IL-10-expressing multi-donor CD4+ T cells and uses thereof

Abstract

The present disclosure provides CD4 T cell clones derived from at least three different T cell donors. + Multi-donor CD4 generated by genetically modifying T cells IL-10 Furthermore, multiple donor CD4 IL-10 Methods for producing cells and multi-donor CD4 T cells for the treatment of immune tolerization, GvHD, cell and organ transplantation, cancer, and other immune disorders IL-10 Methods of using the cells are provided.

Description

[Technical Field]

[0001] 1. CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to PCT application PCT / US2020 / 040372, filed June 30, 2020, which is incorporated herein by reference in its entirety.

[0002] 2. Sequence Listing The present application contains a sequence listing with five sequences. [Background technology]

[0003] 3.Background Regulatory T cells belong to a small but important subset of T cells that maintain immune tolerance to self-antigens and non-pathogenic antigens, thus maintaining immune homeostasis. There are two major populations of regulatory T cells - CD4 + , Foxp3 + CD25 + T cells (Foxp3 + cells) and type 1 regulatory T (Tr1) cells. Foxp3 + Both Tr1 and Tr1 cells downregulate pathogenic T cell responses in various preclinical models of organ and islet transplantation, GvHD, and various autoimmune and inflammatory diseases.

[0004] Tr1 cells have proven effective in clinical studies: administration of cloned, antigen-specific autologous Tr1 cells to patients with active, moderate to severe Crohn's disease resulted in objective, temporary remission (Desreumaux et al., Gastroenterology. 2012;143(5):1207-1217.e2.). Furthermore, donor-derived autologous specific CD4 enriched in Tr1 cells was administered to leukemia patients after allogeneic HSCT. +Adoptive transfer of T cell populations resulted in rapid reconstitution of the immune system and protection against microbial and viral infections without severe GvHD. Responding patients achieved long-term remission and tolerance (>7 years) leading to cure (Bacchetta et al., Front Immunol. 2014;5:16).

[0005] Despite these promising results, the production of donor-derived or autologous Tr1 cells for large-scale treatment of patients with high unmet medical need is not always feasible, is very laborious, and does not allow for the generation of large amounts of pure Tr1 cells.

[0006] Recently, Locafaro et al. used a bidirectional lentiviral vector containing the human IL-10 gene to transfect purified CD4 + Transducing T cells circumvented some of these problems. IL-10 The population shared key features of naturally occurring Tr1 cells. Like Tr1 cells, single-donor CD4 IL-10 The cells produce high levels of IL-10 and express allogeneic CD4 + T cells and CD8 + These single-donor CD4 T cells down-regulate the proliferation of both normal myeloid cells (antigen-presenting cells, APCs, etc.) and myeloid leukemia cells. IL-10 The cells have been shown to be effective in reducing graft-versus-leukemia (GvHD) in a humanized xenogeneic GvHD model while retaining GvL activity. See Locafaro et al. Mol Ther. 2017;25(10):2254-2269 and WO 2016 / 146,542.

[0007] Highly purified single-donor CD4 for therapeutic use IL-10Although it is possible to produce cells, there are still significant limitations due to qualitative and quantitative differences between various individual batches, which are most likely related to inherent differences between various donors in addition to variations in the quality of the buffy coat. Summary of the Invention

[0008] 4. Overview The present disclosure provides a multi-donor CD4 IL-10 We provide a novel Tr1-based therapy that uses a population of cells. Multi-donor CD4 IL-10 The cells were obtained from at least three different T cell donors and then genetically modified to contain an exogenous polynucleotide encoding IL-10. + T cells. T cell donors are multi-donor CD4 IL-10 A third-party donor who is neither the host being treated with the cells nor the HSC or organ transplant donor. IL-10 The cells are not alloantigen-specific, in other words, they have not been primed or stimulated with cells from the host prior to administration.

[0009] The applicant has developed a multi-donor CD4 IL-10 Cells are single-donor CD4 IL-10 We demonstrated that these cells have cytokine production profiles, immunosuppressive and cytotoxic potential comparable to those of single-donor CD4 cells. IL-10 CD4 rather than cells + These multi-donor CD4 T cells are more effective in preventing T cell-mediated xenogeneic GvHD, but they do not induce GvHD by themselves. IL-10 Functional characterization of cells, both in vitro and in vivo, of single-donor CD4 IL-10 The functional properties of the cells were comparable or better.

[0010] Based on these results, the applicants have developed a multi-donor allogeneic CD4 IL-10 It is claimed that the cells may be used for therapeutic purposes in GvHD, cell and organ transplantation, autoimmune and inflammatory diseases.

[0011] Furthermore, by using third-party T cells and eliminating the need for autologous specificity, multiple-donor CD4 IL-10 The cells will make Tr1-based cell therapy available to a larger population of patients with various genetic backgrounds.

[0012] Thus, the present disclosure provides a CD4+ antibody that has been genetically modified to include an exogenous polynucleotide encoding IL-10. + A population of T cells, the CD4 + T cells were collected from at least three different T cell donors (multiple donor CD4 IL-10 The present invention provides a population of cells obtained from a culture of a mammalian cell.

[0013] In some embodiments, CD4 + The T cells are obtained from 3, 4, 5, 6, 7, 8, 9, or 10 different T cell donors. In some embodiments, the CD4 + In total, T cells may have 6, 7, 8, 9, 10, 11, 12, or more different HLA haplotypes.

[0014] In some embodiments, all CD4 + The T cells have at least a 5 / 10, 6 / 10, 7 / 10, 8 / 10, or 9 / 10 match to each other at the HLA-A, HLA-B, HLA-C, HLA-DRB1, and HLA-DQB1 loci. + The T cells have at least a 4 / 8, 5 / 8, 6 / 8, 7 / 8, or 8 / 8 match to each other at the HLA-A, HLA-B, HLA-C, and HLA-DRB1 loci. In some embodiments, all CD4 T cells in the population + The T cells have a 2 / 2 match with each other at the HLA-A locus. In some embodiments, all CD4 T cells in the population + The T cells have a 2 / 2 match with each other at the HLA-B locus. In some embodiments, all CD4 T cells in the population +The T cells have a 2 / 2 match with each other at the HLA-C locus. In some embodiments, all CD4 T cells in the population + The T cells have at least a 3 / 4 or 4 / 4 match to each other at the HLA-DRB1 and HLA-DQB1 loci. In some embodiments, all CD4 T cells in the population + T cells are A * Possess the 02 allele.

[0015] In some embodiments, CD4 + None of the T cells are immortalized. In some embodiments, the exogenous polynucleotide comprises a polynucleotide segment encoding IL-10 operably linked to an expression control element. In some embodiments, the IL-10 is human IL-10. In some embodiments, the IL-10 is viral IL-10. In some embodiments, the polynucleotide segment encoding IL-10 encodes a protein having the sequence of SEQ ID NO: 1. In some embodiments, the polynucleotide segment encoding IL-10 has the sequence of SEQ ID NO: 2. In some embodiments, the expression control element drives constitutive expression of the encoded IL-10. In some embodiments, the expression control element encodes an activated CD4 + In some embodiments, the expression control element drives the expression of IL-10 in T cells. + Drives T cell-specific expression.

[0016] In some embodiments, the exogenous polynucleotide further comprises a sequence encoding a selectable marker. In some embodiments, the selectable marker is ΔNGFR. In some embodiments, ΔNGFR has the sequence of SEQ ID NO:3. In some embodiments, the exogenous polynucleotide comprises the sequence of SEQ ID NO:4. In some embodiments, the exogenous polynucleotide has the sequence of SEQ ID NO:5.

[0017] In some embodiments, the selectable marker is a truncated EGFR polypeptide. In some embodiments, the selectable marker is a truncated human EGFR polypeptide.

[0018] In some embodiments, the exogenous polynucleotide is integrated into the T cell nuclear genome. In some embodiments, the exogenous polynucleotide is not integrated into the T cell nuclear genome. In some embodiments, the exogenous polynucleotide further comprises a lentiviral vector sequence. In some embodiments, the exogenous polynucleotide is not integrated into the T cell nuclear genome.

[0019] In some embodiments, the CD4 + At least 90% of the T cells express IL-10. In some embodiments, the CD4 + At least 95% of the T cells express IL-10. In some embodiments, the CD4 + At least 98% of T cells express IL-10.

[0020] In some embodiments, the genetically modified CD4 + T cells are CD4 + T cells 10 6 In some embodiments, the genetically modified CD4 + T cells are CD4 + T cells 10 6 In some embodiments, the genetically modified CD4 constitutively expresses at least 200 pg, 500 pg, 1 ng, 5 ng, 10 ng, or 50 ng of IL-10 per cell / ml. + After activation with anti-CD3 and anti-CD28 antibodies, T cells expressed CD4 + T cells 10 6 In some embodiments, the genetically modified CD4 + After activation with anti-CD3 and anti-CD28 antibodies, T cells expressed CD4 + T cells 10 6In some embodiments, the genetically modified CD4 expresses at least 2 ng, 5 ng, 10 ng, 100 ng, 200 ng, or 500 ng of IL-10 per cell / ml. + The T cells express IL-10 at levels at least 5-fold higher than unmodified CD4+ T cells. In some embodiments, the genetically modified CD4 + T cells are unmodified CD4 + They express IL-10 at levels at least 10-fold higher than T cells.

[0021] In some embodiments, the CD4 + At least 90% of the T cells express the selectable marker from the exogenous polynucleotide. In some embodiments, the CD4 + At least 95% of the T cells express the selectable marker from the exogenous polynucleotide. In some embodiments, the CD4 + At least 98% of the T cells express the selectable marker from the exogenous polynucleotide.

[0022] In some embodiments, the genetically modified CD4 + The T cells express CD49b. In some embodiments, the genetically modified CD4 + The T cells express LAG-3. In some embodiments, the genetically modified CD4 + The T cells express TGF-β. In some embodiments, the genetically modified CD4 + The T cells express IFNγ. In some embodiments, the genetically modified CD4 + The T cells express GzB. In some embodiments, the genetically modified CD4 + The T cells express perforin. In some embodiments, the genetically modified CD4 + The T cells express CD18. In some embodiments, the T cells express genetically modified CD4 + The T cells express CD2. In some embodiments, the T cells express a genetically modified CD4 + The T cells express CD226. In some embodiments, the genetically modified CD4 + T cells express IL-22.

[0023] In some embodiments, CD4 + The T cells are not anergized in the presence of host-derived peripheral blood mononuclear cells (PBMCs). In some embodiments, CD4 + T cells were not anergized in the presence of recombinant IL-10 protein, and recombinant IL-10 protein inhibited CD4 + In some embodiments, CD4 + T cells are not anergized in the presence of host-derived DC10 cells.

[0024] In some embodiments, CD4 + The T cells are in a frozen suspension. In some embodiments, the CD4 + The T cells are in a liquid suspension. In some embodiments, the liquid suspension is previously frozen.

[0025] In another aspect of the present disclosure, (i) a CD4 according to any one of the preceding claims + A population of T cells, (ii) suspended in a pharmaceutically acceptable carrier Pharmaceutical compositions comprising the populations are provided.

[0026] In yet another aspect, the present disclosure provides a multi-donor CD4 IL-10 1. A method of producing a cell, comprising: (i) Primary CD4 T cells obtained from at least three different T cell donors + pooling the T cells; (ii) pooled CD4 by introducing an exogenous polynucleotide encoding IL-10 + modifying the T cells; This results in genetically modified CD4 + Methods for obtaining T cells.

[0027] In one aspect, the present disclosure provides a multi-donor CD4 IL-10 1. A method of producing a cell, comprising: (i) Primary CD4 T cells from at least three different T cell donors + Obtaining T cells; (ii) Inducing CD4+ IL-10 expression in each donor by introducing an exogenous polynucleotide encoding IL-10 + Separately modifying the T cells, and then (iii) Genetically modified CD4 + T cells are pooled, thereby genetically modifying CD4 + Obtaining T cells; The present invention provides a method comprising:

[0028] In some embodiments, the method comprises adding after step (i) and before step (ii), or after step (ii), or after step (ii) and before step (iii), or after step (iii): First generation CD4 + The method further includes incubating the T cells in the presence of beads coated with anti-CD3 and anti-CD28 antibodies, or anti-CD3 and CD28 antibodies.

[0029] In some embodiments, the method further comprises culturing primary CD4 + In some embodiments, the exogenous polynucleotide is administered to primary CD4 T cells using a viral vector. + In some embodiments, the exogenous polynucleotide comprises an IL-10-encoding segment having the sequence of SEQ ID NO: 1. In some embodiments, the IL-10-encoding polynucleotide segment has the sequence of SEQ ID NO: 2.

[0030] In some embodiments, the exogenous polynucleotide further comprises a segment encoding a selectable marker. In some embodiments, the encoded selectable marker is ΔNGFR. In some embodiments, the encoded selectable marker has the sequence of SEQ ID NO: 3. In some embodiments, the encoded selectable marker is a truncated EGFR polypeptide. In some embodiments, the encoded selectable marker is a truncated human EGFR polypeptide.

[0031] In some embodiments, the method further comprises, after step (ii), Genetically modified CD4 expressing a selectable marker + Isolating T cells and thereby genetically modifying CD4 + The method further includes generating an enriched population of T cells.

[0032] In some embodiments, the genetically modified CD4 + At least 90% or at least 95% of the T cells express IL-10. In some embodiments, the genetically modified CD4 T cells in the enriched population + At least 98% of the T cells express IL-10. In some embodiments, the genetically modified CD4 T cells in the enriched population + At least 90% or at least 95% of the T cells express the selectable marker. In some embodiments, the genetically modified CD4 T cells in the enriched population + At least 98% of the T cells express the selectable marker.

[0033] In some embodiments, the method comprises genetically modifying CD4 + In some embodiments, the method further comprises incubating the enriched population of T cells. + The step of incubating the enriched population of T cells is carried out in the presence of IL-2 and in the presence of beads coated with anti-CD3 and anti-CD28 antibodies, or CD3 and CD28 antibodies.

[0034] In some embodiments, the method comprises genetically modifying CD4+ In some embodiments, step (i) further comprises a subsequent step of freezing the primary CD4 T cells. + The T cells are obtained from 3, 4, 5, 6, 7, 8, 9, or 10 different T cell donors. In some embodiments, at least three T cell donors have at least a 5 / 10, 6 / 10, 7 / 10, 8 / 10, or 9 / 10 match to each other at the HLA-A, HLA-B, HLA-C, HLA-DRB1, and HLA-DQB1 loci. In some embodiments, at least three T cell donors have at least a 4 / 8, 5 / 8, 6 / 8, 7 / 8, or 8 / 8 match to each other at the HLA-A, HLA-B, HLA-C, and HLA-DRB1 loci. In some embodiments, at least three T cell donors have a 2 / 2 match to each other at the HLA-A locus. In some embodiments, at least three T cell donors have a 2 / 2 match to each other at the HLA-B locus. In some embodiments, the at least three T cell donors are 2 / 2 matches to each other at the HLA-C locus. In some embodiments, the at least three T cell donors are at least 3 / 4 or 4 / 4 matches to each other at the HLA-DRB1 and HLA-DQB1 loci. In some embodiments, each of the at least three T cell donors is * Possess the 02 allele.

[0035] In some embodiments, in step (i), primary CD4 + The T cells are obtained from one or more frozen stocks. In some embodiments, in step (i), primary CD4 + T cells are obtained from non-frozen peripheral blood mononuclear cells of at least three different T cell donors.

[0036] In some embodiments, the method comprises isolating CD4 + Further comprising isolating T cells, hi some embodiments, the peripheral blood mononuclear cells are obtained from buffy coat or apheresis.

[0037] In another aspect, the present disclosure provides a method of treating a patient, comprising: The present disclosure of multiple donor CD4 IL-10 Methods are provided that include administering the cells or pharmaceutical composition to a patient in need of immune tolerance.

[0038] In some embodiments, the method comprises administering a multi-donor CD4 IL-10 The method further comprises the preceding step of thawing the frozen suspension of cells.

[0039] In some embodiments, multiple donor CD4 IL-10 The cells or pharmaceutical composition prevent or reduce the severity of a pathogenic T cell response in a patient.

[0040] In some embodiments, the method further comprises administering mononuclear cells from a hematopoietic stem cell (HSC) donor to the patient. IL-10 The cells or pharmaceutical composition and mononuclear cells from the HSC donor are administered simultaneously. In some embodiments, the mononuclear cells from the HSC donor are multi-donor CD4 IL-10 The mononuclear cells are administered either before or after administration of the cells or pharmaceutical composition. In some embodiments, the mononuclear cells are in PBMCs. In some embodiments, the mononuclear cells are in bone marrow. In some embodiments, the mononuclear cells are in umbilical cord blood. In some embodiments, the mononuclear cells are isolated from PBMCs, bone marrow, or umbilical cord blood.

[0041] In some embodiments, the method comprises administering a multi-donor CD4 IL-10 The method further comprises administering hematopoietic stem cells (HSCs) of an HSC donor to the patient either before or after administration of the cells or pharmaceutical composition.

[0042] In some embodiments, the HSC donor is partially HLA-mismatched to the patient. In some embodiments, the HSC donor has less than a 5 / 10, less than a 6 / 10, less than a 7 / 10, less than a 8 / 10, less than a 9 / 10, or less than a 10 / 10 match to the patient at the HLA-A, HLA-B, HLA-C, HLA-DRB1, and HLA-DQB1 loci. In some embodiments, the HSC donor has less than a 4 / 8, less than a 5 / 8, less than a 6 / 8, less than a 7 / 8, or less than a 8 / 8 match to the patient at the HLA-A, HLA-B, HLA-C, and HLA-DRB1 loci. In some embodiments, the HSC donor has less than a 2 / 2 match to the patient at the HLA-A, HLA-B, or HLA-C loci. In some embodiments, the HSC donor has less than a 3 / 4 or less than a 4 / 4 match to the patient at the HLA-DRB1 and HLA-DQB1 loci.

[0043] In some embodiments, one or more T cell donors are HLA-mismatched or partially HLA-mismatched to the patient. In some embodiments, one or more T cell donors have less than a 5 / 10, 6 / 10, 7 / 10, 8 / 10, 9 / 10, or 10 / 10 match to the patient at the HLA-A, HLA-B, HLA-C, HLA-DRB1, and HLA-DQB1 loci. In some embodiments, one or more T cell donors have less than a 4 / 8, less than a 5 / 8, less than a 6 / 8, less than a 7 / 8, or less than a 8 / 8 match to the patient at the HLA-A, HLA-B, HLA-C, and HLA-DRB1 loci. In some embodiments, one or more T cell donors have less than a 2 / 2 match to the patient at the HLA-A, HLA-B, or HLA-C loci. In some embodiments, one or more T cell donors have less than a 2 / 4, less than a 3 / 4, or less than a 4 / 4 match to the patient at the HLA-DRB1 and HLA-DQB1 loci. In some embodiments, one or more T cell donors are HLA-mismatched or partially HLA-mismatched to the HSC donor. In some embodiments, one or more T cell donors have less than a 5 / 10, 6 / 10, 7 / 10, 8 / 10, 9 / 10, or 10 / 10 match to the HSC donor at the HLA-A, HLA-B, HLA-C, HLA-DRB1, and HLA-DQB1 loci. In some embodiments, one or more T cell donors have less than a 4 / 8, 5 / 8, 6 / 8, 7 / 8, or 8 / 8 match to the HSC donor at the HLA-A, HLA-B, HLA-C, and HLA-DRB1 loci. In some embodiments, one or more T cell donors have less than a 2 / 2 match to the HSC donor at the HLA-A, HLA-B, or HLA-C loci. In some embodiments, one or more T cell donors have less than a 3 / 4 or 4 / 4 match to the HSC donor at the HLA-DRB1 and HLA-DQB1 loci.

[0044] In some embodiments, multiple donor CD4 IL-10The cells or pharmaceutical composition prevent or reduce the severity of GvHD caused by transplanted hematopoietic stem cells.

[0045] In some embodiments, multiple donor CD4 IL-10 The cells or pharmaceutical composition prevent or reduce the severity of the pathogenic response of lymphoid cells derived from transplanted hematopoietic stem cells.

[0046] In some embodiments, the patient has cancer. In some embodiments, the patient has neoplastic cells. In some embodiments, the neoplastic cells express CD13, HLA-class I, and CD54. In some embodiments, the neoplastic cells express CD112, CD58, or CD155.

