs appear to express TNFR2 at higher densities than TNFR1. These characteristics make TNFR2 an advantageous molecular target for Tregs.

[0029] Methods for preparing enriched Treg compositions The present invention relates to CD4+ and CD25 hiThe present invention features a method for expanding Tregs in a sample isolated from a patient (e.g., a human), such as a peripheral blood sample or a bone marrow sample, to prepare a Treg-enriched composition characterized as: Methods for promoting the proliferation of Tregs are known in the art and are described, for example, in the following: Brunstein, CG et al., Infusion of ex vivo expanded T regulatory cells in adults transplanted with umbilical cord blood: safety profile and detection kinetics, Blood 117, 1061-1070 (2011); Saas, P. & Perruche, S., (F1000 Immunology, 2012), Tresoldi, E. et al., Stability of human rapamycin-expanded CD4+CD25+ T regulatory cells, Haematologica 96, 1357-1365 (2011); Nadig, SN et al., In vivo prevention of transplant arteriosclerosis by ex vivo-expanded human regulatory T cells. Nat Med 16, 809-813 (2010); Battaglia, M., Stabilini, A. & Tresoldi, E., Expanding human T regulatory cells with the mTOR-inhibitor rapamycin, Methods Mol Biol 821, 279-293 (2012); Pahwa, R. et al., Isolation and expansion of human natural T regulatory cells for cellular therapy, Journal of immunological methods 363, 67-79 (2010); Hoffmann, P., Eder, R., Kunz-Schughart, LA, Andreesen, R. & Edinger, M., Large-scale in vitro expansion of polyclonal human CD4(+)CD25high regulatory T cells, Blood 104, 895-903 (2004); Lin, C.H. & Hunig, T., Efficient expansion of regulatory T cells in vitro and in vivo with a CD28 superagonist, European journal of immunology 33, 626-638 (2003); Lan, Q. et al., Induced FOXP3(+) regulatory T cells: a potential new weapon to treat autoimmune and inflammatory diseases? Journal of molecular cell biology 4, 22-28 (2012); Sagoo, P. et al., Human regulatory T cells with alloantigen specificity are more potent inhibitors of alloimmune skin graft damage than polyclonal regulatory T cells, Science Translational Medicine 83, 1-10 (2011); Edinger, M. & Hoffmann, P., Regulatory T cells in stem cell transplantation: strategies and first clinical experiences, Current opinion in immunology 23, 679-684 (2011); Trzonkowski, P. et al., First-in-man clinical results of the treatment of patients with graft versus host disease with human ex vivo expanded CD4+CD25+CD127-T regulatory cells, Clinical Immunology 133, 22-26 (2009); Di Ianni, M. et al., Tregs prevent GVHD and promote immune reconstitution in HLA-haploidentical transplantation, Blood 117, 3921-3928 (2011); Hippen, K.L. et al, Massive ex vivo expansion of human natural regulatory T cells (T(regs)) with minimal loss of in vivo functional activity. Sci Transl Med 3, 83ra41 (2011); Kim, Y.C. et al., Oligodeoxynucleotides stabilize Helios-expressing Foxp3+ human T regulatory cells during in vitro expansion, Blood 119, 2810-2818 (2012); Bacchetta, R. et al., Interleukin-10 Anergized Donor T Cell Infusion Improves Immune Reconstitution without Severe Graft-Versus-Host-Disease After Haploidentical Hematopoietic Stem Cell Transplantation. ASH Annual Meeting Abstracts 114, 45- (2009); Desreumaux, P. et al., Safety and efficacy of antigen-specific regulatory T-cell therapy for patients with refractory Crohn's disease, Gastroenterology 143, 1207-1217 e1202 (2012); Clerget-Chossat, N. et al., in International Society for Cell Therapy Seattle, Washington, (2012); and Cardenas, PA, Huang, Y. & Ildstad, ST, The role of pDC, recipient T(reg) and donor T(reg) in HSC engraftment: Mechanisms of facilitation, Chimerism 2, 65-70 (2011). Each of these publications, and their methods for expanding Tregs, is incorporated herein by reference.

[0030] The protocols for promoting Treg proliferation described in the above publications generally involve obtaining a fresh sample (e.g., a blood sample) from a patient (e.g., a human patient) containing a population of CD4+ cells. The CD4+ cells can be further purified or enriched in one or more steps prior to Treg proliferation. CD4+ cells can be isolated from the sample using techniques known in the art (e.g., using magnetic beads coupled to anti-CD4+ antibodies, such as Dynabeads® CD4 Positive Isolation Kit (Invitrogen)). During culture, the CD4+ cells can be contacted with one or more reagents to stimulate proliferation. For example, one or more of an anti-CD3 antibody, an anti-CD28 antibody, human IL-2, and rapamycin can be added. However, this method alone results in a heterogeneous population of cells, some of which may release proinflammatory cytokines that may be harmful to the patient. This heterogeneous population is useless for treating immunological or infectious diseases, and Tregs cannot be easily separated from this heterogeneous population without damaging them. The present invention improves on these protocols by using TNFR2 agonists, which preferentially promote the proliferation of Tregs and result in a homogeneous population of Tregs with desirable traits, such as a subpopulation of Tregs expressing FOXP3. TNFR2 agonists and / or NF-κB activators inhibit the proliferation of CD4+CD25 Tregs present within a population of human cells. hi by promoting increased proliferation of Tregs and / or by increasing (e.g., by differentiation or activation) CD4+CD25+ T lymphocytes (e.g., CD4+ cells, CD25+ cells, or CD4+CD25+ cells) present within a population of human cells. hi By increasing the generation of Tregs, CD4+CD25 hi Promoting the Enrichment of Tregs The present invention provides cell populations enriched for Tregs that can be used for the treatment of immunological or infectious diseases as described herein.

[0031] In vitro expansion of Tregs using TNFR2 agonists Generally, the method involves obtaining a starting population of T lymphocytes (e.g., CD4+ cells, CD25+ cells, or CD4+CD25+ cells) from a human sample, such as a blood or bone marrow sample. When using a blood sample, typically 3-4 tubes of blood (approximately 2-10 mL in each tube) can be used in the protocol described below. Those skilled in the art can adjust the amount of blood sample used depending on the scale of the cell culture protocol.

[0032] Generally, anti-CD4 and / or anti-CD25 antibodies coupled to a bead matrix, such as magnetic beads (such as Dynabeads®), can be used to separate CD4+ cells, CD25+ cells, or CD4+CD25+ cells. For example, CD4+ T cells can be separated using commercially available reagents, such as the Dynabeads® CD4 Positive Isolation Kit, and CD25+ cells can be separated using commercially available reagents, such as Dynabeads CD25 and / or DETACHaBEAD CD4 / CD8 (Invitrogen). When CD4+CD25+ cells are used as the starting population, the cells can be separated in a single step using both anti-CD4 and anti-CD25 beads, or in a two-step method by first separating CD4+ cells and then separating CD25+ cells, or vice versa.

[0033] The isolated CD4+ cells, CD25+ cells, or CD4+CD25+ cells are then plated in a suitable cell culture vessel, e.g., a 96-well round-bottom plate (2 × 10 4The starting cells (16, 17, 18, or more cells / well) can be expanded in cell culture for about 16 days (e.g., about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or more days) in the presence of anti-CD3 and / or anti-CD28 antibodies. The amount of cells used in the starting culture depends on the volume of the cell culture vessel. The anti-CD3 and anti-CD28 antibodies can be present throughout the entire cell culture, e.g., every day of culture, or only for a portion of the cell culture (one or several days during culture). Typically, the anti-CD3 and anti-CD28 antibodies can be added in the form of commercially available Dynabead Human Treg Expander (Invitrogen) at a bead-to-cell ratio of 2:1.

[0034] Human IL-2 and / or rapamycin can also be added to the cell culture medium, for example, one or more times during the cell culture period. For example, human IL-2 can be added every two days, for example, on days 2, 4, 7, 9, 11, and 14 of a 16-day cell culture period. Rapamycin can be added on days 0, 2, 4, and 7 of a 16-day cell culture period. Human IL-2 and rapamycin can be added together or alternately every other day. Typically, human IL-2 is added two days after initiating cell culture. Human IL-2 and / or rapamycin can also remain in the cell culture throughout the entire expansion protocol. Cell culture medium can be changed every two to three days by replacing half of the medium with fresh medium; the fresh medium may also contain human IL-2 and / or rapamycin. For example, half of the medium can be changed every two to three days to medium containing rapamycin (up to day 7) and IL-2. Typically, rapamycin is used at a concentration of 0.5 nM to 100 μM (e.g., 0.5 nM, 1 nM, 10 nM, 50 nM, 100 nM, 200 nM, 0.5 μM, 0.75 μM, 1 μM, 1.2 μM, or 2 μM), and human IL-2 may be used at a concentration of 0.05 to 6,000 U / ml (e.g., 0.05 U / ml, 1 U / ml, 2 U / ml, 10 U / ml, 20 U / ml, 50 U / ml, 100 U / ml, 150 U / ml, 200 U / ml, 250 U / ml, or 300 U / ml). During the cell culture process, as described above, cells can be passaged as needed. Protocols for cell passage are known in the art.

[0035] The TNFR2 agonist can be added at the initiation of culture on day 0 or at a subsequent time point after initiation of culture (e.g., any day after initiation of culture, provided that the culture includes contacting the cells with the TNFR2 agonist for at least one or more days (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 days or more) of culture). Additional TNFR2 agonists can be added at several times during the cell culture, for example, on one or more of days 7, 8, 9, 10, 11, or 12. Typically, the TNFR2 agonist is added on day 0, and additional TNFR2 agonists can be added on day 9 of a 16-day cell culture period (see, e.g., Figure 3A). A commonly used TNFR2 agonist is an anti-TNFR2 monoclonal antibody. Additional TNFR2 agonists that can be used in this method are described below. Generally, anti-TNFR2 antibodies can be used at concentrations ranging from 0.05 μg / ml to 500 μg / ml, or higher concentrations if necessary (e.g., 0.05 μg / ml, 0.1 μg / ml, 0.25 μg / ml, 0.5 μg / ml, 1 μg / ml, 1.5 μg / ml, 2 μg / ml, 2.5 μg / ml, 3 μg / ml, 3.5 μg / ml, 4 μg / ml, 4.5 μg / ml, 5 μg / ml, 10 μg / ml, 50 μg / ml, 100 μg / ml, 200 μg / ml, 300 μg / ml, 400 μg / ml, or 500 μg / ml). Anti-TNFR2 antibodies can be bound to a matrix, e.g., beads such as magnetic beads, for removal at the end of the cell culture period.

[0036] To recover the cells and remove the anti-CD3 and anti-CD28 reagents, the Treg Expander beads can be removed, for example, with a Detach-a-bead reagent, or multiple rounds of expansion can be performed to allow the beads to detach. The cells can then be washed in an appropriate medium and allowed to rest. The cells can then be analyzed for expression of various protein markers, appropriately stored, and / or used in methods for treating various diseases, as described below.

[0037] After in vitro expansion, Tregs in the enriched composition comprise at least 60% (e.g., 70%, 80%, 90%, or 100%) of the cells in the composition. The above methods preferably result in a homogenous population of Tregs, e.g., substantially 100% (e.g., at least 90%, 95%, 96%, 97%, 98%, 99%, or more (e.g., all)) of the cells in the composition are Tregs.

[0038] The above method can result in an approximately two-fold (e.g., 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, or more) expansion of Tregs. Tregs prepared by this expansion protocol are characterized by FOXP3 expression; for example, preferably, at least 80% (e.g., 85%, 90%, 95%, 98%, 99%, or substantially 100%) of the cells in the expanded Treg population express FOXP3. Furthermore, Tregs expanded by the methods of the present invention preferably express FOXP3 at high levels. The Treg population also contains a low percentage of cells expressing IFNγ. The Treg population also exhibits an enhanced ability to suppress the activation of CD8+ cells.

