Polydonor CD4+ T cells expressing IL-10 and uses thereof

Abstract

The present disclosure provides CD4 T cell clones from at least two different T cell donors. + Polydonor CD4 generated by genetically modifying T cells IL-10 In addition, polydonor CD4 IL-10 Methods for generating polydonor CD4 cells and for immune tolerance IL-10 Methods of using the cells, methods of treating GvHD, cell and organ transplantation, cancer, autoimmune and inflammatory diseases, and other immune disorders are provided.

Description

[Technical field]

[0001] 1. CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 63 / 295,389, filed December 30, 2021, the entirety of which is incorporated herein by reference.

[0002] 2. Sequence Listing This application has been submitted in XML format and contains a Sequence Listing, which is incorporated herein by reference in its entirety. The XML copy was created on Dec. 23, 2022, is named 37104-49835Sequence-Listing.xml, and is 30.7 kilobytes (KB) in size. [Background technology]

[0003] 3.Background Regulatory T cells belong to a small but important subset of T cells that maintain immune tolerance to self- and non-pathogenic antigens and thus maintain homeostasis of the immune system. There are two main 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 down-regulate pathogenic T cell responses in various preclinical organ and islet transplantation models, graft-versus-host disease (GvHD), and various autoimmune and inflammatory diseases.

[0004] Tr1 cells have been shown to be effective in clinical trials. Administration of cloned antigen-specific autologous Tr1 cells to patients with ongoing moderate to severe Crohn's disease resulted in objective, transient remission (Desreumaux et al., Gastroenterology. 2012;143(5):1207-1217.e2.). In addition, donor-derived allospecific CD4 enriched for Tr1 cells resulted in a 100% remission of the disease (Desreumaux et al., Gastroenterology. 2012;143(5):1207-1217.e2.). +Adoptive transfer of T cell populations into leukemia patients after allogeneic hematopoietic stem cell transplantation (allo-HSCT) resulted in rapid reconstitution of the immune system and protection against microbial and viral infections without causing severe GvHD. Responder patients achieved long-term remission and tolerance (>7 years) leading to cure (Bacchetta et al., Front Immunol. 2014; 5:16).

[0005] Despite these encouraging 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 and is very laborious, and it is not possible to generate large quantities of pure Tr1 cells.

[0006] Recently, Locafaro and coworkers reported purified CD4 + We circumvented some of these problems by transducing T cells with a bidirectional lentiviral vector containing the human IL-10 gene. IL-10 The population shared key features of naturally occurring Tr1 cells. Like Tr1 cells, the single-donor CD4 IL-10 The cells produced high levels of IL-10 and expressed allogeneic CD4 + T cells and allogeneic CD8 + In addition, they are cytotoxic to both normal myeloid cells (including antigen-presenting cells, APCs) and myeloid leukemia cells. In a humanized xeno-GvHD model, these single-donor CD4 IL-10 The cells have been shown to be effective in reducing graft-versus-leukemia (GvHD) in a humanized xeno-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, most likely related to inherent differences between different donors in addition to variability in buffy coat quality. Summary of the Invention

[0008] 4. Overview The present disclosure relates to polydonor CD4 IL-10 The present invention provides a novel Tr1-based therapy using a cell population: polydonor CD4 IL-10 The cells are derived from at least two different T cell donors and then genetically modified to contain an exogenous polynucleotide encoding IL-10. + T cells. T cell donors are polydonor CD4 IL-10 A third party donor who is neither the host to be treated with the cells nor the HSC or organ transplant donor. IL-10 The cells are not alloantigen-specific, ie, they have not been primed or stimulated by cells from the host prior to administration.

[0009] The present applicant provides a polydonor CD4 IL-10 Cells are single-donor CD4 IL-10 We demonstrated that these cells have a cytokine production profile, immunosuppressive and cytotoxic potential equivalent to those of human CD4+ cells. In addition, in vivo, they inhibited CD4+ cells without inducing GvHD alone. + Single-donor CD4 T cell-mediated prevention of xeno GvHD IL-10 Overall, these polydonor CD4 IL-10 The functional characterization of cells both in vitro and in vivo was performed using single-donor CD4 IL-10 The properties of the cells were comparable or better.

[0010] Based on these results, the applicants have developed a polydonor allogeneic CD4 IL-10It is claimed that the cells can 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 requirement for allospecificity, polydonor CD4 IL-10 The cells will make Tr1-based cell therapy available to a larger population of patients with diverse genetic backgrounds.

[0012] Thus, in a first aspect, the present disclosure provides a CD4+ cell line that has been genetically modified to include an exogenous polynucleotide encoding IL-10. + A population of T cells, + T cells were obtained from at least two different T cell donors (polydonor CD4 IL-10 cell)CD4 + Providing a T cell population.

[0013] In some embodiments, CD4 + The T cells are obtained from 2, 3, 4, 5, 6, 7, 8, 9, or 10 different T cell donors. + Collectively, the T cells have 6, 7, 8, 9, 10, 11, 12, or more different HLA haplotypes.

[0014] In some embodiments, all of the CD4 + The T cells have at least a 1 / 10, 2 / 10, 3 / 10, 4 / 10, 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 1 / 8, 2 / 8, 3 / 8, 4 / 8, 5 / 8, 6 / 8, 7 / 8, or 8 / 8 matches to each other at the HLA-A, HLA-B, HLA-C, and HLA-DRB1 loci. +All T cells have a 2 / 2 match to each other at the HLA-A locus. + All T cells have a 2 / 2 match to each other at the HLA-B locus. + All T cells have a 2 / 2 match to each other at the HLA-C locus. + All of the T cells have at least a 3 / 4 or 4 / 4 match to each other at the HLA-DRB1 and HLA-DQB1 loci. + All T cells are A * 02 or A * There are 24 alleles.

[0015] In some embodiments, the CD4 + The T cells all have less than 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. + All of the T cells have less than 4 / 8, 5 / 8, 6 / 8, 7 / 8, or 8 / 8 matches to each other at the HLA-A, HLA-B, HLA-C, and HLA-DRB1 loci. + The T cells have less than a 2 / 2 match to each other at the HLA-A locus. + All of the T cells have less than a 2 / 2 match with each other at the HLA-B locus. + All of the T cells have less than a 2 / 2 match to each other at the HLA-C locus. + All T cells have a 3 / 4 or less than 4 / 4 match with each other at the HLA-DRB1 and HLA-DQB1 loci.

[0016] In some embodiments, the CD4 +All of the T cells are mismatched to each other at the HLA-A, HLA-B, HLA-C, HLA-DRB1, and HLA-DQB1 loci. + The T cells are mismatched to each other at the HLA-A, HLA-B, HLA-C, and HLA-DRB1 loci. + All of the T cells are mismatched to each other at the HLA-A locus. In some embodiments, all of the CD4 + The T cells are dissimilar to each other at the HLA-B locus. In some embodiments, all CD4 + The T cells are mismatched to each other at the HLA-C locus. In some embodiments, the CD4 + All T cells are mismatched to each other at the HLA-DRB1 and HLA-DQB1 loci.

[0017] In some embodiments, CD4 + None of the T cells are immortalized. In some embodiments, the exogenous polynucleotide comprises an IL-10 encoding polynucleotide segment 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 IL-10 is a variant of human IL-10 having the sequence of human IL-10 with 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid modifications. In some embodiments, the 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid modifications are substitutions with an amino acid(s) of viral IL-10 at the corresponding amino acid position. In some embodiments, the variant of human IL-10 has the sequence of SEQ ID NO: 8 or 9.

[0018] In some embodiments, the IL-10-encoding polynucleotide segment encodes a protein 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. In some embodiments, the expression control element drives constitutive expression of the encoded IL-10. In some embodiments, the expression control element drives constitutive expression of the encoded IL-10. In some embodiments, the expression control element drives constitutive expression of the encoded IL-10. + In some embodiments, the expression control element drives the expression of IL-10 in T cells. + Drives T cell specific expression.

[0019] In some embodiments, the exogenous polynucleotide further comprises a sequence encoding a selection marker. In some embodiments, the selection 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.

[0020] In some embodiments, the selectable marker is a truncated EGFR polypeptide. In some embodiments, the selectable marker is a truncated human EGFR polypeptide.

[0021] 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 lentivirus vector sequence. In some embodiments, the exogenous polynucleotide is not integrated into the T cell nuclear genome.

[0022] In some embodiments, the CD4 + At least 70% of the T cells express IL-10. + At least 90% of the T cells express IL-10. +At least 95%, 98% or 99% of T cells express IL-10. In some embodiments, the selection marker is ΔNGFR. In some embodiments, the expression level of IL-10 is linearly correlated with the expression level of the selection marker. In some cases, the expression level of IL-10 can be determined by the expression level of the selection marker.

[0023] 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 express CD4 + T cells 10 6 In some embodiments, the genetically modified CD4 + After activation with anti-CD3 and anti-CD28 antibodies, T cells express CD4 + T cells 10 6 In 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. + T cells are unmodified CD4 + 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.

[0024] In some embodiments, the CD4 + At least 70% of the T cells express the selection marker from the exogenous polynucleotide. In some embodiments, the CD4+ At least 90% of the T cells express the selection marker from the exogenous polynucleotide. In some embodiments, the CD4 + At least 95%, 98% or 99% of the T cells express the selection marker from the exogenous polynucleotide.

[0025] In some embodiments, the genetically modified CD4 + The T cells express CD49b. In some embodiments, the T cells express a 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 T cells express a genetically modified CD4 + The T cells express IFN-γ. In some embodiments, the genetically modified CD4 + The T cells express granzyme B (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 a 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 T cells express a genetically modified CD4 + T cells express IL-22.

[0026] In some embodiments, CD4 + The T cells are not anergized in the presence of peripheral blood mononuclear cells (PBMCs) from the host. In some embodiments, the 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 DC10 cells from the host.

[0027] In some embodiments, CD4 +The T cells are in frozen suspension. In some embodiments, the CD4 + The T cells are in a liquid suspension. In some embodiments, the liquid suspension has been previously frozen.

[0028] In another aspect, the present disclosure provides a method for producing a method for manufacturing a pharmaceutical composition comprising: (i) CD4 as described herein + T cell populations; and (ii) CD4 + A pharma- ceutically acceptable carrier in which the T cell population is suspended. The present invention provides a pharmaceutical composition comprising:

[0029] In yet another aspect, the present disclosure provides a polydonor CD4 IL-10 1. A method of producing a cell, comprising: (i) Primary CD4 T cells from at least two different T cell donors + pooling the T cells; and (ii) IL-10-encoding exogenous polynucleotides to induce IL-10 expression in pooled CD4 + T cells, thereby genetically modifying CD4 + Steps for obtaining T cells The present invention provides a method comprising:

[0030] In one aspect, the present disclosure provides a polydonor CD4 IL-10 1. A method of producing a cell, comprising: (i) Primary CD4 T cells from at least two different T cell donors + Obtaining T cells; and (ii) Immunoglobulin E (IL-10) expression in each donor was increased by introducing an exogenous polynucleotide encoding IL-10. + individually modifying the T cells; (iii) Genetically modified CD4 + T cells are pooled, thereby genetically modifying CD4 + Steps for obtaining T cells The present invention provides a method comprising:

[0031] In some embodiments, the method comprises the steps of: after step (i) and before step (ii); after step (ii); after step (ii) and before step (iii); or after step (iii): Primary culture CD4 + Incubating the T cells in the presence of anti-CD3 and anti-CD28 antibodies, or anti-CD3 and CD28 antibody coated beads. In some embodiments, the polydonor CD4 IL-10 The T cells are cultured in the presence of Miltenyi Biotec's T Cell TransAct™. In some embodiments, polydonor CD4 IL-10 T cells are cultured in the presence of ImmunoCult Human T Cell Activator™ from STEMCELL Technologies.

[0032] In some embodiments, the method comprises culturing primary CD4 + In some embodiments, the exogenous polynucleotide is administered to primary cultured CD4 T cells using a viral vector. + In some embodiments, the exogenous polynucleotide comprises an IL-10-encoding polynucleotide segment having a sequence of SEQ ID NO:1. In some embodiments, the IL-10-encoding polynucleotide segment has a sequence of SEQ ID NO:2.

[0033] In some embodiments, the exogenous polynucleotide further comprises a segment encoding a selection marker. In some embodiments, the encoded selection marker is ΔNGFR. In some embodiments, the encoded selection marker has the sequence of SEQ ID NO: 3. In some embodiments, the encoded selection marker is a truncated EGFR polypeptide. In some embodiments, the encoded selection marker is a truncated human EGFR polypeptide.

[0034] 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 + Further comprising generating an enriched population of T cells.

[0035] In some embodiments, the genetically modified CD4 + At least 70% of the T cells express IL-10. In some embodiments, the genetically modified CD4 + At least 90%, 95%, or 98% of the T cells express IL-10. In some embodiments, the genetically modified CD4 + At least 70% of the T cells express the selection marker. In some embodiments, the genetically modified CD4 + At least 90%, 95%, or 98% of the T cells express the selectable marker.

[0036] In some embodiments, the method comprises genetically modifying CD4 + In some embodiments, the enriched population of T cells is further incubated with a genetically modified CD4 + The step of incubating the enriched population of T cells is carried out in the presence of IL-2, anti-CD3 and anti-CD28 antibodies, or CD3 and CD28 antibody coated beads.

[0037] In some embodiments, the method comprises genetically modifying CD4 +In some embodiments, the method further comprises the subsequent step of freezing the T cells. + The T cells are obtained from 2, 3, 4, 5, 6, 7, 8, 9, or 10 different T cell donors. In some embodiments, at least two T cell donors have at least 1 / 10, 2 / 10, 3 / 10, 4 / 10, 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. In some embodiments, at least two T cell donors have at least 1 / 8, 2 / 8, 3 / 8, 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. In some embodiments, at least two T cell donors have 2 / 2 matches with each other at the HLA-A locus. In some embodiments, at least two T cell donors have a 2 / 2 match to each other at the HLA-B locus. In some embodiments, at least two T cell donors have a 2 / 2 match to each other at the HLA-C locus. In some embodiments, at least two T cell donors have at least a 3 / 4 or 4 / 4 match to each other at the HLA-DRB1 and HLA-DQB1 loci. In some embodiments, each of the at least two T cell donors has an A * 02 or A * There are 24 alleles.

[0038] In some embodiments, at least two T cell donors have less than 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. In some embodiments, at least two T cell donors have less than 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. In some embodiments, at least two T cell donors have less than 2 / 2 matches with each other at the HLA-A locus. In some embodiments, at least two T cell donors have less than 2 / 2 matches with each other at the HLA-B locus. In some embodiments, at least two T cell donors have less than 2 / 2 matches with each other at the HLA-C locus. In some embodiments, at least two T cell donors have a 3 / 4 or less than 4 / 4 match to each other at the HLA-DRB1 and HLA-DQB1 loci.

[0039] In some embodiments, in step (i), primary cultured CD4 + The T cells are obtained from one or more cryopreserved stocks. In some embodiments, in step (i), primary cultured CD4 + T cells are obtained from unfrozen peripheral blood mononuclear cells of at least two different T cell donors.

[0040] In some embodiments, the method comprises isolating CD4 + Further comprising isolating T cells, in some embodiments, peripheral blood mononuclear cells are obtained from buffy coat or apheresis.

[0041] In another aspect, the present disclosure provides a method of treating a patient, comprising: Polydonor CD4 IL-10 Administering the cells or pharmaceutical composition of the present disclosure to a patient in need of immune tolerance. The present invention provides a method comprising:

[0042] In some embodiments, the method comprises the step of:IL-10 It further includes a prior step of thawing the frozen suspension of cells.

[0043] In some embodiments, the polydonor CD4 IL-10 The cells or pharmaceutical composition prevent or reduce the severity of a pathogenic T cell response in a patient.

[0044] In some embodiments, the polydonor CD4 IL-10 The cells or pharmaceutical composition prevent or reduce the severity of an inflammatory or autoimmune response.

[0045] 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 polydonor CD4 IL-10 It is administered either before or after administration of the cells or pharmaceutical composition. In some embodiments, the mononuclear cells are present in PBMC. In some embodiments, the mononuclear cells are present in bone marrow. In some embodiments, the mononuclear cells are present in umbilical cord blood. In some embodiments, the mononuclear cells are isolated from PBMC, bone marrow, or umbilical cord blood.

[0046] In some embodiments, the method comprises: Polydonor CD4 IL-10 administering hematopoietic stem cells (HSCs) of an HSC donor to the patient, either before or after administration of the cells or pharmaceutical composition; Further includes:

[0047] In some embodiments, the HSC donor is partially HLA mismatched to the patient. In some embodiments, the HSC donor has less than 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, the HSC donor has less than 4 / 8, 5 / 8, 6 / 8, 7 / 8 or 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 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 3 / 4 or 4 / 4 match to the patient at the HLA-DRB1 and HLA-DQB1 loci.

[0048] In some embodiments, one or more of the T cell donors are HLA-mismatched or partially HLA-mismatched to the patient. In some embodiments, one or more of the T cell donors have less than 5 / 10, 6 / 10, 7 / 10, 8 / 10, 9 / 10 or 10 / 10 matches to the patient at the HLA-A, HLA-B, HLA-C, HLA-DRB1 and HLA-DQB1 loci. In some embodiments, one or more of the T cell donors have less than 4 / 8, 5 / 8, 6 / 8, 7 / 8 or 8 / 8 matches to the patient at the HLA-A, HLA-B, HLA-C and HLA-DRB1 loci. In some embodiments, one or more of the T cell donors have less than 2 / 2 matches to the patient at the HLA-A, HLA-B or HLA-C loci. In some embodiments, one or more of the T cell donors have less than 2 / 4, 3 / 4, or 4 / 4 matches to the patient at the HLA-DRB1 and HLA-DQB1 loci. In some embodiments, one or more of the T cell donors are HLA-mismatched or partially HLA-mismatched to the HSC donor. In some embodiments, one or more of the T cell donors have less than 5 / 10, 6 / 10, 7 / 10, 8 / 10, 9 / 10, or 10 / 10 matches to the HSC donor at the HLA-A, HLA-B, HLA-C, HLA-DRB1, and HLA-DQB1 loci. In some embodiments, one or more of the T cell donors have less than 4 / 8, 5 / 8, 6 / 8, 7 / 8, or 8 / 8 matches to the HSC donor at the HLA-A, HLA-B, HLA-C, and HLA-DRB1 loci. In some embodiments, 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 loci. In some embodiments, one or more of the 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.

[0049] In some embodiments, the polydonor CD4 IL-10 The cells or pharmaceutical composition prevent or reduce the severity of GvHD due to transplanted hematopoietic stem cells.

[0050] In some embodiments, the polydonor CD4 IL-10 The cells or pharmaceutical composition prevent or reduce the severity of a pathogenic response of lymphoid cells present in the transplanted hematopoietic stem cell population.

[0051] 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.

[0052] In some embodiments, the patient has cancer. In some embodiments, the cancer is a solid or hematological neoplasm. In some embodiments, the patient has cancer of the adrenal gland, 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 family of tumors, eye cancer, gallbladder cancer, gastrointestinal carcinoid tumors, gastrointestinal stromal tumors (GIST), gestational trophoblastic disease, Hodgkin's disease, Kaposi's sarcoma, kidney cancer, laryngo-hypopharyngeal cancer, leukemia, acute lymphocytic leukemia (ALL), acute myeloid leukemia (including AML, myeloid sarcoma, and leukemia cutis), chronic lymphocytic leukemia (CLL), chronic myelogenous (CML) leukemia, chronic myelomonocytic leukemia (CMML), childhood leukemia, liver cancer, lung cancer, The patient has a cancer selected from the group consisting of non-small cell lung cancer, small cell lung cancer, pulmonary carcinoid tumor, lymphoma, cutaneous lymphoma, malignant mesothelioma, multiple myeloma, myelodysplastic syndrome, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, non-Hodgkin's lymphoma, childhood non-Hodgkin's lymphoma, oral 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, thymic cancer, thyroid cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Waldenstrom's macroglobulinemia, and Wilms' tumor.

[0053] In some embodiments, the cancer is a myeloid cancer, hi some embodiments, the cancer is AML or CML.

[0054] In some embodiments, the patient has inflammatory disease or autoimmune disease.In some embodiments, the inflammatory disease or autoimmune disease is selected from the group consisting of type 1 diabetes, autoimmune uveitis, autoimmune hepatitis, vitiligo, alopecia areata, rheumatoid arthritis, psoriasis, psoriatic arthritis, multiple sclerosis, systemic lupus erythematosus, inflammatory bowel disease, Addison's disease, Graves' disease, Sjogren's syndrome, Hashimoto's thyroiditis, myasthenia gravis, autoimmune vasculitis, pernicious anemia, ulcerative colitis, blister disease, scleroderma, Crohn's disease and celiac disease.

[0055] 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.

[0056] In some embodiments, the patient has a disease or disorder associated with hyperactivity of the NLPR3 inflammasome. In some embodiments, the patient has type 2 diabetes, a neurodegenerative disease, a cardiovascular disease, or an inflammatory bowel disease.

[0057] In some embodiments, the patient has a disease or disorder associated with increased IL-1β production by activated monocytes, macrophages, or dendritic cells.

[0058] In some embodiments, the patient has a disease or disorder associated with increased production of IL-18 by activated monocytes, macrophages, or dendritic cells.

[0059] 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.

[0060] In some embodiments, the patient has an allergic or atopic disease. In some embodiments, the allergic or atopic disease is selected from the group consisting of asthma, atopic dermatitis, and rhinitis. In some embodiments, the patient has a food allergy.

[0061] In some embodiments, the method comprises administering to a subject a subject comprising: + The method further comprises administering to the patient a cell or organ transplant either before or after administration of the T cell population or pharmaceutical composition. IL-10 The cells or pharmaceutical compositions prevent or reduce the severity of host rejection of cell and organ transplants.

[0062] In some embodiments, the method comprises administering to a subject a subject comprising: + The method further comprises the step of transplanting iPS cell-derived cells or tissue into the patient, either before or after administration of the T cell population or pharmaceutical composition.

[0063] In some embodiments, the polydonor CD4 IL-10 The cells or pharmaceutical composition prevent or reduce the severity of host rejection of the graft.