[0047] In some embodiments, the patient has cancer. In some embodiments, the cancer is a solid or hematologic neoplasm. In some embodiments, the patient has adrenal gland cancer, anal cancer, bile duct cancer, bladder cancer, bone cancer, adult brain / CNS tumors, pediatric brain / CNS tumors, breast cancer, male breast cancer, cancer of unknown primary site, Castleman's disease, cervical cancer, colon / rectal cancer, endometrial cancer, esophageal cancer, Ewing's tumor, eye cancer, gallbladder cancer, gastrointestinal carcinoid tumors, gastrointestinal stromal tumors (GIST), gestational trophoblastic disease, Hodgkin's disease, Kaposi's sarcoma, kidney cancer, laryngeal and hypopharyngeal cancer, leukemia, acute lymphocytic (ALL), acute myeloid (AML, including myeloid sarcoma and leukemia cutis), chronic lymphocytic (CLL), chronic myeloid (CML) leukemia, chronic myelomonocytic leukemia (CMML), childhood leukemia, liver cancer, lung cancer, non-small cell lung cancer, The patient has a cancer selected from the group consisting of lung cancer, small cell lung cancer, pulmonary carcinoid tumor, lymphoma, lymphoma of the skin, malignant mesothelioma, multiple myeloma, myelodysplastic syndrome, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, non-Hodgkin's lymphoma, pediatric non-Hodgkin's lymphoma, oral cavity and oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, penile cancer, pituitary tumor, prostate cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma - adult soft tissue cancer, skin cancer, skin cancer - basal cell and squamous cell, skin cancer - melanoma, skin cancer - Merkel cell, small intestine cancer, stomach cancer, testicular cancer, thymus cancer, thyroid cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Waldenstrom's macroglobulinemia, and Wilms' tumor.

[0048] In some embodiments, the cancer is a myeloid cancer, hi some embodiments, the cancer is AML or CML.

[0049] In some embodiments, the patient has an inflammatory or autoimmune disease, in some embodiments, the inflammatory or autoimmune disease is selected from the group consisting of type 1 diabetes, autoimmune uveitis, rheumatoid arthritis, psoriasis, psoriatic arthritis, multiple sclerosis, systemic lupus, inflammatory bowel disease, Addison's disease, Graves' disease, Sjogren's syndrome, Hashimoto's thyroiditis, myasthenia gravis, autoimmune vasculitis, pernicious anemia, ulcerative colitis, bullous diseases, scleroderma, and celiac disease.

[0050] In some embodiments, the inflammatory or autoimmune disease is Crohn's disease, ulcerative colitis, celiac disease, type 1 diabetes, lupus, psoriasis, psoriatic arthritis, or rheumatoid arthritis.

[0051] In some embodiments, the patient has a disease or disorder associated with hyperactivity of the NLPR3 inflammasome, hi some embodiments, the patient has type 2 diabetes, a neurodegenerative disease, or an inflammatory bowel disease.

[0052] In some embodiments, the patient has a disease or disorder associated with increased IL-1β production by activated monocytes, macrophages, or dendritic cells.

[0053] In some embodiments, the patient has a disease or disorder associated with increased IL-18 production by activated monocytes, macrophages, or dendritic cells.

[0054] In some embodiments, the patient has a disease or disorder associated with increased production of mature caspase-1 by activated monocytes, macrophages, or dendritic cells.

[0055] In some embodiments, the patient has an allergic or atopic disorder. In some embodiments, the allergic or atopic disorder is selected from the group consisting of asthma, atopic dermatitis, and rhinitis. In some embodiments, the patient has a food allergy.

[0056] In some embodiments, the method comprises administering to a subject a subject a CD4 + In some embodiments, the method further comprises transplanting the cells and organs into the patient either before or after administration of the population of T cells or pharmaceutical composition. IL-10 The cells or pharmaceutical compositions prevent or reduce the severity of host rejection of cell and organ transplants.

[0057] In some embodiments, the method comprises administering to a subject a subject a CD4 + The method further comprises the step of transplanting cells or tissues derived from iPS cells into a patient before or after administration of the population of T cells or pharmaceutical composition.

[0058] In some embodiments, multiple donor CD4 IL-10 The cells or pharmaceutical composition prevent or reduce the severity of host rejection of the transplant.

[0059] In some embodiments, the method comprises administering a multi-donor CD4 IL-10 In some embodiments, the method further comprises administering a recombinant AAV to the patient before or after administration of the cells or pharmaceutical composition. IL-10 The cells or pharmaceutical composition reduce the immune response to the recombinant AAV.

[0060] In some embodiments, the patient has an exaggerated immune response to a viral or bacterial infection, hi some embodiments, the patient has a coronavirus infection.

[0061] In some embodiments, the method detects selectable markers in a biological sample obtained from a patient, thereby detecting multiple donor CD4 IL-10Further comprising detecting the presence or absence of T cells. In some embodiments, the biological sample is a biopsy or blood from the patient.

[0062] In one aspect, the present disclosure provides a method of treating a patient with a malignant tumor, comprising administering to the patient an allogeneic HSCT graft and administering a therapeutically effective amount of multiple-donor CD4 IL-10 and administering the cells.

[0063] In some embodiments, multiple donor CD4 IL-10 Cellular CD4 IL-10 None of the cell donors are donors of the HSCT graft.

[0064] In another aspect, the present disclosure provides a method of treating hematological cancer, comprising administering a sufficient amount of multiple donor CD4 IL-10 administering multiple donor CD4 cells to a patient with hematological cancer; IL-10 CD4 T cells were obtained from at least three different T cell donors and then genetically modified by vector-mediated gene transfer of the coding sequence for human IL-10 under the control of a constitutive promoter. + The method includes administering a T cell to a subject.

[0065] In some embodiments, the method comprises administering a multi-donor CD4 IL-10 In some embodiments, the method further comprises administering an allogeneic HSCT graft to the patient prior to or after administering the cells. IL-10 The amount of cells is still sufficient to suppress graft-versus-host disease (GvHD) without suppressing the graft-versus-leukemia (GvL) or graft-versus-tumor (GvT) efficacy of allogeneic HSCT.

[0066] In some embodiments, the hematological cancer is myeloid leukemia.

[0067] In some embodiments, multiple donor CD4 IL-10 The cells target and kill cancer cells that express CD13. In some embodiments, multi-donor CD4 IL-10The cells target and kill cancer cells that express HLA-class I. In some embodiments, the myeloid leukemia is acute myeloid leukemia (AML).

[0068] In some embodiments, the allogeneic HSCT graft is obtained from a related or unrelated donor to the recipient. IL-10 The cells are non-autologous to the recipient. In some embodiments, multiple donor CD4 IL-10 The cells are allogeneic to the recipient. In some embodiments, multiple donor CD4 IL-10 The cells are not anergized to the host's alloantigens prior to administration to the host.

[0069] In some embodiments, multiple donor CD4 IL-10 The cells are Tr1-like cells.

[0070] In some embodiments, multiple donor CD4 IL-10 The cells are polyclonal. In some embodiments, multiple donor CD4 IL-10 The cells are polyclonal and non-autologous to the recipient.

[0071] In some embodiments, multiple donor CD4 IL-10 The cells are isolated from at least three donors and then genetically modified. In some embodiments, none of the at least three donors is the same donor as the allogeneic HSCT donor. In some embodiments, the allogeneic HSCT graft is obtained from a matched or mismatched donor for the recipient.

[0072] In some embodiments, multiple donor CD4 IL-10 The cells target and kill cells that express CD54. In some embodiments, multi-donor CD4 IL-10 The cells target and kill cancer cells that express HLA-class I and CD54. In some embodiments, multi-donor CD4 IL-10The cells target and kill cancer cells that express CD112. In some embodiments, multi-donor CD4 IL-10 The cells target and kill cancer cells that express CD58. In some embodiments, multi-donor CD4 IL-10 The cells target and kill cancer cells in the host.

[0073] One aspect of the present disclosure is a method of treating hematological cancer by allogeneic hematopoietic stem cell transplantation (allo-HSCT), comprising: administering the allogeneic HSCT graft to the subject (host); Multiple donor CD4 in sufficient quantities to suppress graft-versus-host disease (GvHD) without suppressing graft-versus-leukemia (GvL) or graft-versus-tumor (GvT) efficacy of allogeneic HSCT grafts IL-10 administering the cells to a recipient (host) of an allogeneic HSCT; Multiple donor CD4 IL-10 CD4 T cells were obtained from at least three different T cell donors and genetically modified by vector-mediated gene transfer of the coding sequence for human IL-10 under the control of a constitutive promoter. + Contains T cells, Multiple donor CD4 IL-10 the cells are non-autologous to the recipient and non-autologous to the allogeneic HSCT donor; Multiple donor CD4 IL-10 the cells have not been anergized to the host's alloantigen prior to administration to the host; Multiple donor CD4 IL-10 The method is provided wherein the cells are polyclonal and Tr1-like.

[0074] Another aspect of the present disclosure is a method of treating hematological cancer by allogeneic hematopoietic stem cell transplantation (allo-HSCT), comprising: administering the allogeneic HSCT graft to the subject (host); Multiple donor CD4 in sufficient quantities to suppress graft-versus-host disease (GvHD) without suppressing graft-versus-leukemia (GvL) or graft-versus-tumor (GvT) efficacy of allogeneic HSCT grafts IL-10administering the cells to a recipient (host) of an allogeneic HSCT; Multiple donor CD4 IL-10 CD4 T cells were obtained from at least three different T cell donors and genetically modified by vector-mediated gene transfer of the coding sequence for human IL-10 under the control of a constitutive promoter. + Contains T cells, Multiple donor CD4 IL-10 The cells target and kill cancer cells in the host, Multiple donor CD4 IL-10 the cells have not been anergized to the host's alloantigen prior to administration to the host; Multiple donor CD4 IL-10 The method provides that the cells are non-autologous to the recipient, polyclonal, and Tr1-like. [Brief explanation of the drawings]

[0075] 5. Brief description of the drawings [Figure 1] FIG. 1 shows the partial structure of a bidirectional lentiviral vector for delivering human IL-10 and ΔNGFR coding sequences to CD4+ T cells from multiple donors to generate multi-donor CD4IL-10 cells. [Figure 2] FIG. 2 shows the complete and circular structure of the bidirectional lentiviral vector (hPGK.IL10.WPRE.mhCMV.ΔNGFR.SV40PA) for delivery of human IL-10 and ΔNGFR coding sequences to CD4+ T cells from multiple donors to generate multi-donor CD4IL-10 cells. [Figure 3] FIG. 3 shows an exemplary protocol for generating CD4IL-10 cells. [Figure 4A] Figure 4A shows the percentage of CD4+ΔNGFR+ cells (mean ± SD, n = 10, gray bars) and vector copy number (VCN, mean ± SD, n = 10, orange bars) in human CD4+ T cells transduced with LV-IL-10 / ΔNGFR (a bidirectional lentiviral vector encoding human IL-10 and a truncated human NGF receptor). [Figure 4B] Figure 4B shows FACS analysis of CD4 and ΔNGFR expression in human CD4 T cells from two representative donors (donor B and donor C) transduced with LV-IL-10 / ΔNGFR and purified using anti-CD271 microbeads. [Figure 5] Figure 5 shows the cytokine production profile of single-donor CD4IL-10 cells after a second (TF2) and third (TF3) restimulation. TF2 and TF3CD4IL-10 cells were left unstimulated (orange bars) or stimulated with immobilized anti-CD3 and soluble anti-CD28 mAb (gray bars) for 48 hours. Culture supernatants were collected, and levels of IL-10, IL-4, IL-5, IFN-γ, and IL-22 were measured by ELISA. All samples were tested in triplicate. Mean ± SD, n = 8 donors tested, is shown. [Figure 6] Figure 6A shows the percentage of CD4IL-10 cells expressing granzyme B (GzB) after the second stimulation (TF2) analyzed by FACS. Boxes and whiskers are shown for donors (n=7) and single donors. Figure 6B shows the % dead cells when CD4IL-10 cells (10 / well) were cocultured with K562 and ALL-CM cells (10 / well) at a 1:1 ratio for 3 days. Boxes and whiskers represent data from n=4 donors, and dots represent data from a single donor. [Figure 7A]Figures 7A and 7B show that single-donor CD4IL-10 cells can suppress the proliferation of allogeneic CD4+ T cells. Allogeneic PBMC cells were labeled with eFluor® 670 (10 cells / well) and stimulated with allogeneic mature dendritic (DC) cells (5 x 10 cells / well) and soluble anti-CD3 mAb at a 1:1 responder:suppressor ratio in the absence or presence of CD4IL-10 cells (10 cells / well). After 4 days of culture, CD4+ΔNGFR- T cells were gated and the percentage of proliferating responder cells was determined by eFluor® 670 dilution using flow cytometry. Figure 7A shows the results for donors C, E, and F, and Figure 7B shows the results for donors H, I, and L. Percent proliferation and suppression are shown. Suppression mediated by CD4IL-10 cells was calculated as follows: 100 - ([proliferation of responder cells in the presence of CD4IL-10 cells / proliferation of responder cells alone] x 100). [Figure 7B] Same as above. [Figure 8A] Figures 8A and 8B show that single-donor CD4IL-10 cells can suppress the proliferation of allogeneic CD8+ T cells. Allogeneic PBMC cells were labeled with eFluor® 670 (10 cells / well) and stimulated with allogeneic mature dendritic (DC) cells (5 x 10 cells / well) and soluble anti-CD3 mAb at a 1:1 responder:suppressor ratio in the absence or presence of CD4IL-10 cells (10 cells / well). After 4 days of culture, CD4+ΔNGFR- T cells were gated and the percentage of proliferating responder cells was determined by eFluor® 670 dilution using flow cytometry. Figure 8A shows the results for donors C, E, and F, and Figure 8B shows the results for donors H, I, and L. Percent proliferation and suppression are shown. Suppression mediated by CD4IL-10 cells was calculated as follows: 100 - ([proliferation of responder cells in the presence of CD4IL-10 cells / proliferation of responder cells alone] x 100). [Figure 8B] Same as above. [Figure 9]Figure 9 shows the cytokine production profile of multi-donor CD4IL-10 cells after a third (TF3) restimulation compared to the mean levels (+ / - SD) produced by CD4IL-10 cells from eight individual donors. TF3CD4IL-10 cells from three donors were pooled at a 1:1:1 ratio and stimulated with immobilized anti-CD3 and soluble anti-CD28 mAb for 48 hours. Culture supernatants were collected, and levels of IL-10, IL-4, IL-5, IFN-γ, and IL-22 were measured by ELISA. Points represent the results for multi-donor CD4IL-10 cells, and gray bars represent the mean ± SD for a single donor (n=8). [Figure 10] Figure 10A shows the percentage of multi-donor CD4IL-10 cells (GzB) expressing granzyme B compared to the mean % level (+ / - SD) of granzyme B expression by single-donor CD4IL-10 cells (n=3) used to generate the pool. Cells were analyzed by FACS after the third stimulation (TF3). Figure 10B shows the % cell death when multi-donor CD4IL-10 cells (105 cells / well) were cocultured with K562 cells and ALL-CM cells (105 cells / well) at a 1:1 ratio for 3 days. Residual leukemia cells (CD45+CD33+) were counted by FACS for each target cell type. Points represent multi-donor CD4IL-10 results, and gray bars represent the mean ± SD of the single donors (n=3) used to generate the pool. [Figure 11A]Figures 11A and 11B show that multi-donor CD4IL-10 cells can suppress the proliferation of allogeneic CD4+ and CD8+ T cells. Allogeneic PBMC cells were labeled with eFluor® 670 (10 cells / well) and stimulated with allogeneic mature dendritic (DC) cells (5 × 10 cells / well) and soluble anti-CD3 mAb at a 1:1 responder:suppressor ratio in the absence or presence of multi-donor CD4IL-10 cells (10 cells / well). After 4 days of culture, the percentage of proliferating responder cells was determined by eFluor® 670 dilution using flow cytometry, gating on CD4+ΔNGFR- and CD8+ΔNGFR- T cells. Figure 11A shows the results for multi-donor CD4IL-10 cells containing pooled CD4+ cells from donors C, E, and F. Figure 11B shows the results for multi-donor CD4IL-10 cells containing pooled CD4+ cells from donors H, I, and L. Suppression mediated by CD4IL-10 cells was calculated as follows: 100 - ([proliferation of responder cells in the presence of CD4IL-10 cells / proliferation of responder cells alone] × 100). [Figure 11B] Same as above. [Figure 12] FIG. 12 shows the protocol for testing the induction of GvHD by human PBMC and / or multi-donor CD4IL-10 cells injected on day 0 after irradiation. [Figure 13] Figure 13 shows the % of NSG mice exhibiting GvHD on each day after injection with PBMCs (5 x 10 cells / mouse), multi-donor (3 donors) CD4IL-10 cells (5 x 10 cells / mouse), or PBMCs (5 x 10 cells / mouse) in combination with multi-donor CD4IL-10 cells (3 donors) (5 x 10 cells / mouse). [Figure 14]Figure 14 shows migration of CD4IL-10 cells to the spleen and bone marrow in NSG mice injected with PBMCs (5 x 10 cells / mouse), multi-donor (3 donors) CD4IL-10 cells (5 x 10 cells / mouse), or PBMCs (5 x 10 cells / mouse) combined with multi-donor CD4IL-10 cells (3 donors) (5 x 10 cells / mouse). Boxes and whiskers for donors (n = 8) and single donors are indicated. [Figure 15] FIG. 15 shows the protocol for testing the induction of GvHD by CD4+ T cells and multi-donor or single-donor CD4IL-10 cells injected on day 0 after irradiation. [Figure 16] FIG. 16 shows the % of NSG mice showing GvHD on each day after injection. [Figure 17] Figures 17A and 17B show the graft-versus-leukemia (GvL) effect, assessed by the reduction of circulating leukemia cells and long-term leukemia-free survival. Leukemia was measured as previously reported (Locafaro G. et al., Molecular Therapy, 2017). On day 0, NSG mice were sublethally irradiated and intravenously injected with myeloid leukemia cells (ALL-CM) (5 × 10 cells). Figure 17A shows the leukemia-free survival rate of animals injected with PBMCs (5 × 10 cells) or single-donor CD4, IL-10 cells (2.5 × 10 cells) from donors BC-I and BC-H on day 3. Figure 17B shows the leukemia-free survival rate of animals injected with PBMCs (5 × 10 cells) or multiple-donor CD4, IL-10 cells (2.5 × 10 cells) on day 3. [Figure 18]Figures 18A and 18B show long-term leukemia-free survival measured in NSG mice sublethally irradiated and intravenously injected with ALL-CM cells (5 x 10) on day 0. Figure 18A shows data from animals injected with 5 x 10 PBMCs alone or 5 x 10 PBMCs plus 2.5 x 10 single-donor CD4IL-10 cells (derived from donors BC-H and BC-I) on day 3. Figure 18B shows data from animals injected with 5 x 10 PBMCs alone or 5 x 10 PBMCs plus 2.5 x 10 multi-donor CD4IL-10 cells on day 3. DETAILED DESCRIPTION OF THE INVENTION

[0076] These drawings depict various embodiments of the present invention for purposes of illustration only. Those skilled in the art will readily appreciate from the following description that alternative embodiments of the structures and methods shown herein may be used without departing from the principles of the present invention as described herein.

[0077] 6. Detailed Description 6.1.Definition Unless otherwise defined, all technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this invention belongs. As used herein, the following terms have the meanings ascribed to them:

[0078] "Graft-versus-leukemia effect" or "GvL" refers to the effect that occurs after allogeneic hematopoietic stem cell transplantation (HSCT) or bone marrow transplantation (BMT), in which allograft T lymphocytes eliminate residual malignant host leukemia cells.

[0079] "Graft-versus-tumor effect" or "GvT" refers to the effect that occurs after allogeneic hematopoietic stem cell transplantation (HSCT) or bone marrow transplantation (BMT). Allograft T lymphocytes eliminate malignant residual host cancer cells, such as myeloma and lymphoid and myeloid leukemias, lymphomas, multiple myeloma, and possibly breast cancer cells. The term GvT is a generic term for GvL.

[0080] The terms "treatment," "treating," and the like are used herein in the broadest sense understood in the medical arts. In particular, these terms generally refer to obtaining a desired pharmacological and / or physiological effect. The effect may be prophylactic, in that a disease, condition, or symptoms thereof are completely or partially prevented, and / or may be therapeutic, in that a disease or condition and / or adverse effects, e.g., symptoms resulting from the disease or condition, are partially or completely cured. As used herein, "treatment" encompasses any treatment of a mammalian, particularly a human, disease or condition, including (a) preventing the disease or condition from occurring in a subject who may be susceptible to, but has not yet been diagnosed as having, the disease or condition; (b) inhibiting the disease or condition (e.g., preventing its onset); or (c) alleviating the disease or condition (e.g., causing regression of the disease or condition, resulting in the improvement of one or more symptoms). Improvement in any condition can be readily assessed according to standard methods and techniques known in the art. The population of subjects treated by the disease methods includes those suffering from the undesirable condition or disease, as well as those at risk of developing the condition or disease.