[0039] A disadvantage of previously described Treg expansion protocols was that identification of expanded Tregs within a heterogeneous cell population required post-expansion sorting of Tregs, often multiple rounds of sorting. Such multiple sorting methods had significant and detrimental effects on the viability, function, and yield of Tregs, thereby limiting the subsequent use of sorted cells for therapeutic applications. Furthermore, this sorting could only enrich for Tregs expressing cell surface markers, but not for Tregs expressing the intracellular marker FOXP3. The present invention provides a method for enriching Tregs expressing FOXP3, CD4+, and CD25+. hiThe present invention features the expansion of Tregs by contacting a population of human cells, which may be or include T lymphocytes (e.g., human CD4+ cells, CD25+ cells, or CD4+CD25+ cells), with a TNFR2 agonist to generate a substantially homogeneous population of Tregs expressing TNFR2. Tregs generated by this method do not require post-expansion sorting prior to use in therapeutic applications, a significant advantage over previously described Treg expansion protocols. Tregs prepared by the present invention are also more potent than previously described Treg cell populations, which may be a result of their homogeneity, the subsets of Tregs generated by this method, or both. The enriched Treg populations of the present invention exhibit highly desirable properties similar to those of immunoregulatory Tregs.

[0040] TNFR2 agonists TNFR2 agonists that can be used in the methods of the present invention include agents such as antibodies, peptides, small molecules, and proteins. A TNFR2 agonist is an agent that can bind to TNFR2 and activate TNFR2 signaling. A TNFR2 agonist can be any agent that can stimulate the expression of any one or more proteins selected from the group consisting of FOXP3, TNF, TRAF2, TRAF3, and cIAP2 when contacted with CD4+ T cells.

[0041] In particular, the TNFR2 agonist can be a monoclonal antibody that binds to TNFR2, such as clone MR2-1 (Cell Sciences) or clone MAB2261 (R&D Systems). The TNFR2 agonist can also be a TNF-α mutein that only binds to TNFR2 as an agonist. TNF-α muteins that can be used as TNFR2 agonists include, for example, those described in U.S. Patent Application Publication No. 2008 / 0176796 A1; U.S. Patent Nos. 5,486,463 and 5,422,104; PCT Publication Nos. WO 86 / 02381; WO 86 / 04606; and WO 88 / 06625; and European Patent Nos. 155,549; 168,214; 251,037; 340,333; and 486,908. Each of these publications is incorporated herein by reference.

[0042] Additionally, anti-TNFR2 antibodies capable of acting as TNFR2 agonists have been described in Galloway et al. (Eur. J. Immunol. 22:3045-3048, 1992), Tartaglia et al. (J. Biol. Chem. 268:18542-18548, 1993), Tartaglia et al. (J. Immunol. 151:4637-4641, 1993), Smith et al. (J. Biol. Chem. 269:9898-9905, 1994), and Amrani et al. (Am. J. Respir. Cell. Mol. Biol. 15:55-63, 1996); each of which is incorporated herein by reference. Peptides capable of acting as TNFR2 agonists can include the 11 amino acid TNF receptor agonist peptide described in Laichalk et al. (Infection & Immunity 66:2822-2826, 1998; incorporated herein by reference).

[0043] Since activation of the NF-κB pathway is a downstream effect of TNFR2 agonism, the Treg expansion method can alternatively or additionally include contacting a T lymphocyte population (e.g., CD4+ cells, CD25+ cells, or CD4+CD25+ cells) with one or more activators of the NF-κB pathway instead of a TNFR2 agonist. The NF-κB activator can be a small molecule, peptide, protein, virus, or small non-coding RNA. For example, the NF-κB activator can be any of the small molecules described in Manuvakhova et al., J. Neurosci. Res. 89: 58-72, 2011 (incorporated herein by reference). Alternatively, the NF-κB activator can be betulinic acid. The NF-κB activator can also be the topoisomerase poison VP16. Furthermore, the NF-κB activator can be doxorubicin.

[0044] Characterization of Tregs Tregs in the enriched composition prepared by the above method are CD4+ and CD25 hi and can be characterized by the presence or absence of one or more additional molecular markers. For example, Tregs generated by the methods of the present invention can express one or more proteins selected from the group consisting of FOXP3, CTLA4, TNFR2, CD62L, Fas, HLA-DR, and CD45RO, and are considered "positive" for these markers. Alternatively, Tregs can express no, or low or nearly undetectable amounts of, one or more proteins selected from the group consisting of CD127, CCR5, CCR6, CCR7, CXCR3, IFN-γ, IL10, and ICOS, and are considered "negative" for these markers. Preferably, the methods result in an enriched composition of Tregs, in which at least 90% of the Tregs express HLA-DR and less than 5% of the Tregs express ICOS.

[0045] Treatment with the Enriched Treg Compositions of the Invention The present invention features methods for treating various diseases, e.g., immunological diseases and conditions such as allergies, asthma, autoimmune diseases, GVHD, and transplant rejection, as well as infectious diseases, by administering a composition enriched in Tregs to a patient (e.g., a human) in need thereof. The composition enriched in Tregs can be prepared by the methods described above, for example, by contacting a human sample, e.g., a blood or bone marrow sample, containing T lymphocytes (e.g., CD4+ cells, CD25+ cells, or CD4+CD25+ cells), with a TNFR2 agonist (e.g., a TNFR2 agonist antibody) and / or an NF-κB activator to generate a Treg-enriched composition (e.g., a substantially homogeneous population of Tregs). The TNFR2 agonist and / or NF-κB activator activates the CD4+CD25+ cells present in the population of human cells. hi by promoting increased proliferation of Tregs and / or by increasing (e.g., by differentiation or activation) CD4+CD25+ T lymphocytes (e.g., CD4+ cells, CD25+ cells, or CD4+CD25+ cells) present within a population of human cells. hi By increasing the generation of Tregs, CD4+CD25 hi Promotes the enrichment of Tregs.

[0046] Patients in need of treatment for immunological diseases or conditions, such as allergies, asthma, autoimmune diseases, GVHD, or transplant rejection, or for infectious diseases, may receive Tregs (e.g., CD4+CD25 hi Treg or CD4+CD25 hiCompositions enriched for FOXP3+ Tregs can be administered using the following steps: i) obtaining a cell sample, e.g., a blood or bone marrow sample, from a human patient; ii) isolating T lymphocytes (e.g., CD4+ cells, CD25+ cells, or CD4+CD25+ cells) from the sample as described in the methods above; iii) treating these cells with the Treg expansion methods described above (e.g., CD4+ cells, CD25+ cells, or CD4+CD25+ cells with anti-CD3 antibodies, anti-CD28 antibodies, and T lymphocytes in combination with IL-2 and / or rapamycin). and (iv) introducing the Treg-enriched composition into the patient without post-expansion sorting of the enriched Tregs (or with limited post-expansion sorting) to treat the disease. The steps of the treatment method can be performed in repeated cycles, in which the number of treatment cycles provided to the patient can be determined by the disease being treated, the severity of the disease, and / or the outcome of each treatment cycle (i.e., changes in the disease state). For example, changes in efficacy markers and / or changes in clinical outcomes can be used to determine how frequently blood or bone marrow should be collected from the patient, how frequently Tregs should be enriched from the blood or bone marrow, and / or how frequently the enriched Tregs should be administered to the patient. The enriched Treg composition can also be stored (eg, frozen) for later administration.

[0047] Preferably, Tregs are obtained from the patient's own blood or bone marrow (i.e., autologous cells), but the following treatment methods can also include the use of Tregs from allogeneic or unrelated donors with the best possible HLA compatibility, expanded according to the methods described above. Allogeneic Tregs preferentially share at least 4 / 6 HLA markers in common with the patient receiving the enriched Treg composition.

[0048] Treating Immunological Diseases or Conditions Using the Enriched Treg Compositions of the Invention The enriched Treg composition prepared by the above method can be administered to a patient suffering from an immunological disease or condition, such as allergy, asthma, autoimmune disease, GVHD, or transplant rejection, to treat the immunological disease or condition.

[0049] 1) Allergies: The enriched Treg compositions of the present invention can be used to treat one or more allergic symptoms in a patient, such as an allergy selected from the group consisting of food allergies, seasonal allergies, pet allergies, hives, hay fever, allergic conjunctivitis, poison ivy allergies, oak allergies, mold allergies, drug allergies, dust allergies, cosmetic allergies, and chemical allergies. Administration and dosages of the Treg compositions are described herein below.

[0050] 2) Asthma: The enriched Treg compositions of the present invention can be used to treat asthma by administering the composition to a patient in need thereof.

[0051] 3) Autoimmune diseases: The enriched Treg compositions of the present invention can be used to treat one or more autoimmune diseases selected from the group consisting of type 1 diabetes, alopecia areata, ankylosing spondylitis, antiphospholipid syndrome, autoimmune Addison's disease, autoimmune hemolytic anemia, autoimmune hepatitis, Behcet's disease, bullous pemphigoid, cardiomyopathy, celiac sprue dermatitis, chronic fatigue immune deficiency syndrome (CFIDS), chronic inflammatory demyelinating polyneuropathy, Churg-Strauss syndrome, cicatricial pemphigoid, CREST syndrome, cold agglutinin disease, Crohn's disease, essential mixed cryoglobulinemia, fibromyalgia-fibromyositis, Graves' disease, Guillain-Barré, Hashimoto's thyroiditis, and hypothyroidism. Idiopathic pulmonary fibrosis, idiopathic thrombocytopenic purpura (ITP), IgA nephropathy, juvenile arthritis, lichen planus, systemic lupus erythematosus, Meniere's disease, mixed connective tissue disease, multiple sclerosis, myasthenia gravis, pemphigus vulgaris, pernicious anemia, polyarteritis nodosa, polychondritis, polyglandular syndrome, polymyalgia rheumatica, polymyositis and dermatomyositis, primary agammaglobulinemia, primary biliary cirrhosis, psoriasis, Raynaud's phenomenon, Reiter's syndrome, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma, Sjögren's syndrome, stiff-man syndrome, Takayasu's arteritis, temporal arteritis / giant cell arteritis, ulcerative colitis, uveitis, vasculitis, vitiligo, and Wegener's granulomatosis. Additional autoimmune diseases that can be treated by the methods of the present invention are disclosed in U.S. Patent No. 8,173,129, which is incorporated herein by reference. Administration and dosages of Treg compositions are described herein below.

[0052] 4) Graft rejection or GVHD: The enriched Treg composition of the present invention can be used to reduce or suppress graft rejection or GVHD, which occurs when transplanted tissue is rejected by the recipient's immune system. Graft rejection can be chronic, acute, or hyperacute. Administration and dosage of the Treg composition are described herein below.

[0053] In addition to the enriched Treg composition, other treatments that can be administered to the patient include, for example, steroid treatment, antibody-based treatment, immunosuppressive drugs, blood transfusions, and bone marrow transplants, according to techniques known in the art.