[0064] In some embodiments, the method comprises the step of: IL-10 The method further comprises administering to the patient a recombinant adenovirus, adeno-associated virus (AAV), herpes simplex virus (HSV), retrovirus, lentivirus, alphavirus, flavivirus, rhabdovirus, measles virus, Newcastle disease virus, poxvirus, or picornavirus, either before or after administration of the cells or pharmaceutical composition. IL-10 The cells or pharmaceutical compositions reduce an immune response to a recombinant adenovirus, adeno-associated virus (AAV), herpes simplex virus (HSV), retrovirus, lentivirus, alphavirus, flavivirus, rhabdovirus, measles virus, Newcastle disease virus, poxvirus, or picornavirus.

[0065] In some embodiments, the patient has an exaggerated immune response to a viral or bacterial infection. In some embodiments, the patient has a coronavirus infection. In some embodiments, the patient has organ and / or tissue damage.

[0066] In some embodiments, the method comprises the step of: IL-10 The method further comprises administering an immunogenic therapeutic protein to the patient either before or after administration of the cell population or pharmaceutical composition. IL-10 The cell population or pharmaceutical composition reduces an immune response to an immunogenic therapeutic protein. In some embodiments, the immunogenic therapeutic protein is selected from a therapeutic antibody, a factor VIII replacement, a cytokine, and a cytokine mutein.

[0067] In some embodiments, the method detects a selectable marker in a biological sample obtained from a patient, thereby detecting polydonor CD4 IL-10 Further comprising detecting the presence or absence of T cells, hi some embodiments, the biological sample is a biopsy or blood from the patient.

[0068] In one aspect, the present disclosure provides a method of treating a patient with a malignant disease, comprising administering to the patient an allo-HSCT and administering polydonor CD4 IL-10 In some embodiments, the allo-HSCT comprises administering a therapeutically effective amount of polydonor CD4 IL-10 In some embodiments, the allo-HSCT is administered prior to administration of the cells. IL-10 It is administered after administration of the cells.

[0069] In some embodiments, the polydonor CD4 IL-10 CD4 in cells IL-10 None of the cell donors were HSCT donors.

[0070] In another aspect, the disclosure provides a method of treating hematological cancer, comprising administering to a hematological cancer patient a sufficient amount of polydonor CD4 IL-10 administering polydonor CD4 IL-10 CD4 T cells are derived from at least two different T cell donors and subsequently genetically modified by vector-mediated gene transfer of the coding sequence for human IL-10 under the control of a constitutive or inducible promoter. + The method includes administering a T cell to a subject.

[0071] In some embodiments, the method of treating hematological cancer comprises administering polydonor CD4 + The method includes the steps of: T cells are obtained from individual donors and first individually genetically modified by vector-mediated gene transfer of a coding sequence for human IL-10 under the control of a constitutive or inducible promoter, and then pooled.

[0072] In some embodiments, the method of treating hematological cancer comprises administering polydonor CD4 + The method includes a step in which T cells are obtained from individual donors that are first pooled, and then the pool is genetically modified by vector-mediated gene transfer of a coding sequence for human IL-10 under the control of a constitutive or inducible promoter.

[0073] In some embodiments, the method comprises the step of: IL-10 The method further comprises administering an allo-HSCT to the patient before or after administration of the cells. IL-10 The amount of cells is further sufficient to suppress or prevent graft-versus-host disease (GvHD) without suppressing the graft-versus-leukemia (GvL) or graft-versus-tumor (GvT) efficacy of allo-HSCT.

[0074] In some embodiments, the hematological cancer is myeloid leukemia.

[0075] In some embodiments, the polydonor CD4 IL-10 The cells target and kill cancer cells that express CD13. In some embodiments, the polydonor CD4 IL-10 The cells target and kill cancer cells that express HLA-class I. In some embodiments, the myeloid leukemia is acute myeloid leukemia (AML).

[0076] In some embodiments, the allo-HSCT is obtained from a donor who is related or unrelated to the recipient. IL-10 The cells are non-autologous to the recipient. In some embodiments, polydonor CD4 IL-10 The cells are allogeneic to the recipient. In some embodiments, polydonor CD4 IL-10 The cells are not anergized to host alloantigens prior to administration to the host.

[0077] In some embodiments, the polydonor CD4 IL-10 The cells are Tr1-like cells.

[0078] In some embodiments, the polydonor CD4 IL-10 The cells are polyclonal. In some embodiments, the cells are polydonor CD4 IL-10 The cells are polyclonal and non-autologous to the recipient.

[0079] In some embodiments, the polydonor CD4 IL-10 Cells are genetically modified after being isolated from at least two donors.In some embodiments, none of the at least two donors is the same donor as the allo-HSCT donor.In some embodiments, the allo-HSCT is obtained from a donor that is matched or mismatched with respect to the recipient.

[0080] In some embodiments, the polydonor CD4 IL-10 The cells target and kill cells that express CD54. In some embodiments, the polydonor CD4 IL-10The cells target and kill cancer cells that express HLA-class I and CD54. In some embodiments, the polydonor CD4 IL-10 The cells target and kill cancer cells that express CD112. In some embodiments, the polydonor CD4 IL-10 The cells target and kill cancer cells that express CD58. In some embodiments, the polydonor CD4 IL-10 The cells target and kill cancer cells in the host.

[0081] One aspect of the present disclosure is a method of treating hematological cancer by allogeneic hematopoietic stem cell transplantation (allo-HSCT), comprising: administering allo-HSCT to the subject (host); Sufficient amounts of polydonor CD4 to suppress or prevent graft-versus-host disease (GvHD) without suppressing the graft-versus-leukemia (GvL) or graft-versus-tumor (GvT) efficacy of the allo-HSCT graft. IL-10 Administering the cells to a host Including, Polydonor CD4 IL-10 CD4 T cells are derived from at least two 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 or inducible promoter. + Contains T cells; Polydonor CD4 IL-10 The cells are non-autologous to the host and non-autologous to the allo-HSCT donor; Polydonor CD4 IL-10 the cells are not anergized to host alloantigens prior to administration to the host; and Polydonor CD4 IL-10 The cells are polyclonal and Tr1-like, A method is provided.

[0082] In some embodiments, the allo-HSCT is a polydonor CD4 IL-10 In some embodiments, the allo-HSCT is administered prior to administration of the cells. IL-10 It is administered after administration of the cells.

[0083] Another aspect of the present disclosure is a method of treating hematological cancer by allogeneic hematopoietic stem cell transplantation (allo-HSCT), comprising: administering allo-HSCT to the subject (host); Sufficient amounts of polydonor CD4 to suppress or prevent graft-versus-host disease (GvHD) without suppressing the graft-versus-leukemia (GvL) or graft-versus-tumor (GvT) efficacy of allo-HSCT IL-10 administering the cells to a host; Including, Polydonor CD4 IL-10 CD4 T cells were obtained from at least two 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; Polydonor CD4 IL-10 The cells target and kill cancer cells in the host; Polydonor CD4 IL-10 the cells are not anergized to host alloantigens prior to administration to the host; and Polydonor CD4 IL-10 All cells are non-autologous to the host, polyclonal, and Tr1-like; A method is provided.

[0084] Another embodiment of the present disclosure is a method for the production of CD4 IL-10 from a single donor or multiple donors, wherein the IL-10 is viral IL-10. IL-10 In some embodiments, the viral IL-10 is encoded by a polynucleotide having the sequence of SEQ ID NO: 7. In some embodiments, the IL-10 is human IL-10 in which 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids from human IL-10 are replaced by the corresponding amino acid sequence from the viral IL-10. In some embodiments, the viral IL-10 is encoded by a polynucleotide having the sequence of SEQ ID NO: 6, 19, 20, or 21. In some embodiments, the viral IL-10 is encoded by a polynucleotide having the sequence of SEQ ID NO: 7. In some embodiments, the IL-10 is human IL-10 in which 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids from human IL-10 are replaced by the corresponding amino acid sequence from the viral IL-10. +T cells are transduced with exogenous viral IL-10 under the control of a constitutive promoter. In some embodiments, the expression control element is activated CD4 + Drive the expression of viral IL-10 in T cells. In some embodiments, the exogenous polynucleotide encoding viral IL-10 is integrated into T cell nuclear genome. In some embodiments, the exogenous polynucleotide encoding viral IL-10 is not integrated into T cell nuclear genome. In some embodiments, the exogenous polynucleotide encoding viral IL-10 has the sequence of SEQ ID NO: 7.

[0085] Another aspect of the present disclosure is the use of CD4 IL-10 A CD4+ cell, wherein the IL-10 is IL-10 of a mouse (SEQ ID NO:10), a rat (SEQ ID NO:11), a rhesus monkey (Macaca mulatta) (MACMU) (SEQ ID NO:12), a gorilla (SEQ ID NO:13), a cynomolgus monkey (CYNO) (SEQ ID NO:14), anubis baboon (SEQ ID NO:15), a bonobo (SEQ ID NO:16), a chimpanzee (SEQ ID NO:17), or an EBVB9 (SEQ ID NO:18). IL-10 In some embodiments, the IL-10 is a protein having at least 90%, 95%, 98%, or 99% sequence identity to IL-10 of mouse (SEQ ID NO: 10), rat (SEQ ID NO: 11), rhesus monkey (Macaca mulatta) (MACMU) (SEQ ID NO: 12), gorilla (SEQ ID NO: 13), cynomolgus monkey (CYNO) (SEQ ID NO: 14), Anubis baboon (SEQ ID NO: 15), bonobo (SEQ ID NO: 16), chimpanzee (SEQ ID NO: 17), or EBVB9 (SEQ ID NO: 18).

[0086] Another embodiment of the present disclosure is a CD4+ antibody from a single donor or multiple donors, wherein the IL-10 is a variant of human IL-10. IL-10In some embodiments, the IL-10 is a human IL-10 in which 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids of human IL-10 have been replaced by the corresponding amino acid sequence from another species of IL-10 (e.g., IL-10 of mouse (SEQ ID NO: 10), rat (SEQ ID NO: 11), rhesus monkey (Macaca mulatta) (MACMU) (SEQ ID NO: 12), gorilla (SEQ ID NO: 13), cynomolgus monkey (CYNO) (SEQ ID NO: 14), Anubis baboon (SEQ ID NO: 15), bonobo (SEQ ID NO: 16), chimpanzee (SEQ ID NO: 17), or EBVB9 (SEQ ID NO: 18)). In some embodiments, the IL-10 is a human IL-10 in which 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids of human IL-10 have been replaced by the corresponding amino acid sequence from another species of IL-10 (e.g., IL-10 of mouse (SEQ ID NO: 10), rat (SEQ ID NO: 11), rhesus monkey (Macaca mulatta) (MACMU) (SEQ ID NO: 12), gorilla (SEQ ID NO: 13), cynomolgus monkey (CYNO) (SEQ ID NO: 14), Anubis baboon (SEQ ID NO: 15), bonobo (SEQ ID NO: 16), chimpanzee (SEQ ID NO: 17), or EBVB9 (SEQ ID NO: 18). + The T cells are transduced with an exogenous IL-10 variant under the control of a constitutive promoter. In some embodiments, the expression control element is an activated CD4 + Drive expression of IL-10 variants in T cells. In some embodiments, the exogenous polynucleotide encoding the IL-10 variant is integrated into the T cell nuclear genome. In some embodiments, the exogenous polynucleotide encoding the IL-10 variant is not integrated into the T cell nuclear genome.

[0087] In yet another aspect, the present disclosure provides a method for the preparation of a method for the preparation of a viral IL-10 CD 4IL-10 A method of making a composition comprising the steps of: (i) Primary CD4 T cells from a single donor + Obtaining T cells; and (ii) transfecting donor CD4 by introducing an exogenous polynucleotide encoding viral IL-10 + T cells, thereby genetically modifying CD4 + Steps for obtaining T cells The present invention provides a method comprising:

[0088] In some embodiments, the method further comprises, after step (i) or after step (ii), culturing primary cultured CD4 in the presence of anti-CD3 and anti-CD28 antibodies, or anti-CD3 and CD28 antibody coated beads.+ The method further comprises the step of incubating the T cells.

[0089] In some embodiments, the method further comprises culturing primary CD4 + In some embodiments, the vector is used to incubate the T cells with an exogenous polynucleotide encoding viral IL-10.

[0090] In some embodiments, the exogenous polynucleotide encoding viral IL-10 comprises a segment encoding a selection marker. In some embodiments, the encoded selection marker is ΔNGFR. In some embodiments, the encoded selection marker has the sequence of SEQ ID NO:3. In some embodiments, the encoded selection marker is a truncated EGFR polypeptide. In some embodiments, the encoded selection marker is a truncated human EGFR polypeptide.

[0091] In some embodiments, the method further comprises, after step (ii), administering to the subject a genetically modified CD4 + Isolating T cells and thereby genetically modifying CD4 + Further comprising generating an enriched population of T cells.

[0092] In some embodiments, the method comprises genetically modifying CD4 + In some embodiments, the enriched population of T cells is further incubated with a genetically modified CD4 + The step of incubating the enriched population of T cells is carried out in the presence of IL-2, anti-CD3 and anti-CD28 antibodies, or CD3 and CD28 antibody coated beads.

[0093] In some embodiments, in step (i), primary cultured CD4 + In some embodiments, the T cells are obtained from a cryopreservation. In some embodiments, in step (i), primary cultured CD4 + T cells are obtained from unfrozen peripheral blood mononuclear cells of a single T cell donor.

[0094] In some embodiments, the method comprises isolating CD4 + Further comprising isolating T cells, in some embodiments, peripheral blood mononuclear cells are obtained from buffy coat or apheresis. [Brief description of the drawings]

[0095] [Figure 1] FIG. 1 is a non-limiting illustration of the construction of a bidirectional lentiviral vector for delivering human IL-10 and ΔNGFR coding sequences to CD4+ T cells from multiple donors to generate polydonor CD4IL-10 cells. [Diagram 2] FIG. 2 illustrates the complete circular structure of a bidirectional lentiviral vector to generate a lentiviral vector for delivering human IL-10 and ΔNGFR coding sequences to CD4+ T cells from multiple donors to generate polydonor CD4IL-10 cells. [Diagram 3] FIG. 3 illustrates an exemplary protocol for generating CD4IL-10 cells. [Figure 4-A] Figure 4A shows the percentage of CD4+ΔNGFR+ cells (mean ± SD, n = 10, left grey bar) and vector copy number (VCN, mean ± SD, n = 10, right grey bar) in human CD4+ T cells transduced with LV-IL-10 / ΔNGFR (a bidirectional lentiviral vector encoding a truncated form of human IL-10 and human NGF receptor). 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 4-B] Same as Figure 4-A. [Diagram 5]Figure 5 shows the cytokine production profile of single donor CD4IL-10 cells after the second (TF2) and third (TG3) restimulation. TF2 (left panel) and TF3 (right panel) CD4IL-10 cells were left unstimulated (indicated by arrows) or stimulated with fixed CD3 (10 μg / mL) and soluble CD28 mAb (1 μg / mL) for 48 h. Culture supernatants were collected and levels of IL-10, IL-4, IL-5, IFN-γ and IL-22 were determined by ELISA. All samples were tested in triplicate. Mean values ​​± SD of n=8 donors tested are represented. [Figure 6] Figure 6A shows the percentage of CD4IL-10 cells expressing granzyme B (GzB) after the second round of stimulation (TF2) analyzed by FACS. Box plots of n=7 different single donors are shown. Figure 6B shows the % dead cells in CD4IL-10 cells (105 cells / well) co-cultured with K562 and ALL-CM cells (105 cells / well) at a 1:1 ratio for 3 days. Box plots represent data from n=4 donors and dots represent data from a single donor. [Figure 7-A]Figures 7A and 7B show that single donor CD4IL-10 cells can suppress proliferation of allogeneic CD4+ T cells. Allogeneic PBMC cells were labeled with eFluor® 670 (5x104 cells / well) and stimulated with allogeneic mature dendritic (DC) cells (5x103 cells / well) and soluble anti-CD3 mAb at a 1:1 responder:suppressor ratio in the absence or presence of CD4IL-10 cells (5x104 cells / well). After 3 days of culture, the percentage of proliferative responder cells was determined by dilution of eFluor® 670 by flow cytometry after gating on CD4+ΔNGFR- T cells. Figure 7A shows the results from donors C, E, and F, and Figure 7B shows the results from donors H, I, and L. Percentages of proliferation and suppression are shown. Suppression mediated by CD4IL-10 cells was calculated as follows: 100-([proliferation of responders in the presence of CD4IL-10 cells / proliferation of responders alone] x 100). [Figure 7-B] Same as Figure 7-A. [Figure 8-A] Figures 8A and 8B show that single donor CD4IL-10 cells can suppress proliferation of allogeneic CD8+ T cells. Allogeneic PBMC cells were labeled with eFluor® 670 (5x104 cells / well) and stimulated with allogeneic mature dendritic (DC) cells (5x103 cells / well) and soluble anti-CD3 mAb at a 1:1 responder:suppressor ratio in the absence or presence of CD4IL-10 cells (5x104 cells / well). After 3 days of culture, the percentage of proliferative responder cells was determined by dilution of eFluor® 670 by flow cytometry after gating on CD8+ΔNGFR- T cells. Figure 8A shows the results from donors C, E, and F, and Figure 8B shows the results from donors H, I, and L. Percentages of proliferation and suppression are shown. Suppression mediated by CD4IL-10 cells was calculated as follows: 100-([proliferation of responders in the presence of CD4IL-10 cells / proliferation of responders alone] x 100). [Figure 8-B] Same as Figure 8-A. [Figure 9] Figure 9 shows the cytokine production profile of polydonor CD4IL-10 cells after the third restimulation (TF3) compared to the mean levels (±SD) produced by CD4IL-10 cells from eight individual donors. TF3 CD4IL-10 cells from three donors were pooled at a 1:1:1 ratio and stimulated with fixed CD3 (10 μg / mL) and soluble CD28 mAb (1 μg / mL) for 48 hours. Culture supernatants were collected and IL-10, IL-4, IL-5, IFN-γ and IL-22 levels were determined by ELISA. Dots are the results of polydonor CD4IL-10 cells; grey bars represent the mean ±SD of n=8 single donors. [Figure 10] Figure 10A shows the percentage of polydonor CD4IL-10 cells expressing granzyme B (GzB) compared to the mean % level of granzyme B expression (±SD) by CD4IL-10 cells of n=3 single donors used to generate the pools. Cells were analyzed by FACS after 3 rounds of stimulation (TF3). Figure 10B shows the % dead cells when polydonor CD4IL-10 cells (105 cells / well) were co-cultured with K562 and ALL-CM cells (105 cells / well) at a 1:1 ratio for 3 days. Residual leukemia cells (CD45+, CD3-) were counted by FACS for each target cell. Dots are polydonor CD4IL-10 results and grey bars represent the mean ±SD of n=3 single donors used to generate the pools. [Figure 11-A]Figures 11A and 11B show that polydonor CD4IL-10 cells can suppress the proliferation of allogeneic CD4+ and CD8+ T cells. Allogeneic PBMC cells were labeled with eFluor® 670 (5x104 cells / well) and stimulated with allogeneic mature dendritic (DC) cells (1x104 cells / well) and soluble anti-CD3 mAb at a 1:1 responder:suppressor ratio in the absence or presence of polydonor CD4IL-10 cells (5x104 cells / well). After 3 days of culture, the percentage of proliferative responder cells was determined by dilution of eFluor® 670 by flow cytometry after gating on CD4+ΔNGFR- and CD8+ΔNGFR-T cells. Figure 11A shows the results from polydonor CD4IL-10 cells containing pooled CD4+ cells from donor C, donor E, and donor F. Figure 11B shows results from polydonor CD4IL-10 cells containing pooled CD4+ cells from donor H, donor I, and donor L. Suppression mediated by CD4IL-10 cells was calculated as: 100-([proliferation of responders in the presence of CD4IL-10 cells / proliferation of responders alone] x 100). [Figure 11-B] Same as Figure 11-A. [Figure 12] FIG. 12 illustrates the protocol for testing the induction of GvHD by human PBMC and / or polydonor CD4IL-10 (BC-C / E / F) cells injected on day 0 after radiation. [Figure 13] FIG. 13 shows the % of NSG mice free of GvHD on each day after injection with PBMCs (5×106 cells / mouse), polydonor (3 donors; BC-C / E / F) CD4IL-10 cells (5×106 cells / mouse), or PBMCs (5×106 cells / mouse) in combination with polydonor CD4IL-10 cells (3 donors; BC-C / E / F) (5×106 cells / mouse). [Figure 14]Figure 14 shows migration of CD4IL-10 cells to the spleen (left panel) and bone marrow (right panel) in NSG mice injected with PBMCs (5x106 cells / mouse), polydonor (3 donors; (BC-C / E / F)), CD4IL-10 cells (5x106 cells / mouse), or PBMCs (5x106 cells / mouse) in combination with polydonor CD4IL-10 cells (3 donors; (BC-C / E / F)) (5x106 cells / mouse). Box plots of n=8 animals tested are shown. [Figure 15] FIG. 15 illustrates the protocol for testing the induction of GvHD by CD4+ T cells and polydonor (BC-H / I / L) or single donor (BC-H) CD4IL-10 cells injected 3 days after radiation. [Figure 16] FIG. 16 shows the percentage of NSG mice free of GvHD on each day after injection. [Figure 17-A] Figures 17A-17C show graft-versus-leukemia (GvL) effects tested based on reduction of circulating leukemia cells and long-term leukemia-free survival. Leukemia was measured as previously described (Locafaro G. et al Molecular Therapy 2017). NSG mice were sublethally irradiated and intravenously injected with myeloid leukemia cells (ALL-CM) (2.5x106 cells) on day 0. Figure 17A is illustrative of the experiment. Figure 17B shows leukemia-free survival in animals injected with PBMCs (2.5x106 cells) or single donor (from donors BC-I and BC-H) CD4IL-10 cells (2.5x106 cells) on day 3. FIG. 17C shows leukemia-free survival in animals injected on day 3 with PBMCs (2.5×10 6 ) or polydonor CD4IL-10 cells (from donors BC-I and BC-H) (2.5×10 6 ). [Figure 17-B] Same as Figure 17-A. [Figure 17-C] Same as Figure 17-A. [Figure 18-A]Figures 18A-18C show long-term leukemia-free survival measured in sublethally irradiated NSG mice intravenously injected with ALL-CM cells (2.5x106) on day 0. Figure 18A is illustrative of the experiment. Figure 18B shows data from animals injected on day 3 with mononuclear cells (PBMC) (2.5x106) alone or with mononuclear cells (PBMC) (2.5x106) + single donor (from donors BC-H and BC-I) CD4IL-10 cells (2.5x106). Figure 18C shows data from animals injected on day 3 with mononuclear cells (PBMC) (2.5x106) alone or with mononuclear cells (PBMC) (2.5x106) + polydonor CD4IL-10 cells (BC-I / H) (2.5x106). [Figure 18-B] Same as Figure 18-A. [Figure 18-C] Same as Figure 18-A. [Figure 19-A]Figures 19A-19G show the inhibition of NLPR3 inflammasome activation by CD4IL-10 cells. Figure 19A shows the effect of CD4IL-10 cell supernatant from a single donor (#1) on the production of IL-1β by LPS-activated monocytes. Figure 19B shows the effect of CD4IL-10 cell supernatant from another single donor (#2) on the production of IL-1β by LPS-activated monocytes. Figure 19C shows the effect of CD4IL-10 cell supernatant from a single donor (#1) on the inhibition of LPS-induced IL-1β production enhanced by the NLPR3 inflammasome activator nigericin (NIG). Figure 19D shows the effect of CD4IL-10 cell supernatant from a single donor (#2) on the inhibition of LPS-induced IL-1β production enhanced by the NLPR3 inflammasome activator nigericin (NIG). Figure 19E is a bar graph showing the effect of CD4IL-10 cell supernatants from a single donor (BC-E) and from cells pooled from two different donors (BC-C / E) on LPS-induced IL-1β production by monocytes in the presence or absence of anti-IL-10 receptor (anti-IL-10R) mAb. Figure 19F shows the effect of polydonor CD4IL-10 cell (BC-T / U / V) supernatants on IL-1β production by monocytes in the presence or absence of anti-IL-10R mAb. Figure 19G shows the effect of polydonor CD4IL-10 cell (BC-T / U / V) supernatants on IL-18 production induced by LPS in combination with nigericin in the presence or absence of anti-IL-10R mAb. [Figure 19-B] Same as Figure 19-A. [Figure 19-C] Same as Figure 19-A. [Figure 19-D] Same as Figure 19-A. [Figure 19-E] Same as Figure 19-A. [Figure 19-F] Same as Figure 19-A. [Figure 19-G] Same as Figure 19-A. [Figure 20]Figure 20 illustrates the experimental protocol for testing graft-versus-myeloid leukemia and xeno-GvHD effects. NSG mice were intravenously injected with ALL-CM cells (2.5x106) on day 0. On day 3, mice were divided into 5 groups and treated with (i) no cells as control, (ii) allogeneic mononuclear cells (PBMCs); (iii) allogeneic PBMCs and polydonor CD4IL-10 cells (1:1:1 pooled BC-V / T / E); (iv) allogeneic PBMCs and single donor CD4IL-10 cells (BC-E); or (v) polydonor CD4IL-10 cells (BC-V / T / E) at the concentrations indicated in Figure 20. [Figure 21] FIG. 21 is a bar graph depicting the cytokine secretion profile of single donor (BC-V, BC-T, BC-V, and BC-E) and polydonor CD4IL-10 cells (POOL: 1:1:1 pool of BC-E, BC-V, and BC-T). [Figure 22] Figure 22 shows the suppressive effect of single donor (BC-V and BC-E) and polydonor CD4IL-10 cells (pool of BC-V / T / E) on the in vitro proliferation of allogeneic CD4+ and CD8+ T cells. Allogeneic PBMC cells were labeled with eFluor® 670 (5x104 cells / well) and stimulated with allogeneic mature dendritic (DC) cells (1x104 cells / well) and soluble anti-CD3 mAb at a 1:1 responder:suppressor ratio in the absence or presence of CD4IL-10 cells (5x104 cells / well). After 3 days of culture, the percentage of proliferative responder cells was determined by dilution of eFluor® 670 by flow cytometry after gating on CD4+ ΔNGFR- (top) or CD8+ΔNGFR- (bottom) T cells. Figure 22 shows the results of pooled cells from single donors BC-V and BC-E, and donors BC-V / T / E. The percentage of proliferation and suppression is shown. The suppression mediated by CD4IL-10 cells was calculated as follows: 100-([proliferation of responders in the presence of CD4IL-10 cells / proliferation of responders alone] x 100). [Diagram 23]Figure 23 shows the % of viable cells in co-cultures of single (BC-E and BC-V) or polydonor CD4IL-10 cells (BC-V / T / E) with ALL-CM myeloid tumor cells or K562 cells. The results show the selective cytotoxic effect of single donor and polydonor CD4IL-10 cells against ALL-CM myeloid tumor cells, but not against K562 cells, which lack class I MHC expression. [Figure 24] Figure 24 shows leukemia-free survival measured in NSG mice intravenously injected with ALL-CM cells (2.5x106) on day 0. On day 3, mice were divided into 5 groups and each group was treated with (i) no cells as control, (ii) allogeneic mononuclear cells (PBMCs); (iii) allogeneic PBMCs and polydonor CD4IL-10 cells (BC-V / T / ET); (iv) allogeneic PBMCs and single donor CD4IL-10 cells (BC-E); or (v) polydonor CD4IL-10 cells (BC-V / T / E). The graph shows the leukemia-free survival of animals in each group. [Diagram 25] FIG. 25 shows the % of NSG mice free of GvHD on each day following injection of ALL-CM cells (2.5×106), followed by administration of (i) no cells as a control; (ii) allogeneic mononuclear cells (PBMCs); (iii) allogeneic PBMCs and polydonor CD4IL-10 cells (BC-V / T / E); (iv) allogeneic PBMCs and single donor CD4IL-10 cells (BC-E), or (v) polydonor CD4IL-10 cells on day 3. [Figure 26]Figure 26 shows that all NSG mice administered 2.5E+06 PBMCs (allogeneic for donors C, E, F and H) died on day 22 due to acute, lethal xeno-GvHD. Administration of single-donor CD4IL-10 cells (lot C) or polydonor CD4IL-10 cells (lot CEFH) in combination with PBMCs prevented the development of lethal xeno-GvHD in 75% (3 / 4 mice) and 80% (4 / 5 mice), respectively. In contrast, introduction of 2.5E+06 polydonor CD4IL-10 cells did not induce any signs of GvHD. Taken together, these results indicate that polydonor CD4IL-10 cells from four different donors suppress pathogenic human T cell responses more potently or slightly more potently than single-donor CD4IL-10 cells. PBMCs: peripheral blood mononuclear cells; GvHD: graft-versus-host disease. [Figure 27-A] FIG. 27A shows an alignment of IL-10 protein sequences of various species including human (SEQ ID NO: 1); Mus musculus, "MOUSE" (SEQ ID NO: 10); Rattus norvegicus, "RAT" (SEQ ID NO: 11); Macaca mulatta, "MACMU" (SEQ ID NO: 12); Gorilla gorilla, "GORILLA" (SEQ ID NO: 13); Macaca fascicularis, "CYNO" (SEQ ID NO: 14); Papio Anubis, "OLIVE BABOON" (SEQ ID NO: 15); Pan paniscus, "BONOBO" (SEQ ID NO: 16); Pan troglodytes, "CHIMP" (SEQ ID NO: 17); or EBVB9 (SEQ ID NO: 18). [Figure 27-B]Figure 27B provides sequences of IL-10 variants generated by replacing one or more amino acids of human IL-10 with amino acids of viral IL-10 (EBVB9) at the corresponding positions. Also provided are sequences of exemplary variants potential huIL-10 hybrid #1 (SEQ ID NO: 19) and potential huIL-10 hybrid #2 (SEQ ID NO: 20). "*" indicates one or more amino acid positions that have been replaced. "#" indicates the preferred I105 to A105 amino acid substitution for IL-10 hybrid #2 (SEQ ID NO: 20). [Figure 27-C] Figure 27C shows an alignment of human IL-10 (SEQ ID NO: 1) with IL10 EBVB9 (SEQ ID NO: 18). "*" indicates one or more amino acid positions that are substituted in IL-10 Hybrid #1. "#" indicates the preferred I105 to A105 amino acid substitution for IL-10 Hybrid #2. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0096] The drawings depict various embodiments of the present invention for illustrative purposes only. Those skilled in the art will readily recognize from the following discussion that alternative embodiments of the structures and methods illustrated herein may be used without departing from the principles of the present invention as described herein.