[0081] As used herein, "HLA match" refers to a pair of individuals who have matching HLA alleles at the HLA class I (HLA-A, HLA-B, and HLA-C) and class II (HLA-DRB1 and HLA-DQB1) loci, allowing the individuals to be immunologically compatible with each other. HLA compatibility can be determined using any of the methods available in the art, for example, as described in Tiervy, Haematologica 2016 Volume 101(6):680-687, which is incorporated herein by reference.

[0082] For a given locus, a pair of individuals has a 2 / 2 match if each of the two alleles of one individual matches two alleles of the other individual. A pair of individuals has a 1 / 2 match if only one of the two alleles of one individual matches one of the two alleles of the other individual. A pair of individuals has a 10 / 10 match at the HLA-A, HLA-B, HLA-C, HLA-DRB1, and HLA-DQB1 loci if all 10 alleles of one individual (two for each of the HLA-A, HLA-B, HLA-C, HLA-DRB1, and HLA-DQB1 loci) match all 10 alleles of the other individual.

[0083] In a preferred embodiment, allele-level typing is used to determine HLA compatibility. Allele-level typing can be used to determine HLA compatibility, for example, * It corresponds to the unique nucleotide sequence of the HLA gene, defined by using all the numbers in the first, second, third and fourth segments, such as 02:01:01:01. Functionally, the third and fourth regions, which characterize different alleles due to silent substitutions in coding sequences and substitutions in non-coding sequences, respectively, are irrelevant, except when the substitutions prevent the expression of the HLA allele (e.g., the null allele B*15:01:01:02N). The absence of a null allele often results in a mismatch that is highly likely to be recognized by alloreactive T cells and has adverse clinical consequences. Substitutions in non-coding sequences can affect expression levels (e.g., the A24low allele A*24:02:01:02L). Such variability can also affect anti-HLA non-self recognition.

[0084] As used herein, the term "HLA mismatched" refers to a pair of individuals who have mismatched HLA alleles at the HLA class I (HLA-A, HLA-B, and HLA-C) and class II (HLA-DRB1 and HLA-DQB1) loci, making the individuals immunologically incompatible with each other.

[0085] As used herein, the term "partial HLA mismatch" refers to a pair of individuals who have mismatched HLA alleles at the HLA class I (HLA-A, HLA-B, and HLA-C) and class II (HLA-DRB1 and HLA-DQB1) loci, making the individuals immunologically incompatible with each other to an acceptable degree. Some studies have identified tolerated mismatches. Some HLA class I incompatibilities are considered to be more tolerated.

[0086] "HLA haplotype" refers to the set of HLA locus alleles per chromosome, one inherited from the mother and one inherited from the father. Genotypes at the HLA class I (HLA-A, HLA-B, and HLA-C) and class II (HLA-DRB1 and HLA-DQB1) loci can be used to determine the HLA haplotype.

[0087] The term "therapeutically effective amount" is an amount effective to treat a disease, thus ameliorating the symptoms of the disease. Prevention can be considered treatment, and thus a therapeutically effective amount can be a "prophylactically effective amount."

[0088] The term "ameliorate" refers to any therapeutically beneficial outcome in the treatment of a disease state, for example, a neurodegenerative disease state, including prevention, reduction in severity or progression, remission, or cure.

[0089] 6.2. Other Interpretation Rules Ranges recited herein are understood to be shorthand for all values ​​within the range, inclusive of the recited endpoints. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or subrange from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, and 50.

[0090] 6.3. Summary of experimental observations The present disclosure provides highly purified allogeneic CD4 transduced with a bidirectional lentiviral vector containing the human IL-10 gene and a truncated, non-signaling form of the human NGFR. + Methods for the production and use of successfully transduced CD4 T cells are provided. + T cells were purified using an NGFR-specific monoclonal antibody to identify >95% pure IL-10-producing, NGFR-expressing CD4 + T cells (CD4 IL-10 CD4 cells were obtained from three different allogeneic HLA-mismatched donors. IL-10 Cells were pooled in a 1:1:1 ratio.

[0091] As used herein, multiple donor CD4 IL-10 These pooled populations, also called cells, are single-donor CD4 IL-10 The cytokine production profiles of these CD4 T cells were comparable to those of naturally occurring type 1 regulatory T (Tr1) cells. They produced high levels of IL-10, IL-22, IFN-γ, IL-5, and low levels of IL-4. IL-10 The cells are polyclonal (have multiple antigen specificities) and express allogeneic CD4 + Cells and CD8 + They suppressed the proliferation of both CD4 T cells and CD8 T cells. Furthermore, they specifically killed myeloid leukemia cells in vitro. Multiple donor CD4 T cells were also expressed in a humanized mouse model of graft-versus-host disease (GvHD). IL-10 Adoptive transfer of the cells showed that these cells efficiently homed to the spleen. IL-10 Adoptive transfer of cells using human CD4 + Multi-donor CD4+ / IL-10+ cells inhibited severe xenogeneic GvHD induced by T cells. Importantly, even at high concentrations, multi-donor CD4+ / IL-10+ cells alone did not induce GvHD. These results support the conclusion that multi-donor CD4+ / IL-10+ cells alone did not induce GvHD. IL-10The cells have been shown to be useful for the treatment and / or prevention of GvHD, to be used as an adjunct to allogeneic hematopoietic stem cell transplantation (HSCT) for the treatment of leukemia and other malignancies, to alleviate GvHD while maintaining the GvL or GvT therapeutic effects of HSCT, and to treat cell and organ rejection and autoimmune and inflammatory diseases.

[0092] 6.4. Multiple donor CD4 IL-10 cell In a first aspect, a CD4 gene is genetically modified to contain an exogenous polynucleotide encoding IL-10. + A population of T cells (CD4 IL-10 This population consists of at least three different T cell donors (multiple donor CD4 IL-10 CD4 obtained from + Contains T cells.

[0093] CD4 + T cells and T cell donors Multiple donor CD4 IL-10 CD4 used in population + T cells can be isolated from peripheral blood, umbilical cord blood, or other blood samples from a donor using methods available in the art. In an exemplary embodiment, CD4 + T cells are isolated from peripheral blood. In certain embodiments, CD4 + T cells are isolated from peripheral blood obtained from a third-party blood bank.

[0094] In some embodiments, CD4 + T cells are isolated from pre-frozen stocks of blood or pre-frozen stocks of peripheral blood mononuclear cells (PBMCs). In some embodiments, CD4 + T cells are isolated from peripheral blood or from PBMCs that have not been previously frozen. In some embodiments, CD4 + T cells are isolated separately from blood or PBMCs obtained from multiple donors and then pooled. In some embodiments, CD4 +T cells are isolated from initially pooled blood or PBMCs from multiple donors.

[0095] In some embodiments, CD4 + The T cells are obtained from 3, 4, 5, 6, 7, 8, 9, or 10 different T cell donors.

[0096] In some embodiments, the at least three different T cell donors are selected without regard to genotype. In some embodiments, the at least three different T cell donors are selected based on genotype.

[0097] In certain embodiments, at least three different T cell donors are selected based on their HLA haplotypes.

[0098] In some embodiments, some or all of the at least three different T cell donors have matching HLA haplotypes. In some embodiments, some or all of the at least three different T cell donors have mismatched HLA haplotypes.

[0099] In some embodiments, all CD4 + The T cells have at least a 5 / 10, 6 / 10, 7 / 10, 8 / 10, or 9 / 10 match to each other at the HLA-A, HLA-B, HLA-C, HLA-DRB1, and HLA-DQB1 loci. + The T cells have at least a 4 / 8, 5 / 8, 6 / 8, 7 / 8, or 8 / 8 match to each other at the HLA-A, HLA-B, HLA-C, and HLA-DRB1 loci. In some embodiments, all CD4 T cells in the population + The T cells have a 2 / 2 match with each other at the HLA-A locus. In some embodiments, all CD4 T cells in the population + The T cells have a 2 / 2 match with each other at the HLA-B locus. In some embodiments, all CD4 T cells in the population +The T cells have a 2 / 2 match with each other at the HLA-C locus. In some embodiments, all CD4 T cells in the population + The T cells have at least a 3 / 4 or 4 / 4 match to each other at the HLA-DRB1 and HLA-DQB1 loci. In some embodiments, all CD4 T cells in the population + T cells are A * Possess the 02 allele.

[0100] In a preferred embodiment, at least three different T cell donors each contain CD4 IL-10 In a preferred embodiment, none of the at least three different T cell donors is a CD4 T cell donor in the therapeutic methods described herein. IL-10 Not the donor of stem cells (e.g., HSCs), tissues, or organs used with the cells.

[0101] In some embodiments, one or more T cell donors are HLA-mismatched or partially HLA-mismatched to the patient (host) being treated. In some embodiments, one or more T cell donors have less than a 5 / 10, 6 / 10, 7 / 10, 8 / 10, 9 / 10, or 10 / 10 match to the patient at the HLA-A, HLA-B, HLA-C, HLA-DRB1, and HLA-DQB1 loci. In some embodiments, one or more T cell donors have less than a 4 / 8, less than a 5 / 8, less than a 6 / 8, less than a 7 / 8, or less than a 8 / 8 match to the patient at the HLA-A, HLA-B, HLA-C, and HLA-DRB1 loci. In some embodiments, one or more T cell donors have less than a 2 / 2 match to the patient at the HLA-A, HLA-B, or HLA-C loci. In some embodiments, one or more T cell donors have less than a 2 / 4, less than a 3 / 4, or less than a 4 / 4 match to the patient at the HLA-DRB1 and HLA-DQB1 loci.

[0102] In some embodiments, one or more T cell donors are HLA-mismatched or partially HLA-mismatched to the HSC donor. In some embodiments, one or more T cell donors have less than a 5 / 10, 6 / 10, 7 / 10, 8 / 10, 9 / 10, or 10 / 10 match to the HSC donor at the HLA-A, HLA-B, HLA-C, HLA-DRB1, and HLA-DQB1 loci. In some embodiments, one or more T cell donors have less than a 4 / 8, 5 / 8, 6 / 8, 7 / 8, or 8 / 8 match to the HSC donor at the HLA-A, HLA-B, HLA-C, and HLA-DRB1 loci. In some embodiments, one or more T cell donors have less than a 2 / 2 match to the HSC donor at the HLA-A, HLA-B, or HLA-C loci. In some embodiments, one or more T cell donors have a 3 / 4 or less than 4 / 4 match to the HSC donor at the HLA-DRB1 and HLA-DQB1 loci.

[0103] In a preferred embodiment, CD4 + None of the T cells are immortalized.

[0104] 6.4.2. Exogenous Polynucleotides Encoding IL-10 The present disclosure of multiple donor CD4 IL-10 The CD4 cells have been genetically modified to contain an exogenous polynucleotide encoding IL-10. + The exogenous polynucleotide comprises a polynucleotide segment encoding IL-10 operably linked to an expression control element.

[0105] The polynucleotide segment encoding IL-10 may encode human, bonobo, or rhesus macaque IL-10. In some embodiments, the polynucleotide segment encoding IL-10 encodes human IL-10 having the sequence of SEQ ID NO: 1. In some embodiments, the polynucleotide segment encoding IL-10 encodes a variant of human IL-10 having at least 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 1. In some embodiments, the polynucleotide segment encoding IL-10 has the nucleotide sequence of SEQ ID NO: 2. In some embodiments, the polynucleotide segment encoding IL-10 has at least 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 2.

[0106] In some embodiments, the exogenous polynucleotide encodes viral IL-10. In various embodiments, the exogenous polypeptide encodes IL-10 from HCMV, GMCMV, RhCMV, BaCMV, MOCMV, SMCMV, EBV, Bonobo-HV, BaLCV, OvHV-2, EHV-2, CyHV-3, AngHV-1, ORFV, BPSV, PCPV, LSDV, SPV, GPV, or CNPV. In some embodiments, the exogenous polypeptide encodes viral IL-10 from EBV or ORFV.

[0107] The exogenous polynucleotide is transduced into CD4 + It further comprises an expression control element that directs expression of the encoded IL-10 in T cells.

[0108] In some embodiments, the expression control element is CD4 + In some embodiments, the promoter comprises a promoter capable of directing the expression of IL-10 in T cells. + In some embodiments, the promoter drives constitutive expression of IL-10 in T cells. + Drives IL-10 expression in T cells.

[0109] In some embodiments, an inducible promoter is used to induce expression of IL-10 when therapeutically appropriate. In some embodiments, the IL-10 promoter is used. In some embodiments, a tissue-specific promoter is used. In some embodiments, a lineage-specific promoter is used. In some embodiments, a ubiquitously expressed promoter is used.

[0110] In some embodiments, a native human promoter is used. In some embodiments, the human elongation factor (EF) 1α promoter is used. In some embodiments, the human phosphoglycerate kinase promoter (PGK) is used. In some embodiments, the human ubiquitin C promoter (UBI-C) is used.

[0111] In some embodiments, a synthetic promoter is used. In certain embodiments, a minimal CMV core promoter is used. In certain embodiments, an inducible or constitutive bidirectional promoter is used. In certain embodiments, the synthetic bidirectional promoter disclosed in Amendola et al., Nature Biotechnology, 23(1):108-116 (2005) is used. This promoter can mediate coordinated transcription of two mRNAs in a ubiquitous or tissue-specific manner. In certain embodiments, the bidirectional promoter induces expression of IL-10 and a selectable marker.

[0112] In some embodiments, the exogenous polynucleotide is a polynucleotide that is capable of transducing CD4 +The nucleotide sequence encoding the ΔNGFR selectable marker has the sequence of SEQ ID NO:4. In some embodiments, the nucleotide sequence encoding the ΔNGFR selectable marker has at least 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO:4.

[0113] In some embodiments, the selectable marker is a truncated EGFR polypeptide, optionally huEGFRt, of the human EGFR polypeptide disclosed in Wang et al., "A transgene-encoded cell surface polypeptide for selection, in vivo tracking, and ablation of engineered cells," Blood, v. 118, n. 5 (2011), which is incorporated herein in its entirety.

[0114] In some embodiments, the exogenous polynucleotide further comprises a sequence encoding an antibiotic resistance gene. In some embodiments, the exogenous polynucleotide comprises a sequence encoding an ampicillin resistance gene. In some embodiments, the exogenous polynucleotide comprises a sequence encoding a kanamycin resistance gene.

[0115] In typical embodiments, the exogenous polynucleotide is delivered to the CD4+ T cell using a vector. In some embodiments, the vector is a plasmid vector. In some embodiments, the vector is a viral vector.

[0116] In certain embodiments, the exogenous polynucleotide is delivered to the CD4+ T cell using a lentiviral vector, and the exogenous polynucleotide comprises a lentiviral vector sequence. In certain embodiments, the lentiviral vector disclosed in Matrai et al., Molecular Therapy 18(3):477-490(2010) ("Matrai"), which is incorporated herein by reference, is used.

[0117] In some embodiments, the lentiviral vector can integrate into the T cell nuclear genome. In some embodiments, the lentiviral vector cannot integrate into the T cell nuclear genome. In some embodiments, an integration-deficient lentiviral vector is used. For example, in some embodiments, integration-deficient or other lentiviral vectors disclosed in Matrai are used. In some embodiments, integrase-deficient lentiviruses are used. For example, integrase-deficient lentiviruses containing inactivating mutations in integrase (D64V) can be used as described in Matrai et al., Hepatology 53:1696-1707 (2011), which is incorporated herein by reference.

[0118] In some embodiments, the exogenous polynucleotide is integrated into the T cell nuclear genome. In some embodiments, the exogenous polynucleotide is not integrated into the nuclear genome. In some embodiments, the exogenous polynucleotide is present in the cytoplasm of the T cell.

[0119] In certain embodiments, the exogenous polynucleotide has the sequence of SEQ ID NO: 5. In some embodiments, the exogenous polynucleotide has at least 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO:5.

[0120] 6.4.3. Multiple donor CD4 IL-10 T cell gene expression Multiple donor CD4 IL-10The T cells express IL-10. In some embodiments, multi-donor CD4 IL-10 The T cells constitutively express IL-10. In some embodiments, multi-donor CD4 IL-10 Upon activation, T cells express IL-10.

[0121] In some embodiments, multiple donor CD4 IL-10 T cells are CD4 + T cells 10 6 In some embodiments, the multi-donor CD4 IL-10 T cells are CD4 + T cells 10 6 The cells constitutively express at least 200 pg, 500 pg, 1 ng, 5 ng, 10 ng, or 50 ng of IL-10 per ml of culture medium.

[0122] In some embodiments, multiple donor CD4 IL-10 T cells were activated with a combination of anti-CD3 and anti-CD28 antibodies or beads coated with anti-CD3 and anti-CD28 antibodies, followed by CD4 + T cells 10 6 In some embodiments, the multi-donor CD4 IL-10 T cells were activated with beads coated with anti-CD3 and anti-CD28 antibodies or CD3 and CD28 antibodies, followed by CD4 + T cells 10 6 They express at least 5 ng, 10 ng, 100 ng, 200 ng or 500 ng of IL-10 per ml of culture medium.

[0123] In various embodiments, IL-10 production is measured 12 hours, 24 hours, or 48 hours after activation using various methods for protein detection and measurement, such as ELISA, spectroscopic procedures, colorimetric methods, amino acid analysis, radiolabeling, Edman degradation, HPLC, Western blotting, etc. In a preferred embodiment, IL-10 production is measured by ELISA 48 hours after activation with anti-CD3 and anti-CD28 antibodies.

[0124] In some embodiments, multiple donor CD4 IL-10 T cells are unmodified CD4 + In some embodiments, the multi-donor CD4 IL-10 T cells are unmodified CD4 + express IL-10 at levels at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, or 50 times higher than T cells.

[0125] In some embodiments, multiple donor CD4 IL-10 The T cells further express a selectable marker. In some embodiments, multi-donor CD4 IL-10 The T cells express proteins typically expressed in Tr1 cells. In some embodiments, multi-donor CD4 IL-10 T cells express marker proteins characteristic of Tr1 cells.

[0126] In some embodiments, multiple donor CD4 IL-10 The T cells express CD49b. In some embodiments, multi-donor CD4 IL-10 The T cells express LAG-3. In some embodiments, multi-donor CD4 IL-10 The T cells express TGF-β. In some embodiments, multi-donor CD4 IL-10 The T cells express IFNγ. In some embodiments, multi-donor CD4 IL-10 In some embodiments, the T cells express GzB. IL-10T cells release GzB upon activation with myeloid antigen-presenting cells. In some embodiments, multi-donor CD4 IL-10 The T cells express perforin. In some embodiments, multi-donor CD4 IL-10 T cells release perforin upon activation with myeloid antigen-presenting cells. In some embodiments, multi-donor CD4 IL-10 The T cells express CD18. In some embodiments, multi-donor CD4 IL-10 The T cells express CD2. In some embodiments, multi-donor CD4 IL-10 The T cells express CD226. In some embodiments, multi-donor CD4 IL-10 In some embodiments, the T cells express IL-22. IL-10 T cells express IL-10.

[0127] In some embodiments, multiple donor CD4 IL-10 The T cells exhibit at least one phenotypic function of Tr1 cells, which in various embodiments is through the secretion of IL-10, the secretion of TGF-β, and the specific killing of myeloid antigen-presenting cells by the release of granzyme B (GzB) and perforin.

[0128] 6.4.4. Process Products In an exemplary embodiment, multiple donor CD4 IL-10 T cells transduce CD4 T cells with an exogenous polynucleotide encoding IL-10. + It is obtained by modifying T cells.

[0129] In some embodiments, the exogenous polynucleotide is delivered to CD4 by a viral vector or a plasmid vector. + In certain embodiments, the CD4 + T cells are transduced with a lentivirus containing the coding sequence for IL-10.

[0130] In some embodiments, multiple donor CD4 IL-10T cells were (i) primary CD4 T cells obtained from at least three different T cell donors; + (ii) pooling the pooled CD4 T cells by introducing an exogenous polynucleotide encoding IL-10. + and modifying T cells. In some embodiments, the multi-donor CD4 IL-10 T cells were obtained from (i) primary CD4 T cells from at least three different T cell donors; + (ii) obtaining CD4 T cells from each donor by introducing an exogenous polynucleotide encoding IL-10. + (iii) genetically modifying CD4 T cells; + and pooling T cells.