[0054] Treatment of infectious diseases using the enriched Treg compositions of the present invention The present invention also features a method for treating infectious diseases caused by viruses, bacteria, fungi, or parasites by administering a composition enriched in Tregs. The Treg-enriched composition can be prepared by the methods described above. The method of the present invention can be used to treat viral infections caused by, for example, the following viruses: members of the Flaviviridae family (e.g., members of the genera Flavivirus, Pestivirus, and Hepacivirus) [hepatitis C virus, yellow fever virus]; tick-borne viruses such as Gadget Galli virus, Kadam virus, Kyasanur Forest disease virus, Rangat virus, Omsk hemorrhagic fever virus, Powassan virus, Royal Farm virus, Karsi virus, tick-borne encephalitis virus, Neudoerfl virus, Sofjin virus, Louping ill virus, and Negishi virus; seabird tick-borne viruses such as Meaban virus, Saumarez Reef virus, and the like. Reef virus, and Tyuleniy virus; mosquito-borne viruses, such as Aroa virus, Dengue virus, Kedougou virus, Cacipacore virus, Koutango virus, Japanese encephalitis virus, Mary Valley encephalitis virus, St. Louis encephalitis virus, Ustu virus, West Nile virus, Yaounde virus, Kocobera virus, Bagaza virus, Ilheus virus, Israeli turkey meningoencephalomyelitis virus, Untaya virus, Tembusu virus, Zika virus, Banzi virus, Bouboui virus, Edgehill virus, Jugra virus, Saboya virus, Sepik virus, Uganda S virus, Wesselsbron virus, and yellow fever virus;and viruses with no known arthropod vector, such as Entebbe bat virus, Yokose virus, Apoi virus, Cowbone Ridge virus, Jutiapa virus, Modoc virus, Sal Vieja virus, San Perlita virus, Bukalasa bat virus, Curry Island virus, Dakar bat virus, Montana myositis leukoencephalitis virus, Phnom Penh bat virus, Rio Bravo virus, Tamana bat virus, and viruses causing cell fusion (Cell fusion virus). members of the Arenaviridae family (including Ippy virus, Lassa virus (e.g., Josiah, LP, or GA391 strains), lymphocytic choriomeningitis virus (LCMV), Mobala virus, Mopeia virus, Amapari virus, Flexal virus, Guanarito virus, Junin virus, Latino virus, Machupo virus, Oliveros virus, Parana virus, Pichinde virus, Pirital virus, Sabia virus, Tacaribe virus, Tamiami virus, Whitewater Arroyo virus, Chapare virus, and Lujo virus); members of the Bunyaviridae family - (e.g., members of the genera Hantavirus, Nairovirus, Orthobunyavirus, and Phlebovirus) [including Hantavirus, Sin Nombre virus, Djugbe virus, Bunyamwera virus, Rift Valley fever virus, La Crosse virus, California encephalitis virus, and Crimean-Congo hemorrhagic fever (CCHF) virus]; members of the Filoviridae family [including Ebola virus (e.g., Zaire, Sudan, Côte d'Ivoire, Reston, and Uganda strains) and Marburg virus (e.g., Angola, Ci67, Musoke, Popp, Ravn, and Lake Victoria strains)];members of the Togaviridae family (e.g., members of the genus Alphavirus) [including Venezuelan equine encephalitis virus (VEE), Eastern equine encephalitis virus (EEE), Western equine encephalitis virus (WEE), Sindbis virus, rubella virus, Semliki Forest virus, Ross River virus, Barmah Forest virus, O'nyong-nyong virus, and Chikungunya virus]; members of the Poxviridae family (e.g., Orthopoxvirus members of the genus hopoxvirus) (including smallpox virus, monkeypox virus, and vaccinia virus); members of the family Herpesviridae (including herpes simplex virus (HSV; types 1, 2, and 6), human herpesviruses (e.g., types 7 and 8), cytomegalovirus (CMV), Epstein-Barr virus (EBV), varicella-zoster virus, and Kaposi's sarcoma-associated herpesvirus (KSHV)); members of the family Orthomyxoviridae - [Influenza viruses (A, B, and C), including, for example, H5N1 avian influenza virus or H1N1 swine influenza virus]; members of the Coronaviridae family (including Severe Acute Respiratory Syndrome (SARS) virus); members of the Rhabdoviridae family (including rabies virus and vesicular stomatitis virus (VSV)]; members of the Paramyxoviridae family (including human respiratory syncytial virus (RSV), Newcastle disease virus, members of the Picornaviridae family (including poliovirus, human enteroviruses (A, B, C, and D), hepatitis A virus, and coxsackievirus); members of the Hepadnaviridae family (including hepatitis B virus);Members of the Papillamoviridae family (including human papillomaviruses); members of the Parvoviridae family (including adeno-associated viruses); members of the Astroviridae family (including astroviruses); members of the Polyomaviridae family (including JC virus, BK virus, and SV40 virus); members of the Calciviridae family (including Norwalk virus); members of the Reoviridae family (including rotaviruses); and members of the Retroviridae family (including human immunodeficiency virus (HIV; e.g., types 1 and 2), and human T-lymphotropic virus types I and II (HTLV-1 and HTLV-2, respectively));

[0055] The methods of the present invention can also be used to treat bacterial infections. Examples of bacterial infections that can be treated include, but are not limited to, those caused by bacteria belonging to the following genera: Salmonella, Streptococcus, Bacillus, Listeria, Corynebacterium, Nocardia, Neisseria, Actinobacter, Moraxella, Enterobacteriaceae, Pseudomonas, Escherichia, Klebsiella, Serratia, Enterobacter, Proteus, Salmonella, Shigella, Yersinia, Haemophilus, Bordetella, Legionella, Pasteurella, Francisella, Brucella, Bartonella, Clostridium, Vibrio, Campylobacter, and Staphylococcus.

[0056] The methods of the invention can also be used to treat parasitic infections caused by protozoan parasites (e.g., intestinal, tissue, or blood protozoans), or helminthic parasites (e.g., nematodes, helminths, adenophorea, secementea, trematodes, flukes (schistosomes, liver flukes, intestinal flukes, and lung flukes), or tapeworms). Representative parasitic protozoa include Entamoeba histolytica, Giardia lamblia, Cryptosporidium muris, Trypanosomatida gambiense, Trypanosomatida rhodesiense, Trypanosomatida crusi, Leishmania mexicana, Leishmania braziliensis, Tropical Leishmania, Leishmania donovani, Toxoplasma gondii, Plasmodium vivax, Plasmodium ovale, Plasmodium malariae, Plasmodium falciparum, Trichomonas vaginalis, and Histomonas meleagridis. Representative parasitic helminths include: Trichuris trichiura, Ascaris lumbricoides, Pinworm verruca, Ancylostoma dubili, Ancylostoma americanum, Strongyloides stercoralis, Wuchereria bancrofti, Dracaena lumbricoides, Schistosoma mansoni, Schistosoma haematobium, Schistosoma japonicum, Fasciola hepatica, Fasciola gigantica, Heterophyoma heterophyum, Paragonimus westermani, Taenia solium, Taenia saginata, Taenia dwarfism, and Echinococcus granulosus.

[0057] The methods of the present invention can also be used to treat fungal infections. Examples of fungal infections that can be treated include, but are not limited to, those caused by the genera Aspergillus, Candida, Malassezia, Trichosporon, Fusarium, Acremonium, Rhizopus, Mucor, Pneumocystis, and Absidia. Administration and dosages of Treg compositions in methods for treating infectious diseases are described herein below.

[0058] Treatment with NF-κB activators Because TNFR2 signaling is transmitted through activation of the NF-κB pathway, activators of NF-κB signaling can also be used instead of or in combination with the administration of an enriched Treg composition to treat immunological diseases or conditions and infectious diseases according to the above methods. For example, any of the NF-κB activators described above can be used in the above treatment methods. The NF-κB activator can be used alone or in combination with the enriched Treg composition.

[0059] Methods for preparing Treg-depleted enriched lymphocyte compositions The present invention also features a method for preparing a composition enriched in lymphocytes (and depleted in Tregs) in vitro. Preferably, this method results in a composition in which less than 10% of the cells in the composition (e.g., less than 10%, less than 5%, less than 3%, less than 1%, or less than 0.5%, or none) are Tregs. This method is similar to the method for preparing a composition enriched in Tregs, except that a TNFR2 antagonist, such as an anti-TNFR2 monoclonal antibody, is used instead of a TNFR2 agonist. Additional TNFR2 antagonists that can be used in this method are described below.

[0060] The method generally involves separating T lymphocytes (e.g., CD4+ cells, CD25+ cells, or CD4+CD25+ cells) from a human sample, such as a human blood or bone marrow sample, and then expanding the cells in culture by incubating them with anti-CD3 and anti-CD28 antibodies. During the expansion step, the cells are contacted with a TNFR2 antagonist. The TNFR2 antagonist suppresses the proliferation of Tregs in the culture, thereby producing a composition enriched in lymphocytes and depleted in Tregs. During cell expansion, human IL-2 and / or rapamycin can optionally be added to the cell culture.

[0061] After in vitro lymphocyte enrichment (and Treg depletion), less than 10% (e.g., less than 10%, less than 9%, less than 8%, less than 7%, less than 5%, or less than 2%, or substantially none) of the cells in the composition are Tregs. The above method can result in approximately a 2-fold (e.g., 2.5-fold, 3-fold, 3.5-fold, 4-fold, or more) enrichment of non-Treg lymphocytes (e.g., CD4+ T cells, CD8+ T cells, CD4+CD8+ T cells, B cells, natural killer cells, etc.). The enriched lymphocyte population may also include dendritic cells, monocytes, macrophages, and neutrophils.

[0062] TNFR2 antagonists TNFR2 antagonists that can be used in this method of the invention can include agents such as antibodies, peptides, small molecules, and proteins that can bind to TNFR2 and inhibit TNFR2 signaling. The TNFR2 antagonist can be an agent that, when contacted with CD4+ T cells, can stimulate expression of cIAP, but not TRAF2, TRAF3, or FOXP3.

[0063] The TNFR2 antagonist can be a monoclonal antibody that binds to TNFR2. TNFR2 has two epitopes to which a TNFR2 antagonist antibody can bind. The first epitope includes positions 48-67 (QTAQMCCSKCSPGQHAKVFC) of SEQ ID NO: 1 (the amino acid sequence of human TNFR2). The second epitope includes position 135 (R) of SEQ ID NO: 1 (e.g., positions 135-153 (RLCAPLRKCRPGF) of SEQ ID NO: 1). For example, the TNFR2 antagonist antibody can be either clone MAB726 (R&D Systems) or clone M1 (BD Biosciences). While MAB726 and M1 each bind to the second epitope, the antibody of the present invention can bind to the first epitope or both epitopes. The TNFR2 antagonist antibody or antigen-binding fragment thereof may have a K of less than about 50 nM (e.g., less than about 30 nM, less than about 20 nM, less than about 10 nM, less than about 5 nM, less than about 2 nM, less than about 1 nM, less than about 900 pM, less than about 800 pM, or less than about 700 pM). D The TNFR2 antagonist antibody or antigen-binding fragment thereof can bind to TNFR2 with a K in the range of about 10 pM to about 50 nM (e.g., about 20 pM to about 30 nM, about 50 pM to about 20 nM, about 100 pM to about 5 nM, about 150 pM to about 1 nM, or about 200 pM to about 800 pM). D The avidity of TNFR2 antagonist antibodies can be measured using methods known in the art (e.g., surface plasmon resonance). For example, MAB 726 has a K of 621 pM (as measured by surface plasmon resonance (Pioneer SensiQ®, Oklahoma City, OK)). D The TNFR2 antagonist can also be a TNF-α mutein that is capable of binding to TNFR2 and inhibiting downstream signaling.

[0064] TNFR2 antagonists can function through downstream signal transduction by inhibiting the NF-κB pathway. Therefore, the methods of the present invention can also include contacting a human sample, such as a blood or bone marrow sample, with one or more inhibitors of the NF-κB pathway to achieve the same effect as using a TNFR2 antagonist. The NF-κB inhibitor can be a small molecule, peptide, protein, virus, or small non-coding RNA. In one embodiment, the NF-κB inhibitor that can be used in the present method to create a lymphocyte-enriched composition can be any one or more of the following: 2-(1,8-naphthyridin-2-yl)-phenol, 5-aminosalicylic acid, BAY 11-7082, BAY 11-7085, CAPE (caffeic acid phenethyl ester), diethyl maleate, ethyl 3,4-dihydroxycinnamate, helenalin, gliotoxin, NF-κB activation inhibitor II. JSH-23, NF-κB activation inhibitor III, glucocorticoid receptor modulator, CpdA, PPM-18, pyrrolidine dithiocarbamic acid ammonium salt, (R)-MG-132, rocaglamide, sodium salicylate, QNZ, MG-132 [Z-Leu-Leu-Leu-CHO], astaxanthin, (E)-2-fluoro-4'-methoxystilbene, CHS-828, disulfiram, olmesartan, triptolide, withaferin, celastrol, tanshinone IIA, Ro 106-9920, cardamonin, BAY 11-7821, PSI, HU 211, ML130, PR 39, honokiol, CDI 2858522, andrographolide, and dithiocarbamates. The NF-κB inhibitor may also be a peptide inhibitor, such as a cell-permeable inhibitory peptide as described in May et al., Science, 2000 Sep 1;289(5484):1550-4 and Orange and May, Cell Mol. Life Sci., 2008 Nov;65(22):3564-91, each of which is incorporated herein by reference.Additional NF-κB inhibitors are also described in Gilmore and Herscovitch, Oncogene (2006) 25, 6887-6899; Nam, Mini Rev. Med. Chem., 2006 Aug;6(8):945-51; and U.S. Pat. No. 6,410,516, each of which is incorporated herein by reference.