[0097] 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 below.

[0098] "Graft-versus-leukemia effect" or "GvL" refers to an effect that occurs after allogeneic hematopoietic stem cell transplantation (HSCT) or bone marrow transplantation (BMT): T lymphocytes in the allograft eliminate residual malignant host leukemia cells.

[0099] "Graft-versus-tumor effect" or "GvT" refers to an effect that appears after allogeneic hematopoietic stem cell transplantation (HSCT) or bone marrow transplantation (BMT). T lymphocytes in the allograft eliminate malignant residual host cancer cells, such as myeloma and lymphocytic and myeloid leukemias, lymphomas, multiple myeloma, and possibly breast cancer cells. The term GvT is a general form of GvL.

[0100] The terms "treatment", "treat" and the like are used herein in their broadest sense as understood in the medical arts. In particular, the term generally means to obtain a desired pharmacological and / or physiological effect. "Treatment", as used herein, includes within its scope any treatment of a disease or condition in a mammal, particularly a human, including (a) preventing the disease or condition from occurring in a subject who may be predisposed to the disease or condition but has not yet been diagnosed as having it; (b) inhibiting the disease or condition (e.g., halting its development); or (c) alleviating the disease or condition (e.g., causing regression of the disease or condition, providing improvement of one or more symptoms). Improvement of any condition can be easily assessed according to standard methods and techniques known in the art. The subject population to be treated by the disease method includes subjects suffering from an undesirable condition or disease, as well as subjects at risk of developing a condition or disease.

[0101] "HLA matched" as used herein refers to a pair of individuals with matching HLA alleles at HLA class I (HLA-A, HLA-B, and HLA-C) and class II (HLA-DRB1 and HLA-DQB1) loci, making the individuals immunologically compatible with each other.HLA compatibility can be determined using any method available in the art, such as the method described in Tiervy, Haematologica 2016 Volume 101(6):680-687, which is incorporated herein by reference.

[0102] 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 ten alleles of one individual (two at each of the HLA-A, HLA-B, HLA-C, HLA-DRB1, and HLA-DQB1 loci) match all ten alleles of the other individual.

[0103] In a preferred embodiment, allele level typing is used to determine HLA compatibility. Allele level typing involves typing all digits in the first, second, third and fourth regions, e.g., A * 02:01:01:01 corresponds to a unique nucleotide sequence of an HLA gene, defined by using the nucleotide sequence 02:01:01:01. Functionally, the third and fourth regions, which characterize alleles that differ by silent substitutions in coding sequences and substitutions in non-coding sequences, respectively, are considered to be the only alleles that are expressed when the substitution prevents expression of the HLA allele (e.g., the null allele B * 15:01:01:02N). Deletion of the invalid allele 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., A24low allele A * 24:02:01:02L). Such diversity may also affect anti-HLA allorecognition.

[0104] The term "HLA mismatched," as used herein, 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, rendering the individuals immunologically incompatible with each other.

[0105] The term "partial HLA mismatch" as used herein refers to a pair of individuals with mismatched HLA alleles at HLA class I (HLA-A, HLA-B, and HLA-C) and class II (HLA-DRB1 and HLA-DQB1) loci, making the individuals immunologically incompatible to an acceptable degree. Some studies have identified acceptable mismatches. Some HLA class I mismatches are considered to be more acceptable.

[0106] "HLA haplotype" refers to a set of HLA locus alleles, 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.

[0107] The term "therapeutically effective amount" is an amount that is effective to treat and thus ameliorate the symptoms of a disease.

[0108] The term "prophylactically effective amount" is an amount effective with respect to complete or partial prevention of a disease, condition, or symptoms thereof. The term "ameliorating" refers to any therapeutically beneficial outcome in the treatment of a disease state, such as a neurodegenerative disease state, including prevention, attenuation of the severity or progression, remission, or cure thereof.

[0109] 6.2. Other Interpretive Conventions Ranges recited herein are understood to be shorthand for all values ​​within the range, including the recited endpoints. For example, the range 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.

[0110] 6.3. Polydonor CD4IL-10 cell In a first aspect, a CD4+ antibody that has been genetically modified to include an exogenous polynucleotide encoding IL-10 is provided. + Provides a T cell population (CD4 IL-10 The population consisted of CD4 T cells from at least two different T cell donors. + T cells (polydonor CD4 IL-10 cells).

[0111] 6.3.1.CD4 + T Cells and T Cell Donors Polydonor CD4 IL-10 CD4 used in the 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 + The T cells are isolated from peripheral blood, preferably from a human donor. In certain embodiments, CD4 + T cells are isolated from peripheral blood by leukopheresis. In certain embodiments, CD4 + The T cells are obtained from a third-party blood bank. In one particular embodiment, the CD4 + T cells are obtained from the buffy coat from centrifugation of whole blood.

[0112] In some embodiments, CD4 + The T cells are isolated from pre-frozen stocks of blood or pre-frozen stocks of peripheral blood mononuclear cells (PBMCs). + T cells are isolated from peripheral blood or PBMCs that have not been previously frozen. In some embodiments, CD4 + T cells are individually isolated from blood or PBMCs from multiple donors and then pooled. In some embodiments, CD4 + T cells are isolated from blood or PBMCs that are initially pooled from multiple donors.

[0113] In some embodiments, CD4 +The T cells are obtained from 3, 4, 5, 6, 7, 8, 9, or 10 different T cell donors.

[0114] In some embodiments, the at least two different T cell donors are selected without regard to genotype. In some embodiments, the at least two different T cell donors are selected based on genotype.

[0115] In certain embodiments, the at least two different T cell donors are selected based on their HLA haplotypes.

[0116] In some embodiments, some or all of the at least two different T cell donors have matching HLA haplotypes. In some embodiments, some or all of the at least two different T cell donors have mismatched HLA haplotypes.

[0117] In some embodiments, the CD4 + The T cells all have at least a 1 / 10, 2 / 10, 3 / 10, 4 / 10, 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. + All of the T cells have at least 1 / 8, 2 / 8, 3 / 8, 4 / 8, 5 / 8, 6 / 8, 7 / 8, or 8 / 8 matches to each other at the HLA-A, HLA-B, HLA-C, and HLA-DRB1 loci. + All T cells have a 2 / 2 match to each other at the HLA-A locus. + All T cells have a 2 / 2 match to each other at the HLA-B locus. + All T cells have a 2 / 2 match to each other at the HLA-C locus. + All of the T cells have at least a 3 / 4 or 4 / 4 match to each other at the HLA-DRB1 and HLA DQB1 loci.+ All T cells are A * 02 or A * There are 24 alleles.

[0118] In some embodiments, all of the CD4 + The T cells have less than 5 / 10, 6 / 10, 7 / 10, 8 / 10, or 9 / 10 matches to each other at the HLA-A, HLA-B, HLA-C, HLA-DRB1, and HLA-DQB1 loci. + The T cells have less than 4 / 8, 5 / 8, 6 / 8, 7 / 8, or 8 / 8 matches to each other at the HLA-A, HLA-B, HLA-C, and HLA-DRB1 loci. + The T cells have less than a 2 / 2 match to each other at the HLA-A locus. In some embodiments, all of the CD4 + The T cells have less than a 2 / 2 match to each other at the HLA-B locus. In some embodiments, all of the CD4 + The T cells have less than a 2 / 2 match to each other at the HLA-C locus. In some embodiments, all of the CD4 + The T cells have less than a 3 / 4 or 4 / 4 match to each other at the HLA-DRB1 and HLA DQB1 loci.

[0119] In some embodiments, the CD4 + The T cells all have less than 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. + All of the T cells have less than 4 / 8, 5 / 8, 6 / 8, 7 / 8, or 8 / 8 matches to each other at the HLA-A, HLA-B, HLA-C, and HLA-DRB1 loci. + All of the T cells have less than a 2 / 2 match to each other at the HLA-A locus. +All of the T cells have less than a 2 / 2 match with each other at the HLA-B locus. + All of the T cells have less than a 2 / 2 match to each other at the HLA-C locus. + All T cells have a 3 / 4 or less than 4 / 4 match with each other at the HLA-DRB1 and HLA-DQB1 loci.

[0120] In a preferred embodiment, at least two different T cell donors each have a CD4 IL-10 In a preferred embodiment, at least two different T cell donors are not the host treated by the cells. IL-10 Not the donor of the stem cells (eg, HSCs), tissues or organs used with the cells.

[0121] In some embodiments, one or more of the T cell donors are HLA-mismatched or partially HLA-mismatched to the patient (host) to be treated. In some embodiments, one or more of the T cell donors have less than 5 / 10, 6 / 10, 7 / 10, 8 / 10, 9 / 10, or 10 / 10 matches to the patient at the HLA-A, HLA-B, HLA-C, HLA-DRB1, and HLA-DQB1 loci. In some embodiments, one or more of the T cell donors have less than 4 / 8, 5 / 8, 6 / 8, 7 / 8, or 8 / 8 matches to the patient at the HLA-A, HLA-B, HLA-C, and HLA-DRB1 loci. In some embodiments, one or more of the T cell donors have less than 2 / 2 matches to the patient at the HLA-A, HLA-B, or HLA-C loci. In some embodiments, 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.

[0122] In some embodiments, one or more of the T cell donors are HLA-mismatched or partially HLA-mismatched to the HSC donor. In some embodiments, one or more of the T cell donors have less than 5 / 10, 6 / 10, 7 / 10, 8 / 10, 9 / 10, or 10 / 10 matches to the HSC donor at the HLA-A, HLA-B, HLA-C, HLA-DRB1, and HLA-DQB1 loci. In some embodiments, one or more of the T cell donors have less than 4 / 8, 5 / 8, 6 / 8, 7 / 8, or 8 / 8 matches to the HSC donor at the HLA-A, HLA-B, HLA-C, and HLA-DRB1 loci. In some embodiments, one or more of the T cell donors have less than 2 / 2 matches to the HSC donor at the HLA-A, HLA-B, or HLA-C loci. In some embodiments, 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.

[0123] In a preferred embodiment, CD4 + None of the T cells are immortalized.

[0124] 6.3.2. Exogenous Polynucleotides Encoding IL-10 Polydonor CD4 of the present disclosure IL-10 The cells are CD4+ cells that have been genetically modified to contain an exogenous polynucleotide encoding IL-10. + The exogenous polynucleotide comprises an IL-10-encoding polynucleotide segment operably linked to an expression control element.

[0125] The IL-10-encoding polynucleotide segment may encode human, bonobo, or rhesus IL-10. In some embodiments, the IL-10-encoding polynucleotide segment encodes human IL-10 having the sequence of SEQ ID NO:1. In some embodiments, the IL-10-encoding polynucleotide segment 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 IL-10-encoding polynucleotide segment has the nucleotide 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.

[0126] In some embodiments, the IL-10-encoding polynucleotide segment encodes IL-10 from Mus musculus, "MOUSE" (SEQ ID NO: 10); Rattus norvegicus, "RAT" (SEQ ID NO: 11); Macaca mulatta, "MACMU" (SEQ ID NO: 12); Gorilla gorilla, "GORILLA" (SEQ ID NO: 13); Macaca fascicularis, "CYNO" (SEQ ID NO: 14); Papio Anubis, "OLIVE BABOON" (SEQ ID NO: 15); Pan paniscus, "BONOBO" (SEQ ID NO: 16); Pan troglodytes, "CHIMP" (SEQ ID NO: 17); and EBVB9 (SEQ ID NO: 18). In some embodiments, the IL-10-encoding polynucleotide segment encodes a protein having at least 90%, 95%, 98%, or 99% sequence identity to IL-10 from Mus musculus, "MOUSE" (SEQ ID NO: 10); Rattus norvegicus, "RAT" (SEQ ID NO: 11); Macaca mulatta, "MACMU" (SEQ ID NO: 12); Gorilla gorilla, "GORILLA" (SEQ ID NO: 13); Macaca fascicularis, "CYNO" (SEQ ID NO: 14); Papio Anubis, "OLIVE BABOON" (SEQ ID NO: 15); Pan paniscus, "BONOBO" (SEQ ID NO: 16); Pan troglodytes, "CHIMP" (SEQ ID NO: 17); and EBVB9 (SEQ ID NO: 18).

[0127] 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.

[0128] In some embodiments, the polynucleotide segment encoding IL-10 encodes a variant of human IL-10 having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acid modifications compared to human IL-10 (e.g., SEQ ID NO: 1). In some embodiments, the 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions are substitution(s) with amino acid(s) of viral IL-10 at the corresponding amino acid position(s). In some embodiments, the polynucleotide segment encoding IL-10 encodes a variant of human IL-10 having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acid insertions, deletions, or modifications compared to human IL-10 (e.g., SEQ ID NO: 1). In some embodiments, the variant of human IL-10 has the sequence of SEQ ID NO: 8 or 9.

[0129] In some embodiments, the polynucleotide segment encoding IL-10 encodes a variant of human IL-10 having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid substitutions, insertions, and / or deletions compared to human IL-10 (e.g., SEQ ID NO:1). In some embodiments, the modification is a substitution, insertion, and / or deletion at the corresponding positions with amino acids from Mus musculus, "MOUSE" (SEQ ID NO: 10); Rattus norvegicus, "RAT" (SEQ ID NO: 11); Macaca mulatta, "MACMU" (SEQ ID NO: 12); Gorilla gorilla, "GORILLA" (SEQ ID NO: 13); Macaca fascicularis, "CYNO" (SEQ ID NO: 14); Papio Anubis, "OLIVE BABOON" (SEQ ID NO: 15); Pan paniscus, "BONOBO" (SEQ ID NO: 16); Pan troglodytes, "CHIMP" (SEQ ID NO: 17); and EBVB9 (SEQ ID NO: 18). In some embodiments, the variant of human IL-10 has the sequence of SEQ ID NO: 19 or SEQ ID NO: 20.

[0130] In some embodiments, the polynucleotide segment encoding IL-10 encodes a variant of human IL-10 that has reduced immunostimulatory activity compared to human IL-10. In some embodiments, the variant of human IL-10 comprises an I105A substitution. In some embodiments, the variant of human IL-10 is produced using the method described in A Single Amino Acid Determines the Immunostimulatory Activity of Interleukin 10, J Exp Med, 191, 2, 2000, p.213-223.

[0131] The exogenous polynucleotide is expressed in a transduced CD4 + It further comprises an expression control element that directs expression of the encoded IL-10 in T cells.

[0132] In some embodiments, the expression control element is CD4 + In some embodiments, the promoter comprises a promoter capable of directing expression of IL-10 in T cells. + In some embodiments, the promoter drives constitutive expression of IL-10 in T cells. + Drives expression of IL-10 in T cells.