[0131] In some embodiments, multiple donor CD4 IL-10 T cells are CD4 + In some embodiments, the multi-donor CD4 T cells are cultured in the presence of a protein capable of activating the T cells. IL-10 The T cells are cultured in the presence of anti-CD3 and anti-CD28 antibodies, or beads coated with anti-CD3 and anti-CD28 antibodies. In some embodiments, multi-donor CD4 IL-10 The T cells are cultured in the presence of anti-CD3 antibody, anti-CD28 antibody and IL-2, or anti-CD3 antibody and anti-CD28 antibody coated beads and IL-2. In some embodiments, multi-donor CD4 IL-10 T cells were cultured in the presence of T Cell TransAct™ from Miltenyi Biotec. In some embodiments, multi-donor CD4 IL-10 T cells were cultured in the presence of ImmunoCult Human T Cell Activator™ from STEMCELL Technologies.

[0132] In some embodiments, multiple donor CD4 IL-10 T cells are in frozen stock.

[0133] Pharmaceutical Compositions In another aspect, a pharmaceutical composition is provided. The pharmaceutical comprises a multi-donor CD4 IL-10 It comprises T cells and a pharmaceutically acceptable carrier or diluent.

[0134] The pharmaceutical compositions can be formulated for administration by any route suitable for human or veterinary medicine. In typical embodiments, the compositions are formulated for intravenous (IV) administration. In some embodiments, the compositions are formulated for intravenous (IV) infusion. In embodiments formulated for IV administration, the pharmaceutical composition is in the form of a pyrogen-free, parenterally acceptable aqueous solution having suitable pH, isotonicity, and stability.

[0135] In some embodiments, the pharmaceutically acceptable carrier or diluent is saline, lactated Ringer's solution, or other physiologically compatible solution. In various embodiments, the pharmaceutical composition solution contains 2-20%, preferably 5%, human serum albumin.

[0136] In some embodiments, unit dosage forms of the pharmaceutical composition are provided that are adapted for administration of the pharmaceutical composition by systemic administration, particularly intravenous administration.

[0137] In some embodiments, the unit dosage form comprises 10 4 From 10 11 Multiple donor CD4 IL-10 T cells, 10 4 From 10 10 Multiple donor CD4 IL-10 T cells, 10 4 From 10 9 Multiple donor CD4 IL-10 T cells, 10 5 From 10 10 Multiple donor CD4 IL-10 T cells, 10 5 From 10 9 Multiple donor CD4 IL-10 T cells, 10 5 From 10 8 Multiple donor CD4IL-10 T cells, or 10 5 From 10 7 Multiple donor CD4 IL-10 Contains T cells.

[0138] In an exemplary embodiment, the pharmaceutical composition in unit dosage form is in liquid form.

[0139] 6.6. Multiple donor CD4 IL-10 Cell production method In another aspect, the present disclosure provides a multi-donor CD4 IL-10 A method for producing a cell is provided.

[0140] In some embodiments, the method comprises: (i) primary CD4 T cells obtained from at least three different T cell donors; + (ii) pooling the pooled CD4 T cells by introducing an exogenous polynucleotide encoding IL-10. + In another embodiment, the method comprises (i) modifying primary CD4 T cells from at least three different T cell donors. + (ii) obtaining CD4 T cells from each donor by introducing an exogenous polynucleotide encoding IL-10. + (iii) genetically modifying CD4 T cells; + T cells are pooled, thereby multi-donor CD4 IL-10 and obtaining the cells. Various methods known in the art can be used to transfect an exogenous polynucleotide encoding IL-10 into primary CD4 + It can be introduced into T cells.

[0141] In some embodiments, the method comprises culturing primary CD4+ cells in the presence of beads coated with anti-CD3 and anti-CD28 antibodies, or anti-CD3 and anti-CD28 antibodies. + T cells or genetically modified CD4 +In some embodiments, the method further comprises incubating primary CD4 T cells in the presence of anti-CD3 antibody, anti-CD28 antibody and IL-2, or anti-CD3 antibody and anti-CD28 antibody coated beads and IL-2. + T cells or genetically modified CD4 + In some embodiments, the method further comprises incubating the T cells in the presence of a mixture of feeder cells. + T cells or genetically modified CD4 + In some embodiments, the method further comprises incubating primary CD4 T cells in the presence of a nanopreparation of anti-CD3 and anti-CD28 antibodies. + T cells or genetically modified CD4 + The method further comprises incubating the T cells. In some embodiments, the incubation is performed in the presence of T Cell TransAct™ from Miltenyi Biotec. In some embodiments, the incubation is performed in the presence of ImmunoCult Human T Cell Activator™ from STEMCELL Technologies.

[0142] In some embodiments, the incubation step is performed prior to introducing an exogenous polynucleotide encoding IL-10. In some embodiments, the incubation step is performed using (i) primary CD4 T cells obtained from at least three different T cell donors. + (ii) after the step of pooling T cells, by introducing an exogenous polynucleotide encoding IL-10, + In some embodiments, the incubation step is performed before the step of modifying the T cells. In some embodiments, the incubation step is performed after (i) culturing primary CD4 T cells from at least three different T cell donors. + After the step of obtaining T cells, (ii) the CD4 T cells of each donor are transfected by introducing an exogenous polynucleotide encoding IL-10. + This is done before the step of separately modifying the T cells.

[0143] In some embodiments, the incubation step is performed after step (ii). In other words, in some embodiments, the incubation step is performed after step (ii) inducing pooled CD4 + In some embodiments, the incubation step is performed after (ii) modifying the CD4 T cells of each donor by introducing an exogenous polynucleotide encoding IL-10. + (iii) genetically modified CD4 + T cells are pooled, thereby genetically modifying CD4 + In some embodiments, the incubation step is performed before the step of obtaining T cells. In some embodiments, the incubation step is performed before the step of obtaining T cells. + T cells are pooled, thereby generating multiple donor CD4 IL-10 This is carried out after the step of obtaining the cells.

[0144] In some embodiments, the incubation step is performed two or more times. In some embodiments, the incubation step is performed + This is done both before and after genetic modification of T cells.

[0145] In some embodiments, the exogenous polynucleotide is delivered to primary CD4 +In some embodiments, the exogenous polynucleotide comprises an IL-10-encoding segment having the sequence of SEQ ID NO: 1. In some embodiments, the exogenous polynucleotide comprises an IL-10-encoding segment having at least 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 1. In some embodiments, the IL-10-encoding polynucleotide segment has the sequence of SEQ ID NO: 2. In some embodiments, the IL-10-encoding polynucleotide segment has at least 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 2. In some embodiments, the exogenous polynucleotide is a vector that encodes a gene encoding an IL-10-encoding segment. + The exogenous polynucleotide further comprises a segment encoding a marker that allows for selection of T cells. In some embodiments, the encoded selection marker is ΔNGFR. In certain embodiments, the encoded selection marker has the sequence of SEQ ID NO: 3. In certain embodiments, the exogenous polynucleotide comprises the sequence of SEQ ID NO: 4. In some embodiments, the encoded selection marker is a truncated human EGFR polypeptide.

[0146] In some embodiments, the method comprises genetically modifying CD4 + Isolating T cells and thereby genetically modifying CD4 IL-10 The method further includes generating an enriched population of cells.

[0147] In some embodiments, the genetically modified CD4 + At least 90% of the T cells express the selection marker. In some embodiments, the genetically modified CD4 T cells in the enriched population + At least 95% of the T cells express the selection marker. In some embodiments, the genetically modified CD4 T cells in the enriched population + At least 96, 97, 98, or 99% of the T cells express the selectable marker.

[0148] In some embodiments, the genetically modified CD4 + At least 90% of the T cells express IL-10. In some embodiments, the genetically modified CD4 T cells in the enriched population + At least 95% of the T cells express IL-10. In some embodiments, the genetically modified CD4 T cells in the enriched population + At least 96, 97, 98, or 99% of the T cells express IL-10.

[0149] In some embodiments, the method comprises genetically modifying CD4 + The method further comprises incubating the enriched population of T cells. In some embodiments, the incubation is performed in the presence of anti-CD3 and anti-CD28 antibodies, or beads coated with anti-CD3 and anti-CD28 antibodies. In some embodiments, the incubation is further performed in the presence of IL-2. In some embodiments, the incubation is performed in the presence of feeder cells. In some embodiments, the incubation is performed in the presence of a nanopreparation of anti-CD3 and anti-CD28 antibodies. In some embodiments, the incubation is performed in the presence of T Cell TransAct™ from Miltenyi Biotec. In some embodiments, the incubation is performed in the presence of ImmunoCult Human T Cell Activator™ from STEMCELL Technologies.

[0150] In some embodiments, the method comprises genetically modifying CD4 + The method further comprises freezing the T cells.

[0151] In some embodiments, primary CD4 + The T cells are derived from donors selected based on HLA haplotype. In some embodiments, the method further comprises selecting T cell donors by analyzing their genetic information. In some embodiments, the method comprises analyzing the genetic information or HLA haplotype of candidate T cell donors.

[0152] In some embodiments, primary CD4 + T cells are primary CD4 + The T cells or modifications thereof are derived from a donor that is at least partially HLA-matched to the host to be treated. In some embodiments, primary CD4 + The T cells are derived from a donor that is at least partially HLA-matched to the donor of the stem cells (HSCs), tissue, or organ. In some embodiments, primary CD4 + The T cells are obtained from a third-party donor who is not biologically related to the host. In some embodiments, primary CD4 + The T cells are obtained from a third-party donor who is not biologically related to the donor of the stem cells, tissue, or organs.

[0153] In some embodiments, in step (i), primary CD4 + The T cells are obtained from 3, 4, 5, 6, 7, 8, 9, or 10 different T cell donors. In some embodiments, at least three T cell donors have at least a 5 / 10, 6 / 10, 7 / 10, 8 / 10, or 9 / 10 match to each other at the HLA-A, HLA-B, HLA-C, HLA-DRB1, and HLA-DQB1 loci. In some embodiments, at least three T cell donors have at least a 4 / 8, 5 / 8, 6 / 8, 7 / 8, or 8 / 8 match to each other at the HLA-A, HLA-B, HLA-C, and HLA-DRB1 loci. In some embodiments, at least three T cell donors have a 2 / 2 match to each other at the HLA-A locus. In some embodiments, at least three T cell donors have a 2 / 2 match to each other at the HLA-B locus. In some embodiments, the at least three T cell donors are 2 / 2 matches to each other at the HLA-C locus. In some embodiments, the at least three T cell donors are at least 3 / 4 or 4 / 4 matches to each other at the HLA-DRB1 and HLA-DQB1 loci. In some embodiments, each of the at least three T cell donors is * Possess the 02 allele.

[0154] In some embodiments, in step (i), primary CD4 + The T cells are obtained from one or more frozen stocks. In some embodiments, in step (i), primary CD4 + The T cells are obtained from non-frozen peripheral blood mononuclear cells of at least three different T cell donors. In some embodiments, the method comprises isolating CD4 T cells from peripheral blood mononuclear cells. + In some embodiments, step (i) further comprises isolating primary CD4 T cells. + The T cells are obtained from a liquid suspension. In some embodiments, the liquid suspension is obtained from a previously frozen stock.

[0155] In some embodiments, donor-derived CD4 + T cells are contacted with the patient's antigen-presenting cells (monocytes, dendritic cells, or DC-10 cells) to generate allospecific CD4 + T cells, which subsequently produce high levels of IL-11 (allogeneic CD4 IL-10 The cells are modified to produce

[0156] In some embodiments, the method comprises detecting CD4 + In some embodiments, the method does not include anergizing CD4 T cells in the presence of recombinant IL-10 protein. + The recombinant IL-10 protein does not involve the step of anergizing T cells, and it inhibits CD4 + In some embodiments, the method comprises administering to a subject a CD4 T cell in the presence of DC10 cells derived from the host. + It does not include a step of anergizing T cells.

[0157] 6.7. Multiple donor CD4 IL-10 How to use the cells In yet another aspect, the present disclosure provides a method of treating a patient in need of immune tolerization, comprising administering to the patient a multi-donor CD4 IL-10The present invention provides a method comprising administering a cell or pharmaceutical composition.

[0158] In some embodiments, the method comprises administering a multi-donor CD4 IL-10 The method further comprises the preceding step of thawing the frozen suspension of cells.

[0159] In some embodiments, multiple donor CD4 IL-10 The cells or pharmaceutical composition prevent or reduce the severity of a pathogenic T cell response in a patient.

[0160] In some embodiments, the treatment method comprises administering a multi-donor CD4 IL-10 In some embodiments, the method further comprises detecting a selectable marker in a biological sample obtained from the patient, thereby detecting a multi-donor CD4 IL-10 In some embodiments, the selection marker is a multi-donor CD4 T cell. IL-10 The cells are detected at multiple time points to track changes in their presence. In some embodiments, the biological sample is a biopsy or blood sample from a patient.

[0161] Multiple donor CD4 IL-10 The T cells are administered in a therapeutically effective amount, which can be determined based on body weight and other clinical factors. In some embodiments, 10 3 cells / kg to 10 9 In some embodiments, 10 cells / kg are administered. 3 cells / kg to 10 8 In some embodiments, 10 cells / kg are administered. 3 cells / kg to 10 7 In some embodiments, 10 cells / kg are administered. 3 cells / kg to 10 6 In some embodiments, 10 cells / kg are administered. 3 cells / kg to 10 5 In some embodiments, 10 cells / kg are administered. 3 cells / kg to 104 Cells / kg are administered.

[0162] In various embodiments, multiple donor CD4 IL-10 The T cells are administered in a therapeutically effective schedule. In some embodiments, multiple donor CD4 IL-10 In some embodiments, multiple donor CD4 T cells are administered. IL-10 Cells are administered daily, every 3 days, every 7 days, every 14 days, every 21 days, or monthly.

[0163] Multiple donor CD4 IL-10 T cells can be administered according to different routes of administration, for example, systemically, subcutaneously, or intraperitoneally. In some embodiments, the cells are administered in saline or physiological solution, which may contain 2-20%, preferably 5%, human serum albumin.

[0164] 6.7.1. Methods for reducing or preventing GvHD In some embodiments, multiple donor CD4 IL-10 Cell or multi-donor CD4 IL-10 Pharmaceutical compositions containing the cells are used to treat patients prior to, concurrently with, or following hematopoietic stem cell (HSC) transplantation (HSCT).

[0165] In various embodiments, the HSCT is a matched related HSCT. In various embodiments, the HSCT is a haploidentical HSCT, a mismatched related HSCT, or a mismatched unrelated HSCT.

[0166] In some embodiments, the patient has a hematological malignancy requiring treatment with allogeneic HSCT. In some embodiments, the hematological malignancy is mediated by abnormal bone marrow cells.

[0167] In some embodiments, the T cell donor is a multi-donor CD4 IL-10 The T cell donor is selected based on the genetic information of the patient to be treated with the cells and HSCs and / or the genetic information of the HSC donor. In some embodiments, the T cell donor is a multi-donor CD4IL-10 The cells and HSCs are selected based on the HLA haplotype of the patient to be treated with the HSCs and / or the HLA haplotype of the HSC donor. IL-10 Prior to administering the cells, the method further comprises analyzing the genetic information or HLA haplotype of the T cell donor. In some embodiments, the method further comprises analyzing the genetic information or HLA haplotype of the host. In some embodiments, the method further comprises analyzing the genetic information or HLA haplotype of the HSC donor.

[0168] In some embodiments, the T cell donor, host, and HSC donor are biologically unrelated. In some embodiments, the T cell donor, host, and HSC donor have different HLA haplotypes. In some embodiments, the T cell donor, host, and HSC donor are at least partially mismatched in HLA haplotypes. In some embodiments, T cell donors are selected if they have HLA haplotypes with an HLA match above a threshold.

[0169] In some embodiments, the HSC donor is partially HLA-mismatched to the patient. In some embodiments, the HSC donor has less than a 5 / 10, less than a 6 / 10, less than a 7 / 10, less than a 8 / 10, less than a 9 / 10, or less than a 10 / 10 match to the patient at the HLA-A, HLA-B, HLA-C, HLA-DRB1, and HLA-DQB1 loci. In some embodiments, the HSC donor has less than a 4 / 8, less than a 5 / 8, less than a 6 / 8, less than a 7 / 8, or less than a 8 / 8 match to the patient at the HLA-A, HLA-B, HLA-C, and HLA-DRB1 loci. In some embodiments, the HSC donor has less than a 2 / 2 match to the patient at the HLA-A, HLA-B, or HLA-C loci. In some embodiments, the HSC donor has less than a 3 / 4 or less than a 4 / 4 match to the patient at the HLA-DRB1 and HLA-DQB1 loci.

[0170] In some embodiments, one or more T cell donors are HLA-mismatched or partially HLA-mismatched to the patient. In some embodiments, one or more T cell donors have less than a 5 / 10, 6 / 10, 7 / 10, 8 / 10, 9 / 10, or 10 / 10 match to the patient at the HLA-A, HLA-B, HLA-C, HLA-DRB1, and HLA-DQB1 loci. In some embodiments, one or more T cell donors have less than a 4 / 8, less than a 5 / 8, less than a 6 / 8, less than a 7 / 8, or less than a 8 / 8 match to the patient at the HLA-A, HLA-B, HLA-C, and HLA-DRB1 loci. In some embodiments, one or more T cell donors have less than a 2 / 2 match to the patient at the HLA-A, HLA-B, or HLA-C loci. In some embodiments, one or more T cell donors have less than a 2 / 4, less than a 3 / 4, or less than a 4 / 4 match to the patient at the HLA-DRB1 and HLA-DQB1 loci.

[0171] In some embodiments, one or more T cell donors are HLA-mismatched or partially HLA-mismatched to the HSC donor. In some embodiments, one or more T cell donors have less than a 5 / 10, 6 / 10, 7 / 10, 8 / 10, 9 / 10, or 10 / 10 match to the HSC donor at the HLA-A, HLA-B, HLA-C, HLA-DRB1, and HLA-DQB1 loci. In some embodiments, one or more T cell donors have less than a 4 / 8, 5 / 8, 6 / 8, 7 / 8, or 8 / 8 match to the HSC donor at the HLA-A, HLA-B, HLA-C, and HLA-DRB1 loci. In some embodiments, one or more T cell donors have less than a 2 / 2 match to the HSC donor at the HLA-A, HLA-B, or HLA-C loci. In some embodiments, one or more T cell donors have a 3 / 4 or less than 4 / 4 match to the HSC donor at the HLA-DRB1 and HLA-DQB1 loci. IL-10The cells or pharmaceutical composition prevent or reduce the severity of GvHD caused by transplanted hematopoietic stem cells.

[0172] In some embodiments, multiple donor CD4 IL-10 The cells or pharmaceutical compositions prevent or reduce the severity of pathological T cell responses by transplanted hematopoietic cells. In certain embodiments, multi-donor CD4 IL-10 The cells prevent or reduce GvHD.

[0173] 6.7.2. Cancer Treatment Methods In some embodiments, multiple donor CD4 IL-10 The cells are used for the treatment of cancer. In a preferred embodiment, multi-donor CD4 IL-10 The cells directly mediate an anti-tumor effect (graft versus tumor, GvT), and in certain embodiments, an anti-leukemia effect (graft versus leukemia, GvL).

[0174] In some embodiments, multiple donor CD4 IL-10 The cells are administered in combination with allogeneic mononuclear cells or PBMCs for the treatment of cancer. In some embodiments, multi-donor CD4 IL-10 The cells are administered before or after administration of PBMCs. In some embodiments, multi-donor CD4 IL-10 The cells and allogeneic mononuclear cells or PBMCs are administered simultaneously.

[0175] In some embodiments, multiple donor CD4 IL-10 The cells and allogeneic mononuclear cells or PBMCs are administered in a ratio of 1:3, 1:2, 1:1, 2:1 or 3:1.

[0176] In some embodiments, the neoplastic cells express CD13. In some embodiments, the neoplastic cells express HLA-class I. In some embodiments, the neoplastic cells express CD54. In some embodiments, the neoplastic cells express CD13, HLA-class I, and CD54. In some embodiments, the neoplastic cells express CD112. In some embodiments, the neoplastic cells express CD58. In some embodiments, the neoplastic cells express CD155. In some embodiments, the tumor expresses CD112, CD58, or CD155. In various embodiments, the tumor is a solid tumor or a hematological tumor.