[0065] Methods of Treatment Using Treg-Depleted Lymphocyte-Enriched Compositions and / or Antagonists of the TNFR2 Signaling Pathway The present invention features a method for treating a proliferative disease, such as cancer, by administering a lymphocyte-enriched and Treg-depleted composition to a patient in need thereof. The lymphocyte-enriched composition can be prepared by the methods described above, for example, by contacting cells obtained from a human sample, such as a blood or bone marrow sample, with a TNFR2 antagonist, such as a TNFR2 antagonist antibody, and / or an NF-κB inhibitor to produce a lymphocyte-enriched and Treg-depleted composition. The NF-κB inhibitor can be used instead of or in combination with a lymphocyte-enriched and Treg-depleted composition in methods for treating a proliferative disease, such as cancer. The present invention also features a method for treating a proliferative disease, such as cancer, by administering a composition containing a TNFR2 antagonist (e.g., an anti-TNFR2 antagonist antibody) to a patient in need thereof.

[0066] The present invention features a method for treating infectious diseases by administering a lymphocyte-enriched and Treg-depleted composition to a patient in need thereof. The lymphocyte-enriched composition can be prepared by the methods described above, for example, by contacting cells obtained from a human sample, such as a blood or bone marrow sample, with a TNFR2 antagonist, e.g., a TNFR2 antagonist antibody, and / or an NF-κB inhibitor to produce a lymphocyte-enriched and Treg-depleted composition. The NF-κB inhibitor can be used instead of or in combination with the lymphocyte-enriched and Treg-depleted composition in the method for treating infectious diseases. The present invention also features a method for treating infectious diseases by administering a composition containing a TNFR2 antagonist (e.g., an anti-TNFR2 antagonist antibody) alone to a patient in need thereof.

[0067] Treatment of Proliferative Disorders with Enriched Lymphocyte Compositions of the Invention To treat a proliferative disease (e.g., cancer), non-Treg lymphocytes in the patient's blood can be expanded and administered back to the patient. The enriched lymphocyte composition can be administered alone or in combination with one or more anti-cancer agents known in the art.

[0068] The enriched lymphocyte composition can be prepared by: i) obtaining a sample, such as a blood or bone marrow sample, from a human patient and isolating nucleated cells present therein (e.g., lymphocytes, e.g., T lymphocytes such as CD4+ cells, CD25+ cells, or CD4+CD25+ cells); ii) subjecting these cells to the lymphocyte expansion method described above to produce a lymphocyte-enriched and Treg-depleted composition (e.g., a substantially homogenous population of lymphocytes in which Tregs constitute less than 10% of the cells in the composition, preferably less than 5% of the cells in the composition, or in which Tregs are absent from the expanded composition); and iii) introducing the lymphocyte-enriched composition into the patient without post-expansion sorting of the lymphocytes. The above steps of this treatment method can be performed in repeated cycles, in which case the number of treatment cycles provided to the patient can be determined by the proliferative disease being treated, the severity of the disease, and / or the outcome of each treatment cycle, i.e., changes in the disease state. For example, changes in efficacy markers and / or changes in clinical outcome can be used to determine how often blood or bone marrow should be collected from a patient, how often the blood should be enriched for lymphocytes (and Tregs depleted), and how often the enriched lymphocytes should be administered to the patient. Enriched lymphocyte compositions can also be prepared and stored (e.g., frozen) for later use.

[0069] Preferably, the lymphocytes are obtained from the patient's own blood or bone marrow (i.e., autologous cells), but the following treatment methods can also include the use of lymphocytes from allogeneic or unrelated donors with the highest possible HLA compatibility, expanded according to the methods described above. Allogeneic lymphocytes preferentially share at least 4 / 6 HLA markers in common with the patient receiving the enriched lymphocyte composition.

[0070] The treatment of a proliferative disease according to the present invention can include inhibiting the NF-κB signaling pathway in combination with the administration of a Treg-depleted enriched lymphocyte composition. Because TNFR2 signaling is transmitted via the NF-κB pathway, the treatment of a proliferative disease according to the present invention can also include administering an NF-κB pathway inhibitor to a patient. For example, any of the NF-κB inhibitors described above can be administered to treat a proliferative disease. The NF-κB inhibitor can be administered alone or in combination with the lymphocyte-enriched composition. The NF-κB inhibitor can also function independently of the TNFR2 signaling pathway.

[0071] Proliferative disorders that can be treated by administering the lymphocyte enriched / Treg depleted compositions include one or more cancers selected from the group consisting of acute lymphoblastic leukemia, acute lymphocytic leukemia, acute myeloid leukemia, adrenocortical carcinoma, AIDS-related lymphoma, AIDS-related malignancies, anal cancer, astrocytoma, bile duct cancer, bladder cancer; bone cancer, osteosarcoma / malignant fibrous histiocytoma, brain stem glioma, visual pathway and hypothalamic glioma, breast cancer, bronchial adenoma / carcinoid, chronic lymphocytic leukemia, chronic myelogenous leukemia, chronic myeloproliferative disorder. Cancer of the gallbladder, gastric cancer, hairy cell leukemia, head and neck cancer, hepatocellular (liver) cancer, Hodgkin lymphoma, hypopharyngeal cancer, Kaposi's sarcoma, kidney cancer, laryngeal cancer, pituitary cancer, plasma cell neoplasm / multiple myeloma, pleuropulmonary blastoma, skin cancer, small cell lung cancer, small intestine cancer, sarcoma of the soft tissue, squamous cell cervical cancer, testicular cancer, thyroid cancer, urethral cancer, uterine sarcoma, and vaginal cancer. Proliferative disorders can also include solid tumors, including malignancies of various organ systems (e.g., sarcomas, adenocarcinomas, and carcinomas), such as solid tumors of the brain, lung, breast, lymphatic system, gastrointestinal (e.g., colon) and genitourinary tract (e.g., renal, urothelial, or testicular tumors), pharynx, prostate, ovaries, etc. Representative adenocarcinomas include colorectal carcinoma, renal cell carcinoma, liver cancer, non-small cell carcinoma of the lung, and cancer of the small intestine. The administration and dosage of the enriched lymphocyte composition in methods for treating proliferative disorders is described herein below.

[0072] Treatment of infectious diseases using the enriched lymphocyte compositions of the present invention The invention also features a method for treating an infectious disease caused by a virus, bacteria, fungus, or parasite, comprising administering a composition enriched in lymphocytes (e.g., CD8+ T cells, B cells, or natural killer cells) and depleted in Tregs. The composition can be prepared by the methods described above. The methods of the invention can be used to treat viral infections caused by, for example, the following viruses: members of the Flaviviridae family (e.g., members of the genera Flavivirus, Pestivirus, and Hepacivirus) [hepatitis C virus, yellow fever virus; tick-borne viruses such as Gadgett Gallie virus, Kadam virus, Kyasanur Forest disease virus, Rangat virus, Omsk hemorrhagic fever virus, Powassan virus, Royal Farm virus, Karsi virus, tick-borne encephalitis virus, Neudorf virus, Sofjin virus, Louping ill virus, and Negishi virus; seabird tick-borne viruses such as Meaban virus, Saumare's Reef virus, and Tyureni virus; mosquito-borne viruses such as Aroa virus, Dengue virus, Kedougo virus, Cacipaca virus, Koutango virus, Japanese encephalitis virus, Murray Valley encephalitis virus, Viruses with no known arthropod vector, such as Entebbe bat virus, Yokose virus, Apoi virus, Cowbone Ridge virus, Jutiapa virus, Modoc virus, Salvieha virus, San Pelita virus, Bukarassa bat virus, Curry Island virus, Dakar bat virus, Montana myositis leukoencephalitis virus, Phnom Penh bat virus, Rio Bravo virus, Tamana bat virus, and viruses that cause cell fusion.Members of the Arenaviridae family [including Yippee virus, Lassa virus (e.g., Josiah, LP, or GA391 strains), lymphocytic choriomeningitis virus (LCMV), Mobara virus, Mopeia virus, Amapari virus, Flexar virus, Guanarito virus, Junin virus, Latino virus, Machupo virus, Oliveros virus, Parana virus, Pichinde virus, Piritar virus, Sabia virus, Tacaribe virus, Tamiami virus, Whitewater Arroyo virus, Chapare virus, and Lujo virus] members of the Bunyaviridae family (e.g., members of the genera Hantavirus, Nairovirus, Orthobunyavirus, and Phlebovirus) [including Hantaan virus, Sin Nombre virus, Djugbe virus, Bunyamwera virus, Rift Valley fever virus, La Crosse virus, California encephalitis virus, and Crimean-Congo hemorrhagic fever (CCHF) virus]; members of the Filoviridae family [Ebola virus (e.g., Zaire, Sudan, Côte d'Ivoire, Reston, and Uganda strains) and Marburg virus (e.g., , Angola, Ci67, Musoke, Popp, Ravn, and Lake Victoria strains)]; members of the Togaviridae family (e.g., members of the genus Alphavirus) [including Venezuelan equine encephalitis virus (VEE), Eastern equine encephalitis virus (EEE), Western equine encephalitis virus (WEE), Sindbis virus, rubella virus, Semliki Forest virus, Ross River virus, Barmah Forest virus, O'nyong-nyong virus, and Chikungunya virus]; members of the Poxviridae family (e.g., members of the genus Orthopoxvirus) [variola virus , monkeypox virus, and vaccinia virus]; members of the Herpesviridae family [including herpes simplex virus (HSV; types 1, 2, and 6), human herpesviruses (e.g., types 7 and 8), cytomegalovirus (CMV), Epstein-Barr virus (EBV), varicella-zoster virus, and Kaposi's sarcoma-associated herpesvirus (KSHV)]; members of the Orthomyxoviridae family [including influenza viruses (A, B, and C), e.g., H5N1 avian influenza virus or H1N1 swine influenza virus];Members of the Coronaviridae family (including Severe Acute Respiratory Syndrome (SARS) virus); members of the Rhabdoviridae family (including rabies virus and vesicular stomatitis virus (VSV)); members of the Paramyxoviridae family (including human respiratory syncytial virus (RSV), Newcastle disease virus, Hendra virus, Nipah virus, measles virus, rinderpest virus, canine distemper virus, Sendai virus, human parainfluenza viruses (e.g., 1, 2, 3, and 4), rhinovirus, and mumps virus); members of the Picornaviridae family (including poliovirus, human enteroviruses (A, B, C, and D), hepatitis A virus, and coxsackievirus); Members of the Padnaviridae family (including hepatitis B virus), Papillomaviridae family (including human papillomavirus), Parvoviridae family (including adeno-associated virus), Astroviridae family (including astrovirus), Polyomaviridae family (including JC virus, BK virus, and SV40 virus), Caliciviridae family (including Norwalk virus), Reoviridae family (including rotavirus), and Retroviridae family (including human immunodeficiency virus (HIV; e.g., types 1 and 2), and human T-lymphotropic virus types I and II (HTLV-1 and HTLV-2, respectively)).

[0073] The methods of the present invention can also be used to treat bacterial infections. Examples of bacterial infections that can be treated include, but are not limited to, those caused by bacteria belonging to the following genera: Salmonella, Streptococcus, Bacillus, Listeria, Corynebacterium, Nocardia, Neisseria, Actinobacter, Moraxella, Enterobacteriaceae, Pseudomonas, Escherichia, Klebsiella, Serratia, Enterobacter, Proteus, Salmonella, Shigella, Yersinia, Haemophilus, Bordetella, Legionella, Pasteurella, Francisella, Brucella, Bartonella, Clostridium, Vibrio, Campylobacter, and Staphylococcus.

[0074] The methods of the present invention can also be used to treat parasitic infections caused by protozoan parasites (e.g., intestinal, tissue, or blood protozoans) or helminthic parasites (e.g., nematodes, helminths, Diptera, Distringer, trematodes, flukes (schistosomes, liver flukes, intestinal flukes, and lung flukes), or cestodes. Representative protozoan parasites include Entamoeba histolytica, Giardia lamblia, Cryptosporidium murinus, Trypanosoma gambiense, Trypanosoma rhodesiense, Trypanosoma cruzi, Leishmania mexicanus, Leishmania braziliensis, Leishmania tropica, Leishmania donovani, Toxoplasma gondii, Plasmodium vivax, Plasmodium ovale, Plasmodium malariae, Plasmodium falciparum, Trichomonas vaginalis, and Histomonas meleagridis. Representative parasitic helminths include: Trichuris trichiura, Ascaris lumbricoides, Pinworm verruca, Ancylostoma dubili, Ancylostoma americanum, Strongyloides stercoralis, Wuchereria bancrofti, Dracaena lumbricoides, Schistosoma mansoni, Schistosoma haematobium, Schistosoma japonicum, Fasciola hepatica, Fasciola gigantica, Heterophyoma heterophyum, Paragonimus westermani, Taenia solium, Taenia saginata, Taenia dwarfism, and Echinococcus granulosus.