[0133] 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.

[0134] In some embodiments, a native human promoter is used. In some embodiments, a human elongation factor (EF) 1 alpha promoter is used. In some embodiments, a human phosphoglycerate kinase promoter (PGK) is used. In some embodiments, a human ubiquitin C promoter (UBI-C) is used.

[0135] 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 a specific embodiment, a synthetic bidirectional promoter is used as disclosed in Amendola et al., Nature Biotechnology, 23(1):108-116(2005). This promoter can mediate the coordinated transcription of two mRNAs in a ubiquitous or tissue-specific manner. In certain embodiments, the bidirectional promoter induces the expression of IL-10 and a selection marker.

[0136] In some embodiments, the exogenous polynucleotide comprises a polypeptide that is capable of transducing successfully CD4+ The nucleotide sequence encoding the ΔNGFR selection marker further comprises a segment that allows for selection of the T cell. In some embodiments, the selection marker is ΔNGFR. In certain embodiments, the selection marker is a polypeptide having the sequence of SEQ ID NO:3. In certain embodiments, the selection marker is a polypeptide having at least 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO:3. In certain embodiments, the nucleotide sequence encoding the ΔNGFR selection marker has the sequence of SEQ ID NO:4. In some embodiments, the nucleotide sequence encoding the ΔNGFR selection marker has at least 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO:4.

[0137] In some embodiments, the expression of the selection marker is correlated with the expression of IL-10 from the exogenous polynucleotide. In some embodiments, the expression of the selection marker is linearly correlated with the expression of IL-10 from the exogenous polynucleotide. Thus, in some embodiments, the expression of the selection marker is measured to estimate the expression of IL-10 from the exogenous polynucleotide.

[0138] In some embodiments, the selection marker is a truncated form of an EGFR polypeptide. In some embodiments, the selection marker is a truncated form of a human EGFR polypeptide, huEGFR, as 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 hereby incorporated by reference in its entirety.

[0139] 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.

[0140] In an exemplary embodiment, the exogenous polynucleotide is delivered to a CD4 + In some embodiments, the vector is a plasmid vector. In some embodiments, the vector is a viral vector.

[0141] In certain embodiments, the exogenous polynucleotide is delivered to CD4 + The exogenous polynucleotide delivered to the T cell 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.

[0142] In some embodiments, the lentiviral vector is capable of integrating into the T cell nuclear genome. In some embodiments, the lentiviral vector is not capable of integrating into the T cell nuclear genome. In some embodiments, an integration-deficient lentiviral vector is used. For example, in some embodiments, an integration-deficient or other lentiviral vector disclosed in Matrai is used. In some embodiments, an integrase-deficient lentivirus is used. For example, an integrase-deficient lentivirus containing an inactivating mutation (D64V) in integrase, as described in Matrai et al., Hepatology 53:1696-1707 (2011), which is incorporated herein by reference, can be used.

[0143] 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.

[0144] 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.

[0145] Polydonor CD4 IL-10 T cell gene expression Polydonor CD4 IL-10 The T cells express IL-10. In some embodiments, the polydonor CD4 IL-10 The T cells constitutively express IL-10. In some embodiments, the polydonor CD4 IL-10 Upon activation, T cells express IL-10.

[0146] In some embodiments, the polydonor CD4 IL-10 T cells are CD4 + T cells 10 6 In some embodiments, the polydonor CD4 IL-10 T cells are CD4 + T cells 10 6 Constitutively express at least 200 pg, 500 pg, 1 ng, 5 ng, 10 ng, or 50 ng of IL-10 per cell / mL culture medium.

[0147] In some embodiments, the polydonor CD4 IL-10 T cells were activated with a combination of anti-CD3 and anti-CD28 antibodies or with a combination of anti-CD3 and anti-CD28 antibody-coated beads, followed by CD4 + T cells 10 6 In some embodiments, the polydonor CD4 IL-10 T cells were activated with anti-CD3 and anti-CD28 antibodies, or CD3 and CD28 antibody-coated beads, followed by CD4 + T cells 10 6 Express at least 5 ng, 10 ng, 100 ng, 200 ng, or 500 ng of IL-10 per cell / mL culture medium.

[0148] In various embodiments, IL-10 production is determined 12 hours, 24 hours, or 48 hours after activation using various methods for protein detection and measurement, such as ELISA, spectroscopic procedures, colorimetry, amino acid analysis, radiolabeling, Edman degradation, HPLC, Western blotting, etc. In a preferred embodiment, IL-10 production is determined by ELISA 48 hours after activation with anti-CD3 and anti-CD28 antibodies.

[0149] In some embodiments, the polydonor CD4 IL-10 T cells are unmodified CD4 + In some embodiments, polydonor 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.

[0150] In some embodiments, the polydonor CD4 IL-10 The T cells further express a selection marker. In some embodiments, the polydonor CD4 IL-10 The T cells express proteins typically expressed in Tr1 cells. In some embodiments, the polydonor CD4 IL-10 T cells express marker proteins characteristic of Tr1 cells.

[0151] In some embodiments, the polydonor CD4 IL-10 The T cells express CD49b. In some embodiments, the polydonor CD4 IL-10 The T cells express LAG-3. In some embodiments, the polydonor CD4 IL-10 The T cells express TGF-β. In some embodiments, the polydonor CD4 IL-10 The T cells express IFNγ. In some embodiments, the polydonor CD4 IL-10 The T cells express granzyme B (GzB). In some embodiments, the polydonor CD4 IL-10T cells release granzyme B (GzB) when activated by myeloid antigen-presenting cells or myeloid tumor cells. In some embodiments, polydonor CD4 IL-10 The T cells express perforin. In some embodiments, the polydonor CD4 IL-10 T cells release perforin when activated by myeloid antigen-presenting cells or myeloid tumor cells. In some embodiments, polydonor CD4 IL-10 The T cells express CD18. In some embodiments, the polydonor CD4 IL-10 The T cells express CD2. In some embodiments, the polydonor CD4 IL-10 The T cells express CD226. In some embodiments, the polydonor CD4 IL-10 The T cells express IL-22. In some embodiments, the polydonor CD4 IL-10 T cells express IL-10.

[0152] In some embodiments, the polydonor CD4 IL-10 The T cells exhibit at least one phenotypic function of Tr1 cells, which in various embodiments is the secretion of IL-10, the secretion of TGF-β, and the specific killing of myeloid antigen-presenting cells through the release of granzyme B (GzB) and perforin.

[0153] 6.3.4. Process Products In an exemplary embodiment, polydonor CD4 IL-10 T cells are CD4 + This is achieved by modifying T cells with an exogenous polynucleotide encoding IL-10.

[0154] 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.

[0155] In some embodiments, the polydonor CD4IL-10 T cells were (i) primary CD4 + (ii) pooling the pooled CD4 + In some embodiments, the polydonor CD4 T cells are generated by modifying the T cells by introducing an exogenous polynucleotide encoding IL-10. IL-10 T cells were (i) primary CD4 + (ii) obtaining CD4 T cells from each donor; + T cells by introducing an exogenous polynucleotide encoding IL-10, and then (iii) genetically modifying CD4 + T cells.

[0156] In some embodiments, the polydonor CD4 IL-10 T cells are CD4 + In some embodiments, the polydonor CD4 IL-10 The T cells are cultured in the presence of anti-CD3 and anti-CD28 antibodies, or anti-CD3 and anti-CD28 antibody coated beads. 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. IL-10 The T cells are cultured in the presence of Miltenyi Biotec's T Cell TransAct™. In some embodiments, polydonor CD4 IL-10 T cells are cultured in the presence of ImmunoCult Human T Cell Activator™ from STEMCELL Technologies.

[0157] In some embodiments, the polydonor CD4 IL-10 The T cells are in frozen storage.

[0158] Pharmaceutical Compositions In another aspect, a pharmaceutical composition is provided. The pharmaceutical composition comprises a polydonor CD4 IL-10 Comprising T cells and a pharma- ceutically acceptable carrier or diluent.

[0159] The pharmaceutical composition can be formulated for administration by any route of administration suitable for human or veterinary medicine. In a typical embodiment, the composition is formulated for intravenous (IV) administration. In some embodiments, the composition is formulated for intravenous (IV) infusion. In the embodiment formulated for IV administration, the pharmaceutical composition is in the form of a pyrogen-free, parenterally acceptable aqueous solution with suitable pH, isotonicity, and stability.

[0160] In some embodiments, the pharma- ceutically 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.

[0161] In some embodiments, a unit dosage form of the pharmaceutical composition is provided that is adapted for administering the pharmaceutical composition by systemic administration, in particular intravenous administration.

[0162] In some embodiments, the unit dosage form comprises 10 4 ~10 11 Polydonor CD4 IL-10 T cells, 10 4 ~10 10 Polydonor CD4 IL-10 T cells, 10 4 ~10 9 Polydonor CD4 IL-10 T cells, 10 5 ~10 10 Polydonor CD4 IL-10 T cells, 10 5 ~10 9 Polydonor CD4 IL-10 T cells, 10 5 ~10 8 Polydonor CD4IL-10 T cells, or 10 5 ~10 7 Polydonor CD4 IL-10 Contains T cells.

[0163] In an exemplary embodiment, the pharmaceutical composition in unit dosage form is in liquid form.

[0164] 6.5. Polydonor CD4 IL-10 How to make cells In another aspect, the present disclosure provides a polydonor CD4 IL-10 A method for producing a cell is provided.

[0165] In some embodiments, the method comprises: (i) determining whether primary cultured CD4 T cells are present in a human T cell donor; + (ii) pooling the pooled CD4 + In another embodiment, the method comprises: (i) modifying T cells by introducing an exogenous polynucleotide encoding IL-10. + (ii) obtaining CD4 T cells from each donor; + (iii) individually modifying the T cells by introducing an exogenous polynucleotide encoding IL-10; and then (iv) genetically modifying the CD4 + T cells are pooled, thereby generating polydonor CD4 IL-10 The step of obtaining cells includes administering an exogenous polynucleotide encoding IL-10 to primary cultured CD4 + It can be introduced into T cells.

[0166] In some embodiments, the method comprises culturing primary CD4 + T cells or genetically modified CD4 + In some embodiments, the method further comprises incubating the T cells in the presence of anti-CD3 and anti-CD28 antibodies, or anti-CD3 and anti-CD28 antibody coated beads. +T cells or genetically modified CD4 + In some embodiments, the method further comprises incubating the T cells in the presence of an anti-CD3 antibody, an anti-CD28 antibody and IL-2, or an anti-CD3 antibody and an anti-CD28 antibody coated bead 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 feeder cell mixture. + T cells or genetically modified CD4 + The method further comprises incubating the T cells in the presence of a nanopreparation of anti-CD3 and anti-CD28 antibodies. In some embodiments, the incubation is performed in the presence of Miltenyi Biotec's T Cell TransAct™. In some embodiments, the incubation is performed in the presence of STEMCELL Technologies' ImmunoCult Human T Cell Activator™.

[0167] 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 prior to introducing an exogenous polynucleotide encoding IL-10. In some embodiments, the incubation step is performed prior to introducing an exogenous polynucleotide encoding IL-10. + (ii) after pooling of T cells; + In some embodiments, the incubation step is performed before modifying the T cells by introducing an exogenous polynucleotide encoding IL-10. + (ii) after T cells are obtained from at least two different T cell donors; and + This is performed before the T cells are individually modified by introducing an exogenous polynucleotide encoding IL-10.

[0168] 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) of inducing pooled CD4 + In some embodiments, the incubation step is performed after the step of modifying the T cells. In some embodiments, the incubation step is performed after the step of (ii) modifying the CD4 T cells of each donor by introducing an exogenous polynucleotide encoding IL-10. + After individually modifying T cells, (iii) genetically modified CD4 + T cells are pooled, thereby + In some embodiments, the incubation step is performed prior to obtaining the T cells. + T cells are pooled, thereby generating polydonor CD4 IL-10 This is performed after obtaining the cells.

[0169] In some embodiments, the incubation step is performed more than once. + This is performed both before and after genetic modification of the T cells.

[0170] In some embodiments, the exogenous polynucleotide is delivered to primary cultured CD4 +In some embodiments, the exogenous polynucleotide comprises a segment encoding IL-10 having the sequence of SEQ ID NO:1. In some embodiments, the exogenous polynucleotide comprises a segment encoding 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 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. In some embodiments, the exogenous polynucleotide comprises a segment encoding IL-10 having the sequence of SEQ ID NO:2. In some embodiments, the exogenous polynucleotide comprises a segment encoding IL-10 having at least 90%, 95%, 98%, or 99% sequence identity to ... + The exogenous polynucleotide further comprises a segment encoding a marker that allows for the selection of T cells. In some embodiments, the encoded selectable marker is ΔNGFR. In certain embodiments, the encoded selectable 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 selectable marker is a truncated form of the human EGFR polypeptide.

[0171] In some embodiments, the method comprises the step of: + Isolating T cells and thereby genetically modifying CD4 IL-10 The method further comprises the step of generating an enriched population of cells.

[0172] In some embodiments, the genetically modified CD4 + At least 70% of the T cells express the selection marker. In some embodiments, the genetically modified CD4 + At least 95% of the T cells express the selection marker. In some embodiments, the genetically modified CD4 + At least 96, 97, 98, or 99% of the T cells express the selectable marker.

[0173] 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 anti-CD3 and anti-CD28 antibody coated beads. 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 nano preparation 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.

[0174] In some embodiments, the method comprises genetically modifying CD4 + The method further comprises the step of freezing the T cells.

[0175] In some embodiments, primary cultured CD4 + The T cells are derived from a donor selected based on its HLA haplotype. In some embodiments, the method further comprises selecting a T cell donor by analyzing its genetic information. In some embodiments, the method comprises analyzing the genetic information or HLA haplotype of a potential T cell donor.

[0176] In some embodiments, primary cultured CD4 + T cells were cultured in primary CD4 + In some embodiments, the primary CD4 + The T cells are derived from a donor with at least a partial HLA match to the stem cell (HSC), tissue, or organ donor. In some embodiments, primary cultured 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 biologically unrelated to the stem cell, tissue, or organ donor.

[0177] In some embodiments, in step (i), primary cultured CD4 + The T cells are obtained from 2, 3, 4, 5, 6, 7, 8, 9, or 10 different T cell donors. In some embodiments, at least two T cell donors have at least 1 / 10, 2 / 10, 3 / 10, 4 / 10, 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. In some embodiments, at least two T cell donors have at least 1 / 8, 2 / 8, 3 / 8, 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. In some embodiments, at least two T cell donors have 2 / 2 matches with each other at the HLA-A locus. In some embodiments, at least two T cell donors have a 2 / 2 match to each other at the HLA-B locus. In some embodiments, at least two T cell donors have a 2 / 2 match to each other at the HLA-C locus. In some embodiments, at least two T cell donors have at least a 3 / 4 or 4 / 4 match to each other at the HLA-DRB1 and HLA-DQB1 loci. In some embodiments, each of the at least two T cell donors has an A * 02 or A * There are 24 alleles.

[0178] In some embodiments, at least two T cell donors have less than 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. In some embodiments, at least two T cell donors have less than 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. In some embodiments, at least two T cell donors have less than 2 / 2 matches with each other at the HLA-A locus. In some embodiments, at least two T cell donors have less than 2 / 2 matches with each other at the HLA-B locus. In some embodiments, at least two T cell donors have less than 2 / 2 matches with each other at the HLA-C locus. In some embodiments, at least two T cell donors have a 3 / 4 or less than 4 / 4 match to each other at the HLA-DRB1 and HLA-DQB1 loci.

[0179] In some embodiments, in step (i), primary cultured CD4 + The T cells are obtained from one or more cryopreservations. In some embodiments, in step (i), primary cultured CD4 + The T cells are obtained from unfrozen peripheral blood mononuclear cells of at least two different T cell donors. In some embodiments, the method comprises: + In some embodiments, the method further comprises isolating T cells from the peripheral blood mononuclear cells. + The T cells are obtained from a liquid suspension. In some embodiments, the liquid suspension is obtained from a previously frozen stock.

[0180] In some embodiments, CD4 from the donor + The T cells are contacted with the patient's antigen-presenting cells (monocytes, dendritic cells, or DC-10 cells) to generate allospecific CD4 + Generate T cells that are then engineered to produce high levels of IL-10 (allo-CD4 IL-10 cell).

[0181] In some embodiments, the method comprises detecting CD4 + In some embodiments, the method does not include anergizing the T cells. + The recombinant IL-10 protein does not include a step of anergizing T cells and inhibits the activation of CD4 + In some embodiments, the method comprises the step of determining whether a CD4 T cell is expressed in the presence of DC10 cells from a host. + It does not include a step of anergizing T cells.

[0182] 6.6. Polydonor CD4 IL-10 How to use the cells In yet another aspect, the present disclosure provides a method of treating a patient, comprising administering to a patient a polydonor CD4 IL-10 Methods are provided that include administering the cells or pharmaceutical composition to a patient in need of immune tolerance.

[0183] In some embodiments, the method comprises the step of: IL-10 It further includes a prior step of thawing the frozen suspension of cells.

[0184] In some embodiments, the polydonor CD4 IL-10 The cells, or pharmaceutical compositions prevent or reduce the severity of a pathogenic T cell response in a patient. In some embodiments, the polydonor CD4 IL-10 The cells or pharmaceutical compositions reduce inflammation. In some embodiments, the polydonor CD4 IL-10 The cells or pharmaceutical compositions enhance tissue repair. In some embodiments, the polydonor CD4 IL-10 The cells or pharmaceutical compositions enhance immune tolerance to self and non-pathogenic antigens and maintain homeostasis of the immune system. In some embodiments, the polydonor CD4 IL-10 The cells or pharmaceutical compositions down-regulate pathogenic T cell responses associated with organ transplantation, GvHD, and various autoimmune and inflammatory diseases. In some embodiments, the polydonor CD4 IL-10The cells or pharmaceutical compositions treat an autoimmune disease. In some embodiments, the polydonor CD4 IL-10 The cells or pharmaceutical compositions reduce hyperactivity of the NLPR3 inflammasome or reduce symptoms associated with hyperactivity of the NLPR3 inflammasome. IL-10 The cells or pharmaceutical compositions induce tumor cell death or reduce tumor growth. In some embodiments, the polydonor CD4 IL-10 The cells or pharmaceutical compositions increase disease-free survival (e.g., the absence of minimal residual disease). In some embodiments, the polydonor CD4 IL-10 The cells or pharmaceutical composition induce wound healing or tissue repair.

[0185] In some embodiments, the polydonor CD4 IL-10 The cells or pharmaceutical compositions are administered in an amount effective to prevent or reduce the severity of a pathogenic T cell response in a patient. IL-10 The cells or pharmaceutical compositions are administered in an amount effective to reduce inflammation. IL-10 The cells or pharmaceutical compositions are administered in an amount effective to enhance tissue repair. In some embodiments, the polydonor CD4 IL-10 The cells or pharmaceutical compositions are administered in an amount effective to enhance immune tolerance to self and pathogenic antigens and to maintain homeostasis of the immune system. In some embodiments, polydonor CD4 IL-10 The cells or pharmaceutical compositions are administered in an amount effective to downregulate pathogenic T cell responses associated with organ transplantation, GvHD and various autoimmune or inflammatory diseases. In some embodiments, polydonor CD4 IL-10 The cells or pharmaceutical compositions are administered in an amount effective to treat an autoimmune disease. In some embodiments, the polydonor CD4 IL-10 The cells or pharmaceutical compositions are administered in an amount effective to reduce NLPR3 inflammasome hyperactivity or to reduce symptoms associated with NLPR3 inflammasome hyperactivity.IL-10 The cells or pharmaceutical compositions are administered in an amount effective to induce tumor cell death or reduce tumor growth. In some embodiments, the polydonor CD4 IL-10 The cells or pharmaceutical composition are administered in an amount effective to increase disease-free survival (eg, the absence of minimal residual disease).

[0186] In some embodiments, the method of treatment comprises administering to the patient a polydonor CD4 IL-10 In some embodiments, the method further comprises detecting a selectable marker in a biological sample obtained from a patient, thereby detecting a polydonor CD4 IL-10 In some embodiments, the selection marker comprises detecting the presence or absence of polydonor CD4 T cells in the patient. IL-10 The cells are detected at multiple time points to track changes in the presence of the cells. In some embodiments, the biological sample is a biopsy or a blood sample from the patient.

[0187] Polydonor CD4 IL-10 The T cells are administered in a therapeutically effective amount. The amount can be determined based on body weight and other clinical factors. In some embodiments, the cells 10 3 ~10 9 In some embodiments, 10 cells / kg are administered. 3 ~10 8 In some embodiments, 10 cells / kg are administered. 3 ~10 7 In some embodiments, 10 cells / kg are administered. 3 ~10 6 In some embodiments, 10 cells / kg are administered. 3 ~10 5 In some embodiments, 10 cells / kg are administered. 3 ~10 4 Pieces / kg are administered.

[0188] In various embodiments, polydonor CD4 IL-10The T cells are administered in a therapeutically effective schedule. IL-10 The T cells are administered once. In some embodiments, the polydonor CD4 IL-10 The cells are administered daily, every 3 days, every 7 days, every 14 days, every 21 days, or monthly.

[0189] Polydonor CD4 IL-10 The T cells may 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.

[0190] In some embodiments, the polydonor CD4 IL-10 The administration of is prophylactic in that it prevents, either completely or partially, a disease, condition, or a symptom thereof.

[0191] 6.6.1. Ways to reduce or prevent GvHD In some embodiments, the polydonor CD4 IL-10 Cell or polydonor CD4 IL-10 Pharmaceutical compositions comprising the cells are used to treat patients prior to, concurrently with, or following hematopoietic stem cell (HSC) transplantation (HSCT).

[0192] 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.

[0193] In some embodiments, the patient has a hematological malignancy that requires treatment with allo-HSCT. In some embodiments, the hematological malignancy is mediated by abnormal myeloid cells.

[0194] In some embodiments, the T cell donor is a polydonor CD4 IL-10The 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 polydonor CD4 IL-10 The HLA haplotype of the patient to be treated with the cells and HSCs and / or the HLA haplotype of the HSC donor are selected. IL-10 The method further comprises analyzing the genetic information or HLA haplotype of the T cell donor before administering the cells. 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.

[0195] In some embodiments, the T cell donor, the host and the HSC donor are biologically unrelated.In some embodiments, the T cell donor, the host and the HSC donor have different HLA haplotypes.In some embodiments, the T cell donor, the host and the HSC donor have at least partial mismatch in HLA haplotype.In some embodiments, the T cell donor is selected if it has HLA haplotype with HLA match above threshold.