[0177] In some embodiments, the patient is diagnosed with adrenal gland cancer, anal cancer, bile duct cancer, bladder cancer, bone cancer, adult brain / CNS tumors, pediatric brain / CNS tumors, breast cancer, male breast cancer, cancer of unknown primary, Castleman's disease, cervical cancer, colon / rectal cancer, endometrial cancer, esophageal cancer, Ewing's tumor, eye cancer, gallbladder cancer, gastrointestinal carcinoid tumors, gastrointestinal stromal tumors (GIST), gestational trophoblastic disease, Hodgkin's disease, Kaposi's sarcoma, kidney cancer, laryngeal and hypopharyngeal cancer, leukemia, acute lymphocytic (ALL), acute myeloid (AML including myeloid sarcoma and leukemia cutis), chronic lymphocytic (CLL), chronic myeloid (CML) leukemia, chronic myelomonocytic leukemia (CMML), childhood leukemia, liver cancer, lung cancer, non-small cell lung cancer, The patient has a cancer selected from the group consisting of lung cancer, small cell lung cancer, pulmonary carcinoid tumor, lymphoma, lymphoma of the skin, malignant mesothelioma, multiple myeloma, myelodysplastic syndrome, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, non-Hodgkin's lymphoma, pediatric non-Hodgkin's lymphoma, oral cavity and oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, penile cancer, pituitary tumor, prostate cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma - adult soft tissue cancer, skin cancer, skin cancer - basal cell and squamous cell, skin cancer - melanoma, skin cancer - Merkel cell, small intestine cancer, stomach cancer, testicular cancer, thymus cancer, thyroid cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Waldenstrom's macroglobulinemia, and Wilms' tumor.

[0178] In some embodiments, the cancer is a myeloid tumor. In particular embodiments, the cancer is AML or CML. In some embodiments, the cancer is a myeloid tumor.

[0179] In some embodiments, the methods are used to treat hematological cancers that affect the blood, bone marrow, and lymph nodes. In various embodiments, the hematological cancer is lymphoma (e.g., Hodgkin's lymphoma), lymphocytic leukemia, or myeloma. In various embodiments, the hematological cancer is acute or chronic myeloid (bone marrow) leukemia (AML, CML), or myelodysplastic syndrome.

[0180] In some embodiments, the cancer is refractory or resistant to therapeutic intervention.

[0181] In some embodiments, multiple donor CD4 IL-10 The cells are used in combination with therapeutic interventions, which may be administered simultaneously or at different times. Preferably, the therapeutic intervention is selected from the group consisting of chemotherapy, radiation therapy, allogeneic HSCT, immunosuppression, blood transfusion, bone marrow transplantation, growth factors, and biological agents.

[0182] In some embodiments, multiple donor CD4 IL-10 The cells induce cell death of tumor-infiltrating myeloid cells (e.g., monocytes, macrophages, neutrophils).

[0183] 6.7.3. Methods for treating other disorders In some embodiments, multiple donor CD4 IL-10 The cells are administered to treat an autoimmune disease.

[0184] In some embodiments, the autoimmune disease is selected from the group consisting of type 1 diabetes, autoimmune uveitis, rheumatoid arthritis, psoriasis, psoriatic arthritis, multiple sclerosis, systemic lupus, inflammatory bowel disease, Addison's disease, Graves' disease, Sjögren's syndrome, Hashimoto's thyroiditis, myasthenia gravis, autoimmune vasculitis, pernicious anemia, ulcerative colitis, bullous disease, scleroderma, and celiac disease. In some embodiments, the autoimmune disease is Crohn's disease, ulcerative colitis, celiac disease, type 1 diabetes, lupus, psoriasis, psoriatic arthritis, or rheumatoid arthritis. In some embodiments, the patient has an allergic or atopic disease. The allergic or atopic disease may be selected from the group consisting of asthma, atopic dermatitis, and rhinitis. In some embodiments, the patient has a food allergy.

[0185] In some embodiments, multiple donor CD4 IL-10 The cells are administered to prevent or reduce the severity of pathogenic T cell responses to cell transplants and organ transplants other than HSCT. In some embodiments, the method involves administering multiple donor CD4 IL-10 The method includes the step of transplanting an organ into the patient either before or after administration of the T cells or pharmaceutical composition. In certain embodiments, the organ is a kidney, heart, or pancreatic islet cells. In a preferred embodiment, multiple donor CD4 IL-10 The cells or pharmaceutical composition prevent or reduce the severity of host rejection of an organ transplant.

[0186] In some embodiments, multiple donor CD4 IL-10 The cells are administered to prevent or reduce an immune response associated with gene therapy, e.g., administration of recombinant AAV (rAAV). In these embodiments, the method involves administering multiple donor CD4 IL-10 The method further includes administering a recombinant AAV to the patient before or after administration of the cells or pharmaceutical composition.

[0187] In some embodiments, multiple donor CD4 IL-10The cells are administered to prevent or reduce immune responses associated with transplantation of iPS-derived tissues or cells, including, but not limited to, cardiomyocytes, hepatocytes, epithelial cells, cartilage, bone and muscle cells, and neurons.

[0188] In some embodiments, multiple donor CDs 4IL-10 The cells are administered to treat inflammation, which may be associated with, but is not limited to, coronary artery disease (CAD), type 2 diabetes, neurodegenerative diseases, or inflammatory bowel disease.

[0189] In some embodiments, multiple donor CDs 4IL-10 The cells are administered to treat a disease or disorder associated with hyperactivity of the NLPR3 inflammasome. 4IL-10 The cells are administered to treat a disease or disorder associated with increased IL-1β production by activated monocytes, macrophages, or dendritic cells. In some embodiments, multi-donor CD4+ cells are administered. 4IL-10 The cells are administered to treat a disease or disorder associated with increased IL-18 production by activated monocytes, macrophages, or dendritic cells. In some embodiments, multi-donor CD4+ cells are administered to treat a disease or disorder associated with increased IL-18 production by activated monocytes, macrophages, or dendritic cells. 4IL-10 The cells are administered to treat a disease or disorder associated with increased production of mature caspase-1 by activated monocytes, macrophages, or dendritic cells.

[0190] In some embodiments, multiple donor CDs 4IL-10 The cells are administered to reduce IL-1β production by activated monocytes, macrophages, or dendritic cells. In some embodiments, multi-donor CD4+ 4IL-10 The cells are administered to reduce IL-18 production by activated monocytes, macrophages, or dendritic cells. In some embodiments, multi-donor CD4+ cells are administered to reduce IL-18 production by activated monocytes, macrophages, or dendritic cells. 4IL-10 The cells are administered to reduce mature caspase-1 production by activated monocytes, macrophages, or dendritic cells.

[0191] In some embodiments, multiple donor CD4 IL-10The cells are administered to reduce a patient's overactive immune response to a viral infection. In some embodiments, the virus is SARS-CoV-2. In some embodiments, multi-donor CD4 IL-10 The cells are administered to reduce an overly active immune response to bacterial infection, such as toxic shock and cytokine storm. [Example]

[0192] 6.8.Example The following examples are offered by way of illustration and not by way of limitation.

[0193] 6.8.1. Example 1: Multiple Donor CD4 IL-10 Cell preparation and vector production As described in WO 2016 / 146542, which is incorporated by reference in its entirety, transduction with a lentiviral vector containing the coding sequence for both human IL-10 and a truncated form of NGFR (ΔNGFR) (FIGS. 1 and 2) has been shown to be effective in generating multiple donor CD4+ ... IL-10 The cells were then produced. The sequence of the vector is shown as SEQ ID NO: 5. Briefly, the lentiviral vector was generated by ligating the coding sequence for human IL-10 from a 549 bp fragment of pH15C (ATCC 68192) into plasmid #1074.1071.hPGK.GFP.WPRE.mhCMV.dNGFR.SV40PA. The presence of a bidirectional promoter (human PGK promoter plus minimal core elements of the CMV promoter in opposite orientations) allows for simultaneous expression of two transgenes. The plasmid further contains the coding sequence for an antibiotic resistance gene (e.g., ampicillin or kanamycin).

[0194] Lentiviral vectors were produced by transient four-plasmid cotransfection into 293T cells with Ca3PO4, concentrated by ultracentrifugation, and 1 μM sodium butyrate was added to the culture for vector recovery. Titers on 293T cells were estimated by limiting dilution, and vector particles were measured by HIV-1 Gag p24 antigen immunocapture (NEN Life Science Products, Waltham, MA). Vector infectious titers were calculated as the ratio of titer to particles. For concentrated vectors, titers were 5 × 10 8 From 6 x 10 9 transducing units / ml, with an infectious titer of 5 x 10 4 From 5 x 10 5 The transducing units / ng range.

[0195] CD4 IL-10 Cell production Figure 3 shows the CD4 IL-10 Schematic diagram of the cell production process. + Purified human CD4 T cells + T cells were activated with soluble anti-CD3 mAb, soluble anti-CD28 mAb, and rhIL-2 (50 U / mL) for 48 h and then transduced with a bidirectional lentiviral vector encoding human IL-10 and a truncated human NGF receptor (LV-IL-10 / ΔNGFR) at a multiplicity of infection (MOI) of 20.

[0196] After 11 days, transduced cells were analyzed by FACS for expression of ΔNGFR and vector copy number (VCN) was quantified by digital droplet PCR (ddPCR).

[0197] CD4 from 10 different donors + The mean transduction efficiency of T cells was 45 ± 17%, and the VCN was 2.7 ± 0.6%. Figure 4A shows the transduction efficiency of human CD4 T cells transduced with LV-IL-10 / ΔNGFR (a bidirectional lentiviral vector encoding human IL-10 and a truncated human NGF receptor). + CD4 on T cells + ΔNGFR +Percentages of CD4 cells (mean ± SD, n = 10, left bar) and vector copy numbers (VCN, mean ± SD, n = 10, right bar) are shown. + ΔNGFR + The cell frequency and vector copy number were measured using CD4 IL-10 Quantification was performed by digital droplet PCR (ddPCR) in cells.

[0198] ΔNGFR + T cells were purified using anti-CD271 mAb-coated microbeads to obtain >95% pure CD4 IL-10 A cell population was obtained. After purification, the cells were stained with CD4 and ΔNGFR markers and analyzed by FACS. The data showed that the purity obtained from the purification process was greater than 98%. Figure 4B shows FACS data obtained from two representative donors (donor B and donor C) out of the 10 donors tested. Purified CD4 IL-10 Cells were restimulated three times at 14 day intervals and their in vitro and in vivo function was tested after the second restimulation (TF2) and / or third restimulation (TF3).

[0199] Resting CD4 IL-10 The cells constitutively produced IL-10, and upon activation, the levels of IL-10 produced were strongly enhanced.

[0200] CD4 IL-10 The cells have a cytokine production profile that is comparable to that of naturally occurring Tr1 cells. After the second (TF2) and third (TF3) restimulations, single-donor CD4 IL-10 The cytokine production profile of the cells was analyzed and the results are shown in FIG. Specifically, CD4 IL-10 cells (2 x 10 in 200 μl 5The CD4+ T cells were restimulated as previously described (Andolfi et al. Mol Ther. 2012;20(9):1778-1790 and Locafaro et al. Mol Ther. 2017;25(10):2254-2269). On day 14, after the second (TF2) and third (TF3) restimulations, CD4+ T cells were obtained. IL-10 Cells were left unstimulated (orange bars) or stimulated with immobilized anti-CD3 and soluble anti-CD28 mAbs (gray bars) for 48 hours. Culture supernatants were collected, and levels of IL-10, IL-4, IL-5, IFN-γ, and IL-22 were measured by ELISA. All samples were tested in triplicate. The mean ± SD of donors tested (n=8) is shown. The results shown in Figure 5 show that CD4 IL-10 Figure 1 shows that the cells exhibit a Tr1 cell cytokine production profile.

[0201] Although considerable variation was observed between different donors, the overall cytokine production profile after the second (TF2) or third (TF3) restimulation was comparable and mirrored that of Tr1 cells (Roncarolo et al., Immunity, 2018). Similar to Tr1 cells, CD4 IL-10 The cells produced high levels of IL-10, IL-5, IFN-γ, and IL-22, but low levels of IL-4 and undetectable levels of IL-2.

[0202] CD4 IL-10 The cells express high levels of granzyme B, which selectively kills myeloid leukemia cells. CD4 IL-10 The cells were further analyzed for granzyme B (GzB) expression after a second restimulation (TF2). The data in Figure 6A show that most CD4 IL-10 Total CD4 cells from seven different donors were shown to express GzB. IL-10 More than 95% of the cells expressed high levels of granzyme B.

[0203] CD4 obtained from the second (TF2) restimulationIL-10 The cells were further analyzed for their cytotoxic effect against myeloid leukemia cells (ALL-CM) and erythroid leukemia cell lines (K562). IL-10 cells (10 5 cells / well) were added to K562 cells and ALL-CM cells (10 5 The cells were co-cultured with the residual leukemia cell line (CD45 low CD33 + ) were counted by FACS for each target cell.

[0204] CD4 IL-10 The cells selectively killed myeloid leukemia cells (ALL-CM) as shown in Figure 6B. The percentage of ALL-CM cells killed varied between 62% and 100%, whereas killing of the erythroid leukemia cell line K562 (highly sensitive to nonspecific cytotoxic activity) varied between 0 and 27% (tested in four different donors). Collectively, these data support the conclusion that CD4 IL-10 These results confirm that the cells express granzyme B and efficiently kill myeloid leukemia cells. As expected, CD4 IL-10 Some variability in cell killing ability was observed.

[0205] CD4 IL-10 The cells were allogeneic CD4 + T cells and CD8 + suppresses both T cell proliferation responses CD4 IL-10 The cells were then transfected with allogeneic CD4 + T cells or CD8 + Their effects on T cells were also analyzed. Specifically, allogeneic PBMC cells were treated with eFluor® 670 (10 5 cells / well) and labeled with CD4 IL-10 In the absence or presence of cells (10 5 cells / well), and allogeneic mature dendritic (DC) cells (5 × 10 4 After 4 days of culture, the percentage of proliferative responding cells was determined by CD4 + ΔNGFR- T cells or CD8 + ΔNGFR - Figures 7A and 7B show CD4 T cells from six different unpooled donors (donors C, E, and F in Figure 7A and donors H, I, and L in Figure 7B) and CD4 T cells from six different unpooled donors (donors C, E, and F in Figure 7A and donors H, I, and L in Figure 7B) were measured by eFluor® 670 dilution using flow cytometry after gating on T cells. IL-10 Cellular CD4 + The effects on T cells are shown as percentages of proliferation and suppression. Figures 8A and 8B show CD4 T cells from six different unpooled donors (donors C, E, and F in Figure 8A and donors H, I, and L in Figure 8B). IL-10 Cellular CD8 + Shows the effect on T cells.

[0206] These results are not pooled and are based on CD4 counts from six different donors tested separately. IL-10 The cells are allogeneic CD4 + T cells and CD8 + showed that CD4 down-regulated both the proliferation and proliferation responses of T cells. + The inhibitory effect on T cells varied between 51% and 96%, but the CD8 + The inhibitory effect on T cells varied between 62% and 73%.

[0207] Multiple donor CD4 IL-10 Cell generation and characterization CD4 from multiple donors + Use CD4 cells as described above and in Figure 3. IL-10 CD4 cells were generated from each donor. IL-10 The cells were stimulated by a second (TF2) and third (TF3) restimulation. After the third stimulation, CD4 IL-10 Cells were pooled in a 1:1:1 ratio and stimulated with immobilized anti-CD3 and soluble anti-CD28 mAb for 48 hours.

[0208] Multiple donor CD4 IL-10 The cells were CD4 Il-10The cytokine production profile is comparable to that of the cells. Culture supernatants were collected and levels of IL-10, IL-4, IL-5, IFN-γ, and IL-22 were measured by ELISA. The results, presented in Figure 9, show pooled (1:1:1) multi-donor CD4 IL-10 Cytokine production of cells (red dots) was measured using CD4 IL-10 Cell-derived CD4 IL-10 The cytokine production of CD4+ cells (gray bars) was comparable to that of CD4+ cells (gray bars). IL-10 The cells produced high levels of IL-10, IL-5, IFN-γ, and IL-22, as well as low levels of IL-4 and undetectable levels of IL-2 (not shown). IL-10 It is feasible to pool cells, and these multi-donor CD4 IL-10 The cells were derived from a single donor. IL-10 These results demonstrate that the pooled homogeneous cell populations maintain the cytokine production signature of Tr1 and Tr1 cells. Importantly, the pooled homogeneous cell populations contained over 95% viable cells, indicating that they did not kill each other.

[0209] Multiple donor CD4 IL-10 The cells express high levels of granzyme B and are capable of killing myeloid leukemia cell lines. Multiple donor CD4 IL-10 Cells were further analyzed after the third restimulation (TF3) for expression of granzyme B (GzB). Data in Figure 10A show that most multi-donor CD4 IL-10 Showing that the cells express GzB. Multi-donor CD4 IL-10 Over 95% of cells express granzyme B and are CD4 IL-10 This was comparable to the GzB expression in the cells.

[0210] CD4 obtained from the third (TF3) restimulation IL-10 The cells were further analyzed for their cytotoxic effect against myeloid leukemia cells (ALL-CM cell line) or K562.IL-10 cells (10 5 cells / well) were added to K562 cells and ALL-CM cells (10 5 The cells were co-cultured with the residual leukemia cell line (CD45 low CD33 + ) were counted by FACS for each target cell. The results shown in Figure 10B demonstrate some degree of cytotoxicity against K562 cells, which are highly sensitive to nonspecific cytotoxicity. Nevertheless, a level of selectivity for myeloid leukemia cells (ALL-CM) was obtained, which was achieved by using CD4 IL-10 This is comparable to cell selectivity.

[0211] Multiple donor CD4 IL-10 The cells suppress the proliferative responses of both allogeneic CD4+ and CD8+ T cells. Multiple donor CD4 IL-10 The cells were then transfected with allogeneic CD4 + T cells or CD8 + Their effects on T cells were also analyzed. Specifically, allogeneic PBMC cells were treated with eFluor® 670 (10 5 cells / well) and labeled with multiple donor CD4 IL-10 In the absence or presence of cells (10 5 cells / well), and allogeneic mature dendritic (DC) cells (5 × 10 4 After 4 days of culture, the percentage of proliferative responding cells was determined by CD4 + ΔNGFR - T cells and CD8 + ΔNGFR - After gating on T cells, eFluor® 670 dilution was determined using flow cytometry. Figure 11A shows CD4+ T cells from donors C, E, and F. IL-10 Multiple donor CD4 containing cells IL-10 Figure 11B shows the results of cells from donors H, I, and L that were frozen, stored, and thawed before testing. IL-10 Multiple donor CD4 containing cells IL-10The results for cells are shown.

[0212] Figure 11A shows a multi-donor CD4 IL-10 Cells (from three different donors) were CD4 + and CD8 + A second, different batch of multi-donor CD4 T cells was tested after freezing, storing, and thawing the cells prior to testing. IL-10 Comparable results were obtained using CD4 cells (Fig. 11B). + and CD8 + The suppression of T cell proliferation was 68% and 75%, respectively. These data are consistent with the multi-donor CD4 IL-10 This demonstrates that cells can be frozen and preserved without loss of function.

[0213] In summary, multiple donor CD4 IL10 Data obtained with cells indicate that these cell preparations can be pooled successfully. They contain >95% viable cells and are single-donor CD4 IL-10 Maintains all relevant functions of the cells (cytokine production, cytotoxicity, and suppression of allogeneic T cell responses). IL-10 The use of larger pools of cells allows for the identification of CD4 IL-10 This should reduce the natural variability observed between cell lots and allow for the production of large quantities of uniform CD4 IL-10 The cells should be provided.

[0214] Multiple donor CD4 IL-10 Cellular products have the significant advantage of being more homogeneous products, which will allow for the determination of well-defined potency and release criteria with less lot-to-lot variability, and will also enable the development of continuous large-scale cell manufacturing processes.

[0215] Multiple donor CD4 IL-10 Other methods of producing cells Pool buffy coats from a minimum of 3–5 different donors prior to lentiviral transduction. + Cells are isolated from the buffy coat by positive selection using anti-CD4 antibodies. + Confirm cell purity by FACS. Alternatively, use frozen human CD4 + Cells are obtained from a minimum of 3-5 normal healthy donors. Frozen human CD4 + Thaw cells before use. CD4 cells from buffy coat or frozen stocks + Cells are activated with a combination of CD3 and CD28 antibodies or beads coated with CD3 and CD28 antibodies in the presence of IL-2 for 24-48 hours. Optionally, CD4 antibodies from buffy coats or frozen stocks are used. + The cells were activated with soluble anti-CD3, soluble anti-CD28 mAb and rhIL-2 (50 U / mL) for 48 hours, and CD4 IL-10 For the production of cells, transduce with the bidirectional lentiviral vector encoding human IL-10 as described above.