[0075] The methods of the present invention can also be used to treat fungal infections. Examples of fungal infections that can be treated include, but are not limited to, those caused by the genera Aspergillus, Candida, Malassezia, Trichosporon, Fusarium, Acremonium, Rhizopus, Mucor, Pneumocystis, and Absidia. The administration and dosage of lymphocyte-enriched compositions in methods for treating infectious diseases are described herein below.

[0076] Dosage The Treg-enriched composition can be administered to a patient in need thereof daily, weekly, monthly (e.g., once every two weeks or more), or once a year or more, depending on the severity of the disease and changes in the patient's condition during treatment. Generally, a typical dosage is 5×10 5 ~5×1012 pieces (e.g., 5 x 10 5 , 5×10 6 , 5×10 7 , 5×10 8 , 5×10 9 , 5×10 10 , 5×10 11 , or 5 × 10 12 Disease metrics, such as the severity of symptoms, changes in symptoms, the patient's response to treatment, any adverse effects of treatment, and / or the effectiveness of any additional treatment(s), can be used to determine the frequency and dosage of treatment, i.e., the number of Tregs administered to the patient.

[0077] The lymphocyte-enriched (and Treg-depleted) composition can be administered daily, weekly, monthly (e.g., once every two weeks or more), or once a year or more, depending on the severity of the disease and changes in the patient's condition during treatment. Preferably, less than 10% of the cells in the lymphocyte-enriched composition (e.g., less than 9%, less than 8%, less than 7%, less than 5%, less than 1%, or none) are Tregs. Generally, a typical dosage is 5×10 Tregs in the enriched lymphocyte composition. 5 ~5×10 12 pieces (e.g., 5 x 10 5 , 5×10 6 , 5×10 7 , 5×10 8 , 5×10 9 , 5×10 10 , 5×10 11 , or 5 × 10 12 Disease indicators, such as the severity of symptoms, changes in symptoms, the patient's response to treatment, any adverse effects of treatment, and / or the effectiveness of any additional treatment(s), can be used to determine the frequency and dosage of treatment, i.e., the number of lymphocytes administered to the patient.

[0078] Administration In general, the compositions of the invention (e.g., Treg-enriched compositions or lymphocyte-enriched and Treg-depleted compositions) can be administered in any medically useful form. For example, such compositions may optionally include added compounds, such as adjuvants, preservatives, carriers, excipients, diluents, antibacterial or antifungal agents, anti-inflammatory agents, and / or anticancer agents. The compositions of the invention can be administered intravenously, intramuscularly, orally, by inhalation, parenterally, intraperitoneally, intraarterially, transdermally, sublingually, nasally, bucally, liposomally, adiposally, ophthalmically, intraocularly, subcutaneously, intrathecally, orally, orally, and will be formulated accordingly for the selected route of administration.

[0079] Administration of antibodies of the present invention Pharmaceutical compositions containing the anti-TNFR2 antibodies (e.g., anti-TNFR2 antagonist antibodies) of the present invention are prepared for storage in the form of an aqueous solution, lyophilized formulation, or other dry formulation by mixing the antibody having the desired purity with any physiologically acceptable carrier, excipient, or stabilizer. Acceptable excipients or carriers are selected based on the mode and route of administration. Suitable pharmaceutical carriers and pharmaceutical essentials for use in pharmaceutical formulations are described in well-known reference texts in the field, such as Remington: The Science and Practice of Pharmacy, 21st Edition, edited by Gennaro; Lippincott, Williams & Wilkins (2005), and USP / NF (United States Pharmacopeia and National Formulary). Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed and include: buffers, such as phosphate, citrate, histidine, and other organic acids; antioxidants, such as ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl, or benzyl alcohol; alkyl parabens, such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, such as glucose, mannose, or dextrins; chelating agents, such as EDTA; sugars, such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions, such as sodium; metal complexes (e.g., zinc-protein complexes); and / or non-ionic surfactants, such as TWEEN™, PLURONICS™, or polyethylene glycol (PEG).Other representative pharmaceutical excipients are described in Handbook of Pharmaceutical Excipients, 6th Edition, edited by Rowe et al., Pharmaceutical Press (2009).

[0080] The compositions of the present invention can be prepared in a pharmaceutically acceptable carrier or excipient. Such suitable carriers or excipients can be selected from, for example, water, saline (e.g., phosphate-buffered saline (PBS) or acetate-buffered saline (ABS), or Ringer's solution), dextrose, glycerol, ethanol, etc., and combinations thereof. Furthermore, if necessary, compositions for administration to mammals can contain minor amounts of auxiliary substances, such as wetting agents, emulsifying agents, or pH buffering agents that enhance the effectiveness of the compositions. The compositions of the present invention can also be prepared into acceptable salt formulations. Other additives that can be used to prepare the compositions of the present invention include, for example, adjuvants, preservatives, diluents, antibacterial or antifungal agents, anti-inflammatory agents, and / or anticancer agents, if necessary.

[0081] The compositions may also contain more than one active compound as necessary for the particular condition being treated, preferably those with complementary activities that do not adversely affect each other, such molecules being suitably present in combination in amounts that are effective for the purpose intended.

[0082] The active ingredient can also be entrapped in microcapsules (e.g., hydroxymethylcellulose or gelatin microcapsules, and poly(methyl methacrylate) microcapsules, respectively) prepared by coacervation techniques or interfacial polymerization, in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules), or in macroemulsions. Such techniques are disclosed in Remington: The Science and Practice of Pharmacy, 21st Edition, edited by Gennaro; Lippincott, Williams & Wilkins (2005).

[0083] Compositions to be used for in vivo administration must be sterile, which is readily accomplished by filtration through sterile filtration membranes.

[0084] Sustained-release preparations can be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the immunoglobulins of the present invention, which matrices are in the form of shaped articles, e.g., films, or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (e.g., poly(2-hydroxyethyl-methacrylate) or poly(vinyl alcohol)), polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and gamma-ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers, such as LUPRON DEPOT™ (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(-)-3-hydroxybutyric acid. While polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid enable release of molecules for over 100 days, certain hydrogels release proteins for shorter time periods.

[0085] Compositions containing one or more anti-TNFR2 antibodies (e.g., anti-TNFR2 antagonist antibodies) may be administered to a patient before the onset of symptoms of a proliferative or infectious disease, or the compositions may be administered to a patient after one or more (e.g., 1, 2, 3, 4, or 5) symptoms of the disease have appeared and the patient has been diagnosed with a proliferative or infectious disease. The dosage of the anti-TNFR2 antibody will vary depending on the patient's health, but is generally in the range of about 0.1 mg to about 400 mg of antibody per dose (e.g., 1 mg, 5 mg, 10 mg, 20 mg, 50 mg, 100 mg, 200 mg, 300 mg, 400 mg, or more per dose).

[0086] The composition can be administered to a patient in one or more doses (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more doses). When multiple doses are administered, the doses can be administered by the same mode of administration (e.g., intravenous administration) or by different modes of administration (e.g., intravenous administration and intramuscular administration). A patient can also receive different doses at different times. For example, a patient can receive a higher initial dose and lower subsequent doses over the course of treatment, or vice versa.

[0087] The composition can be administered daily, weekly, monthly, or yearly. For example, the composition can be administered twice daily, twice weekly, twice a year, three times a year, or four times a year. The dosage of the composition can be determined by a skilled physician, taking into account the subject's clinical symptoms and / or physical condition (e.g., weight, sex, height, and the severity of proliferative or infectious diseases). The composition can be administered intravenously, intradermally, parenterally, intraarterially, subcutaneously, intramuscularly, intraorbitally, topically, intracerebroventricularly, intrathecally, intraperitoneally, intranasally, intracranially, or orally.

[0088] Kits of the Invention The present invention features a kit for preparing a composition enriched in Tregs. The kit can include a TNFR2 agonist (e.g., a TNFR2 agonist antibody) or an NF-κB activator (e.g., one or more of the NF-κB activators described above), reagents and / or equipment for collecting a human sample, such as a blood or bone marrow sample, reagents and / or equipment for separating blood or bone marrow cells (e.g., CD4+CD25+ cells) from the sample, and reagents for culturing the blood or bone marrow cells (e.g., an anti-CD3 antibody, an anti-CD28 antibody, interleukin-2, and / or rapamycin). Furthermore, the kit can also include instructions for carrying out the methods of the invention, such as instructions for collecting a blood or bone marrow sample and separating blood or bone marrow cells from the sample, instructions for contacting blood or bone marrow cells with a TNFR2 agonist, and / or instructions for culturing, collecting, and / or storing the enriched Tregs. The kit can also include reagents and instructions for assaying the expression of various marker genes that can be used to characterize Tregs. For example, these can include reagents and instructions for detecting mRNA or protein levels of one or more of FOXP3, CTLA4, TNFR2, CD62L, Fas, HLA-DR, CD45RO, CD127, CCR5, CCR6, CCR7, CXCR3, IFN-γ, IL10, and ICOS.

[0089] The present invention features a kit for preparing a composition enriched in lymphocytes and depleted in Tregs. The kit can include a TNFR2 antagonist (e.g., a TNFR2 antagonist antibody) or an NF-κB inhibitor (e.g., one or more of the NF-κB inhibitors described above), reagents and / or equipment for collecting a human blood or bone marrow sample, reagents and / or equipment for separating blood or bone marrow cells (e.g., T lymphocytes such as CD4+ cells, CD25+ cells, or CD4+CD25+ cells) from the sample, and reagents for culturing the blood cells (e.g., an anti-CD3 antibody, an anti-CD28 antibody, interleukin-2, and / or rapamycin). Furthermore, the kit can also include instructions for carrying out the method of the present invention, such as instructions for collecting a blood or bone marrow sample and separating blood or bone marrow cells from the sample, instructions for contacting blood cells with a TNFR2 antagonist, and / or instructions for culturing, collecting, and / or storing the enriched lymphocytes. The kits of the invention may also include reagents and instructions for assaying the expression of various marker genes that can be used to characterize lymphocytes, for example, they may include reagents and instructions for detecting the mRNA or protein levels of one or more of FOXP3, TRAF2, TRAF3, and cIAP.

[0090] The present invention also features a kit that includes a composition containing an anti-TNFR2 antibody (e.g., an anti-TNFR2 antagonist antibody), a pharmaceutically acceptable carrier or excipient, and, optionally, other agents described herein; the composition contains an effective amount of the anti-TNFR2 antibody for treating a proliferative or infectious disease. The kit can include instructions that describe how a practitioner (e.g., a doctor, nurse, or patient) should administer the composition included therein. Furthermore, the kit can also include additional components, such as one or more of the additional components described above, instructions or a dosing schedule for a patient suffering from a proliferative or infectious disease, and, optionally, a device (e.g., a syringe) for administering the composition.

[0091] The following examples are intended to illustrate the present invention. They are not intended to limit the invention in any way. (Example) material and method TNF-α induction in human subjects and by BCG vaccine Two doses of BCG vaccination were used to induce TNF-α, and BCG administration was approved by the Massachusetts General Hospital Human Studies Committee and the FDA (NCT00607230).

[0092] In a double-blind, placebo-controlled study, 1.6–3.2 × 10 6 Subjects were injected with BCG at a dose of 1000 cfu, while placebo subjects received saline injections. BCG or saline injections were administered intradermally twice, 4 weeks apart. All blood samples were blinded and sent simultaneously to laboratories for monitoring of TNF-α and Treg levels.