[0196] In some embodiments, the HSC donor is partially HLA mismatched to the patient. In some embodiments, the HSC donor has less than 5 / 10, 6 / 10, 7 / 10, 8 / 10, 9 / 10, or 10 / 10 matches 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 4 / 8, 5 / 8, 6 / 8, 7 / 8, or 8 / 8 matches to the patient at the HLA-A, HLA-B, HLA-C, and HLA-DRB1 loci. In some embodiments, the HSC donor has less than 2 / 2 matches to the patient at the HLA-A, HLA-B, or HLA-C loci. In some embodiments, the HSC donor has less than 3 / 4 or 4 / 4 matches to the patient at the HLA-DRB1 and HLA-DQB1 loci.

[0197] In some embodiments, one or more of the T cell donors are HLA-mismatched or partially HLA-mismatched to the patient. In some embodiments, one or more of the T cell donors have a match of less than 5 / 10, 6 / 10, 7 / 10, 8 / 10, 9 / 10, or 10 / 10 to the patient at the HLA-A, HLA-B, HLA-C, HLA-DRB1, and HLA-DQB1 loci. In some embodiments, one or more of the T cell donors have a match of less than 4 / 8, 5 / 8, 6 / 8, 7 / 8, or 8 / 8 to the patient at the HLA-A, HLA-B, HLA-C, and HLA-DRB1 loci. In some embodiments, one or more of the T cell donors have a match of less than 2 / 2 to the patient at the HLA-A, HLA-B, or HLA-C loci. In some embodiments, 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.

[0198] In some embodiments, one or more of the T cell donors are HLA-mismatched or partially HLA-mismatched to the HSC donor. In some embodiments, one or more of the T cell donors have less than 5 / 10, 6 / 10, 7 / 10, 8 / 10, 9 / 10, or 10 / 10 matches to the HSC donor at the HLA-A, HLA-B, HLA-C, HLA-DRB1, and HLA-DQB1 loci. In some embodiments, one or more of the T cell donors have less than 4 / 8, 5 / 8, 6 / 8, 7 / 8, or 8 / 8 matches to the HSC donor at the HLA-A, HLA-B, HLA-C, and HLA-DRB1 loci. In some embodiments, one or more of the T cell donors have less than 2 / 2 matches to the HSC donor at the HLA-A, HLA-B, or HLA-C loci. In some embodiments, 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. IL-10 The cells or pharmaceutical composition prevent or reduce the severity of GvHD due to transplanted hematopoietic stem cells.

[0199] In some embodiments, the polydonor CD4 IL-10 The cells or pharmaceutical compositions prevent or reduce the severity of pathogenic T cell responses by transplanted hematopoietic cells. In certain embodiments, polydonor CD4 IL-10 The cells prevent or reduce GvHD.

[0200] In some embodiments, the polydonor CD4 IL-10 The cells or pharmaceutical compositions prevent or reduce the severity of tissue damage induced by pathogenic T cells or inflammation.

[0201] 6.6.2. Methods of Treating Cancer In some embodiments, the polydonor CD4 IL-10 The cells are used to treat cancer. In a preferred embodiment, the polydonor CD4 IL-10 The cells directly mediate an anti-tumor effect, and in certain embodiments, an anti-leukemic effect.

[0202] In some embodiments, the polydonor CD4 IL-10 The cells are administered in combination with allogeneic mononuclear cells or PBMCs to treat cancer. In some embodiments, the polydonor CD4 IL-10 The cells are administered before or after administration of the PBMCs. In some embodiments, the polydonor CD4 IL-10 The cells and allogeneic mononuclear cells or PBMCs are administered simultaneously.

[0203] In some embodiments, the polydonor 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.

[0204] 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.

[0205] 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 site, Castleman's disease, cervical cancer, colon / rectal cancer, endometrial cancer, esophageal cancer, Ewing's family of tumors, eye cancer, gallbladder cancer, gastrointestinal carcinoid tumors, gastrointestinal stromal tumors (GIST), gestational trophoblastic disease, Hodgkin's disease, Kaposi's sarcoma, kidney cancer, laryngo-hypopharyngeal cancer, acute lymphoblastic leukemia (ALL), acute myeloid leukemia (including AML, myeloid sarcoma, and leukemia cutis), chronic lymphocytic leukemia (CLL), chronic myeloid (CML) leukemia, chronic myelomonocytic leukemia (CMML), childhood leukemia, liver cancer, lung cancer, non-small cell lung cancer, pulmonary fibrosis ... The patient has a cancer selected from the group consisting of: small cell lung cancer, small cell lung cancer, pulmonary carcinoid tumor, lymphoma, cutaneous lymphoma, malignant mesothelioma, multiple myeloma, myelodysplastic syndrome, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, non-Hodgkin's lymphoma, childhood non-Hodgkin's lymphoma, oral 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, skin cancer - melanoma, skin cancer - Merkel cell, small intestine cancer, gastric cancer, testicular cancer, thymic cancer, thyroid cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Waldenstrom's macroglobulinemia, and Wilms' tumor.

[0206] In some embodiments, the cancer is a myeloid tumor. In certain embodiments, the cancer is AML or CML. In some embodiments, the cancer is a myeloid tumor.

[0207] In some embodiments, the method is used to treat hematological cancers that affect blood, bone marrow, and lymph nodes. In various embodiments, the hematological cancer is lymphoma (e.g., Hodgkin's lymphoma), lymphocytic leukemia, myeloma. In various embodiments, the hematological cancer is acute or chronic myelogenous (myeloid) leukemia (AML, CML), or myelodysplastic syndrome.

[0208] In some embodiments, the cancer is refractory or resistant to therapeutic intervention.

[0209] In some embodiments, the polydonor CD4 IL-10 The cells are used in combination with therapeutic intervention. The combination can be performed simultaneously or at different times.Preferably, the therapeutic intervention is selected from the group consisting of chemotherapy, radiotherapy, allo-HSCT immunosuppression, blood transfusion, bone marrow transplantation, growth factors, and biological agents.

[0210] In some embodiments, the polydonor CD4 IL-10 The cells induce cell death of tumor-infiltrating and tumor growth-promoting myeloid lineage cells (eg, monocytes, macrophages, neutrophils).

[0211] 6.6.3. Methods of Treating Inflammatory or Autoimmune Diseases In some embodiments, the polydonor CD4 IL-10 The cells are administered to treat an inflammatory disease or an autoimmune disease. In some embodiments, the polydonor CD4 IL-10 The cells are administered to treat a disease or disorder associated with hyperactivity of the NLPR3 inflammasome.

[0212] The NOD-like receptor family (NLR) protein NLRP3 is an intracellular signaling molecule that senses danger signals from pathogens, the environment, or endogenous sources. After activation, NLPR3 interacts with caspase-1 to form a complex called the inflammasome. This leads to the activation of caspase-1, which cleaves the pro-inflammatory cytokines IL-1β and IL-18 into their active forms and mediates a type of inflammatory cell death known as pyroptosis.

[0213] In some embodiments, the polydonor CD4 IL-10 The cells are administered to treat an inflammatory disease selected from Muckle-Wells Syndrome (MWS), Familial Cold Autoinflammatory Syndrome (FCAS) and Neonatal-onset Multisystem Inflammatory Disease (NOMID). IL-10 The cells are administered to treat a chronic disease selected from metabolic syndrome, type 2 diabetes, atherosclerosis, Alzheimer's disease, Parkinson's disease, ALS, non-alcoholic steatohepatitis, osteoarthritis, silicosis, asbestosis, gout, and pulmonary fibrosis. In some embodiments, the polydonor CD4 IL-10 The cells are administered to treat Crohn's disease, ulcerative colitis, multiple sclerosis and systemic lupus erythematosus, or inflammatory eye diseases such as diabetic retinopathy, acute glaucoma, and age-related macular degeneration.

[0214] In some embodiments, the polydonor CD4 IL-10 The cells are administered to treat diseases associated with NLRP3. The diseases can be selected from the group consisting of CAPS, NASH, Alzheimer's disease, Parkinson's disease, cardiovascular disease, osteoarthritis, gout, pseudogout, nephrocalcinosis, type II diabetes, Sjogren's syndrome, sickle cell disease (SCD), AMD, infectious diseases, cerebral malaria, asbestosis, contact hypersensitivity, sunburn, silicosis, cystic fibrosis, inflammatory bowel disease, nephrocalcinosis, ALS, myelodysplastic syndrome, and uveitis.

[0215] In some embodiments, the disease is a brain disorder selected from Parkinson's disease, Alzheimer's disease, age-related cognitive impairment, frontotemporal dementia, traumatic brain injury, intracerebral hemorrhage, sepsis-related encephalopathy, cerebral ischemia, subarachnoid hemorrhage, epilepsy, acrylamide poisoning, opioid-induced neuroinflammation, chronic migraine, perioperative neurocognitive impairment, post-stroke cognitive impairment, post-cardiac arrest cognitive impairment, social isolation-induced cognitive impairment, anxiety, and post-traumatic stress disorder.

[0216] In some embodiments, the disease is a pulmonary disorder selected from asthma, IR lung injury, ARDS / COPD, particulate matter induced lung injury, radiation pneumonitis, pulmonary hypertension, sarcoidosis, cystic fibrosis, and allergic rhinitis.

[0217] In some embodiments, the disease is a cardiac disorder selected from atherosclerosis, heart failure, hypertension, myocardial infarction, atrial fibrillation, cardiac damage induced by metabolic dysfunction, and endothelial dysfunction.

[0218] In some embodiments, the disease is a gastrointestinal disease, such as colitis. In some embodiments, the disease is a liver disorder selected from acute liver failure, immune circadian rhythm regulation, NASH, diabetic cognitive dysfunction, IR liver injury, idiosyncratic drug-induced liver injury, and liver fibrosis. In some embodiments, the disease is a pancreatic or renal disorder selected from diabetic encephalopathy, diabetic atherosclerosis, insulin resistance, islet transplant rejection, chronic crystal nephropathy, renal fibrosis, I / R kidney injury, obesity-related kidney disease, and renal hypertension. In some embodiments, the disease is a skin or eye disorder selected from psoriasis and retinal neovascularization. In some embodiments, the disease is a reproductive disorder, such as premature birth. In some embodiments, the disease is an immune disorder selected from primary dysmenorrhea, innate immunity, innate to adaptive immunity, systemic lupus erythematosus-lupus nephritis, and multiple sclerosis. In some embodiments, the disease is a genetic disorder selected from Muckle-Wells syndrome, rheumatoid arthritis, sickle cell disease, and VCP-related disease. In some embodiments, the disease is a pain disorder selected from multiple sclerosis-related neuropathic pain, chronic prostatitis / chronic pelvic pain, cancer-induced bone pain, and hyperalgesia. In some embodiments, the disease is cancer, such as human squamous cell carcinoma of the head and neck. In some embodiments, the disease is an infectious disorder, such as a bacterial, viral, or parasitic infection.

[0219] In some embodiments, the polydonor CD4 IL-10 The cells are used in combination with currently available treatments for NLRP3-associated diseases, such as biologic agents that target IL-1, including the recombinant IL-1 receptor antagonist anakinra, the neutralizing IL-1β antibody canakinumab, and the soluble decoy IL-1 receptor rilonacept.

[0220] In some embodiments, the polydonor CD4 IL-10The cells are administered to treat a disease selected from type 2 diabetes, metabolic syndrome, cardiovascular disease, SLE, MS, CD, ulcerative colitis (UC), osteoarthritis, nonalcoholic steatohepatitis (Nash), Parkinson's disease, ALS, pulmonary fibrosis, silicosis, asbestosis, diabetic retinopathy, and age-related macular degeneration.

[0221] In some embodiments, the polydonor CD4 IL-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.

[0222] In some embodiments, the polydonor CD4 IL-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, the polydonor CD4 IL-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, the polydonor CD4 IL-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.

[0223] In some embodiments, the polydonor CD4 IL-10 The cells are administered to reduce IL-1β production by activated monocytes, macrophages, or dendritic cells. In some embodiments, the polydonor CD4 IL-10 The cells are administered to reduce IL-18 production by activated monocytes, macrophages, or dendritic cells. In some embodiments, the polydonor CD4 IL-10 The cells are administered to reduce mature caspase-1 production by activated monocytes, macrophages, or dendritic cells.

[0224] 6.6.4. Methods for Treating Other Disorders In some embodiments, the polydonor CD4 IL-10 The cells are administered to treat an autoimmune disease.

[0225] In some embodiments, the autoimmune disease is selected from the group consisting of type 1 diabetes, autoimmune uveitis, autoimmune hepatitis, vitiligo, alopecia areata, rheumatoid arthritis, psoriasis, psoriatic arthritis, multiple sclerosis, systemic lupus erythematosus, inflammatory bowel disease, Addison's disease, Graves' disease, Sjogren'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 can be selected from the group consisting of asthma, atopic dermatitis, and rhinitis. In some embodiments, the patient has a food allergy.

[0226] In some embodiments, the polydonor CD4 IL-10 The cells are administered to prevent or reduce the severity of pathogenic T cell responses to cell and organ transplants other than HSCT. In some embodiments, the method comprises administering polydonor CD4 IL-10 The method includes administering an organ transplant to the patient either before or after administration of the T cells or pharmaceutical composition. In certain embodiments, the organ is a kidney, a heart, or pancreatic islet cells. In a preferred embodiment, the polydonor CD4 IL-10 The cells or pharmaceutical composition prevent or reduce the severity of host rejection of an organ transplant.

[0227] In some embodiments, the polydonor CD4 IL-10 The cells are administered to prevent or reduce immune responses associated with gene therapy, such as administration of recombinant AAV (rAAV). In these embodiments, the methods include administering polydonor CD4 IL-10 The method further includes administering a recombinant AAV to the patient, either before or after administration of the cells or pharmaceutical composition.

[0228] In some embodiments, the polydonor CD4 IL-10 The cells are administered to prevent or reduce immune responses associated with transplantation of iPS cell-derived tissues or cells. iPS cell-derived tissues and cells include, but are not limited to, cardiomyocytes, hepatocytes, epithelial cells, cartilage, bone and muscle cells, and neurons.

[0229] In some embodiments, the polydonor CD4 IL-10 The cells are administered to reduce an overactive immune response of a patient to a viral infection. In some embodiments, the virus is SARS-coV-2. In some embodiments, the polydonor CD4 IL-10 The cells are administered to reduce an overactive immune response to bacterial infection, such as toxic shock and cytokine storm.

[0230] In some embodiments, the method comprises the step of: IL-10 In some embodiments, the method further comprises administering an immunotherapeutic protein to the patient either before or after administration of the cell population or pharmaceutical composition. IL-10 The cell population, or pharmaceutical composition reduces an immune response to an immunogenic therapeutic protein. In some embodiments, the immunotherapeutic protein is selected from a therapeutic antibody, a factor VIII replacement, a cytokine, and a cytokine mutein. EXAMPLES

[0231] 6.7. Working Example The following examples are offered by way of illustration and not by way of limitation.

[0232] 6.7.1. Summary of experimental findings The present disclosure provides highly purified allogeneic CD4+ cells 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 produce >95% pure IL-10-producing, NGFR-expressing CD4 + T cells (CD4 IL-10 The CD4+ cells from three different allogeneic HLA-mismatched donors were used to generate IL-10 Cells were pooled in a 1:1:1 ratio.

[0233] These pooled populations are referred to herein as polydonor CD4 IL-10 Also called single-donor CD4 IL-10 The polydonor CD4 T cells had a cytokine production profile comparable to that of naturally occurring type 1 regulatory T (Tr1) cells. They produced high levels of IL-10 and IL-22, variable levels of IFN-γ and IL-5, and low levels of IL-4. IL-10 The cells are polyclonal (have multiple antigen specificities) and contain allogeneic CD4 + and CD8 + In addition, they specifically killed myeloid leukemia cells in vitro. IL-10 The cells inhibited NLPR3 inflammasome activation and proinflammatory IL-1β and IL-18 production by human monocytes in vitro.

[0234] Polydonor CD4 in a humanized mouse graft-versus-host disease (GvHD) model IL-10 Adoptive transfer of the cells showed that these cells homed efficiently to the spleen and bone marrow. Polydonor CD4 in a humanized mouse GvHD model IL-10 Adoptive transfer of cells using human CD4 + Polydonor CD4IL-10 cells alone, even at high concentrations, inhibited severe xeno-GvHD induced by T cells or PBMCs. Importantly, polydonor CD4IL-10 cells alone did not induce GvHD, even at high concentrations. IL-10The cells had a cytotoxic effect against cancer cells in NSG mice that were intravenously injected with ALL-CM cells. Single-donor and poly-donor CD4 IL-10 Injection of the cells 3 days after administration of ALL-CM cells (at which point massive expansion of these cells is already underway) resulted in inhibition of tumor growth. These results are consistent with the results of polydonor CD4 IL-10 We demonstrate that single-donor and poly-donor CD4 cells have a direct therapeutic antimyeloid leukemia effect in vivo. IL-10 When cells were administered together with PBMCs, CD4 IL-10 The cells further downregulated xeno-GvHD induced by allogeneic PBMCs.

[0235] These results suggest that polydonor CD4 IL-10 It is demonstrated that the cells can be used for the treatment and / or prevention of GvHD; can be used as an adjunct to allogeneic hematopoietic stem cell transplantation (HSCT) for the treatment of leukemia and other malignancies to reduce GvHD while retaining the GvL or GvT therapeutic effects of HSCT; and can be used to treat cell and organ rejection, as well as autoimmune and inflammatory diseases.

[0236] [Example 1] Polydonor CD4 IL-10 Cell generation Vector production Polydonor CD4 IL-10The cells were produced by transducing a lentiviral vector (LV-IL-10 / ΔNGFR) containing the coding sequence of both human IL-10 and a truncated form of NGFR (ΔNGFR) as described in WO 2016 / 146542, which is incorporated by reference in its entirety (FIGS. 1 and 2). The sequence of the plasmid encoding human IL-10 and ΔNGFR (pLVIL-10) used to produce LV-IL-10 / ΔNGFR is provided in SEQ ID NO:5. Briefly, pLVIL-10 was generated by ligating the coding sequence of human IL-10 from a 549 bp fragment of pH15C (ATCC68192) into plasmid #1074.1071.hPGK.GFP.WPRE.mhCMV.dNGFR.SV40PA. The presence of a bidirectional promoter (the human PGK promoter plus the minimal core elements of the CMV promoter in opposite orientations) allows for co-expression of the two transgenes. The plasmid further contains a coding sequence for an antibiotic resistance gene (eg, ampicillin or kanamycin).

[0237] Lentiviral vectors were produced by Ca3PO4 transient four-plasmid co-transfection into 293T cells and concentrated by ultracentrifugation. 1 μM sodium butyrate was added to the culture for vector harvest. Titers were estimated by limiting dilution in 293T cells, and vector particles were measured by HIV-1 Gag p24 antigen immunocapture (NEN Life Science Products; Waltham, MA). Vector infectivity was calculated as the ratio between titer and particles. For concentrated vectors, titers were 5×10 8 ~6×10 9 Transducing units / mL, and infectivity is 5 × l0 4 ~5×10 5 The transducing units / ng range.

[0238] CD4 IL-10 Cell production Figure 3 shows CD4 IL-10 Schematic diagram of the cell production process.+ T cells were purified. Human CD4 + T cells were activated with soluble anti-CD3, soluble anti-CD28 mAb, and rhIL-2 (50 U / mL) for up to 48 h and then transduced with a bidirectional lentiviral vector encoding human IL-10 and a truncated form of the human NGF receptor (LV-IL-10 / ΔNGFR) at a multiplicity of infection (MOI) of 20.

[0239] After 9-11 days, transduced cells were analyzed by FACS for expression of ΔNGFR and vector copy number (VCN) was quantified by digital droplet PCR (ddPCR).

[0240] 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 human CD4 T cells transduced with LV-IL-10 / ΔNGFR (a bidirectional lentiviral vector encoding a truncated form of human IL-10 and the human NGF receptor). + CD4 on T cells + ΔNGFR + The percentage of cells (mean ± SD, n = 10, left bar graph) and vector copy number (VCN, mean ± SD, n = 10, right bar graph) are shown. CD4 + ΔNGFR + Cell frequency and vector copy number were determined by CD4 IL-10 Quantification was performed in cells by digital droplet PCR (ddPCR).

[0241] 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 step was greater than 98%. Figure 4B shows FACS data from two representative donors (donor B and donor C) out of the 10 donors tested. The CD4 IL-10The cell purities were 98.3% and 99.2%, respectively. IL-10 Cells were restimulated three times at 14-day intervals, and their in vitro and in vivo functions were tested after the second (TF2) and / or third restimulation (TF3) functions.

[0242] Dormant CD4 IL-10 The cells constitutively produced IL-10, and upon activation, the levels of IL-10 produced were strongly enhanced.

[0243] CD4 IL-10 The cells have a cytokine production profile comparable to that of naturally occurring Tr1 cells

[0244] Single donor CD4 IL-10 The cytokine production profile of the cells was analyzed after the second (TF2) and third (TF3) restimulation, and the results are presented in Figure 5. Specifically, CD4 IL-10 Cells (2 × 10 in 200 μl 5 ) 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) rounds of restimulation, CD4 IL-10 Cells were left unstimulated or activated with fixed CD3 (10 μg / mL) and soluble CD28 mAb (1 μg / mL) for 48 h. Culture supernatants were collected and levels of IL-10, IL-4, IL-5, IFN-γ and IL-22 were determined by ELISA. All samples were tested in triplicate. Mean values ​​± SD of n=8 donors tested are shown. Results provided in Figure 5 show that CD4+ cells stimulated with fixed anti-CD3 and soluble anti-CD28 mAb were significantly increased in 10% of ... IL-10 1 shows that the cells display the cytokine production profile of Tr1 cells.

[0245] Although variability between different donors was observed, the overall cytokine production profiles after the second (TF2) (Figure 5, left panel) or third (TF3) (Figure 5, right panel) restimulation were comparable and mirrored those of Tr1 cells (Roncarolo et al., Immunity, 2018). Similar to Tr1 cells, CD4 IL-10 The cells produced high levels of IL-10 and IL-22, variable levels of IL-5 and IFN-γ, relatively low levels of IL-4, and undetectable levels of IL-2 (not shown).

[0246] CD4 IL-10 The cells express high levels of granzymes and selectively kill myeloid leukemia cells.