[0216] In some cases, the T cell donor (or CD4 + First, the HLA haplotype of the CD4 + Cells are selectively pooled for use.

[0217] Multiple donor CD4 IL-10 The cells were activated with CD4 + It is generated by transducing cells with a lentiviral vector containing the human IL-10 and ΔNGFR coding sequences described above.

[0218] On days 7-11, 5-9 days after transduction, cells were harvested and successfully transduced T cells were purified using anti-NGFR antibodies. This process typically yields 95% pure multi-donor CD4 IL-10 resulting in a cell population.

[0219] Purified multi-donor CD4 IL-10Cells are counted and restimulated for another 8-10 days with a mixture of CD3 and CD28 antibodies, beads coated with CD3 and CD28 antibodies, in the presence of IL-2, and optionally in the presence of feeder cells. IL-10 The cells are restimulated in the presence of feeder cells.

[0220] After a total culture period of 14–18 days, CD4 IL-10 Cells are harvested, counted, and tested for their ability to spontaneously produce IL-10 or their ability to produce IL-10 after activation with CD3 and CD28 antibodies or beads coated with CD3 and CD28 antibodies. In addition, levels of GrzB and perforin are measured. Human T cells (PBMCs) and purified CD4 + and CD8 + Their ability to suppress T cell proliferation is also tested.

[0221] In addition, IL-22 production was observed constitutively and in a 200 microliter volume of 200,000 CD4 cells using a combination of CD3 and CD28 antibodies, as previously described for the production of other cytokines, e.g., IFNγ, IL-10, IL-4, and IL-5. IL-10 The levels of IL-22 produced are measured after cell activation using an IL-22-specific ELISA, as described for other cytokines in WO 2016 / 146542. Prior to storage, pooled CD4 IL-10 Freeze the cells.

[0222] 6.8.2. Example 2: Multiple Donor CD4 IL-10 Treatment or prevention of GvHD using cells In vivo multiple donor CD4 IL-10 Cellular effects Multiple donor CD4 IL-10The cell populations were tested for their effect on GvHD induced by human PBMCs in the NSG mouse model, a humanized xeno-GvHD disease model, as shown in Figure 12. NSG mice were sublethally irradiated and injected with human PBMCs (5x10 6 cells / mouse), multiple donor (3 donors) CD4 IL-10 cells (5×10 6 cells / mouse), or multiple donor CD4 IL-10 Cells (3 donors) (5 × 10 6 cells / mouse) combined with human PBMCs (5 × 10 6 GvHD was assessed based on weight loss (>20% weight loss), skin lesions, coat condition, activity, and posture as previously reported (Bondanza et al. Blood 2006).

[0223] Figure 13 shows the % of NSG mice showing GvHD on each day after injection. 6 Administration of 5 × 10 human PBMCs unexpectedly resulted in unusually excitable GvHD. All mice died by day 10, reflecting highly lethal GvHD. 6 Multiple donor CD4 IL-10 Although co-administration of cells delayed this fulminant form of GvHD, mice were sacrificed on day 14 after reaching the pre-specified human 20% weight loss criterion for sacrifice (Figure 13). However, these results are consistent with the results of multiple-donor CD4 IL-10 Importantly, multi-donor CD4+ PBMCs administered alone at the same dose as PBMCs have been shown to delay very severe GvHD. IL-10 cells (5×10 6 cells) did not induce any signs of GvHD.

[0224] In addition, human PBMCs (5 × 10 6 cells / mouse), multiple donor (3 donors) CD4 IL-10 cells (5×10 6 cells / mouse) or human PBMCs (5 × 10 6cells / mouse) multiple donor CD4 IL-10 Cells (3 donors) (5 × 10 6 Human CD4 in the spleen (FIG. 14, left panel) and bone marrow (FIG. 14, right panel) of NSG mice injected with a combination of IgG1 and IgG2-associated IgG1 (IgG1-associated IgG1) ... cells / mouse. IL-10 The presence of cells was examined 14 days after injection. The results shown in Figure 14 showed that multiple donor CD4 IL-10 These results indicate that the cells migrated to the spleen and bone marrow. 14 days after cell injection, these cells were found to be present in low proportions. These results support the conclusion that multi-donor CD4 IL-10 These results demonstrate that the cells delay the fulminant GvHD induced by human PBMCs and do not themselves induce any xenogeneic GvHD.

[0225] Multiple donor CD4 IL-10 Cells were purified CD4 + Inhibition of severe xenogeneic GvHD by cells. As shown in Figure 15, 2.5 x 10 6 In a humanized xenogeneic GvHD model in which GvHD disease was induced by the administration of purified human CD4+ T cells, multiple donor CD4 IL-10 NSG mice were sublethally irradiated on day 0 and human CD4 + T cells (2.5×10 6 cells / mouse) single or multiple donor CD4 IL-10 Cells (three different donors) (2.5 × 10 6 CD4 from a single donor in combination with or from a pool (cells / mouse) IL-10 cells (2.5×10 6 GvHD was assessed based on weight loss (>20% weight loss), skin lesions, coat condition, activity, and posture as previously reported (Bondanza et al. Blood 2006).

[0226] Figure 16 shows the % of NSG mice showing GvHD on each day after injection. These results are consistent with multi-donor CD4 IL-10 Cells are human allogeneic CD4 +This indicates that T cell-mediated GvHD can be inhibited. In this particular experiment, the heterologous GvHD was very severe, as all mice in the control group that received CD4+ T cells died by day 20. In contrast, 2.5 × 10 6 Multiple donor CD4 IL-10 Coadministration of single donor CD4 inhibited GvHD by 75%. IL-10 The cells were also protective, but the effect was not as strong.

[0227] Other experiments Multiple donor CD4 IL-10 The therapeutic effect of the cells was evaluated in four different groups of mice: (i) CD4 IL-10 (ii) mice receiving human PBMCs from a donor unrelated to the cells (GvHD positive control); (iii) mice receiving multiple donor CD4 IL-10 mice receiving cells (negative control); (iii) PBMC and multiple donor CD4 IL-10 Mice receiving the cell combination in a 1:1 ratio, and (iv) PBMC and multiple donor CD4 IL-10 Cell combinations are tested in mice receiving PBMC and multi-donor CD4 IL-10 Among animals receiving cell combinations, some animals received PBMC and multi-donor CD4 IL-10 The animals were simultaneously administered with PBMCs and some animals developed multiple-donor CD4 IL-10 Some animals received multiple donor CD4 IL-10 The cells are administered.

[0228] PBMC and / or multiple donor CD4 IL-10 After cell administration, mice are monitored for the development of GvHD by measuring body weight at weeks 1, 2, 3, 4, and optionally at week 5. In addition to weight loss, mice are examined for skin lesions, coat condition, and activity. Mice in treatment groups are monitored over an extended period to assess the efficacy of multi-donor CD4 T cells for long-term survival.IL-10 Examine the effects on cells.

[0229] Multiple donor CD4 IL-10 The quantity and localization of cells will also be monitored in peripheral blood and tissues after administration. Specifically, multi-donor CD4 cells in peripheral blood and at sites of inflammation, i.e., lymph nodes, spleen, intestine, and bone marrow. IL-10 The presence of cells is monitored. Mice in the treatment group are monitored for an additional 3 weeks to ensure long-term survival.

[0230] These results are consistent with multiple donor CD4 IL-10 These results demonstrate that the cells are effective in reducing and preventing xenogeneic GvHD.

[0231] 6.8.3. Example 3: Inhibition of GvHD and Treatment of Cancer Multiple donor CD4 IL-10 The cell populations will be tested for their efficacy against xenogeneic GvHD induced by human PBMCs and anti-tumor effects in an NSG mouse model implanted with human PBMCs and AML tumor cells. AML cells (ALL-CM) will be administered intravenously as previously described in WO 2016 / 146542. PBMCs or multi-donor CD4 IL-10 The cells or a combination thereof are administered three days later.

[0232] As described in Example 1, multiple donor CD4 IL-10 Obtain cells from multiple donor CD4 IL-10 The therapeutic effect of cells was evaluated by irradiating 5 × 10 cells on day 0. 6 Four different groups of mice (AML mice) with ALL-CM cells were treated: (i) AML mice without further treatment; (ii) multiple donor CD4 IL-10 Cells were derived from unrelated donors and consisted of 5 × 10 6 AML mice administered 2.5 × 10 human PBMCs - PBMCs induce severe xenogeneic GvHD, (iii) 2.5 × 10 6 Multiple donor CD4 IL-10 AML mice administered cells, and (iv) PBMCs and multiple donor CD4IL-10 Combinations with ALL-CML cells will be tested in AML mice treated with ALL-CML cells at a 1:1 or 2:1 ratio, or at different ratios. One additional group of mice will not receive ALL-CML cells, but will receive 5x10 cells on day 3 post-irradiation. 6 The mice are administered human PBMCs.

[0233] Multi-donor CD4 for xenogeneic GvHD induced by human PBMC IL-10 The efficacy of the cells will be tested based on weight loss, skin lesions, coat condition, viability, mortality and long-term survival. IL-10 The anti-tumor or graft-versus-leukemia (GvL) effect of the cells is tested based on the reduction of circulating tumor cells and long-term tumor-free survival.

[0234] Some mice are monitored for up to 7 weeks to monitor for long-term survival and complete tumor remission.

[0235] The results were from multiple donor CD4 IL-10 The cells have been shown to be effective in both inhibiting xenogeneic GvHD and treating cancer.

[0236] 6.8.4. Example 4: Multiple Donor CD4 IL-10 Cell-based cancer treatment Multiple donor CD4 IL-10 The population of cells will be tested in an ALL-CM leukemia model of T cell therapy in NSG mice.

[0237] On day 0, NSG mice were sublethally irradiated and injected with myeloid leukemia cells (ALL-CM) (5 × 10 6 In the first group of animals, PBMCs (5 × 10 6 CD4 counts of 100 individuals or from a single donor (donors BC-I and BC-H) IL-10 cells (2.5×10 6 ) were injected on day 3. In the second group of animals, PBMCs (5 × 10 6 ) or multiple donor CD4 IL-10 cells (2.5×10 6The mice were injected with 100 mg of 10 ...

[0238] As shown in Figures 17A and 17B, all of the mice injected with ALL-CM myeloid leukemia cells had extensive leukemia progression by day 17. 6 Administration of PBMCs resulted in potent inhibition of leukemia progression. Interestingly, a similar level of inhibition of leukemia progression was observed with a lower number (2.5 x 10 6 (units) single donor CD4 IL10 (Figure 17A) or multiple donor CD4 IL10 These data were obtained using single-donor and multiple-donor CD4 IL10 These results indicate that IL-16 has a strong direct anti-leukemia effect.

[0239] Single donor CD4 IL10 and multiple donor CD4 IL10 The graft-versus-leukemia (GvL) effect of was further tested in combination with PBMCs in mice injected with ALL-CM myeloid leukemia cells. 6 Administration of PBMCs resulted in potent inhibition of leukemia progression, and single-donor CD4 IL10 (2.5×10 6 5×10 6 Administration of 2.5 x 10 PBMCs had a synergistic effect (Figure 18A). 6 Multiple donor CD4 IL10 5×10 combined with 6 Administration of multiple PBMCs had a comparable synergistic GvL effect (Figure 18B). These data support the results of multiple-donor CD4 IL10 These results demonstrate that PBMCs synergize with each other to mediate potent GvL effects.

[0240] 6.8.5. Example 5: Multiple Donor CD4 IL-10Cell-based treatment of chronic inflammatory and autoimmune diseases

[0241] NLPR3 inflammasome activation is involved in many chronic inflammatory and autoimmune diseases. NLPR3 inflammasome can be activated by "danger signals" leading to caspase-1-mediated production of the pro-inflammatory cytokines IL-1β and IL-18 by monocytes / macrophages. Multiple-donor CD4 receptors for NLPR3 inflammasome and IL-1β / IL-18 production by human monocytes. IL-10 A series of in vitro experiments are performed to examine the effects on cells.

[0242] First, human PBMCs are isolated from peripheral blood by standard density centrifugation using Ficoll / Paque (Sigma-Aldrich). Monocytes are isolated from human PBMCs by negative selection using the Monocyte Isolation Kit II (Miltenyi) according to the manufacturer's instructions. Negative selection is preferred because positive selection or adhesion can result in unwanted activation of the cells. 2 x 10 cells per 200 μL of monocytes are cultured in a 96-well microtiter plate in culture medium containing 3% toxin-free human AB serum. 5 1 x 10 5 Multiple donor CD4 IL-10 Where cells are present, isolated monocytes are added at a concentration of 5 x 10 4 Seed cells / 200 μl.

[0243] Table 1 summarizes the treatment conditions applied to 17 sets of monocytes, each set containing 6 wells of cells. It is known that LPS alone can activate human monocytes without a second signal provided by ATP. [Table 1]

[0244] After treatment as outlined in Table 1, supernatants are collected from six wells per group and IL-1β / IL-18 production is measured by ELISA specific for mature IL-1β or IL-18 (Biolegend). Cells collected from six wells for groups 3, 10, 13, 14, and 17 are analyzed by Western blot to examine levels of activated caspase 1.

[0245] Data from the experiment showed that multiple donor CD4 IL-10 We further demonstrate that multi-donor CD4 IL-10 We show that multiple donor CD4 cells downregulate mature caspase-1 production in activated monocytes. IL-10 and multiple donor CD4 IL-10 IL-10 produced by IL-10 downregulates inflammasomes.

[0246] Similar experiments were performed using human macrophages or dendritic cells instead of monocytes. The results of the experiments were compared between multiple donor CD4 IL-10 These results demonstrate that IL-1β, IL-18, and mature caspase-1 production from activated macrophages and dendritic cells are further downregulated.

[0247] These results are consistent with multiple donor CD4 IL-10 These results suggest that multi-donor CD4 cells may be used to treat diseases or disorders associated with overactivation of the NLPR3 inflammasome. IL-10 The cells can be used to treat chronic inflammatory and autoimmune diseases. The NLPR3 inflammasome can be activated by exogenous or endogenous "danger signals," such as pathogen-associated molecular patterns (PAMPs), silica, asbestos, damaged mitochondria, danger-associated molecular pattern (DAMP)-like products from necrotic and stressed cells, and uremic acid crystals.

[0248] 6.8.6. Experimental methods and materials Cell preparations and cell lines Peripheral blood mononuclear cells (PBMCs) were prepared by centrifugation on a Ficoll-Hypaque gradient. + T cells were purified with a CD4 T cell isolation kit (Miltenyi Biotec, Bergisch Gladbach, Germany) to a purity of >95%. Mature dendritic cells (DCs) were isolated from CD14 + Peripheral blood CD14 was positively selected using MicroBeads (Miltenyi Biotech, Germany) according to the manufacturer's instructions. + They were generated from monocytes and cultured in RPMI 1640 (Lonza, Italy) supplemented with 10% fetal bovine serum (FBS, Lonza, Italy), 100 U / mL penicillin / streptomycin (Lonza, Italy), and 2 mM L-glutamine (Lonza, Italy) in the presence of 10 ng / mL recombinant human (rh)IL-4 (R&D Systems, Minneapolis, MN, USA) and 100 ng / mL rhGM-CSF (Genzyme, Seattle, WA) at 37°C for 5 days, and then matured with 1 mg / mL lipopolysaccharide (LPS, Sigma, CA, USA) for an additional 2 days.

[0249] Plasmid Construction: The coding sequence for human IL-10 was excised from pH15C (ATCC no. 68192), and a 549 bp fragment was cloned into the multiple cloning site of pBluKSM (Invitrogen) to obtain pBluKSM-hIL-10. hIL-10 was excised from pBluKSM-hIL-10 and ligated into 1074.1071.hPGK.GFP.WPRE.mhCMV.dNGFR.SV40PA (referred to here as LV-ΔNGFR) to obtain a 555 bp fragment, LV-IL-10 / ΔNGFR. The presence of a bidirectional promoter (human PGK promoter plus minimal core elements of the CMV promoter in opposite orientations) allows for simultaneous expression of two transgenes (Locafaro et al. Mol Ther. 2017;25(10):2254-2269). The sequence of LV-IL-10 / ΔNGFR was verified by pyrosequencing (Primm).

[0250] Vector Production and Titration: VSV-G-pseudotyped third-generation bidirectional lentiviral vectors were produced by Ca3PO4 transient four-plasmid cotransfection into 293T cells and concentrated by ultracentrifugation as previously described (Locafaro et al. Mol Ther. 2017;25(10):2254-2269). Titers were estimated by limiting dilution, and vector particles were measured by HIV-1 Gag p24 antigen immunocapture (NEN Life Science Product, Waltham, MA). Vector infectious titers were calculated as the ratio of titer to particles. Titers were 5 × 10 8 From 6 x 10 9 Transducing units / mL range, infectious titer is 5 x 10 4 From 10 5 The transducing units / ng of p24 were in the range.

[0251] CD4 IL-10 Polyclonal CD4 transduced cells were obtained as previously described (Andolfi et al. Mol Ther. 2012;20(9):1778-1790). Briefly, CD4 purified T cells were activated with soluble anti-CD3 monoclonal antibody (mAb, 30 ng / mL, OKT3, Janssen-Cilag, Raritan, NJ, USA), anti-CD28 mAb (1 μg / mL, BD), and rhIL-2 (50 U / mL, PROLEUKIN, Novartis, Italy) for 48 h. T cells were transfected with LV-IL-10 / ΔNGFR (CD4 IL-10 On day 11, CD271 + CD4 microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany) were used. + ΔNGFR +Cells were bead-sorted and expanded in X-VIVO15 medium containing 5% human serum (BioWhittaker-Lonza, Washington), 100 U / mL penicillin-streptomycin (BioWhittaker), and 50 U / mL rhIL-2 (PROLEUKIN, Novartis, Italy). On days 7 and 10, the medium was replaced with fresh medium supplemented with 50 U / mL rhIL-2. On day 14, cells were harvested, washed, and restimulated with an allogeneic feeder mixture as previously described (Andolfi et al. Mol Ther. 2012;20(9):1778-1790). After 14 days, cells were harvested and frozen. Thawed CD4 IL-10 Cells were restimulated and after a second and third round of restimulation and expansion, were functionally characterized in vitro and used for in vivo experiments.

[0252] Vector copy number analysis: Cells were cultured for 11 days post-transduction to remove non-integrated vector forms. Genomic DNA was isolated using the QIAamp DNA Blood Mini Kit (QIAgen, 51106) according to the manufacturer's instructions. Vector integration was quantified using a QX200 Droplet Digital PCR System (Bio-Rad) according to the manufacturer's instructions.

[0253] Cytokine Measurements: Single-donor and multiple-donor CD4 counts were measured after the second and third restimulations to measure cytokine production. IL-10 Cells were either left unstimulated or plated in a final volume of 200 μl of medium (96-well round-bottom plate, 2 × 10 5 Cells were stimulated with immobilized anti-CD3 (10 μg / mL) and soluble anti-CD28 (1 μg / mL) mAbs in 100 μL / well. After 48 h of culture, supernatants were collected and the levels of IL-10, IL-4, IL-5, IFN-γ, and IL-22 were measured by ELISA according to the manufacturer's instructions (BD Biosciences).

[0254] Flow cytometry analysis was performed for the expression of granzyme B (clone MHGB04, Invitrogen, USA) after surface staining with CD4 IL-10 Cells were fixed, permeabilized, and stained using the BD Cytofix / Cytoperm™ kit according to the manufacturer's instructions (Cat. No. 554714, Biolegend, USA). Stained cells were washed twice with PBS supplemented with 1% FBS and analyzed on a BD LSRFortessa using FlowJo10 software.

[0255] Killing assay: Single-donor and multiple-donor CD4 counts after second and third restimulations. IL-10 The cytotoxicity of the cells was analyzed in co-culture experiments. Briefly, non-myeloid leukemia and myeloid leukemia cell lines, K562 and ALL-CM, respectively, were used as target cells, and CD4 IL-10 Cells were cultured at a 1:1 ratio (10 5 target cells and 10 5 CD4 IL-10 At the end of the co-culture, cells were harvested and K562 and ALL-CM cells were analyzed and counted by FACS.

[0256] Suppression assays single-donor and multi-donor CD4 IL-10 To measure the suppressive capacity of the cells, allogeneic PBMCs were labeled with Cell Proliferation Dye eFluor® 670 (Invitrogen, CA, USA) according to the manufacturer's instructions, followed by the addition of allogeneic mature DCs (5 × 10 4 PBMCs and suppressor cells were stimulated with 1:1 ratio (10 cells / well) and soluble anti-CD3 (50 ng / mL) mAb. 5 PBMCs and 10 5 CD4 IL-10 After 3 days of culture, CD4 + ΔNGFR - or CD8 + ΔNGFR -Responder cell proliferation was determined by analyzing eFluor670 dilutions of T cells by FACS.