[0093] Reagents and flow cytometry Recombinant human TNF-α was purchased from Leinco Technologies (St. Louis, MO), and recombinant human IL-2 was purchased from Sigma-Aldrich (St. Louis, MO). Monoclonal antibodies against TNFR1 and TNFR2 used for screening purposes were obtained from internal sources and external suppliers (Table 1). External suppliers included R&D Systems, Hycult-Biotechnology, BD-Pharmingen, Accurate, Abcam, and Sigma. All other antibodies were purchased from BD-Biosciences. Intracellular staining of FOXP3 and CD152 was performed using either the FOXP3 Fix / Perm Buffer Set (Biolegend) or the Human FOXP3 Buffer Set (BD Biosciences). The binding affinity of the MAB726 and M1 antibodies to TNFR2 was measured using surface plasmon resonance on a Pioneer SensiQ (SensiQ Technologies, Oklahoma City, OK). [Table 1]

[0094] CD4+ cell isolation, FOXP3 induction, and CD4 + CD25 + Cell proliferation CD4+ T cells were isolated using a Dynal CD4 Positive Isolation Kit (Invitrogen). CD25+ cells were then extracted using Dynabeads CD25 and DETACHaBEAD CD4 / CD8 (Invitrogen) after CD4+ isolation. After isolation, 2 × 10 4Cells were cultured in 96-round-bottom well plates. Dynabeads for human Treg Expander (Invitrogen) (Dynabeads conjugated with anti-CD3 and anti-CD28 monoclonal antibodies) were added at a bead-to-cell ratio of 2:1. TNF-α (20 ng / ml), TNFR2 mAb (2.5 μg / ml), and rapamycin (1 μM, EMD Biosciences, San Diego, CA) were added to designated wells. After 2 days, IL-2 (200 U / ml) was added to the cultures. Half of the medium was replaced every 2–3 days with medium containing rapamycin (until day 7) and 100 U / ml IL-2. On day 9, additional TNF-α or TNFR2 mAb was added to the medium. On day 16, cells were harvested, the Dynabeads Human Treg Expander was removed, and the cells were washed and quiescent. The following day, the cells were analyzed.

[0095] Intracellular staining Expanded CD4+CD25+ cells were stimulated with phorbol myristate acetate (PMA) (2 ng / ml) and ionomycin (500 ng / ml) (Sigma) for 24 hours. Monensin (GolgiStop, BD Biosciences) was added for the final 4 hours of incubation. Cells were fixed and permeabilized with Human FOXP3 Buffer Set and then stained with fluorochrome-conjugated IFNγ and IL-10 mAbs.

[0096] mRNA isolation Isolated CD4+ cells were incubated with or without TNFR2 mAb (2.5 μg / ml) in the presence of IL-2 (50 U / ml). After 3 hours, cells were harvested and total RNA was isolated using the RNAqueous-4 PCR kit (Ambion, Austin, TX). The extracted RNA was reverse transcribed using the High Capacity cDNA Reverse Transcription Kit (Applied-Biosystems, Foster City, CA).

[0097] Cell proliferation and inhibition assays For CD4 proliferation experiments, CD4 cells were stained with 1 μM carboxyfluorescein diacetate succinimidyl ester (CFSE). Cells were plated in 96-well plates containing anti-CD3 mAb at 2 × 10 5 Cells were plated at a density of 1000 cells / well. After 4 days, cells were harvested and analyzed.

[0098] For the Treg suppression assay, autologous PBMCs were used as responders. PBMCs were collected using Ficoll-Paque, frozen at -80°C, thawed the day before, mixed with Tregs, and quiescent overnight in RPMI 1640 and 10 U / ml IL-2. The next day, responder cells were stained with CFSE (1 μM). Responder cells (5 × 10 4 The cells and expanded Tregs were mixed at various ratios and stimulated with anti-CD3 mAb and IL-2. After 4 days, the cells were harvested and analyzed.

[0099] statistical analysis All data analyses were performed using GraphPad Prism-5 software (GraphPad Software, La Jolla, CA) by paired Student's t-test. A two-tailed p-value of 0.05 was considered significant. [Example]

[0100] Induction of human Treg clinical trials Unexpanded, naturally occurring human Tregs are heterogeneous and rare in the blood. Homogeneous populations of Tregs are difficult to expand in vitro, even with a mixture of multiple ligands. With the goal of expanding a sufficient number of homogeneous populations of human Treg cells with TNF-α, we first attempted to confirm or refute the increase in Treg concentrations by inducing with natural TNF-α. Due to the lack of an FDA-approved form of TNF-α and the difficulty in producing a stable form, we administered Bacillus Calmette-Guerin (BCG), a well-known and potent inducer of TNF-α; a common vaccine for tuberculosis and bladder cancer that has been on the market for decades. This method of inducing endogenous TNF-α eliminated the problem of producing TNF-α, which forms non-natural monomers, dimers, and trimers that exhibit different cellular effects.

[0101] A small, double-blind, placebo-controlled clinical trial enrolled two subjects. One human subject received a BCG injection (1.6–3.2 × 10 6 cfu / injection) and placebo subjects received saline twice, 4 weeks apart. Both were monitored weekly for 20 weeks to study the pharmacokinetics of TNF-α and the induction of Tregs. After each injection, TNF-α induced Tregs in a bimodal fashion with slightly delayed kinetics (Fig. 1A, left panel). After 20 weeks of observation, saline injection induced neither TNF-α nor Tregs. Total CD4+ cell counts were measured using the CD4+CD25 hi Other than the change in the proportion of FOXP3+ cells, there were no changes in BCG-treated or placebo-treated patients. This in vivo evidence confirms that endogenous TNF-α increases Treg numbers in vivo, but because TNF-α binds to both TNFR1 and TNFR2 receptors, it is unclear which receptor is more central to the Treg effect. [Example]

[0102] Functional effects of TNFR monoclonal antibodies and signaling pathways Freshly isolated human CD4+ cells from 14 human subjects were cultured with TNF-α alone for 16 hours (Figure 1B). Although no Treg induction was observed, as assessed by inducible FOXP3, a significant increase in Tregs was observed after the addition of IL-2. Co-incubation of TNF-α and IL-2 resulted in a significant increase in Tregs compared with IL-2 alone (Figure 1B). IL-2 is important for the induction and maintenance of Tregs in mice. Co-incubation significantly increased CD4+CD25 expression in blood-derived human cultured cells. hi The finding of increasing the proportion of FOXP3 cells was confirmed by flow cytometry (Figure 1C). Thus, the data in Figures 1B and 1C confirm that TNF-α can induce a homogenous population of Tregs in vitro.

[0103] In freshly isolated CD4+ cells, we examined the expression levels of each TNFR in relation to CD25+ expression. TNFR1 expression on CD4+ cells did not change using flow cytometry, regardless of the CD25+ expression level (Fig. 2A, middle panel), whereas TNFR2 expression was significantly higher in CD4+ cells than in CD4+ cells (Fig. 2B). + CD25 hi It was preferentially expressed approximately 10-fold on Tregs (Fig. 2A, right panel), confirming previous studies that TNFR2 is more densely expressed on Tregs.

[0104] Screening several TNFR1 and TNFR2 monoclonal antibodies (mAbs) on CD4+ cells isolated from fresh human blood allowed for selective study of each TNF receptor, unlike studies of TNF-α, which acts through both receptors and presents anticipated manufacturing challenges from the use of E. coli and yeast systems. While most of the screened TNFR1 or TNFR2 mAbs failed to induce or suppress FOXP3+ Tregs after 16 hours of stimulation in the presence of IL-2 (Figures 7 and 8), we found two TNFR2 mAbs with significant, yet opposing, effects on FOXP3 induction (Figure 2B). Studying freshly cultured cells from 10 subjects, one TNFR2 antibody significantly induced FOXP3 expression in CD4+ human T cells (designated a "TNFR2 agonist"), whereas the other TNFR2 monoclonal suppressed intracellular FOXP3 expression (designated a "TNFR2 antagonist") (Figure 2B). Thus, the present inventors have identified M1 and MAB726 as TNFR2 antagonists.

[0105] Having identified two functionally opposing TNFR2 mAbs, we measured their effects on isolated CD4+ T cells by examining the downstream mRNA expression of signaling proteins specific to TNFR2 activation. After 24 hours of stimulation with a TNFR2 agonist or antagonist, the relative mRNA expression was significantly different. The TNFR2 agonist stimulated the expression of TNF, TRAF2, TRAF3, cIAP2, and FOXP3. In contrast, the TNFR2 antagonist stimulated the expression of cIAP1 but not TRAF2, TRAF3, or FOXP3 (Figure 2C).

[0106] The effects of TNFR2 agonists and antagonists were examined in purified human CD4+ T cells cocultured with anti-CD3 or IL-2. When CD4+ proliferation was examined using anti-CD3 in combination with a TNFR2 agonist, the agonist demonstrated the highest level of proliferation. In contrast, TNFR2 antagonists (e.g., M1 or MAB726) suppressed CD4+ proliferation, even compared with the control anti-CD3 alone (Figure 2D, leftmost panel). Similar experimental results were observed 4 days later by measuring CD4+ proliferation directly by flow cytometry and by measuring CD4+ proliferation by CFSE dilution (Figure 2D, right three panels). Thus, the opposing effects of TNFR2 agonist and TNFR2 antagonist treatment on Tregs were demonstrated, as shown in Figures 2B-2D.

[0107] Despite the high expression of TNFR2 on Tregs, some TNFR2 expression also occurs on CD4+ T cells that are not true Tregs because they express only intermediate levels of CD25, i.e., CD4 + CD25 mid Therefore, the present inventors investigated the effects of IL-2 alone, IL-2 and TNF-α, IL-2 and a TNFR2 agonist, or IL-2 and a TNFR2 antagonist on CD25 mid The effect of overnight incubation on cell subpopulations was examined. Stimulation with IL-2 and TNF-α alone, or with IL-2 and a TNFR2 agonist alone, resulted in CD25 expression similar to effector cells. hi FOXP3 - An increase in the percentage of CD25 cells was observed (Figure 8). However, the inventors observed a suppression by IL-2 and TNFR2 antagonist compared to the other three groups. Therefore, TNFR2 agonists and antagonists tested in the same assay have the same CD25 + The opposite trend was observed for the FOXP3- cell population: addition of TNFR2 antagonists (e.g., M1 or MAB726) inhibited Foxp3 expression on CD4+CD25+ T cells.

[0108] The present inventors separated fresh human blood and analyzed CD4+ and CD25 hi We obtained pure Tregs co-expressing CD4+CD25+ Tregs (Figure 3). These Tregs were purified and expanded in vitro for 16 days using a standard protocol of anti-CD3 and anti-CD28 plus IL-2 (Figure 3A), after which they were rested overnight before cell counting. We added rapamycin (until day 7) because it selectively expands the highest number of Tregs with the greatest ability to suppress CD8+ cells. This method allows for the selective expansion of CD4+CD25+ Tregs. hi Tregs were successfully generated (Figure 3B). We evaluated Treg proliferation by treatment group: no treatment, treatment with TNF-α, TNFR2 agonist, or TNFR2 antagonist. TNFR2 agonist treatment outperformed all other groups, proliferating Tregs at least two-fold more than no treatment or antagonist treatment. Antagonist treatment suppressed proliferation, as its effect was less than that of no treatment. Because rapamycin is known to inhibit proliferation, we examined the effect of treatment without rapamycin. However, we found a similar, opposite effect between agonist and antagonist treatment, although the mean absolute values ​​were smaller (Figure 9B). Although the yield of expanded cells tended to be lower without rapamycin, the agonist still proliferated Tregs. [Example]

[0109] TNFR2 agonist expansion and homogeneity of Tregs Next, we investigated whether Tregs treated with TNFR2 agonists possessed more homogeneous Treg cell surface markers than those treated with antagonists in vitro. Phenotype comparisons of 14 cell surface markers revealed that all treatment groups highly expressed FOXP3 and CD25, markers characteristic of Tregs (Fig. 4A). The expression level of FOXP3 was similar to that before treatment. However, CD25+ expression was much higher after agonist treatment, which could be attributed to a proliferation effect rather than an antagonist effect (data not shown). The proliferation of CD25+ within each group was significantly higher than that of the control group (Fig. 4B). hiNearly 100% of Tregs were positive for CTLA4, TNFR2, CD62L, and Fas, and negative for CD127 (Figure 4A). Tregs treated with TNFR2 antagonists also maintained the expression of these markers. In contrast, several other surface markers, such as HLA-DR, ICOS, CD45RO, and chemokine receptors, were differentially expressed between agonist and antagonist treatments (Figures 4B and 4C, Figure 10). Similar results were observed with Tregs expanded without rapamycin (Figure 10). Tregs expanded with TNFR2 agonists maintained this phenotype: CD4 + CD25 hi FOXP3 + CTLA4 + TNFR2 + CD127 - CD62L + Fas + HLA-DR + CD45RO + CCR5 - CCR6 - CCR7 - CXCR3 - ICOS - This resulted in a surprisingly homogeneous population of cells with CD45RO. Mean fluorescence intensity (MFI), a direct measure of the average density of the protein per cell, similarly revealed that for most surface markers, TNFR2 agonist-treated cells showed opposite expression levels compared to TNFR2 antagonist-treated cells (Figure 12). Further studies will be required to define whether these expanded cells maintain phenotypic homogeneity over time, but evidence from this conventional expansion protocol indicates that they were more homogeneous than the other groups. Before treatment, Treg markers were more heterogeneous (Figure 11). Unexpanded, naturally occurring human Tregs are a heterogeneous population. CD45RO + FOXP3 low In vitro studies of mixed Treg populations with T cells result in the production of proinflammatory cytokines, and this particular phenotype is found in up to 50% of FOXP3+ T cells.