[0247] CD4 IL-10 Cells were further analyzed for expression of granzyme B (GzB) after a second round of restimulation (TF2). Data in Figure 6A show that all CD4 IL-10 It is shown that more than 95% of the cells expressed high levels of granzyme B.

[0248] CD4 from second round (TF2) restimulation IL-10 The cells were further analyzed for their cytotoxic effect against human myeloid leukemia cell lines (ALL-CM) and erythroleukemia cell lines (K562). IL-10 cells (10 5 cells / well) in K562 and ALL-CM cells (10 5 The cells were co-cultured with a residual leukemia cell line (CD45 low , CD3 - ) were counted by FACS for each target cell.

[0249] CD4 IL-10The cells selectively killed myeloid leukemia cells (ALL-CM) as shown in Figure 6B. The % of ALL-CM cells killed varied between 62% and 100%, whereas killing of the erythroleukemia cell line K562 (highly susceptible to nonspecific cytotoxicity and natural killer (NK) cell activity) varied between 0 and 27% (four different donors tested). Taken together, these data suggest that CD4 IL-10 We confirm that the cells express granzyme B and effectively kill myeloid leukemia cells. As expected, CD4 IL-10 Some variation in cell killing capacity was observed.

[0250] CD4 IL-10 The cells were allogeneic CD4 + and CD8 + Suppresses both proliferative responses of T cells

[0251] CD4 IL-10 The cells were also transfected with allogeneic CD4 + T cells or CD8 + Its effect on T cells was also analyzed. Specifically, allogeneic PBMC cells were incubated with eFluor® 670 (5×10 4 CD4 IL-10 cells (5×10 4 Allogeneic mature dendritic (mDC) cells (5 × 10 cells / well) were administered in the presence or absence of 5 × 10 3 Cells were stimulated with 1:1 responder:suppressor ratios of CD4+ / -100 cells / well and soluble anti-CD3 mAb. After 3 days of culture, the percentage of proliferative responder cells was determined by flow cytometry. + ΔNGFR - T cells or CD8 + ΔNGFR - After gating on T cells, the dilution of eFluor® 670 was determined. + CD4 T cells from six different unpooled donors (donors C, E, and F in FIG. 7A, and donors H, I, and L in FIG. 7B). IL-10The effect of the cells is shown as percentage of proliferation and inhibition. + The effect of CD4+ from six different single donors (donors C, E, and F in FIG. 8A, and donors H, I, and L in FIG. 8B) on T cell proliferation. IL-10 The effect of cells is shown.

[0252] Results are based on unpooled, individually tested CD4 IL-10 The cells are allogeneic CD4 + and CD8 + We demonstrated that the CD4 T cell proliferation response was downregulated by both + The inhibitory effect on T cells varied between 51% and 96%, but not CD8 + The inhibitory effect on T cells varied between 62% and 73%.

[0253] Polydonor CD4 IL-10 Cell production and characterization

[0254] CD4 IL-10 The cells were then purified using CD4 + Cells were used to generate CD4 from each donor as described above and in Figure 3. 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 fixed CD3 (10 μg / mL) and soluble CD28 mAb (1 μg / mL) for 48 hours.

[0255] Polydonor CD4 IL-10 The cells were isolated from individual donor CD4 Il-10 have a cytokine production profile comparable to that of Tr1 and Tr2 cells

[0256] Culture supernatants were collected and levels of IL-10, IL-4, IL-5, IFN-γ and IL-22 were determined by ELISA. The results presented in Figure 9 show the results of a polydonor CD4 IL-10 Cytokine production (black dots) of CD4 IL-10 CD4 from cells IL-10 The results are comparable to those of polydonor CD4 cells (gray bars). IL-10 The cells produced high levels of IL-10 and IL-22, variable levels of IL-5, IFN-γ, and low levels of IL-4, and undetectable levels of IL-2 (not shown). IL-10 It is feasible to pool cells and these polydonor CD4 IL-10 The cells are CD4 IL-10 These results demonstrate that the pooled homogenous cell populations maintained the cytokine production signature of Tr1 and Tr2 cells. Importantly, the pooled homogenous cell populations contained >95% viable cells, indicating that they did not kill each other.

[0257] Polydonor CD4 IL-10 The cells express high levels of granzyme B and kill myeloid leukemia cell lines.

[0258] Polydonor CD4 IL-10 Cells were further analyzed for expression of granzyme B (GzB) after the third round of restimulation (TF3). Data in Figure 10A show that most polydonor CD4 IL-10 It has been shown that the cells express GzB. Polydonor CD4 IL-10 More than 95% of cells were CD4 from a single donor IL-10 They expressed granzyme B, which was comparable to the cells' GzB expression (Fig. 10A ).

[0259] CD4 from the third round (TF3) restimulation IL-10The cells were further analyzed for their cytotoxic effect on myeloid leukemia cells (ALL-CM cell line) or K562. IL-10 cells (10 5 cells / well) in K562 and ALL-CM cells (10 5 The cells were co-cultured with the residual leukemia cell line (CD45 low , CD3 - ) were counted by FACS for each target cell. The results presented in Figure 10B show some level of cytotoxicity against K562 cells, which are highly sensitive to non-specific cytotoxicity. Nevertheless, single-donor derived CD4 IL-10 Polydonor CD4 to myeloid leukemia cells (ALL-CM) levels are comparable to those of ALL-CM cells (white bars). IL-10 Selective killing levels of cells (black dots) were obtained.

[0260] Polydonor CD4 IL-10 The cells suppress the proliferative responses of both allogeneic CD4+ and CD8+ T cells. Polydonor CD4 IL-10 The cells were also transfected with allogeneic CD4 + T cells or CD8 + The effect of eFluor® 670 (5×10 4 Polydonor CD4 IL-10 cells (5×10 4 cells / well) in the presence or absence of allogeneic mature dendritic (DC) cells (5 × 10 3 Cells were stimulated with 1:1 responder:suppressor ratios of CD4+ / -100 cells / well and soluble anti-CD3 mAb. After 3 days of culture, the percentage of proliferative responder cells was determined by flow cytometry. + ΔNGFR - T cells or CD8 + ΔNGFR - After gating on T cells, the eFluor® 670 dilution was determined. Figure 11A shows CD4 T cells from donors C, E, and F (CEF).IL-10 Polydonor CD4 containing cells IL-10 FIG. 11B shows the results of cells from donors H, I, and L (HIL) that had been frozen, stored, and thawed prior to testing. IL-10 Polydonor CD4 containing cells IL-10 The results of cells are shown.

[0261] FIG. 11A shows a polydonor CD4 IL-10 Cells (from three different donors) were CD4 + and CD8 + The polydonor CD4 T cells were tested after the cells had been frozen, stored, and thawed prior to testing. IL-10 Comparable results were obtained with a second different batch of cells (Figure 11B). + and CD8 + The inhibition of T cell proliferation was 68% and 75%, respectively. These data are consistent with those of polydonor CD4 IL-10 It has been shown that cells can be frozen, stored and thawed without loss of function.

[0262] Collectively, polydonor CD4 IL10 The data obtained for the cells indicates that these cell preparations can be pooled without any problems. They contain >95% viable cells and are single-donor CD4 IL-10 Maintains all relevant functions of the cell (cytokine production, cytotoxicity, and suppression of allogeneic T cell responses). Polydonor CD4 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 provide large amounts of off-the-shelf CD4 IL-10 The cells should be provided.

[0263] Polydonor CD4 IL-10Cellular products have significant advantages in terms of a more homogenous product that allows for well-defined, low lot-to-lot variability, determination of potency, and release criteria. In addition, this allows for the development of continuous large-scale cell production processes.

[0264] Polydonor CD4 IL-10 Other methods for producing cells

[0265] Pool buffy coats from at least 3-5 different donors prior to lentiviral transduction. + Cells are isolated from the buffy coat by positive selection using anti-CD4 antibodies. + Check cell purity by FACS. Alternatively, use frozen human CD4 + Cells are obtained from at least 3-5 healthy donors. Frozen human CD4 + Cells are thawed before use. CD4 + Cells are activated with a combination of CD3 and CD28 antibodies, or CD3 and CD28 antibody coated beads, in the presence of IL-2 for 24-48 hours. In some cases, CD4 + Cells were activated with soluble anti-CD3, soluble anti-CD28 mAb, and rhIL-2 (50 U / mL) for 48 h and CD4 IL-10 To produce cells, transduce a bidirectional lentiviral vector encoding human IL-10 as described above.

[0266] In some cases, the T cell donor (or CD4 + First, the HLA haplotype of the CD4 + The cells are selectively pooled for use.

[0267] Polydonor CD4 IL-10 The cells were incubated with activated CD4 +The cells are generated by transducing with a lentiviral vector containing the human IL-10 and ΔNGFR coding sequences described above.

[0268] On days 7–11, 5–9 days after transduction, cells are harvested and successfully transduced T cells are purified using anti-NGFR antibodies. This process is typically performed using polydonor CD4 IL-10 Results in a 95% pure population of cells.

[0269] Purified polydonor CD4 IL-10 Cells are counted and restimulated with a mixture of CD3 and CD28 antibodies, CD3 and CD28 antibody coated beads, optionally in the presence of feeder cells, and in the presence of IL-2 for an additional 8-10 days. In some cases, purified polydonor CD4 IL-10 The cells are restimulated in the presence of feeder cells.

[0270] After a total culture period of 5 weeks, CD4 IL-10 Cells are harvested, counted, and tested for their ability to produce IL-10, either naturally or after activation with CD3 and CD28 antibodies, or CD3 and CD28 antibody-coated beads. In addition, levels of GrzB and perforin are measured. They are expressed in human T cells (PBMCs) and purified CD4 + and CD8 + The ability to suppress T cell proliferation is also tested.

[0271] In addition, production of IL-22 was constitutively and, as previously described for the production of other cytokines such as IFN-γ, IL-10, IL-4 and IL-5, was achieved by inducing IL-22 production in a volume of 200 microliters using a combination of CD3 and CD28 antibodies in 200,000 CD4+ cells. IL-10 The levels of IL-22 production are measured after activation of the cells in an IL-22-specific ELISA as described for other cytokines in WO 2016 / 146542. IL-10 The cells are frozen and then stored.

[0272] [Example 2] Polydonor CD4 IL-10 Treatment or prevention of GvHD using cells Polydonor CD4 IL-10 In vivo effects on cells Polydonor CD4 IL-10 The cell populations were tested in a humanized xeno GvHD disease model, the NSG mouse model, for their effect on xeno-GvHD induced by human PBMCs as illustrated in Figure 12. NSG mice were sublethally irradiated and (i) injected with human PBMCs (5x10 6 (ii) polydonor (three donors; B-C-C / E / F) CD4 IL-10 cells (5×10 6 cells / mouse), or (iii) human PBMCs (5 × 10 6 (cells / mouse) and polydonor CD4 IL-10 Cells (BC-C / E / F) (5×10 6 Xeno-GvHD was assessed based on survival, weight loss (>20% weight loss), skin lesions, coat condition, activity, and curling up as previously described (Bondanza et al. Blood 2006).

[0273] FIG. 13 shows the percentage of NSG mice that do not show xeno-GvHD on each day after injection. 6 Administration of 5 × 10 human PBMCs to irradiated NSG mice unexpectedly led to an unusually fulminant xeno-GvHD. All mice died by day 10, reflecting highly lethal xeno-GvHD. 6 Polydonor CD4 IL-10 Co-administration of cells delayed this fulminant xeno-GvHD, but mice were sacrificed on day 14 due to reaching the defined humane 20% weight loss criterion for sacrifice (Figure 13). Nonetheless, these results are consistent with the polydonor CD4 IL-10Importantly, the same dose of PBMC (5 × 10 6 Polydonor CD4 IL-10 The cells did not induce any signs of xeno-GvHD.

[0274] Human CD4 IL-10 The presence of cells was also confirmed by using human PBMCs (5 × 10 6 individual / mouse), polydonor (three donors; BC-C / E / F) CD4 IL-10 cells (5×10 6 cells / mouse), or human PBMCs (5 × 10 6 (cells / mouse) and polydonor CD4 IL-10 Cells (three donors; BC-C / E / F) (5 × 10 6 Combinations of polydonor CD4 (100 / mouse) were also tested in the spleen (FIG. 14, left panel) and bone marrow (FIG. 14, right panel) of injected NSG mice 14 days after injection. The results presented in FIG. 14 show that polydonor CD4 IL-10 It has been shown that the cells migrated to the spleen and bone marrow. A low percentage of these cells was found to be present 14 days after cell injection. These results support the conclusion that polydonor CD4 IL-10 These results indicate that the cells delayed fulminant xeno-GvHD induced by human PBMCs but did not induce any xeno-GvHD by themselves.

[0275] Polydonor CD4 IL-10 The cells were purified CD4 + Inhibits cell-induced severe xeno-GvHD Polydonor CD4 IL-10 The cells were cultured at 2.5×10 6 Purified human CD4 + We tested a humanized xeno-GvHD model induced by administration of T cells. NSG mice were sublethally irradiated on day 0 and transfected with human CD4 T cells on day 3. + T cells (2.5×10 6 100 / mouse) alone or in polydonor CD4 IL-10Cells (three different donors; BC-H / I / L) (2.5 × 10 6 CD4 from a single donor (BC-H) or from a pool IL-10 cells (2.5×10 6 Xeno-GvHD was assessed based on survival, weight loss (>20% weight loss), skin lesions, coat condition, activity, and curling up as previously described (Bondanza et al. Blood 2006).

[0276] FIG. 16 shows the percentage of NSG mice free of GvHD on each day after injection. The results are IL-10 (BC-H / I / L) cells were cloned into human allogeneic CD4 + This shows that the CD4+ T cell-mediated xeno-GvHD can be inhibited. In this experiment, xeno-GvHD was so severe that all mice in the control group that received CD4+ T cells died by day 20. In contrast, 2.5 × 10 6 Polydonor CD4 IL-10 Coadministration of single-donor CD4 IL-10 Cells were also protective, but to a lesser extent.

[0277] Other Experiments Polydonor CD4 IL-10 The therapeutic effect of the cells is tested in four different groups of mice: (i) CD4 IL-10 (ii) mice administered human PBMCs from an unrelated donor (xeno-GvHD positive control); (iii) polydonor CD4 IL-10 (iii) mice administered PBMCs and polydonor CD4 IL-10 Mice administered a 1:1 ratio combination of PBMCs and polydonor CD4 IL-10 Mice were administered a 2:1 ratio of cells or a combination of different ratios. PBMC and polydonor CD4 IL-10 In animals receiving the cell combination, some animals received both PBMC and polydonor CD4 IL-10Cells were administered simultaneously, and some animals were then transfected with polydonor CD4 IL-10 Cells were administered, and some animals were depleted several days (e.g., 5 days) prior to administration of PBMCs. IL-10 The cells are administered.

[0278] Mice were cultured using PBMC and / or polydonor 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, if necessary, 5. In addition to weight loss, mice are examined for skin lesions, coat condition, and activity. Mice in the treatment groups are cultured with polydonor CD4 IL-10 Cells are monitored for an additional period of time to determine the effect on long term survival.

[0279] Polydonor CD4 IL-10 The amount and localization of cells will also be monitored in peripheral blood and tissues following administration. IL-10 The presence of polydonor CD4 cells is monitored in peripheral blood and in the sites of inflammation: spleen and bone marrow. IL-10 The presence of cells at other sites is monitored. Mice in the treatment group(s) are monitored for an additional 3 weeks to determine long-term survival.

[0280] The results are polydonor CD4 IL-10 The cells demonstrate efficacy in reducing and preventing xeno-GvHD.

[0281] [Example 3] Inhibition of GvHD and treatment of cancer Polydonor CD4 IL-10 The cell populations are tested for their effect on xeno-GvHD induced by human PBMCs and anti-tumor effects in NSG mouse models engrafted with human PBMCs and AML tumor cells. AML cells (ALL-CM) are administered iv as previously described in WO 2016 / 146542. PBMCs or polydonor CD4 IL-10The cells, or the combination, are administered three days later.

[0282] Polydonor CD4 IL-10 Cells are obtained as described in Example 1. Polydonor CD4 IL-10 The therapeutic effect of the cells was evaluated by irradiating each with 5 × 10 6 Four different groups of mice (AML mice) administered ALL-CM cells are tested: (i) AML mice without additional treatment; (ii) polydonor CD4 IL-10 5 x 10 cells from an unrelated donor 6 AML mice administered 2.5 × 10 human PBMCs induce severe xeno-GvHD; (iii) 2.5 × 10 6 Polydonor CD4 IL-10 AML mice administered cells; and (iv) PBMCs and polydonor CD4 IL-10 AML mice were administered 1:1 or 2:1 ratios of cells, or a combination of different ratios. One additional group of mice did not receive ALL-CML cells, but instead received 5×10 6 10 human PBMCs are administered.

[0283] Polydonor CD4 IL-10 The effect of the cells on xeno-GvHD induced by human PBMCs will be examined based on weight loss, skin lesions, coat condition, activity, mortality, and long-term survival. IL-10 The anti-tumor or graft-versus-leukemia (GvL) effects of the cells are examined based on the reduction of circulating tumor cells and long-term tumor-free survival.

[0284] Some mice will be monitored for up to 7 weeks to monitor for long-term survival and complete tumor regression.

[0285] The results are polydonor CD4 IL-10 The cells demonstrate efficacy in both inhibiting xeno-GvHD and treating cancer.

[0286] [Example 4] Polydonor CD4 IL-10 Cell-Based Cancer Treatment Polydonor CD4 IL-10 The cell populations were tested in an ALL-CM leukemia model of T cell therapy in NSG mice.

[0287] NSG mice were sublethally irradiated and then inoculated with myeloid leukemia cells (ALL-CM) (2.5 × 10 6 In the first group of animals, no additional cells were administered. In the second group of animals, PBMCs (2.5 × 10 6 In the third group of animals, polydonor CD4 IL-10 cells (2.5×10 6 ) were injected on day 3. In the fourth group of animals, single donor (donor BC-I) CD4 IL-10 cells (2.5×10 6 ) on day 3. In the fifth group of animals, single donor (donor BC-H) CD4 IL-10 cells (2.5×10 6 ) were injected on day 3. Graft-versus-leukemia (GvL) effects were tested in animals based on reduction of circulating leukemia cells and long-term leukemia-free survival. Leukemia was measured as previously described (Locafaro G. et al Molecular Therapy 2017). See Figure 17A.

[0288] As provided in Figures 17B and 17C, all mice injected with ALL-CM myeloid leukemia cells alone showed extensive leukemia progression by day 17. 6 Administration of single-donor CD4 PBMCs resulted in robust inhibition of leukemia progression. Interestingly, a comparable level of leukemia progression inhibition was observed in single-donor CD4 IL10 (Figure 17B) or polydonor CD4 IL10 (Figure 17C) These data were obtained with both single-donor and polydonor CD4 IL10 have been shown to have a strong direct anti-leukemic effect.

[0289] Single donor CD4IL10 and polydonor CD4 IL10 The graft-versus-leukemia (GvL) effect of was further examined in combination with PBMCs in mice injected with ALL-CM myeloid leukemia cells (Figure 18A). 6 Administration of 2.5 × 10 PBMCs resulted in a strong inhibition of leukemia progression. 6 PBMCs (2.5 × 10 6 (single donor CD4 IL10 Administration in combination with 2.5×10 cells resulted in even stronger inhibition of leukemia progression ( FIG. 18B ). 6 2.5 x 10 PBMCs 6 Polydonor CD4 IL10 When administered in combination with cells, single-donor CD4 IL10 (2.5×10 6 These data support the idea that polydonor CD4 IL10 These results indicate that the cells do not interfere with the protective GVL effect of PBMCs, but rather act synergistically with PBMCs to mediate a robust GvL effect.

[0290] [Example 5] Polydonor CD4 IL-10 Treatment of chronic inflammation and autoimmune diseases using cells - Patents.com Activation of the NLPR3 inflammasome has been implicated in many chronic inflammatory and autoimmune diseases. The NLPR3 inflammasome can be activated by "danger signals" that result in caspase-1-mediated production of the proinflammatory cytokines IL-1β and IL-18 by monocytes / macrophages. A series of in vitro experiments was performed to investigate the effect of polydonor CD4 on the NLPR3 inflammasome and IL-1β / IL-18 production by human monocytes. IL-10 Examine the effects on cells.

[0291] First, human PBMCs are isolated from peripheral blood by standard density gradient centrifugation on 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, as positive selection or adhesion can result in undesired activation of the cells. Isolated monocytes are cultured at 2×10 in culture medium containing 3% toxin-free human AB serum in 96-well microtiter plates. 5 or 1×10 5 Polydonor CD4 IL-10 5 × 10 cells in the presence of 200 μl / well 4 Seed at 200 μl per cell.

[0292] Table 1 summarizes the treatment conditions applied to nine sets of monocytes, with each set containing six wells of cells.

[0293] [Table 1]

[0294] Following treatment as outlined in Table 1, supernatants are collected from six wells for each 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 of selected groups are analyzed by Western blot to determine levels of activated caspase 1.

[0295] Experimental data are from polydonor CD4 IL-10 They further show that polydonor CD4 downregulates IL-1β and IL-18 production by activated monocytes. IL-10 In addition, polydonor CD4 downregulates mature caspase-1 production by activated monocytes. IL-10 and polydonor CD4 IL-10 IL-10 produced by B. cerevisiae downregulates the inflammasome.

[0296] Similar experiments are performed with human macrophages or dendritic cells rather than monocytes. IL-10 We demonstrate that the cells further downregulate IL-1β, IL-18, and mature caspase-1 production from activated macrophages and dendritic cells.

[0297] These data suggest that polydonor CD4 IL-10 These results suggest that polydonor CD4 cells can 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.NLPR3 inflammasome can be activated by exogenous or endogenous "danger signals", such as pathogen-associated molecular patterns (PAMPs), silica, asbestos, damaged mitochondria, necrotic cells, and danger-associated molecular patterns (DAMPs)-like products from stressed cells, and uric acid crystals.

[0298] [Example 6] Polydonor CD4 IL-10 Cell supernatants inhibit NLPR3 inflammasome activation and IL-1β and IL-18 production by human monocytes CD14 + Monocytes were isolated from PBMCs using a pan-monocyte isolation kit (Miltenyi Biotec, Bergisch Gladbach, Germany) and plated at 2 × 10 5 Cells were seeded at 200 μL / well and cultured in the presence of LPS. Cells were treated with Z-YVADfmk (20 micromolar), MMC950 (10 micromolar), IL-10 (10 ng / mL), or various concentrations of single or pooled donor CD4 IL-10 Further incubation was carried out in the presence of cell supernatant.