[0257] Graft-versus-host disease model: In all experiments, 6 / 8-week-old female NSG mice were used. On day 0, mice received a single dose of total body irradiation from a linear accelerator at 175–200 cGy depending on the mouse weight, and then were inoculated with PBMC cells (5 × 10 6 ), or CD4 IL-10 Cells (single donor or multiple donor (pool of 3 donors) 5 x 10 6 or 2.5 x 10 6 ), or CD4 IL-10 cells (5×10 6 or 2.5 x 10 6 PBMCs (5 × 10 6 Mice were intravenously administered 100 mg of ...

[0258] Alternatively, on day 0, mice received total body irradiation as described above. On day 3, CD4 + T cells (2.5×10 6 individuals), single and multiple donor (pool of 3 donors) CD4 IL-10 cells (2.5×10 6 ), or CD4 + T cells (2.5×10 6 (individuals) single and multiple donor (pool of 3 donors) CD4 IL-10 cells (2.5×10 6 Mice were injected with a combination of IgG1 and IgG2a. GvHD induction was monitored as described above.

[0259] 7. Incorporation by Reference All publications, patents, patent applications, and other documents cited in this application are herein incorporated by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application, or other document was individually indicated to be incorporated by reference for all purposes. The present invention may include the following aspects. [1] CD4 that has been genetically modified to contain an exogenous polynucleotide encoding IL-10 + a population of T cells, the CD4 + T cells were obtained from at least three different T cell donors, CD4 + T cell population (multiple donor CD4 IL-10 cell). [2] CD4 + 2. The CD4 T cells of claim 1, wherein the T cells are obtained from 3, 4, 5, 6, 7, 8, 9, or 10 different T cell donors. + A population of T cells. [3] The CD4 + 3. The CD4 T cells of claim 1 or claim 2, wherein the T cells have a total of 6, 7, 8, 9, 10, 11, 12 or more different HLA haplotypes. + A population of T cells. [4] All of the CD4 + 4. The CD4 T cells of claim 1, wherein the T cells have at least 5 / 10, 6 / 10, 7 / 10, 8 / 10, or 9 / 10 matches with each other at the HLA-A, HLA-B, HLA-C, HLA-DRB1, and HLA-DQB1 loci. + A population of T cells. [5] All of the CD4 + 5. The CD4 T cell of claim 1, wherein the T cells have at least 4 / 8, 5 / 8, 6 / 8, 7 / 8, or 8 / 8 matches with each other at the HLA-A, HLA-B, HLA-C, and HLA-DRB1 loci. + A population of T cells. [6] All of the CD4 + 6. The CD4 T cells of any one of claims 1 to 5, wherein the T cells have a 2 / 2 match with each other at the HLA-A locus. + A population of T cells. [7] All of the CD4 + 7. The CD4 T cells of claim 1, wherein the T cells have a 2 / 2 match with each other at the HLA-B locus. + A population of T cells. [8] All of the CD4+ 8. The CD4 T cells of any one of claims 1 to 7, wherein the T cells have a 2 / 2 match with each other at the HLA-C locus. + A population of T cells. [9] All of the CD4 + 9. The CD4 T cells of claim 1, wherein the T cells have at least a 3 / 4 or 4 / 4 match with each other at the HLA-DRB1 and HLA-DQB1 loci. + A population of T cells.

[10] All CD4 + T cells are A * 10. The CD4 according to any one of claims 1 to 9, having the 02 allele. + A population of T cells.

[11] Any CD4 + The CD4 T cells of any one of claims 1 to 10 are also not immortalized. + A population of T cells.

[12] The CD4 of any one of claims 1 to 11, wherein the exogenous polynucleotide comprises a polynucleotide segment encoding IL-10 operably linked to an expression control element. + A population of T cells.

[13] 13. The CD4 of claim 1, wherein the IL-10 is human IL-10. + A population of T cells.

[14] 14. The CD4 immunoglobulin of claim 1, wherein the IL-10 is a viral IL-10. + A population of T cells.

[15] The CD4 according to any one of claims 1 to 14, wherein the polynucleotide segment encoding IL-10 encodes a protein having the sequence of SEQ ID NO: 1. + A population of T cells.

[16] 16. The CD4 of claim 15, wherein the polynucleotide segment encoding IL-10 has the sequence of SEQ ID NO: 2. + A population of T cells.

[17] 17. The CD4 vector of any one of claims 12 to 16, wherein the expression control element drives constitutive expression of the encoded IL-10. + A population of T cells.

[18] 18. The CD4 gene of any one of claims 1 to 17, wherein the exogenous polynucleotide further comprises a sequence encoding a selectable marker. + A population of T cells.

[19] 19. The CD4 of claim 18, wherein the selection marker is ΔNGFR. + A population of T cells.

[20] 20. The CD4 of claim 19, wherein the ΔNGFR has the sequence of SEQ ID NO: 3. + A population of T cells.

[21] 20. The CD4 of claim 19, wherein the exogenous polynucleotide comprises the sequence of SEQ ID NO: 4. + A population of T cells.

[22] 19. The CD4 of claim 18, wherein the selection marker is a truncated EGFR polypeptide. + A population of T cells.

[23] 23. The CD4 antibody of any one of claims 1 to 22, wherein the exogenous polynucleotide has the sequence of SEQ ID NO: 5. + A population of T cells.

[24] 24. The CD4+ T cell of claim 1, wherein the exogenous polynucleotide is integrated into the T cell nuclear genome. + A population of T cells.

[25] 24. The CD4+ antibody of any one of claims 1 to 23, wherein the exogenous polynucleotide is not integrated into the T cell nuclear genome. + A population of T cells.

[26] 26. The CD4 of claim 24 or 25, wherein the exogenous polynucleotide further comprises a lentiviral vector sequence. + A population of T cells.

[27] 27. The CD4+ antibody of any one of claims 1 to 26, wherein the exogenous polynucleotide is not integrated into the T cell nuclear genome. + A population of T cells.

[28] The CD4 + 28. The CD4 T cell of any one of claims 1 to 27, wherein at least 90% of the T cells express IL-10. + A population of T cells.

[29] The CD4 + 29. The CD4 T cell of claim 28, wherein at least 95% of the T cells express IL-10. + A population of T cells.

[30] The CD4 + 30. The CD4 T cell of claim 29, wherein at least 98% of the T cells express IL-10. + A population of T cells.

[31] The genetically modified CD4+ T cells are CD4 + T cells 10 6 31. The CD4+ antibody of claim 1, which constitutively expresses at least 100 pg of IL-10 per cell / ml of culture medium. + A population of T cells.

[32] The genetically modified CD4 + T cells are CD4 + T cells 10 6 32. The CD4 antibody of claim 31, which constitutively expresses at least 100 pg, 200 pg, 500 pg, 1 ng, 5 ng, 10 ng, or 50 ng of IL-10 per cell / ml. + A population of T cells.

[33] The genetically modified CD4 + T cells express CD4 after activation with anti-CD3 and anti-CD28 antibodies. + T cells 10 6 33. The CD4 of any one of claims 1 to 32, which expresses at least 1 ng of IL-10 per cell / ml. + A population of T cells.

[34] The genetically modified CD4 + T cells express CD4 after activation with anti-CD3 and anti-CD28 antibodies. + T cells 10 6 34. The CD4 of claim 33, which expresses at least 2 ng, 5 ng, 10 ng, 100 ng, 200 ng, or 500 ng of IL-10 per cell / ml. + A population of T cells.

[35] The genetically modified CD4 + T cells are non-modified CD4 + 35. The CD4+ T cell of claim 1, wherein the CD4+ T cell expresses IL-10 at a level at least 5 times higher than T cells. + A population of T cells.

[36] The genetically modified CD4 + T cells are non-modified CD4 + 36. The CD4+ T cells of claim 35, which express IL-10 at a level at least 10 times higher than T cells. + A population of T cells.

[37] The CD4 + 37. The CD4 T cell of any one of claims 1 to 36, wherein at least 90% of the T cells express the selection marker from the exogenous polynucleotide. + A population of T cells.

[38] The CD4 + 38. The CD4 T cell of claim 37, wherein at least 95% of the T cells express the selectable marker from the exogenous polynucleotide. + A population of T cells.

[39] The CD4 + 39. The CD4 T cell of claim 38, wherein at least 98% of the T cells express the selectable marker from the exogenous polynucleotide. + A population of T cells.

[40] The genetically modified CD4 + 40. The CD4 T cell of any one of claims 1 to 39, wherein the T cell expresses CD49b. + A population of T cells.

[41] The genetically modified CD4 + 41. The CD4 T cell of claim 1, wherein the T cell expresses LAG-3. + A population of T cells.

[42] The genetically modified CD4 + 42. The CD4 T cell of claim 1, wherein the T cell expresses TGF-β. + A population of T cells.

[43] Genetically modified CD4 + 43. The CD4 T cell of any one of claims 1 to 42, wherein the T cell expresses IFNγ. + A population of T cells.

[44] The genetically modified CD4 + 44. The CD4 T cell of any one of claims 1 to 43, wherein the T cell expresses GzB. + A population of T cells.

[45] The genetically modified CD4 + 45. The CD4 T cell of any one of claims 1 to 44, wherein the T cell expresses perforin. + A population of T cells.

[46] The genetically modified CD4 + 46. ​​The CD4 T cell of any one of claims 1 to 45, wherein the T cell expresses CD18. + A population of T cells.

[47] The genetically modified CD4 + 47. The CD4 T cell of claim 1, wherein the T cell expresses CD2. + A population of T cells.

[48] The genetically modified CD4 + 48. The CD4 T cell of any one of claims 1 to 47, wherein the T cell expresses CD226. + A population of T cells.

[49] The genetically modified CD4 + 49. The CD4 T cell of any one of claims 1 to 48, wherein the T cell expresses IL-22. + A population of T cells.

[50] CD4 + 50. The CD4 T cell of any one of claims 1 to 49, wherein the T cell is not anergized in the presence of peripheral blood mononuclear cells (PBMCs) from the host. + A population of T cells.

[51] CD4 + T cells are not anergized in the presence of recombinant IL-10 protein, and said recombinant IL-10 protein inhibits said CD4 + 51. The CD4 of any one of claims 1 to 50, which is not expressed by T cells. + A population of T cells.

[52] CD4 + 52. The CD4 T cell of any one of claims 1 to 51, wherein the T cell is not anergized in the presence of host-derived DC10 cells. + A population of T cells.

[53] CD4 + 53. The CD4 T cell of any one of claims 1 to 52, wherein the T cell is in a frozen suspension. + A population of T cells.

[54] CD4 + 53. The CD4 T cell of any one of claims 1 to 52, wherein the T cell is in liquid suspension. + A population of T cells.

[55] 55. The CD4 of claim 54, wherein the liquid suspension is pre-frozen. + A population of T cells.

[56] (i) a CD4 antibody according to any one of claims 1 to 55 + A population of T cells, (ii) suspended in a pharmaceutically acceptable carrier A pharmaceutical composition comprising the population.

[57] Multiple donor CD4 IL-10 1. A method of producing a cell, comprising: (i) Primary CD4 T cells obtained from at least three different T cell donors + pooling the T cells; (ii) introducing an exogenous polynucleotide encoding IL-10 into the pooled CD4 + modifying the T cells; Thereby, the multiple donor CD4 IL-10 Methods for obtaining cells.

[58] Multiple donor CD4 IL-10 1. A method of producing a cell, comprising: (i) Primary CD4 T cells from at least three different T cell donors + Obtaining T cells; (ii) Inducing CD4+ IL-10 expression in each donor by introducing an exogenous polynucleotide encoding IL-10 + separately modifying the T cells; and then (iii) the genetically modified CD4 + T cells are pooled, thereby IL-10 and obtaining the cells.

[59] after step (i) and before step (ii), or after step (ii), or after step (ii) and before step (iii), or after step (iii); The primary CD4 + 59. The method of claim 57 or claim 58, further comprising incubating the T cells in the presence of beads coated with anti-CD3 and anti-CD28 antibodies, or anti-CD3 and CD28 antibodies.

[60] The primary CD4 + 60. The method of claim 59, wherein the T cells are further incubated in the presence of IL-2.

[61] The exogenous polynucleotide is delivered to the primary CD4 + 61. The method of any one of claims 57 to 60, wherein the method is introduced into a T cell.

[62] 62. The method of claim 61, wherein the viral vector is a lentiviral vector.

[63] 63. The method of any one of claims 57 to 62, wherein the exogenous polynucleotide comprises a segment encoding IL-10 having the sequence of SEQ ID NO: 1.

[64] 59. The method of any one of claims 53 to 58, wherein the polynucleotide segment encoding IL-10 has the sequence of SEQ ID NO:2.

[65] 65. The method of any one of Claims 57 to 64, wherein the exogenous polynucleotide further comprises a segment encoding a selectable marker.

[66] 66. The method of claim 65, wherein the encoded selectable marker is ΔNGFR.

[67] 67. The method of claim 66, wherein the encoded selectable marker has the sequence of SEQ ID NO:3.

[68] After step (ii), the genetically modified CD4 + Isolating T cells and thereby genetically modifying CD4 + 68. The method of any one of Claims 65 to 67, further comprising generating an enriched population of T cells.

[69] the genetically modified CD4 + 68. The method of claim 67, wherein at least 90% or at least 95% of the T cells express IL-10.

[70] the genetically modified CD4 + 69. The method of claim 68, wherein at least 98% of the T cells express IL-10.

[71] the genetically modified CD4 + 70. The method of any one of claims 68 to 69, wherein at least 90% or at least 95% of T cells express the selection marker.

[72] the genetically modified CD4 + 71. The method of claim 70, wherein at least 98% of the T cells express the selectable marker.

[73] The genetically modified CD4 + 73. The method of any one of Claims 68 to 72, further comprising incubating the enriched population of T cells.

[74] The genetically modified CD4 + 74. The method of claim 73, wherein the step of incubating the enriched population of T cells is carried out in the presence of IL-2 and in the presence of beads coated with anti-CD3 and anti-CD28 antibodies, or CD3 and CD28 antibodies.

[75] The genetically modified CD4 + 75. The method of any one of Claims 57 to 74, further comprising the subsequent step of freezing the T cells.

[76] In step (i), the primary CD4 + 76. The method of any one of claims 57 to 75, wherein the T cells are obtained from 3, 4, 5, 6, 7, 8, 9 or 10 different T cell donors.

[77] 77. The method of any one of claims 76, wherein the at least three T cell donors have at least a 5 / 10, 6 / 10, 7 / 10, 8 / 10, or 9 / 10 match with each other at the HLA-A, HLA-B, HLA-C, HLA-DRB1, and HLA-DQB1 loci.

[78] 78. The method of any one of claims 57 to 77, wherein the at least three T cell donors have at least a 4 / 8, 5 / 8, 6 / 8, 7 / 8 or 8 / 8 match with each other at the HLA-A, HLA-B, HLA-C and HLA-DRB1 loci.

[79] 79. The method of any one of claims 57 to 78, wherein the at least three T cell donors have a 2 / 2 match with each other at the HLA-A locus.

[80] 80. The method of any one of claims 57 to 79, wherein the at least three T cell donors have a 2 / 2 match with each other at the HLA-B locus.

[81] 81. The method of any one of claims 57 to 80, wherein the at least three T cell donors have a 2 / 2 match with each other at the HLA-C locus.

[82] 82. The method of any one of claims 57 to 81, wherein the at least three T cell donors have at least a 3 / 4 or 4 / 4 match with each other at the HLA-DRB1 and HLA-DQB1 loci.

[83] Each of the at least three T cell donors is A * 83. The method of any one of claims 57 to 82, wherein the genotype has the 02 allele.

[84] In step (i), the primary CD4 + 84. The method of any one of claims 57 to 83, wherein the T cells are obtained from one or more frozen stocks.

[85] In step (i), the primary CD4 + 84. The method of any one of claims 57 to 83, wherein the T cells are obtained from non-frozen peripheral blood mononuclear cells of said at least three different T cell donors.

[86] 1. CD4 + 86. The method of Claim 85, further comprising isolating the T cells.

[87] 1. A method of treating a patient, comprising: 56. The multi-donor CD4 IL-10 57. A method comprising administering a cell or the pharmaceutical composition of claim 56 to a patient in need of immune tolerization.

[88] Multiple donor CD4 IL-10 88. The method of claim 87, further comprising the preceding step of thawing the frozen suspension of cells.

[89] The multiple donor CD4 IL-10 89. The method of claim 87 or 88, wherein the cells or said pharmaceutical composition prevent or reduce the severity of a pathogenic T cell response in said patient.

[90] 90. The method of any one of Claims 87 to 89, further comprising administering mononuclear cells to said patient.

[91] The multiple donor CD4 IL-10 91. The method of claim 90, wherein the cells or pharmaceutical composition and the mononuclear cells are administered simultaneously.

[92] The mononuclear cells are IL-10 91. The method of claim 90, wherein the administration is either before or after administration of the cells or the pharmaceutical composition.

[93] 5. Pooled donor CD4 IL-10 administering hematopoietic stem cells (HSCs) from an HSC donor to said patient either before or after administration of the cells or pharmaceutical composition. 93. The method of any one of claims 87 to 92, further comprising:

[94] 94. The method of claim 93, wherein the HSC donor is partially HLA-mismatched to the patient.

[95] 95. The method of claim 94, wherein the HSC donor has less than a 5 / 10, 6 / 10, 7 / 10, 8 / 10, 9 / 10, or 10 / 10 match to the patient at the HLA-A, HLA-B, HLA-C, HLA-DRB1, and HLA-DQB1 loci.

[96] 95. The method of claim 94, wherein the HSC donor has a 4 / 8, 5 / 8, 6 / 8, 7 / 8, or less than 8 / 8 match to the patient at the HLA-A, HLA-B, HLA-C, and HLA-DRB1 loci.

[97] 95. The method of claim 94, wherein the HSC donor has less than a 2 / 2 match to the patient at the HLA-A, HLA-B, or HLA-C locus.

[98] 95. The method of claim 94, wherein the HSC donor has less than a 3 / 4 or less than a 4 / 4 match to the patient at the HLA-DRB1 and HLA-DQB1 loci.

[99] 99. The method of any one of claims 87 to 98, wherein one or more of the T cell donors are HLA-mismatched or partially HLA-mismatched to the patient.

[0100] 100. The method of claim 99, wherein one or more of the T cell donors have less than a 5 / 10, 6 / 10, 7 / 10, 8 / 10, 9 / 10, or 10 / 10 match to the patient at the HLA-A, HLA-B, HLA-C, HLA-DRB1, and HLA-DQB1 loci.

[0101] 100. The method of claim 99, wherein one or more of the T cell donors have a 4 / 8, 5 / 8, 6 / 8, 7 / 8 or less than 8 / 8 match to the patient at the HLA-A, HLA-B, HLA-C and HLA-DRB1 loci.

[0102] 100. The method of claim 99, wherein one or more of the T cell donors have less than a 2 / 2 match to the patient at the HLA-A, HLA-B, or HLA-C locus.

[0103] 100. The method of claim 99, wherein one or more of the T cell donors have a 2 / 4, 3 / 4, or less than 4 / 4 match to the patient at the HLA-DRB1 and HLA-DQB1 loci.

[0104] 104. The method of any one of claims 87 to 103, wherein one or more of the T cell donors are HLA-mismatched or partially HLA-mismatched to the HSC donor.

[0105] 105. The method of claim 104, wherein one or more of the T cell donors have less than a 5 / 10, 6 / 10, 7 / 10, 8 / 10, 9 / 10, or 10 / 10 match to the HSC donor at the HLA-A, HLA-B, HLA-C, HLA-DRB1, and HLA-DQB1 loci.

[0106] 105. The method of claim 104, wherein one or more of the T cell donors have a 4 / 8, 5 / 8, 6 / 8, 7 / 8, or less than 8 / 8 match to the HSC donor at the HLA-A, HLA-B, HLA-C, and HLA-DRB1 loci.

[0107] 105. The method of claim 104, wherein one or more of the T cell donors have less than a 2 / 2 match to the HSC donor at the HLA-A, HLA-B, or HLA-C locus.

[0108] 105. The method of claim 104, wherein one or more of the T cell donors have a 3 / 4 or less than 4 / 4 match to the HSC donor at the HLA-DRB1 and HLA-DQB1 loci.