[0110] One of the most upregulated markers by Tregs treated with TNFR2 agonists was HLA-DR, which has been reported to have greater suppressive activity against CD8+ T cells, suggesting effector Tregs. In contrast to HLA-DR, all four chemokine receptors were strongly downregulated. While the lack of chemokine receptors may result in failure to migrate to inflammatory sites, another homing receptor, CD62L (Figure 4), which was highly expressed in all treatment groups, is essential for pathogenic T cells to enter sites of acute GVHD. The fact that agonist-treated Tregs were CD45RO+ and CCR7- and showed significantly higher expression levels of Fas, as measured by MFI (Figure 12), supports the view that they are activated effector Tregs. [Example]

[0111] Suppression of Tregs and CD8+ cells treated with TNFR2 agonists One important function of Tregs, particularly in autoimmunity, is to suppress the function of autoreactive cytotoxic CD8+ T cells. To assess this ability, Tregs from each treatment group were stimulated with anti-CD3 mAb and IL-2 for 4 days and then mixed with CFSE-stained autologous PBMCs. Responder cells, autologous CD8+ T cells, were tested for suppression by observing the responder-to-Treg ratio. Dilution ratios allow for dose-dependent studies. All groups of Tregs exhibited suppressive function against CD8+ T cells, although the extent of this function varied among treatment groups (Figure 5A, left panel). For example, Tregs treated with TNFR2 agonists exhibited the strongest suppressive ability (by preserving the minimum number of CD8+ cells) at a 1:1 ratio, which then gradually weakened at higher ratios. However, cells treated with antagonists exhibited weak suppressive ability, which was essentially unchanged from that without treatment. With a suppression index of 2:1, the TNFR2 agonist-treated group showed greater suppression than the antagonist-treated and untreated groups (Figure 5A, right panel). Similar results were observed with Tregs treated without rapamycin (Figure 13A). These results are consistent with the known phenotype and proliferation capacity of functional Tregs. [Example]

[0112] TNFR2 agonist-treated Tregs and cytokine production We found that all treatment groups had a relatively limited ability to produce intracellular IFNγ and IL-10 after PMA and ionomycin stimulation. However, Tregs treated with TNFR2 agonists and TNF alone produced the lowest percentage of IFNγ+ cells (Figure 5B, lower left panel). Tregs treated with antagonists showed significantly higher IFNγ production than agonists (Figure 5B, lower left panel). In a similar experiment without rapamycin, the TNFR2 agonist-treated group not only produced lower IFNγ but also lower IL-10 and TNF production compared with TNF treatment or no treatment, respectively (Figure 13B). Agonist-treated Tregs also showed the lowest number of TH1 transcription factor (T-bet)+ Tregs (Figure 5C), consistent with their lower IFNγ production (Figure 5C). One of the reasons why these Tregs exhibit high suppressive ability against CD8+ T cells may be due to their lack of IFNγ production.

[0113] Other embodiments The disclosures of U.S. Provisional Patent Application No. 61 / 762,136, filed February 7, 2013, and U.S. Provisional Patent Application No. 61 / 763,217, filed February 11, 2013, are incorporated herein by reference in their entireties. While the invention has been described in relation to particular embodiments thereof, it will be understood that further modifications are possible, and this application is intended to cover any variations, uses, or adaptations of the invention which generally follow the principles of the invention, including such departures from the present disclosure as come within custom or practice in the art to which this invention pertains and which may be applied to the essential features described hereinabove. All publications and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference in its entirety. Other embodiments are within the scope of the following claims.

Claims

1. CD4+CD25 hi A method for preparing a composition enriched in T regulatory cells (Tregs), comprising contacting in vitro a population of human cells comprising T lymphocytes obtained from a human blood or bone marrow sample from a patient with a tumor necrosis factor receptor 2 (TNFR2) agonist or an NF-κB activator, thereby preparing the composition enriched in Tregs, wherein the Tregs comprise at least 60% of the cells in the composition.

2. 2. The method of claim 1, wherein the population comprises CD4+ cells, CD25+ cells, or CD4+CD25+ cells, and the method further comprises isolating the CD4+ cells, CD25+ cells, or CD4+CD25+ cells, respectively, from the sample prior to said contacting.

3. The method of claim 1, wherein the Tregs comprise at least 70% of the cells in the composition.

4. The method of claim 1, wherein the Tregs comprise at least 80% of the cells in the composition.

5. The method of claim 1, wherein the Tregs comprise at least 90% of the cells in the composition.

6. The method of claim 1, wherein the Tregs comprise substantially 100% of the cells in the composition.

7. 7. The method of any one of claims 1 to 6, wherein the Tregs are further characterized as being positive for expression of one or more proteins selected from the group consisting of FOXP3, CTLA4, TNFR2, CD62L, Fas, HLA-DR, and CD45RO.

8. 8. The method of any one of claims 1 to 7, wherein the Tregs are further characterized as having low or negative expression of one or more proteins selected from the group consisting of CD127, CCR5, CCR6, CCR7, CXCR3, IFN-γ, IL10, and ICOS.

9. The method of any one of claims 1 to 8, wherein the TNFR2 agonist is selected from the group consisting of an antibody, a peptide, a small molecule, and a protein.

10. The method of claim 9, wherein the agonist is an antibody.

11. The method of claim 10, wherein the antibody is a monoclonal anti-TNFR2 agonist antibody.

12. 9. The method of any one of claims 1 to 8, wherein the NF-κB activator is selected from the group consisting of a small molecule, a peptide, a protein, a virus, and a small non-coding RNA.

13. The method of claim 12, wherein the NF-κB activator is a small molecule.

14. 14. The method of claim 13, wherein the NF-κB activator is selected from the group consisting of betulinic acid, topoisomerase poison VP16, and doxorubicin.

15. 15. The method of any one of claims 1 to 14, wherein the method further comprises contacting the population of human cells with any one or more of interleukin-2 (IL2), rapamycin, anti-CD3, and / or anti-CD28.

16. The method includes at least 5×10 6 16. The method of any one of claims 1 to 15, wherein the method results in Tregs.

17. CD4+CD25 hi A composition enriched for Tregs, wherein the Tregs comprise at least 60% of the cells in the composition, and the composition contains at least 5×10 6 A composition comprising Tregs.

18. The composition of claim 17, wherein the Tregs comprise at least 70% of the cells in the composition.

19. The composition of claim 18, wherein the Tregs comprise at least 80% of the cells in the composition.

20. 20. The composition of claim 19, wherein the Tregs comprise at least 90% of the cells in the composition.

21. 21. The composition of claim 20, wherein the Tregs comprise substantially 100% of the cells in the composition.

22. 22. The composition of any one of claims 17 to 21, wherein the Tregs are further characterized as being positive for expression of one or more proteins selected from the group consisting of FOXP3, CTLA4, TNFR2, CD62L, Fas, HLA-DR, and CD45RO.

23. 23. The composition of any one of claims 17 to 22, wherein the Tregs are further characterized as being negative for expression of one or more proteins selected from the group consisting of CD127, CCR5, CCR6, CCR7, CXCR3, IFN-γ, IL10, and ICOS.

24. CD4+ and CD25 prepared by the method according to any one of claims 1 to 16. hi 1. A composition enriched for Tregs, said composition comprising at least 5×10 6 A composition comprising Tregs.

25. 25. The composition of claim 24, wherein the Tregs comprise at least 70% of the cells in the composition.

26. 26. The composition of claim 25, wherein the Tregs comprise at least 80% of the cells in the composition.

27. 27. The composition of claim 26, wherein the Tregs comprise at least 90% of the cells in the composition.

28. 28. The composition of claim 27, wherein the Tregs comprise substantially 100% of the cells in the composition.

29. 29. The composition of any one of claims 24 to 28, wherein the Tregs are further characterized as being positive for expression of one or more proteins selected from the group consisting of FOXP3, CTLA4, TNFR2, CD62L, Fas, HLA-DR, and CD45RO.

30. 30. The composition of any one of claims 24 to 29, wherein the Tregs are further characterized as having low or negative expression of one or more proteins selected from the group consisting of CD127, CCR5, CCR6, CCR7, CXCR3, IFN-γ, IL10, and ICOS.

31. 31. A method of treating an immunological disease in a patient, comprising administering to said patient a composition according to any one of claims 17 to 30, wherein said composition treats said immunological disease.

32. 32. The method of claim 31, wherein the immunological disease is selected from the group consisting of allergies, asthma, autoimmune diseases, graft-versus-host disease (GVHD), and transplant rejection.

33. 33. The method of claim 32, wherein the allergy is selected from the group consisting of food allergies, seasonal allergies, pet allergies, hives, hay fever, allergic conjunctivitis, poison ivy allergies, oak allergies, mold allergies, drug allergies, dust allergies, cosmetic allergies, and chemical allergies.

34. The autoimmune disease is type 1 diabetes, alopecia areata, ankylosing spondylitis, antiphospholipid syndrome, autoimmune Addison's disease, autoimmune hemolytic anemia, autoimmune hepatitis, Behçet's disease, bullous pemphigoid, cardiomyopathy, celiac sprue dermatitis, chronic fatigue immune deficiency syndrome (CFIDS), chronic inflammatory demyelinating polyneuropathy, Churg-Strauss syndrome, cicatricial pemphigoid, CREST syndrome, cold agglutinin disease, Crohn's disease, essential mixed cryoglobulinemia, fibromyalgia-fibromyositis, Graves' disease, Guillain-Barré syndrome, Hashimoto's thyroiditis, hypothyroidism, idiopathic pulmonary fibrosis, idiopathic thrombocytopenic purpura (ITP), IgA nephropathy, or juvenile arthritis. , lichen planus, systemic lupus erythematosus (Lupus), Meniere's disease, mixed connective tissue disease, multiple sclerosis, myasthenia gravis, pemphigus vulgaris, pernicious anemia, polyarteritis nodosa, polychondritis, polyglandular syndrome, polymyalgia rheumatica, polymyositis and dermatomyositis, primary agammaglobulinemia, primary biliary cirrhosis, psoriasis, Raynaud's phenomenon, Reiter's syndrome, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma, Sjogren's syndrome, stiff-man syndrome, Takayasu's arteritis, temporal arteritis / giant cell arteritis, ulcerative colitis, uveitis, vasculitis, vitiligo, and Wegener's granulomatosis.

35. 31. A method of treating an infectious disease in a patient, comprising administering to said patient a composition according to any one of claims 17 to 30, wherein said composition treats said infectious disease.

36. 36. The method of claim 35, wherein the infectious disease is selected from the group consisting of a bacterial infection, a viral infection, a fungal infection, and a parasitic infection.

37. The composition is at least 5×10 6 The method of any one of claims 31 to 36, comprising Tregs.

38. The composition is at least 5×10 7 38. The method of claim 37, comprising Tregs.

39. The composition is at least 5×10 8 39. The method of claim 38, comprising Tregs.

40. The composition is at least 5×10 9 40. The method of claim 39, comprising Tregs.

41. 17. A method of treating an immunological disease in a patient, comprising administering to said patient a composition prepared by the method of any one of claims 1 to 16, wherein said composition treats said immunological disease.

42. 42. The method of claim 41, wherein the immunological disease is selected from the group consisting of allergy, asthma, autoimmune disease, GVHD, and transplant rejection.

43. 43. The method of claim 42, wherein the allergy is selected from the group consisting of food allergies, seasonal allergies, pet allergies, hives, hay fever, allergic conjunctivitis, poison ivy allergies, oak allergies, mold allergies, drug allergies, dust allergies, cosmetic allergies, and chemical allergies.