[0299] Supernatants were purified using single-donor or pooled-donor CD4+ cells activated for 72 h with a combination of CD3 and CD28 antibodies as previously described (Andolfi et al. 2012, Mol. Therapy Vol. 20, 1778-1790, Locafaro et al. Mol Ther 2017, 25, 2254). IL-10 In some cases (FIGS. 19C and 19D), monocytes were incubated with LPS in combination with the NLPR3 inflammasome activator nigericin ("NIG"), which was added during the final 30 minutes of LPS activation.

[0300] Activation of the NLPR3 inflammasome by LPS led to the production of mature caspase 1 and the bioactive forms of IL-1β and IL-18. Monocytes plated in the absence of LPS activation did not produce detectable levels of IL-10 during the incubation period (not shown).

[0301] Single donor CD4 IL-10 Addition of cell supernatants (containing 1769 pg IL-10 / mL) dose-dependently inhibited IL-1β production by LPS-activated monocytes from donors #1 and #2 at concentrations of 50%, 25% and 12.5%, respectively (Figures 19A and 19B). Supernatants from GFP-transduced CD4+ cells were used as controls. Complete inhibition of IL-1β production was observed at concentrations of 50% and 25%, whereas supernatants from control GFP-transduced CD4+ cells were ineffective at a concentration of 50%.

[0302] Various concentrations of CD4 IL-10 T cell supernatants (50%, 25%, or 12.5%), Z-YVADfmk, or MCC950 were further tested on monocytes activated with LPS and nigericin ("NIG"). Single donor (BC-E) or pooled donor CD4 IL-10 Supernatants from single-donor CD4 cells contained 5295 or 3532 pg IL-10 / mL, respectively. IL-10The cell supernatants were also highly effective in inhibiting LPS-induced IL-1β production, which was enhanced by the NLPR3 inflammasome activator nigericin (Figures 19C and 19D).

[0303] Data are CD4 IL-10 We demonstrated that supernatants from cells, at 50% concentration, were as effective as the irreversible caspase-1 inhibitor Z-YVADfmk (Guo et al. 2015, Nature Med 21, 677), the selective NLPR3 inflammasome inhibitor MCC950 (Coll et al. 2019, Nature Chem. Biol 15,556), and recombinant IL-10, indicating that IL-10-containing supernatants inhibit NLPR3 inflammasome activation and mature caspase-1 production, resulting in robust inhibition of production of the pro-inflammatory cytokine IL-1β (Figures 19A-19D).

[0304] Comparable results were obtained with pooled CD4 from a single donor (BC-E) and from two different donors (BC-C / E). IL-10 In a second series of experiments, the supernatant of cells was obtained. CD4 IL-10 Cells were activated with a combination of CD3 and CD28 antibodies as described (Andolfi et al. 2012). After 3 days, CD4 IL-10 Supernatants from the cells were collected. These supernatants contained 5295 and 3532 pg IL-10 / mL, respectively, and dose-dependently inhibited LPS-induced IL-1β production by monocytes from donor #3 (Figure 19E). At 50% concentration, the supernatants were as effective as Z-YVADfmk and MCC950. Pooled CD4 IL-10The inhibitory effect of the cell supernatant was completely neutralized by anti-IL-10 receptor antibody. Similarly, pooled supernatant from three different donors containing 2589 pg IL-10 / mL dose-dependently inhibited IL-1β production by monocytes from donor #4 (Figure 19F). The inhibitory effect of the supernatant was completely neutralized by IL-10 receptor antibody, demonstrating that NLPR3 activation is mediated by IL-10. The results show that the production of IL-1β, a pro-inflammatory cytokine, is suppressed by polydonor CD4 IL-10 As expected, anti-IL-10 receptor antibodies had no inhibitory effect on IL-1β production mediated by Z-VADfmk and MCC950 ( FIG. 19E ).

[0305] CD4 IL-10 T cells were further tested on monocytes from donor #4 activated with LPS and nigericin. Various concentrations of single-donor (BC-V) or polydonor (three donors; BC-T / U / V) CD4 IL-10 Cell supernatants, ZYVADfmk, or MCC950 were tested. The data presented in Figure 19G show that pooled supernatants of three different donors (BC TUV) down-regulate IL-18 production induced by the combination of LPS and nigericin.

[0306] Collectively, these data support the notion that single-donor and polydonor CD4 IL-10 We show that IL-10 produced by cells strongly downregulates the NLPR3 inflammasome, leading to strong inhibition of the pro-inflammatory cytokines IL-1β and IL-18.

[0307] [Example 7] Single-donor and polydonor CD4 IL-10 Cells inhibit xeno GvHD and myeloid tumor growth in vivo Single-donor (BC-T, BC-V, and BC-E) or polydonor (BC-V / T / E) CD4IL-10 The functional properties and quality of the cells were tested as described in Andolfi et al. Mol Ther 2012, 20, 177 and Locafaro et al. Mol Ther 2017, 25, 2254. Single-donor and polydonor CD4 IL-10 The cells produced high levels of IL-10, variable levels of IFN-γ, very low levels of IL-4 and undetectable IL-2 (the latter not shown), reflecting the characteristic cytokine production profile of Tr1 cells (Figure 21).

[0308] In addition, single-donor (BC-T, BC-V, and BC-E) or polydonor (BC-V / T / E) CD4 IL-10 The suppressive ability of the cells on CD4+ and CD8+ T cell proliferation was measured in vitro on allogeneic PBMCs. PBMCs were labeled with eFLuor670 (Invitrogen). Labeled PBMCs (1 × 10 5 ) were activated with fixed CD3 (10 mg / mL) and soluble CD28 antibody (1 mg / mL). Single-donor and polydonor CD4 IL-10 Cells were added at a 1:1 ratio in a final volume of 0.2 mL in 96-well round-bottom plates. After 4 days of co-culture, their inhibitory effect on the proliferation of eFluor670-labeled responder cells was determined by dilution of eFluor670 using flow cytometry as described (Locafaro et al. Mol Ther 2017,25,2254). Figure 22 provides the flow cytometry results. Single-donor and polydonor CD4 IL-10 The cells potently inhibited the in vitro proliferation of both allogeneic CD4+ and CD8+ T cells by greater than 80% (FIG. 22).

[0309] CD4 IL-10 The cells were further analyzed for their cytotoxic effect against myeloid leukemia cells (ALL-CM) and erythroleukemia cell lines (K562). Single-donor (BC-E and BC-V) or polydonor (BC-V / T / E) CD4 IL-10The cells were co-cultured with ALL-CM or K562 cells at a 1:1 ratio. After 3 days, the cells were harvested and the viable CD45 low CD3- target cells were counted and analyzed by FACS as described (Locafaro et al. Mol Ther 2017, 25, 2254). Single-donor and polydonor CD4 IL-10 The cells also mediated a strong direct cytotoxic effect against ALL-CM myeloid tumor cells, but they failed to kill sensitive K562 cells that lack class I MHC expression required for their cytotoxic activity (Figure 23). Single-donor (BC-E and BC-V) or polydonor (BC-V / T / E) CD4 IL-10 The cells had comparable cytotoxic activity against these two target cell lines (ALL-CM and K562).

[0310] Single donor (BC-E) and polydonor (BC-V / T / E) CD4 IL-10 The cytotoxicity of the cells was also examined using a humanized xeno-GvHD disease model, i.e., ALL-CM cells (2.5 × 10 6 It was also tested in vivo using NSG mice intravenously injected with 100 μg / mL of 10 ...

[0311] Female NOD scid gamma (NSG) mice aged 8–10 weeks were obtained from Charles-River Italia (Calco, Italy). The experimental protocol was approved by the Institutional Animal Care and Use Committee (IACUC) of Ospedale San Raffaele. On day 0, mice were subjected to total body irradiation from a linear accelerator. ALL-CM cells (2.5 × 10 6 On day 0, mice in different groups were injected with either no cells, allogeneic PBMCs (2.5 × 10 6 ), single donor (BC-E, 2.5 × 106 ), or polydonor CD4 pooled in a 1:1:1 ratio from three different donors IL-10 Cells (BC-V / T / E, 2.5×10 6 ) and allogeneic PBMCs (2.5 × 10 6 ) or polydonor CD4 IL-10 cells (2.5×10 6 ) combination was injected on day 3. All cells were administered iv in a volume of 250 μl of Iscove's modified Dulbecco's medium. Mice were monitored 3-4 times per week.

[0312] NSG mice were divided into five cohorts of five mice, and each group was treated with either: (i) no cells as a control; (ii) allogeneic mononuclear cells (PBMCs); or (iii) allogeneic PBMCs and polydonor CD4 IL-10 (iv) allogeneic PBMC and single-donor CD4 IL-10 cells (BC-E); or (v) polydonor CD4 IL-10 Treatment was by administration of (BC-V / T / E) cells on day 3. Myeloid leukemia progression was measured as previously described (Locafaro et al. Mol Ther 2017, 25, 2254).

[0313] Administration of ALL-CM cells to NSG mice led to a rapid expansion of these cells, and all mice died or had to be sacrificed on day 20. Injection of PBMCs prevented leukemia progression, as expected. Single-donor and polydonor CD4 IL10 Administration of both cells in combination with allogeneic PBMCs did not interfere with the anti-myeloid leukemia effect of the PBMCs.

[0314] Polydonor CD4 IL-10 Injection of (BC-V / T / E) cells 3 days after administration of ALL-CM cells (at which point massive expansion of these cells is already underway) resulted in inhibition of tumor growth. These results were consistent with the results of polydonor CD4 IL-10 The results show that the cells have a direct therapeutic anti-myeloid leukemia effect in vivo (Figure 24).

[0315] However, despite their beneficial antimyeloid leukemia effects, PBMCs induced very severe xeno-GvHD and all mice died by day 24 (Figure 25). IL-10 The cells were tested for their ability to inhibit xeno-GvHD induced by PBMCs after administration to NSG mice. On day 0, NSG mice were inoculated with ALL-CM cells (2.5 × 10 6 Mice were divided into five groups, each of which was injected with: (i) no cells as a control; (ii) allogeneic mononuclear cells (PBMCs); (iii) allogeneic PBMCs and polydonor (BC-V / T / E) CD4 IL-10 (iv) allogeneic PBMC and single-donor CD4 IL-10 Cell (BC-E) or polydonor (BC-V / T / E) CD4 IL-10 The cells were treated by injecting on day 3. In animals, xeno-GvHD was measured by survival and weight loss. In addition, curling, coat condition and skin integrity were monitored as described (Bondanza et al, Blood, 2006, 107, 1828). Mice were sacrificed for ethical reasons when weight loss reached more than 20%. Figure 25 shows the effect of ALL-CM cells (2.5×10 6 ) on day 1, and single-donor or polydonor CD4 IL-10 The percentage of NSD mice free of GvHD on each day after treatment with PBMC in the presence or absence of cells is shown.

[0316] The results are polydonor CD4 IL-10 Collectively, these results indicate that polydonor CD4 cells did not induce xeno-GvHD and down-regulated xeno-GvHD induced by allogeneic PBMCs. IL-10 We show that the cells down-regulate severe xeno-GvHD, have a direct anti-myeloid leukemia effect in a therapeutic setting, and do not interfere with the protective anti-myeloid leukemia effect of PBMCs.

[0317] [Example 8] Adoptive transfer of polydonor CD4IL-10 cells derived from four different donors Polydonor CD4 derived from four different donors IL-10 Adoptive transfer of cells was tested for the ability of the transfer to inhibit PBMC-induced xeno-GvHD.

[0318] In these experiments, single-donor CD4 IL-10 cells (Donor C; Lot C) and polydonor CD4 derived from four different donors IL-10 The cells (donors C, E, F, and H; lot CEFH) were tested in a humanized mouse GvHD model induced by allogeneic PBMCs, in which NSG mice were sublethally irradiated on day 0 and treated on day 3 with (i) 2.5E+06 allogeneic PBMCs, (ii) 2.5E+06 allogeneic PBMCs plus 2.5E+06 single-donor CD4s. 4IL-10 (iii) a combination of 2.5E+06 allogeneic PBMCs and 2.5E+06 polydonor CD4 IL-10 (iv) a combination of 2.5E+06 cells polydonor CD4 IL-10 Cells (lot CEFH) alone were injected (slow bolus iv). Xeno-GvHD was determined using a composite score of weight loss, coat appearance, skin appearance, curling, and activity (see Bondanza A, et al. Blood 2006;107:1828-36). As shown in Figure 26, polydonor CD4 IL-10 Only mice that received cells showed no xeno-GvHD (100%) at the end of the study.

[0319] In summary, this data represents a polydonor CD4 IL-10 We demonstrated that adoptive transfer of cells inhibits PBMC-induced xeno-GvHD but does not induce xeno GvHD.

[0320] [Example 9] Generation of IL-10 variants Variants of human IL-10 are generated by introducing amino acid modification(s) (e.g., substitutions, insertions, deletions) with respect to the IL-10 sequences of other species. The modification sites are determined by the sequence alignment provided in Figure 27A. Amino acid positions that have amino acids that differ between the species are identified from the alignment and modified by introducing amino acid substitutions, insertions, or deletions.

[0321] Two examples of variants of human IL-10 are provided in Figure 27B. Potential huIL-10 Hybrid #1 (SEQ ID NO: 19) is generated by replacing three amino acids (D, I and A) of human IL-10 with three different amino acids (E, A and D) of viral IL-10 (EBVB9) at the corresponding positions. Potential huIL-10 Hybrid #2 (SEQ ID NO: 20) is generated by replacing one amino acid (I105) of human IL-10 with another amino acid (A105) of viral IL-10 (EBVB9) at the corresponding position. Figure 27C shows the alignment of human IL-10 (SEQ ID NO: 1) with IL10 EBVB9 (SEQ ID NO: 18) and indicates " * " indicates the amino acid position or positions that are substituted in IL-10 Hybrid #1, and "#" indicates the preferred I105 to A105 amino acid substitution for IL-10 Hybrid #2.

[0322] The variants of human IL-10 are cloned into expression vectors as described in the above sections and tested for expression and function of the variant proteins. Selected variants of human IL-10 are used to stimulate CD4 IL-10 Generates cells. CD4 IL-10 The efficacy of the cells is tested as provided herein.

[0323] Experimental methods and materials Cell preparation and cell lines. Peripheral blood mononuclear cells (PBMCs) were prepared by centrifugation on a Ficoll-Hypaque gradient. CD4 +T cells were purified by CD4 T cell isolation kit (Miltenyi Biotec, Bergisch Gladbach, Germany) and the purity obtained was >95%. Mature dendritic cells (DCs) were isolated by CD14 + Peripheral blood CD14 cells were positively selected using microbeads (Miltenyi Biotech, Germany) according to the manufacturer's instructions. + They were generated from monocytes and cultured in RPMI1640 (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) at 37°C 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, USA) for 5 days and matured with 1 mg / mL lipopolysaccharide (LPS, Sigma, CA, USA) for an additional 2 days.

[0324] Construction of plasmids. The coding sequence of human IL-10 was excised from pH15C (ATCC number 68192) and a 549 bp fragment was cloned into the multiple cloning site of pBluKSM (Invitrogen) to obtain pBluKSM-hIL-10. A 555 bp fragment was obtained by excising hIL-10 from pBluKSM-hIL-10 and ligating with 1074.1071.hPGK.GFP.WPRE.mhCMV.dNGFR.SV40PA (herein named LV-ΔNGFR) to obtain pLVIL-10. The presence of a bidirectional promoter (human PGK promoter plus the minimal core elements of the CMV promoter in the opposite orientation) allows for the co-expression of two transgenes (Locafaro et al. Mol Ther. 2017;25(10):2254-2269). The sequence of pLVIL-10 was confirmed by pyrosequencing (Primm).

[0325] Vector production and titration. VSV-G-pseudotyped third generation bidirectional lentiviral vectors were produced by Ca3PO4 transient four-plasmid co-transfection into 293T cells and concentrated by ultracentrifugation as described (Locafaro et al. Mol Ther. 2017;25(10):2254-2269). Titers were estimated by limiting dilution, vector particles were measured by HIV-1 Gag p24 antigen immunocapture (NEN Life Science Products; Waltham, MA), and vector infectivity was calculated as the ratio between titer and particles. Titers were 5×10 8 ~6×10 9 Transducing units / mL, infectivity is 5 x 10 4 ~10 5 transducing units / ng p24.

[0326] CD4 IL-10 Generation of cell lines. Polyclonal CD4 transduced cells were obtained as previously described (Andolfi et al. Mol Ther. 2012;20(9):1778-1790, Locafaro et al. Mol Ther 2017, 25, 2254). Briefly, CD4 purified T cells were activated for 48 h 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). T cells were transduced with LV-IL-10 / ΔNGFR (CD4 IL-10 ) were transduced at a multiplicity of infection (MOI) of 20. On day 11, CD4 + ΔNGFR + Cells were transfected with CD271 +They were bead-sorted using microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany) and expanded in X-VIVO15 medium with 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 allogeneic feeder mixture as previously described (Locafaro et al. Mol Ther 2017, 25, 2254). After 14 days, cells were harvested and frozen. Thawed CD4 IL-10 Cells were restimulated and after the second and third restimulation, expansion was functionally characterized in vitro and used for in vivo experiments.

[0327] Vector copy number analysis. Cells were cultured for 11 days after transduction to exclude non-integrated vector forms. Genomic DNA was isolated by QIAamp DNA Blood Mini Kit (QIAGEN, 51106) according to the manufacturer's instructions. Vector integration was quantified by QX200 Droplet Digital PCR System (Bio-Rad) according to the manufacturer's instructions.

[0328] Cytokine determination. Single-donor and polydonor CD4 IL-10 Cells were left unstimulated or stimulated with immobilized anti-CD3 (10 μg / mL) and soluble anti-CD28 (1 μg / mL) mAbs in a final volume of 200 μL of medium (96-well round-bottom plates, 2 × 10 5 Supernatants were harvested after 48 h of culture and levels of IL-10, IL-4, IL-5, IFN-γ, and IL-22 were determined by ELISA according to the manufacturer's instructions (BD Biosciences).

[0329] Flow cytometry analysis. 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 by BD LSRFortessa using FlowJo 10 software.

[0330] Killing assay. Single-donor and polydonor CD4 IL-10 The cytotoxicity of the cells was analyzed in co-culture experiments (Locafaro et al. Mol Ther 2017, 25, 2254). Briefly, non-myeloid and myeloid leukemia cell lines, K562 and ALL-CM, respectively, were used as target cells to inhibit CD4 IL-10 Together with the cells, a 1:1 ratio (10 5 target cells and 10 5 CD4 IL-10 Cells) were seeded for 3 days. At the end of the co-culture, cells were harvested and K562 and ALL-CM cells were transfected with CD45 + , CD3 - Analyzed based on expression and counted by FACS.

[0331] Suppression assays. Single-donor and polydonor CD4 IL-10 To measure the suppressive capacity of cells, allogeneic PBMCs were labeled with the cell proliferation dye eFluor® 670 (Invitrogen, CA, USA) according to the manufacturer's instructions. +Allogeneic mature dendritic cells (DCs) from 100 cells were activated in the presence of GM-CSF and IL-4 in the presence of anti-CD3 mAb (50 ng / mL). Peripheral blood CD14 monocytes were positively selected using CD14 microbeads (Miltenyi Biotec) according to the manufacturer's instructions. Cells were cultured for 5 days in the presence of 10 ng / mL recombinant human (rh) IL-4 (R&D Systems) and 100 ng / mL recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) (Genzyme) at 37°C in RPMI1640 (Lonza) supplemented with 10% fetal bovine serum (FBS), 100 U / mL penicillin / streptomycin (Lonza), 2 mM L-glutamine (Lonza). To generate mature dendritic cells (mDCs), on day 5 the cells were stimulated with 1 mg / mL lipopolysaccharide (LPS; Sigma) for an additional 2 days. On day 7, DCs were harvested, phenotyped, and used to stimulate T cells. The purity and maturation status of DCs were checked by flow cytometry to determine the expression of CD1a, CD14, CD86, CD83, and HLA-DR.

[0332] Labeled cells were seeded in 96-well round-bottom plates in a final volume of 200 μL and incubated for 3 days as follows: (i) 5 × 10 labeled PBMC alone 4 (ii) labeled PBMCs 5 x 10 4 5 x 10 mature DCs per well 3 cells / well + anti-CD3 mAb (50ng / mL); (iii) labeled PBMC 5×10 4 5 x 10 cells / well + single-donor or poly-donor CD4IL-10 cells 4 5 x 10 mature DCs per well 3 / well + anti-CD3 mAb (50 ng / mL).

[0333] After 3 days of culture, the cells were harvested and transferred to 96-well V-bottom plates for immunofluorescence staining. + eFluor670 + and CD8 + eFluor670+ Cells were analyzed by FACS gating. The percentage of inhibition was calculated by measuring the dilution of eFluor670 label as previously described (Locafaro et al. Mol Ther 2017,25,2254).

[0334] Model of graft-versus-host disease: In all experiments, 6 / 8-week-old female NSG mice were used. On day 0, mice were irradiated whole body with a single dose of 175–200 cGy from a linear accelerator according to the mouse weight. In some experiments, mice were irradiated with a single dose of 350 cGy. Mice were inoculated with PBMC cells (5 × 10 6 pcs or 2.5×10 6 ), or CD4 IL-10 Cells (5 × 10 of a single donor or a polydonor that is a pool of three donors) 6 pcs or 2.5×10 6 cells), or PBMCs (5 × 10 6 pcs or 2.5×10 6 ) and CD4 IL-10 cells (5×10 6 pcs or 2.5×10 6 Mice were intravenously injected with a combination of 100 mg / kg / day ...

[0335] Alternatively, on day 0, mice were totally irradiated as above. On day 3, mice were transfected with CD4 + T cells (2.5×10 6 individual), single-donor and polydonor (pooled from three donors) CD4 IL-10 cells (2.5×10 6 ), or CD4 + T cells (2.5×10 6 (individuals) were collected from single-donor and polydonor (pooled from three donors) CD4 IL-10 cells (2.5×10 6The induction of GvHD was monitored as described above.

[0336] 7. INCORPORATION BY REFERENCE All publications, patents, patent applications, and other documents cited in this application are 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 herein for all purposes.

[0337] 8. Equivalents While various specific embodiments have been illustrated and described, the above specification is not limiting. It will be recognized 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.