[0109] The multiple donor CD4 IL-10 109. The method of any one of claims 79 to 108, wherein the cells or pharmaceutical composition prevent or reduce the severity of GvHD caused by the transplanted hematopoietic stem cells.

[0110] The multiple donor CD4 IL-10 110. The method of any one of claims 93 to 109, wherein the cells or pharmaceutical composition prevent or reduce the severity of a pathogenic response of lymphoid cells derived from the transplanted hematopoietic cells.

[0111] 111. The method of any one of claims 87 to 110, wherein the patient has neoplastic cells.

[0112] The method of claim 111, wherein the neoplastic cells express CD13, HLA-class I and CD54.

[0113] 113. The method of any one of claims 111 to 112, wherein the neoplastic cells express CD112, CD58 or CD155.

[0114] 114. The method of any one of claims 111 to 113, wherein the patient has cancer, optionally wherein the cancer is a solid or hematological neoplasm.

[0115] The patient may have adrenal gland cancer, anal cancer, bile duct cancer, bladder cancer, bone cancer, adult brain / CNS tumors, pediatric brain / CNS tumors, breast cancer, male breast cancer, cancer of unknown primary site, Castleman's disease, cervical cancer, colon / rectal cancer, endometrial cancer, esophageal cancer, Ewing's tumor, eye cancer, gallbladder cancer, gastrointestinal carcinoid tumors, gastrointestinal stromal tumors (GIST), gestational trophoblastic disease, Hodgkin's disease, Kaposi's sarcoma, kidney cancer, laryngeal and hypopharyngeal cancer, leukemia, acute lymphocytic (ALL), acute myeloid (AML including myeloid sarcoma and leukemia cutis), chronic lymphocytic (CLL), chronic myeloid (CML) leukemia, chronic myelomonocytic leukemia (CMML), childhood leukemia, liver cancer, lung cancer, non-small cell lung cancer, small cell lung cancer, pulmonary carcinoid tumors. 115. The method of any one of claims 87 to 114, wherein the patient has cancer selected from the group consisting of: tumor, lymphoma, lymphoma of the skin, malignant mesothelioma, multiple myeloma, myelodysplastic syndrome, nasal cavity and paranasal sinus cancer, nasopharyngeal carcinoma, neuroblastoma, non-Hodgkin's lymphoma, pediatric non-Hodgkin's lymphoma, oral cavity and oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, penile cancer, pituitary tumor, prostate cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma - adult soft tissue cancer, skin cancer, skin cancer - basal cell and squamous cell, skin cancer - melanoma, skin cancer - Merkel cell, small intestine cancer, gastric cancer, testicular cancer, thymus cancer, thyroid cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Waldenstrom's macroglobulinemia, and Wilms' tumor.

[0116] 116. The method of claim 115, wherein the patient has a myeloid cancer.

[0117] 116. The method of claim 115, wherein the patient has AML or CML.

[0118] 88. The method of any one of claims 87, wherein the patient has an inflammatory disease or an autoimmune disease.

[0119] 119. The method of claim 118, wherein the inflammatory or autoimmune disease is selected from the group consisting of type 1 diabetes, autoimmune uveitis, rheumatoid arthritis, psoriasis, psoriatic arthritis, multiple sclerosis, systemic lupus, inflammatory bowel disease, Addison's disease, Graves' disease, Sjogren's syndrome, Hashimoto's thyroiditis, myasthenia gravis, autoimmune vasculitis, pernicious anemia, ulcerative colitis, bullous diseases, scleroderma, and celiac disease.

[0120] 120. The method of claim 119, wherein the inflammatory or autoimmune disease is Crohn's disease, ulcerative colitis, celiac disease, type 1 diabetes, lupus, psoriasis, psoriatic arthritis, or rheumatoid arthritis.

[0121] 121. The method of any one of claims 87-113 or 118-120, wherein the patient has a disease or disorder associated with hyperactivity of the NLPR3 inflammasome.

[0122] 122. The method of any one of claims 87-113 or 118-121, wherein the patient has type 2 diabetes, a neurodegenerative disease, a cardiovascular disease, or an inflammatory bowel disease.

[0123] 122. The method of any one of claims 87-113 or 118-121, wherein the patient has a disease or disorder associated with increased IL-1β production by activated monocytes, macrophages, or dendritic cells.

[0124] 122. The method of any one of claims 87-113 or 118-121, wherein the patient has a disease or disorder associated with increased IL-18 production by activated monocytes, macrophages, or dendritic cells.

[0125] 122. The method of any one of claims 87-113 or 118-121, wherein the patient has a disease or disorder associated with increased production of mature caspase 1 by activated monocytes, macrophages, or dendritic cells.

[0126] 114. The method of any one of claims 87 to 113, wherein the patient has an allergic or atopic disease.

[0127] 127. The method of claim 126, wherein the allergic or atopic disease is selected from the group consisting of asthma, atopic dermatitis, and rhinitis.

[0128] 114. The method of any one of claims 87 to 113, wherein the patient has a food allergy.

[0129] CD4 + 114. The method of any one of Claims 87 to 113, further comprising the step of transplanting an organ into said patient either before or after administration of the population of T cells or said pharmaceutical composition.

[0130] The multiple donor CD4 IL-10 130. The method of claim 129, wherein the cells or pharmaceutical composition prevent or reduce the severity of host rejection of the organ transplant.

[0131] CD4+ 114. The method of any one of Claims 87 to 113, further comprising transplanting iPS cell-derived cells or tissue into the patient either before or after administration of the population of T cells or the pharmaceutical composition.

[0132] The multiple donor CD4 IL-10 132. The method of claim 131, wherein the cells or pharmaceutical composition prevent or reduce the severity of host rejection of the cell transplant.

[0133] The multiple donor CD4 IL-10 114. The method of any one of Claims 87 to 113, further comprising administering a recombinant AAV to the patient before or after administration of cells or the pharmaceutical composition.

[0134] The multiple donor CD4 IL-10 The method of claim 133, wherein the cells or the pharmaceutical composition reduce the immune response to the recombinant AAV.

[0135] 114. The method of any one of claims 87 to 113, wherein the patient has an exaggerated immune response to a viral or bacterial infection.

[0136] 136. The method of claim 135, wherein the patient has a coronavirus infection.

[0137] detecting a selectable marker in a biological sample obtained from said patient; This allows for multiple donor CD4 IL-10 137. The method of any one of Claims 87 to 136, further comprising detecting the presence or absence of T cells.

[0138] 138. The method of claim 137, wherein the biological sample is a biopsy or blood from the patient.

[0139] 1. A method of treating a patient having a malignant tumor, comprising: administering an allogeneic HSCT graft to said patient; Therapeutic doses of multiple donor CD4 IL-10 and administering the cells.

[0140] The multiple donor CD4 IL-10 CD4 cells IL-10 140. The method of claim 139, wherein none of the donors of the cells is the donor of the HSCT graft.

[0141] 1. A method of treating hematological cancer, comprising: Multiple donor CD4 in sufficient quantities to induce anti-cancer effects IL-10 administering the cells to a patient with hematological cancer; The multiple donor CD4 IL-10 CD4 T cells were obtained from at least three different T cell donors and genetically modified by vector-mediated gene transfer of the coding sequence for human IL-10 under the control of a constitutive promoter. + The method comprises T cells.

[0142] The multiple donor CD4 IL-10 142. The method of claim 141, further comprising administering an allogeneic HSCT graft to the patient before or after administration of the cells.

[0143] The multiple donor CD4 IL-10 143. The method of claim 142, wherein the amount of cells is sufficient to suppress graft-versus-host disease (GvHD) without suppressing the graft-versus-leukemia (GvL) or graft-versus-tumor (GvT) efficacy of allogeneic HSCT.

[0144] 144. The method of any one of claims 141 to 143, wherein the hematological cancer is myeloid leukemia.

[0145] The multiple donor CD4 IL-10 143. The method of any one of claims 141 to 142, wherein the cells target and kill cancer cells that express CD13.

[0146] The multiple donor CD4 IL-10 146. A method according to any one of claims 141 to 145, wherein the cells target and kill cancer cells that express HLA-class I.

[0147] 147. The method of any one of claims 141 to 146, wherein the myeloid leukemia is acute myeloid leukemia (AML).

[0148] 148. The method of any one of claims 141 to 147, wherein the allogeneic HSCT graft is obtained from a donor that is related or unrelated to the recipient.

[0149] The multiple donor CD4 IL-10 149. The method of any one of claims 141 to 148, wherein the cells are non-autologous to the recipient.

[0150] 149. The method of any one of claims 141 to 148, wherein the multiple-donor CD4IL-10 cells are allogeneic to the recipient.

[0151] The multiple donor CD4 IL-10 149. The method of any one of claims 141 to 148, wherein the cells have not been anergized to the host's alloantigen prior to administration to the host.

[0152] The multiple donor CD4 IL-10 149. The method of any one of claims 141 to 148, wherein the cell is a Tr1-like cell.

[0153] The multiple donor CD4 IL-10 149. The method of any one of claims 141 to 148, wherein the cells are polyclonal.

[0154] The multiple donor CD4 IL-10 149. The method of any one of claims 141 to 148, wherein the cells are polyclonal and non-autologous to the recipient.

[0155] The multiple donor CD4 IL-10 149. The method of any one of claims 141 to 148, wherein the cells are genetically modified after isolation from at least three donors.

[0156] 156. The method of claim 155, wherein none of the at least three donors is the same donor as the allogeneic HSCT donor.

[0157] 157. The method of any one of claims 141 to 156, wherein the allogeneic HSCT graft is obtained from a donor that is matched or mismatched to the recipient.

[0158] The multiple donor CD4 IL-10 158. The method of any one of claims 141 to 157, wherein the cell targets and kills cells expressing CD54.

[0159] The multiple donor CD4 IL-10 159. A method according to any one of claims 141 to 158, wherein the cells target and kill cancer cells that express HLA-class I and CD54.

[0160] The multiple donor CD4 IL-10 160. The method of any one of claims 141 to 159, wherein the cells target and kill cancer cells that express CD112.

[0161] The multiple donor CD4 IL-10 161. The method of any one of Claims 141 to 160, wherein the cells target and kill cancer cells that express CD58.

[0162] The multiple donor CD4 IL-10 162. The method of any one of claims 141 to 161, wherein the cells target and kill cancer cells in the host.

[0163] 1. A method of treating hematological cancers by allogeneic hematopoietic stem cell transplantation (allo-HSCT), comprising: administering the allogeneic HSCT graft to the subject (host); Multiple donor CD4 CD4 in amounts sufficient to suppress graft-versus-host disease (GvHD) without suppressing graft-versus-leukemia (GvL) or graft-versus-tumor (GvT) efficacy of the allogeneic HSCT graft. IL-10 administering the cells to a recipient (host) of the allogeneic HSCT; The multiple donor CD4 IL-10 CD4 T cells were obtained from at least three different T cell donors and genetically modified by vector-mediated gene transfer of the coding sequence for human IL-10 under the control of a constitutive promoter. + Contains T cells, The multiple donor CD4 IL-10 the cells are non-autologous to the recipient and non-autologous to the allogeneic HSCT donor; The multiple donor CD4 IL-10 the cells have not been anergized to the host's alloantigen prior to administration to said host; The multiple donor CD4 IL-10 The method wherein the cells are polyclonal and Tr1-like.

[0164] 1. A method of treating hematological cancers by allogeneic hematopoietic stem cell transplantation (allo-HSCT), comprising: administering the allogeneic HSCT graft to the subject (host); Multiple donor CD4 CD4 in amounts sufficient to suppress graft-versus-host disease (GvHD) without suppressing graft-versus-leukemia (GvL) or graft-versus-tumor (GvT) efficacy of the allogeneic HSCT graft. IL-10 administering the cells to a recipient (host) of the allogeneic HSCT; The multiple donor CD4 IL-10 CD4 T cells were obtained from at least three different T cell donors and genetically modified by vector-mediated gene transfer of the coding sequence for human IL-10 under the control of a constitutive promoter. + Contains T cells, The multiple donor CD4 IL-10 the cells target and kill cancer cells in the host; The multiple donor CD4 IL-10 the cells have not been anergized to the host's alloantigen prior to administration to said host; The multiple donor CD4 IL-10 The method, wherein the cells are non-autologous to the recipient, polyclonal, and Tr1-like.

[0260] 8. Equivalents While various specific embodiments have been shown and described, the above specification is not intended to be limiting. It will be understood that various changes can be made without departing from the spirit and scope of the invention. Many variations will become apparent to those skilled in the art upon review of this specification. 9. Arrays SEQ ID NO: 1 (amino acid sequence of human IL-10) JPEG0007772391000002.jpg23118 SEQ ID NO: 2 (an exemplary nt sequence of human IL-10) JPEG0007772391000003.jpg4199 SEQ ID NO: 3 (amino acid sequence of ΔNGFR) JPEG0007772391000004.jpg31118 SEQ ID NO: 4 (Exemplary nt sequence of ΔNGFR) JPEG0007772391000005.jpg7187 SEQ ID NO: 5 (nucleotide sequence of bd.ΔNGFR.PGK.IL-10) JPEG0007772391000006.jpg87118 JPEG0007772391000007.jpg172119 JPEG0007772391000008.jpg80118

Claims

1. CD4 genetically modified to contain an exogenous polynucleotide encoding IL-10 ＋ A population of T cells, (a) the CD4 ＋ T cells were obtained from at least three different T cell donors (multi-donor CD4 ＩＬ－１０ cells); and (b) the exogenous polynucleotide is integrated into the T cell nuclear genome; CD4 ＋ A population of T cells.

2. The CD4 ＋ 2. The CD4 T cells of claim 1, wherein the T cells have a total of 6, 7, 8, 9, 10, 11, 12 or more different HLA haplotypes. ＋ A population of T cells.

3. (a) determining whether all of the CD4 ＋ T cells, (i) have at least a 5 / 10, 6 / 10, 7 / 10, 8 / 10, or 9 / 10 match with each other at the HLA-A, HLA-B, HLA-C, HLA-DRB1, and HLA-DQB1 loci; (ii) have at least a 4 / 8, 5 / 8, 6 / 8, 7 / 8, or 8 / 8 match with each other at the HLA-A, HLA-B, HLA-C, and HLA-DRB1 loci; (iii) have a 2 / 2 match with each other at the HLA-A locus; (iv) have a 2 / 2 match with each other at the HLA-B locus; (v) have a 2 / 2 match with each other at the HLA-C locus; and / or (vi) all of the CD4 ＋ the T cells have at least a 3 / 4 or 4 / 4 match to each other at the HLA-DRB1 and HLA-DQB1 loci; and / or (b) all CD4 ＋ T cells are A ＊ 02 allele, CD4 according to claim 1 ＋ A population of T cells.

4. the exogenous polynucleotide (a) further comprising a sequence encoding a selectable marker; and / or (b) further comprising a lentiviral vector sequence; The CD4 of claim 1 or 2 ＋ A population of T cells.

5. A population of CD4 + T cells described in claim 4, wherein the selection marker is ΔNGFR.

6. (a) the CD4 ＋ At least 90% of T cells express IL-10; (b) the genetically modified CD4 ＋ T cells are CD4 ＋ T cell 10 ６ constitutively express at least 100 pg, 200 pg, 500 pg, 1 ng, 5 ng, 10 ng, or 50 ng of IL-10 per cell / ml of culture medium; and / or (c) the genetically modified CD4 ＋ T cells express CD4 after activation with anti-CD3 and anti-CD28 antibodies. ＋ T cell 10 ６ expressing at least 1 ng, 2 ng, 5 ng, 10 ng, 100 ng, 200 ng, or 500 ng of IL-10 per cell / ml; The CD4 of any one of claims 1 to 5. ＋ A population of T cells.

7. The CD4 ＋ 6. The CD4 T cell of claim 4(a) or claim 5, wherein at least 90%, at least 95%, or at least 98% of T cells express the selectable marker from the exogenous polynucleotide. ＋ A population of T cells.

8. The genetically modified CD4 ＋ 8. The CD4 T cell of claim 1, wherein the T cell expresses CD49b, LAG-3, TGF-β, IFNγ, GzB, perforin, CD18, CD2, CD226, and / or IL-22. ＋ A population of T cells.

9. The CD4 ＋ T cells (a) in a frozen suspension; or (b) in a liquid suspension; The CD4 of any one of claims 1 to 8 ＋ A population of T cells.

10. A population of CD4 + T cells as described in claim 9, wherein the liquid suspension has been previously frozen.

11. Multiple donor CD4 ＩＬ－１０ 1. A method of producing a cell, comprising: (i) Primary CD4 T cells obtained from at least three different T cell donors ＋ A step of modifying T cells, the CD4 T cells of each donor by introducing an exogenous polynucleotide encoding IL-10. ＋ Separately modifying the T cells; (ii) the genetically modified CD4 ＋ T cells are pooled, thereby ＩＬ－１０ and obtaining the cells.

12. before step (i), after step (i), after step (i) and before step (ii), or after step (ii); The primary CD4 ＋ T cells, (a) an anti-CD3 antibody and an anti-CD28 antibody, or (b) Beads coated with anti-CD3 and anti-CD28 antibodies 12. The method of claim 11, further comprising incubating in the presence of 13. The method of claim 12, further comprising incubating the primary CD4 + T cells in the presence of IL-2.

14. 14. The method of any one of claims 11 to 13, wherein the exogenous polynucleotide further comprises a segment encoding a selectable marker.

15. The method described in claim 14, wherein the encoded selectable marker is ΔNGFR.

16. The method described in claim 14 or 15, further comprising, after step (i), isolating the genetically modified CD4 + T cells that express the selection marker, thereby generating an enriched population of genetically modified CD4 + T cells.

17. In step (i), the primary CD4 ＋ 17. The method of any one of claims 11 to 16, wherein the T cells are obtained from one or more frozen stocks.

18. 1. A pharmaceutical composition for use in a method of treating a patient in need of immune tolerization, comprising: The pharmaceutical composition comprises a CD4 ＋ comprising a population of T cells, The method comprises administering to a subject a multi-donor CD4 ＩＬ－１０ A pharmaceutical composition comprising the step of administering the cells to a patient.

19. The method comprises: ＩＬ－１０ administering hematopoietic stem cells (HSCs) of an HSC donor to said patient either before or after administering the cells.

20. The pharmaceutical composition of claim 18, further comprising:

20. The patient (a) have an inflammatory or autoimmune disease; (b) have a disease or disorder associated with hyperactivity of the NLPR3 inflammasome; (c) have type 2 diabetes, neurodegenerative disease, cardiovascular disease or inflammatory bowel disease; and / or (d) have an allergic or atopic disease; 19. The pharmaceutical composition of claim 18.

21. The pharmaceutical composition of claim 20, wherein the inflammatory or autoimmune disease is selected from the group consisting of type 1 diabetes, Crohn's disease, autoimmune uveitis, rheumatoid arthritis, psoriasis, psoriatic arthritis, multiple sclerosis, systemic lupus, inflammatory bowel disease, Addison's disease, Graves' disease, Sjogren's syndrome, Hashimoto's thyroiditis, myasthenia gravis, autoimmune vasculitis, pernicious anemia, ulcerative colitis, bullous diseases, scleroderma, and celiac disease.

22. The pharmaceutical composition of claim 20, wherein the allergic or atopic disease is selected from the group consisting of asthma, atopic dermatitis, and rhinitis.

23. The method further comprises: ＋ further comprising the step of transplanting an organ into said patient either before or after administration of the population of T cells.

19. The pharmaceutical composition of claim 18.

24. 1. A pharmaceutical composition for use in a method of treating hematological cancer, comprising: The pharmaceutical composition comprises a CD4 ＋ comprising a population of T cells, The method comprises administering to a subject a sufficient amount of multiple donor CD4 ＩＬ－１０ administering the cells to a patient with hematological cancer; The multiple donor CD4 ＩＬ－１０ A pharmaceutical composition in which cells are genetically modified by vector-mediated gene transfer of a coding sequence for human IL-10 under the control of a constitutive promoter.

25. (a) the multiple donor CD4 ＩＬ－１０ The cells are administered to the patient before or after administration of an allogeneic hematopoietic stem cell transplant (allo-HSCT), and the multiple donor CD4 ＩＬ－１０ the amount of cells is sufficient to suppress GvHD without suppressing the graft-versus-leukemia (GvL) or graft-versus-tumor (GvT) efficacy of allogeneic HSCT; and / or (b) the hematological cancer is myeloid leukemia; 25. The pharmaceutical composition of claim 24.

26. 19. The pharmaceutical composition of claim 18, wherein the patient has Crohn's disease.

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