44. The autoimmune disease is type 1 diabetes, alopecia areata, ankylosing spondylitis, antiphospholipid syndrome, autoimmune Addison's disease, autoimmune hemolytic anemia, autoimmune hepatitis, Behçet's disease, bullous pemphigoid, cardiomyopathy, celiac sprue dermatitis, chronic fatigue immune deficiency syndrome (CFIDS), chronic inflammatory demyelinating polyneuropathy, Churg-Strauss syndrome, cicatricial pemphigoid, CREST syndrome, cold agglutinin disease, Crohn's disease, essential mixed cryoglobulinemia, fibromyalgia-fibromyositis, Graves' disease, Guillain-Barré syndrome, Hashimoto's thyroiditis, hypothyroidism, idiopathic pulmonary fibrosis, idiopathic thrombocytopenic purpura (ITP), IgA nephropathy, or juvenile arthritis. , lichen planus, systemic lupus erythematosus, Meniere's disease, mixed connective tissue disease, multiple sclerosis, myasthenia gravis, pemphigus vulgaris, pernicious anemia, polyarteritis nodosa, polychondritis, polyglandular syndrome, polymyalgia rheumatica, polymyositis and dermatomyositis, primary agammaglobulinemia, primary biliary cirrhosis, psoriasis, Raynaud's phenomenon, Reiter's syndrome, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma, Sjogren's syndrome, stiff-man syndrome, Takayasu's arteritis, temporal arteritis / giant cell arteritis, ulcerative colitis, uveitis, vasculitis, vitiligo, and Wegener's granulomatosis.

45. 17. A method of treating an infectious disease in a patient, comprising administering to said patient a composition prepared by the method of any one of claims 1 to 16, wherein said composition treats said infectious disease.

46. 46. ​​The method of claim 45, wherein the infectious disease is selected from the group consisting of a bacterial infection, a viral infection, a fungal infection, and a parasitic infection.

47. The composition is at least 5×10 6 47. The method of any one of claims 41 to 46, comprising Tregs.

48. The composition is at least 5×10 7 48. The method of claim 47, comprising Tregs.

49. The composition is at least 5×10 8 49. The method of claim 48, comprising Tregs.

50. The composition is at least 5×10 9 50. The method of claim 49, comprising Tregs.

51. An isolated antibody or antigen-binding fragment thereof that selectively binds to a first epitope of TNFR2, wherein the first epitope comprises positions 48-67 of SEQ ID NO: 1, and wherein the antibody or antigen-binding fragment thereof has antagonistic activity against TNFR2 upon binding.

52. 52. The antibody or antigen-binding fragment thereof of claim 51, which further binds to a second epitope of TNFR2, wherein the second epitope comprises position 135 of SEQ ID NO:

1.

53. 53. The antibody or antigen-binding fragment thereof of claim 52, wherein the second epitope comprises positions 135-147 of SEQ ID NO:

1.

54. 54. The antibody or antigen-binding fragment thereof of claim 53, wherein the second epitope comprises positions 130-149 of SEQ ID NO:

1.

55. 54. The antibody or antigen-binding fragment thereof of claim 53, wherein the second epitope comprises positions 128-147 of SEQ ID NO:

1.

56. 54. The antibody or antigen-binding fragment thereof of claim 53, wherein the second epitope comprises positions 135-142 of SEQ ID NO:

1.

57. 54. The antibody or antigen-binding fragment thereof of claim 53, wherein the second epitope comprises positions 135-153 of SEQ ID NO:

1.

58. The antibody or antigen-binding fragment thereof may be a monoclonal antibody or antigen-binding fragment thereof, a polyclonal antibody or antigen-binding fragment thereof, a Fab, a humanized antibody or antigen-binding fragment thereof, a bispecific antibody or antigen-binding fragment thereof, a monovalent antibody or antigen-binding fragment thereof, a chimeric antibody or antigen-binding fragment thereof, a single chain Fv molecule, a bispecific single chain Fv ((scFv') 2 ) molecule, domain antibody, diabody, triabody, affibody, domain antibody, SMIP, nanobody, Fv fragment, Fab fragment, F(ab') 2 58. The antibody or antigen-binding fragment thereof of any one of claims 51 to 57, selected from the group consisting of: a tandem scFv (taFv) fragment, a fusion protein, a fusion protein fragment ...

59. The equilibrium dissociation constant ("K") of the binding of the antibody or antigen-binding fragment thereof to TNFR2 D 59. The antibody or antigen-binding fragment thereof of any one of claims 51 to 58, wherein the .times. ...

60. The equilibrium dissociation constant ("K") of the binding of the antibody or antigen-binding fragment thereof to TNFR2 D 60. The antibody or antigen-binding fragment thereof of claim 59, wherein the .times. ...

61. The equilibrium dissociation constant ("K") of the binding of the antibody or antigen-binding fragment thereof to TNFR2 D 61. The antibody or antigen-binding fragment thereof of claim 60, wherein the .times. ...

62. A method for preparing a composition enriched in lymphocytes, comprising contacting in vitro a population of human cells comprising said lymphocytes obtained from a human blood or bone marrow sample from a patient with a tumor necrosis factor receptor 2 (TNFR2) antagonist or an NF-κB inhibitor that suppresses the proliferation of T regulatory cells (Tregs), thereby preparing said composition enriched in lymphocytes, wherein said Tregs comprise less than 10% of the cells in said composition.

63. 63. The method of claim 62, further comprising isolating said human cells from a human blood sample or a human bone marrow sample prior to said contacting.

64. 63. The method of claim 62, wherein the Tregs comprise less than 5% of the cells in the composition.

65. 65. The method of any one of claims 62 to 64, wherein the TNFR2 antagonist is selected from the group consisting of an antibody or antigen-binding fragment thereof, a peptide, a small molecule, and a protein.

66. 66. The method of claim 65, wherein the antagonist is an antibody or an antigen-binding fragment thereof.

67. 67. The method of claim 66, wherein the antibody or antigen-binding fragment thereof is a monoclonal anti-TNFR2 antagonist antibody or antigen-binding fragment thereof.

68. The method of claim 65 or 66, wherein the antibody or antigen-binding fragment thereof binds to a first epitope of TNFR2, and the first epitope comprises positions 48-67 of SEQ ID NO:

1.

69. 69. The method of claim 68, wherein the antibody or antigen-binding fragment thereof binds to a second epitope of TNFR2, and the second epitope comprises position 135 of SEQ ID NO:

1.

70. 70. The method of claim 69, wherein the second epitope comprises positions 135-147 of SEQ ID NO:

1.

71. The antibody or antigen-binding fragment thereof may be a monoclonal antibody or antigen-binding fragment thereof, a polyclonal antibody or antigen-binding fragment thereof, a Fab, a humanized antibody or antigen-binding fragment thereof, a bispecific antibody or antigen-binding fragment thereof, a monovalent antibody or antigen-binding fragment thereof, a chimeric antibody or antigen-binding fragment thereof, a single chain Fv molecule, a bispecific single chain Fv ((scFv') 2 ) molecule, domain antibody, diabody, triabody, affibody, domain antibody, SMIP, nanobody, Fv fragment, Fab fragment, F(ab') 2 71. The method of any one of claims 66 to 70, wherein the antibody is selected from the group consisting of a tandem scFv (taFv) fragment, a soluble antibody molecule, and a tandem scFv (taFv) fragment.

72. The equilibrium dissociation constant ("K") of the binding of the antibody or antigen-binding fragment thereof to TNFR2 D 72. The method of any one of claims 66-71, wherein the ATP is less than about 50 nM.

73. The equilibrium dissociation constant ("K") of the binding of the antibody or antigen-binding fragment thereof to TNFR2 D 73. The method of claim 72, wherein the .alpha.-aspartate is less than about 10 nM.

74. The equilibrium dissociation constant ("K") of the binding of the antibody or antigen-binding fragment thereof to TNFR2 D 74. The method of claim 73, wherein the ATP is less than about 1 nM.

75. 65. The method of any one of claims 62 to 64, wherein the NF-κB inhibitor is selected from the group consisting of a small molecule, a peptide, a protein, a virus, and a small non-coding RNA.

76. 76. The method of claim 75, wherein the NF-κB inhibitor is a small molecule.

77. The NF-κB inhibitor is 2-(1,8-naphthyridin-2-yl)-phenol, 5-aminosalicylic acid, BAY 11-7082, BAY 11-7085, CAPE (caffeic acid phenethyl ester), diethyl maleate, ethyl 3,4-dihydroxycinnamate, helenalin, gliotoxin, or NF-κB activation inhibitor II. JSH-23, NF-κB activation inhibitor III, glucocorticoid receptor modulator, CpdA, PPM-18, pyrrolidine dithiocarbamic acid ammonium salt, (R)-MG-132, rocaglamide, sodium salicylate, QNZ, MG-132 [Z-Leu-Leu-Leu-CHO], astaxanthin, (E)-2-fluoro-4'-methoxystilbene, CHS-828, disulfiram, olmesartan, triptolide, withaferin, celastrol, tanshinone IIA, Ro 106-9920, cardamonin, BAY 11-7821, PSI, HU 211, ML130, PR 39, honokiol, CDI 2858522, andrographolide, and dithiocarbamates.

78. 78. The method of claim 77, wherein the NF-κB inhibitor is a peptide.

79. 79. The method of claim 78, wherein the peptide is a cell-permeable inhibitory peptide.

80. 80. A composition prepared by the method of any one of claims 62-79, wherein the composition is enriched for lymphocytes and wherein Tregs in the composition comprise less than 10% of the cells in the composition.

81. The composition of claim 80, wherein the Tregs comprise less than 5% of the cells in the composition.

82. 82. A method of treating a proliferative disease in a patient, comprising administering to said patient a composition of claim 80 or 81.

83. The proliferative disease is selected from the group consisting of acute lymphoblastic leukemia, acute lymphocytic leukemia, acute myeloid leukemia, adrenocortical carcinoma, AIDS-related lymphoma, AIDS-related malignancies, anal cancer, astrocytoma, bile duct cancer, bladder cancer; bone cancer, osteosarcoma / malignant fibrous histiocytoma, brain stem glioma, visual pathway and hypothalamic glioma, breast cancer, bronchial adenoma / carcinoid, chronic lymphocytic leukemia, chronic myelogenous leukemia, chronic myeloproliferative disorder, clear cell sarcoma of tendon sheath, colon cancer, colorectal cancer, cutaneous T-cell lymphoma, endometrial cancer, epithelial carcinoma, esophageal cancer, uterine cancer, thyroid ...

83. The method of claim 82, wherein the cancer is selected from the group consisting of: thyroid cancer, extracranial germ cell tumor, extragonadal germ cell tumor, extrahepatic bile duct cancer, eye cancer, intraocular melanoma, retinoblastoma, gallbladder cancer, gastric cancer, hairy cell leukemia, head and neck cancer, hepatocellular (liver) cancer, Hodgkin's lymphoma, hypopharyngeal cancer, Kaposi's sarcoma, kidney cancer, laryngeal cancer, pituitary cancer, plasma cell neoplasm / multiple myeloma, pleuropulmonary blastoma, skin cancer, small cell lung cancer, small intestine cancer, sarcoma of soft tissue, squamous cell cervical carcinoma, testicular cancer, thyroid cancer, urethral cancer, uterine sarcoma, and vaginal cancer.

84. 83. The method of claim 82, wherein the proliferative disease is a solid tumor of the brain, lung, breast, lymphatic system, gastrointestinal tract, genitourinary tract, pharynx, prostate, or ovary.

85. 82. A method of treating an infectious disease in a patient, comprising administering to said patient a composition of claim 80 or 81.

86. 86. The method of claim 85, wherein the infectious disease is selected from the group consisting of a bacterial infection, a viral infection, a fungal infection, and a parasitic infection.

87. A composition according to any one of claims 17 to 30 for use in a method for the treatment of an immunological or infectious disease.

88. 82. The composition of claim 80 or 81 for use in a method for the treatment of a proliferative disease.

89. 62. A method of treating an infectious disease in a patient, comprising administering to said patient an effective amount of the antibody or antigen-binding fragment thereof of any one of claims 51 to 61.

90. 62. A method of treating a proliferative disease in a patient, comprising administering to said patient an effective amount of the antibody or antigen-binding fragment thereof of any one of claims 51 to 61.

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