[0338] 9. Arrays SEQ ID NO:1 (Human IL-10 amino acid sequence-protein sequence: Ref P22301)

[0339] [ka]

[0340] SEQ ID NO:2 (an exemplary nucleic acid sequence for human IL-10)

[0341] [ka] SEQ ID NO:3 (ΔNGFR amino acid sequence)

[0342] [ka] SEQ ID NO:4 (an exemplary nucleic acid sequence of ΔNGFR)

[0343] [ka] SEQ ID NO:5 (nucleotide sequence of pLVIL-10):

[0344] [ka]

[0345] [ka]

[0346] [ka]

[0347] [ka] SEQ ID NO:6 (Viral Interleukin-10 Homolog, aka Interleukin-10 BCRF1, aka IL10H_EBVB9) Protein sequence: Ref:P03180

[0348] [ka] SEQ ID NO: 7 (Viral Interleukin-10 Homolog cDNA Sequence) Nucleotide sequence (cDNA): Ref:NC_007605.1

[0349] [ka] SEQ ID NO:8 (Exemplary human IL-10 variant with amino acid substitutions based on viral IL-10)

[0350] [ka] SEQ ID NO:9 (Exemplary human IL-10 variant with amino acid substitutions based on viral IL-10)

[0351] [ka] SEQ ID NO:10 (Mus musculus; "MOUSE")

[0352] [ka] SEQ ID NO:11 (Rattus norvegicus; "RAT")

[0353] [ka] SEQ ID NO:12 (Macaca mulatta; "MACMU")

[0354] [ka] SEQ ID NO:13 (Gorilla gorilla; "GORILLA")

[0355] [ka] SEQ ID NO:14 (Macaca fascicularis; "CYNO")

[0356] [ka] SEQ ID NO:15 (Papio Anubis; "OLIVE BABOON")

[0357] [ka] SEQ ID NO:16 (Pan paniscus; "BONOBO")

[0358] [ka] SEQ ID NO:17 (Pan troglodytes; "CHIMP")

[0359] [ka] SEQ ID NO: 18 (EBVB9)

[0360] [ka] SEQ ID NO: 19 huIL-10 hybrid #1

[0361] [ka] SEQ ID NO: 20 huIL-10 hybrid #2

[0362] [ka]

Claims

1. A population of CD4 + T cells (polydonor CD4 IL-10 cells) that have been genetically modified to contain an exogenous polynucleotide encoding IL-10, wherein the CD4 + T cells are obtained from at least two different T cell donors. (a) the CD4+ T cells are obtained from 2, 3, 4, 5, 6, 7, 8, 9, or 10 different T cell donors; (b) the CD4 + T cells in said population collectively have 6, 7, 8, 9, 10, 11, 12 or more different HLA haplotypes; (c)(i) all CD4 + T cells in said population: (A) have at least 1 / 10, 2 / 10, 3 / 10, 4 / 10, 5 / 10, 6 / 10, 7 / 10, 8 / 10, or 9 / 10 matches to each other at the HLA-A, HLA-B, HLA-C, HLA-DRB1, and HLA-DQB1 loci; (B) have at least a 1 / 8, 2 / 8, 3 / 8, 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; (C) have a 2 / 2 match to each other at the HLA-A locus; (D) have a 2 / 2 match to each other at the HLA-B locus; (E) have a 2 / 2 match to each other at the HLA-C locus; and / or (F) have at least a 3 / 4 or 4 / 4 match to each other at the HLA-DRB1 and HLA-DQB1 loci; or (ii) all CD4 + T cells in said population have less than 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; optionally, all CD4 + T cells in said population: (A) have less than 4 / 8, 5 / 8, 6 / 8, 7 / 8, or 8 / 8 matches to each other at the HLA-A, HLA-B, HLA-C, and HLA-DRB1 loci; (B) have less than a 2 / 2 match to each other at the HLA-A locus; (C) have less than a 2 / 2 match to each other at the HLA-B locus; (D) have less than a 2 / 2 match to each other at the HLA-C locus; and / or (E) having less than a 2 / 4, 3 / 4, or 4 / 4 match to each other at the HLA-DRB1 and HLA-DQB1 loci; (d) all CD4 + T cells in said population have the A*02 or A*24 allele; (e) none of the CD4 + T cells are immortalized; (f) the exogenous polynucleotide comprises an IL-10-encoding polynucleotide segment operably linked to an expression control element; optionally, the expression control element drives constitutive expression of the encoded IL-10; (g) the IL-10 is: (i) is human IL-10; or (ii) is a viral IL-10; optionally, the IL-10-encoding polynucleotide segment encodes a protein having the sequence of SEQ ID NO:6; optionally, the IL-10-encoding polynucleotide segment has the sequence of SEQ ID NO:7; and / or (h) the IL-10-encoding polynucleotide segment encodes a protein having the sequence of SEQ ID NO:1; optionally, the IL-10-encoding polynucleotide segment has the sequence of SEQ ID NO:2; The CD4 + T cell population of claim 1 .

3. The exogenous polynucleotide of claim 2, wherein: (a) further comprising a polynucleotide segment encoding a selectable marker; optionally, the selectable marker is: (i) is ΔNGFR; optionally, (A) the ΔNGFR has the sequence of SEQ ID NO: 3; or (B) the polynucleotide segment comprises the sequence of SEQ ID NO:4; or (ii) is a truncated form of the EGFR polypeptide; (b) having the sequence of SEQ ID NO: 5; and / or (c)(i) integrated into the T cell nuclear genome; or (ii) not integrated into the T-cell nuclear genome; wherein, optionally, the exogenous polynucleotide further comprises a lentiviral vector sequence. The CD4 + T cell population of claim 1 . (a) at least 70% of CD4 + T cells in said population express IL-10; optionally, at least 90% of CD4 + T cells in said population express IL-10; optionally, at least 95% or 98% of CD4 + T cells in said population express IL-10; (b) the genetically modified CD4 + T cells constitutively express at least 100 pg of IL-10 per 10 6 CD4 + T cells / mL of culture medium; optionally, the genetically modified CD4 + T cells constitutively express at least 100 pg, 200 pg, 500 pg, 1 ng, 5 ng, 10 ng, or 50 ng of IL-10 per 10 6 CD4 + T cells / mL; (c) the genetically modified CD4 + T cells express IL-10 at a level at least 5-fold higher than unmodified CD4 + T cells; optionally, the genetically modified CD4 + T cells express IL-10 at a level at least 10-fold higher than unmodified CD4 + T cells; (d) at least 70% of CD4 + T cells in said population express the selection marker from the exogenous polynucleotide; optionally, at least 90% of CD4 + T cells in said population express the selection marker from the exogenous polynucleotide; optionally, at least 95% or 98% of CD4 + T cells in said population express the selection marker from the exogenous polynucleotide; (e) the genetically modified CD4 + T cells express CD49b, LAG-3, TGF-β, IFN-γ, GzB, perforin, CD18, CD2, CD226, and / or IL-22; and / or (f) the CD4 + T cells: (i) is in a frozen suspension; and / or (ii) present in a liquid suspension; The CD4 + T cell population of claim 1 . (ii) suspended in a pharmaceutically acceptable carrier; (i) a CD4 + T cell population according to any one of claims 1 to 4.

10. A pharmaceutical composition comprising:

6. A method for producing polydonor CD4 IL-10 cells, comprising: （A） (i) pooling primary CD4 + T cells obtained from at least two different T cell donors; and (ii) modifying pooled CD4 + T cells by introducing an exogenous polynucleotide encoding IL-10; thereby obtaining polydonor CD4 IL-10 cells; or （B） (i) modifying primary CD4 + T cells obtained from at least two different T cell donors, wherein the CD4 + T cells of each donor are individually modified by introducing an exogenous polynucleotide encoding IL-10; and (ii) pooling the genetically modified CD4 + T cells; Thereby obtaining polydonor CD4 IL-10 cells. A method comprising:

7. (a) before or after the step of modifying CD4 + T cells, or after the step of pooling the genetically modified CD4 + T cells, Incubating the primary cultured CD4 + T cells in the presence of anti-CD3 and anti-CD28 antibodies, or anti-CD3 and CD28 antibody coated beads. optionally, the primary cultured CD4 + T cells are further incubated in the presence of IL-2; (b) the exogenous polynucleotide is introduced into the primary CD4 + T cells using a viral vector; optionally, the viral vector is a lentiviral vector; (c) the exogenous polynucleotide is: (i) SEQ ID NO: 1; or (ii) SEQ ID NO: 2 or 7 a segment encoding IL-10 having the sequence: (d) the exogenous polynucleotide further comprises a segment encoding a selectable marker; optionally; (i) the encoded selectable marker is ΔNGFR; optionally, the encoded selectable marker has the sequence of SEQ ID NO: 3; and / or (ii) the method further comprises, after step (ii), Optionally, further comprising isolating said genetically modified CD4 + T cells that express said selection marker, thereby generating an enriched population of genetically modified CD4 + T cells; (A) at least 70% of the genetically modified CD4 + T cells in the enriched population express IL-10; optionally, at least 90%, 95%, or 98% of the genetically modified CD4 + T cells in the enriched population express IL-10; (B) at least 70% of the genetically modified CD4 + T cells in the enriched population express a selectable marker; optionally, at least 90%, 95%, or 98% of the genetically modified CD4 + T cells in the enriched population express a selectable marker; and / or (C) the method further comprises incubating the enriched population of genetically modified CD4 + T cells; optionally, the incubating the enriched population of genetically modified CD4 + T cells is performed in the presence of anti-CD3 antibody and anti-CD28 antibody, or CD3 antibody and CD28 antibody coated beads, in the presence of IL-2; (e) the method further comprises a subsequent step of freezing the genetically modified CD4 + T cells; (f) in step (i), the primary CD4 + T cells are obtained from 2, 3, 4, 5, 6, 7, 8, 9, or 10 different T cell donors; optionally; (i) the at least two T cell donors are: (A) have at least a 1 / 10, 2 / 10, 3 / 10, 4 / 10, 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; (B) have at least a 1 / 8, 2 / 8, 3 / 8, 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; (C) have a 2 / 2 match to each other at the HLA-A locus; (D) have a 2 / 2 match to each other at the HLA-B locus; (E) have a 2 / 2 match to each other at the HLA-C locus; and / or (F) have at least a 3 / 4 or 4 / 4 match to each other at the HLA-DRB1 and HLA-DQB1 loci; or (ii) the at least two T cell donors; (A) have less than 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; (B) have less than 4 / 8, 5 / 8, 6 / 8, 7 / 8, or 8 / 8 matches to each other at the HLA-A, HLA-B, HLA-C, and HLA-DRB1 loci; (C) have less than a 2 / 2 match to each other at the HLA-A locus; (D) have less than a 2 / 2 match to each other at the HLA-B locus; (E) have less than a 2 / 2 match to each other at the HLA-C locus; and / or (F) having a 3 / 4 or less than 4 / 4 match to each other at the HLA-DRB1 and HLA-DQB1 loci; (g) each of said at least two T cell donors has an A*02 or A*24 allele; and / or (h) in step (i), the primary cultured CD4 + T cells: (i) obtained from one or more cryopreservations; or (ii) obtained from unfrozen peripheral blood mononuclear cells of said at least two different T cell donors; optionally, said method further comprises isolating CD4 + T cells from said peripheral blood mononuclear cells; The method of claim 6.

8. A pharmaceutical composition for use in a method for treating a patient in need of immune tolerance, comprising a CD4 + T cell population described in any one of claims 1 to 4, the method comprising administering polydonor CD4 IL-10 cells, or a pharmaceutical composition, to the patient. (a) the method further comprises the preliminary step of thawing the frozen suspension of polydonor CD4 IL-10 cells; (b) the polydonor CD4 IL-10 cells or the pharmaceutical composition are: (i) preventing or reducing the severity of a pathogenic T cell response in said patient; (ii) reducing inflammation or enhancing immune tolerance; or (iii) enhancing tissue repair; (c) the method further comprises administering mononuclear cells to said patient; optionally: (i) the polydonor CD4 IL-10 cells or the pharmaceutical composition and the mononuclear cells are administered simultaneously; or (ii) the mononuclear cells are administered either before or after administration of the polydonor CD4 IL-10 cells or the pharmaceutical composition; (d) the method further comprises administering hematopoietic stem cells (HSCs) of an HSC donor to said patient either before or after administration of said polydonor CD4 IL-10 cells or pharmaceutical composition; optionally: (i) the HSC donor is partially HLA-mismatched to the patient; optionally, the HSC donor: (A) has less than a 5 / 10, 6 / 10, 7 / 10, 8 / 10, 9 / 10, or 10 / 10 match to said patient at the HLA-A, HLA-B, HLA-C, HLA-DRB1, and HLA-DQB1 loci; (B) has less than a 4 / 8, 5 / 8, 6 / 8, 7 / 8, or 8 / 8 match to said patient at the HLA-A, HLA-B, HLA-C, and HLA-DRB1 loci; (C) has less than a 2 / 2 match to said patient at the HLA-A, HLA-B, or HLA-C locus; or (D) having a 3 / 4 or less than 4 / 4 match to said patient at the HLA-DRB1 and HLA-DQB1 loci; and / or (ii) the polydonor CD4 IL-10 cells or the pharmaceutical composition prevent or reduce the severity of a lymphoid cell pathogenic response from the transplanted hematopoietic cells; (e) one or more of said T cell donors are HLA-mismatched or partially HLA-mismatched to said patient; optionally, one or more of said T cell donors are: (i) has less than a 5 / 10, 6 / 10, 7 / 10, 8 / 10, 9 / 10, or 10 / 10 match to said patient at the HLA-A, HLA-B, HLA-C, HLA-DRB1, and HLA-DQB1 loci; (ii) has less than a 4 / 8, 5 / 8, 6 / 8, 7 / 8, or 8 / 8 match to said patient at the HLA-A, HLA-B, HLA-C, and HLA-DRB1 loci; (iii) has less than a 2 / 2 match to said patient at the HLA-A, HLA-B, or HLA-C locus; or (iv) having a 2 / 4, 3 / 4, or less than 4 / 4 match to said patient at the HLA-DRB1 and HLA-DQB1 loci; (f) one or more of said T cell donors are HLA-mismatched or partially HLA-mismatched to said HSC donor; optionally, one or more of said T cell donors are: (i) has less than a 5 / 10, 6 / 10, 7 / 10, 8 / 10, 9 / 10, or 10 / 10 match to said HSC donor at the HLA-A, HLA-B, HLA-C, HLA-DRB1, and HLA-DQB1 loci; (ii) has less than a 4 / 8, 5 / 8, 6 / 8, 7 / 8, or 8 / 8 match to said HSC donor at the HLA-A, HLA-B, HLA-C, and HLA-DRB1 loci; (iii) has less than a 2 / 2 match to the HSC donor at the HLA-A, HLA-B, or HLA-C locus; or (iv) having a 3 / 4 or less than 4 / 4 match to said HSC donor at the HLA-DRB1 and HLA-DQB1 loci; (g) the polydonor CD4 IL-10 cells or the pharmaceutical composition prevent or reduce the severity of GvHD caused by the transplanted hematopoietic stem cells; and / or (h) the patient has neoplastic cells; optionally, (i) the neoplastic cells express CD13, HLA-class I, and CD54; (ii) the neoplastic cells express CD112, CD58, or CD155; and / or (iii) the patient has cancer, optionally wherein the cancer is a solid or hematologic neoplasm; and / or (i) the exogenous polynucleotide further comprises a segment encoding a selectable marker, and the method detects the selectable marker in a biological sample obtained from the patient; thereby detecting the presence or absence of polydonor CD4 IL-10 T cells; optionally, said biological sample is a biopsy or blood from said patient. The pharmaceutical composition of claim 8.

10. The patient is one of the following cancers: 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 family of tumors, eye cancer, gallbladder cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor (GIST), gestational trophoblastic disease, Hodgkin's disease, Kaposi's sarcoma, kidney cancer, laryngo-hypopharyngeal cancer, leukemia, acute lymphoblastic leukemia (ALL), acute myeloid leukemia (including AML, myeloid sarcoma, and leukemia cutis), chronic lymphocytic leukemia (CLL), chronic myeloid (CML) leukemia, chronic myelomonocytic leukemia (CMML), childhood leukemia, liver cancer, lung cancer, non-small cell lung cancer, small cell lung cancer, alveolar lung cancer, pulmonary carcinoid tumor, lymphoma, cutaneous lymphoma, malignant mesothelioma, multiple myeloma, myelodysplastic syndrome, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, non-Hodgkin's lymphoma, childhood non-Hodgkin's lymphoma, oral 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, skin cancer - melanoma, skin cancer - Merkel cell, small intestine cancer, stomach cancer, testicular cancer, thymic cancer, thyroid cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Waldenstrom's macroglobulinemia, and Wilms' tumor; optionally, the patient has: (a) have cancer of the myeloid system; or (b) have AML or CML; The pharmaceutical composition of claim 8.

11. The patient has an inflammatory disease or an autoimmune disease; optionally, the inflammatory or autoimmune disease is selected from the group consisting of type 1 diabetes, autoimmune uveitis, autoimmune hepatitis, vitiligo, alopecia areata, 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 disease, scleroderma, and celiac disease; optionally, the inflammatory disease or autoimmune disease is Crohn's disease, ulcerative colitis, celiac disease, type 1 diabetes, lupus, psoriasis, psoriatic arthritis, or rheumatoid arthritis; optionally: (a) the patient has a disease or disorder associated with hyperactivity of the NLPR3 inflammasome; (b) the patient has type 2 diabetes, a neurodegenerative disease, a cardiovascular disease, or an inflammatory bowel disease; (c) the patient has a disease or disorder associated with increased IL-1β production by activated monocytes, macrophages, or dendritic cells; (d) the patient has a disease or disorder associated with increased IL-18 production by activated monocytes, macrophages, or dendritic cells; or (e) the patient has a disease or disorder associated with increased production of mature caspase-1 by activated monocytes, macrophages, or dendritic cells; The pharmaceutical composition of claim 8.

12. (a) The patient has a disease or disorder associated with hyperactivity of the NLPR3 inflammasome; (b) the patient has type 2 diabetes, a neurodegenerative disease, a cardiovascular disease, or an inflammatory bowel disease; (c) the patient has a disease or disorder associated with increased IL-1β production by activated monocytes, macrophages, or dendritic cells; (d) the patient has a disease or disorder associated with increased IL-18 production by activated monocytes, macrophages, or dendritic cells; (e) the patient has a disease or disorder associated with increased production of mature caspase-1 by activated monocytes, macrophages, or dendritic cells; (f) the patient has an allergic or atopic disease; optionally, the allergic or atopic disease is selected from the group consisting of asthma, atopic dermatitis, and rhinitis; (g) the patient has a food allergy; (h) the method further comprises administering an organ transplant to the patient either before or after administering the population of CD4 + T cells or the pharmaceutical composition; optionally, the polydonor CD4 IL-10 cells or the pharmaceutical composition prevents or reduces the severity of host rejection of the organ transplant; (i) the method further comprises transplanting iPS cell-derived cells or tissue into the patient either before or after administration of the CD4 + T cell population or the pharmaceutical composition; optionally, the polydonor CD4 IL-10 cells or the pharmaceutical composition prevents or reduces the severity of host rejection of the cell transplant; (j) the method further comprises administering a recombinant AAV to the patient either before or after administration of the polydonor CD4 IL-10 cells or the pharmaceutical composition; optionally: (i) the patient has organ and / or tissue damage; (ii) the polydonor CD4 IL-10 cells or the pharmaceutical composition reduce an immune response to the recombinant AAV; optionally, the method further comprises administering to the patient an immunogenic therapeutic protein either before or after administration of the CD4 IL-10 cells, the CD4 IL-10 cell population, or the pharmaceutical composition; and optionally: (A) the CD4 IL-10 cells, the CD4 IL-10 cell population, or the pharmaceutical composition reduces the immune response to an immunogenic therapeutic protein; and / or (B) the immunogenic therapeutic protein is selected from a therapeutic antibody, a Factor VIII replacement, a cytokine, and a cytokine mutein; or (k) the patient has an exaggerated immune response to a viral or bacterial infection; optionally, the patient has a coronavirus infection; optionally, the patient has organ and / or tissue damage. The pharmaceutical composition of claim 8.

13. A pharmaceutical composition comprising polydonor CD4 IL-10 cells for use in a method of treating a patient having a malignant disease, said method comprising: administering allo-HSCT to said patient; and administering a therapeutically effective amount of polydonor CD4 IL-10 cells optionally, The pharmaceutical composition, wherein none of the donors of the CD4 IL-10 cells in the polydonor CD4 IL-10 cells is a donor of the HSCT.

14. A pharmaceutical composition comprising the CD4+ T cell population of claim 1 for use in a method of treating a hematological cancer in a patient, said method comprising: administering to the patient polydonor CD4 IL-10 cells in an amount sufficient to induce an anti-cancer effect; A pharmaceutical composition, wherein said polydonor CD4 IL-10 cells are genetically modified by vector-mediated gene transfer of a coding sequence for human IL-10 under the control of a constitutive or inducible promoter.

15. (a) the method further comprises administering allo-HSCT to the patient before or after administration of the polydonor CD4 IL-10 cells; optionally, the amount of polydonor CD4 IL-10 cells is further sufficient to inhibit or prevent graft-versus-host disease (GvHD) without inhibiting graft-versus-leukemia (GvL) or graft-versus-tumor (GvT) efficacy of the allo-HSCT; (b) the hematological cancer is myeloid leukemia; (c) the polydonor CD4 IL-10 cells target and kill cancer cells that express CD13 and / or HLA-class I; (d) the hematological cancer or the myeloid leukemia is acute myeloid leukemia (AML); (e) the allo-HSCT is obtained from a donor that is related or unrelated with respect to the recipient; (f) the polydonor CD4 IL-10 cells are non-autologous to the recipient; (g) the polydonor CD4 IL-10 cells are: (i) are not anergized to host alloantigens prior to administration to the host; (ii) polyclonal; (iii) is polyclonal and non-autologous to the recipient; or (iv) isolated from at least two donors and subsequently genetically modified; optionally, none of the at least two donors is the same as the allo-HSCT donor; (h) the allo-HSCT is obtained from a donor that is matched or mismatched with respect to the recipient; (i) the polydonor CD4 IL-10 cells target and kill cells expressing CD54; (j) the polydonor CD4 IL-10 cells are: (i) HLA-class I and CD54; (ii) CD112; and / or (iii) CD58 and / or targeting and killing cancer cells expressing (k) the polydonor CD4 IL-10 cells target and kill cancer cells in the host. The pharmaceutical composition of claim 14.

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