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

Abstract

The present disclosure provides a multi-donor CD4IL-10 cell population that is generated by genetic modification of CD4 + T cells from at least two different T cell donors. Further provided are methods of generating the multi-donor CD4IL-10 cells and methods of using the multi-donor CD4IL-10 cells for immune tolerance, treatment of GvHD, cell and organ transplantation, cancer, autoimmune and inflammatory diseases, and other immune disorders.

Description

1. Cross-reference to related applications

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 295,389, filed on December 30, 2021, which is hereby incorporated by reference in its entirety. 2. Sequence Listing

[0004] This application contains a sequence listing, which has been submitted in XML format and is hereby incorporated by reference in its entirety. The XML copy was created on December 23, 2022, named 37104-49835Sequence-Listing.xml, and is 30.7 kilobytes (KB) in size. 3. Background technology

[0005] Regulatory T cells belong to a small but important subset of T cells that maintain immune tolerance to self and non-pathogenic antigens and maintain immune homeostasis. There are two major populations of regulatory T cells: CD4 + 、FOXP3 + CD25 + T cells (FOXP3 + cells) and type 1 regulatory T (Tr1) cells. FOXP3 + Both Tr1 and Tr1 cells downregulate pathogenic T cell responses in various preclinical models of organ and islet transplantation, graft-versus-host disease (GvHD), and various autoimmune and inflammatory diseases.

[0006] Tr1 cells have been shown to be effective in clinical studies. Administration of cloned antigen-specific autologous Tr1 cells to patients with persistent moderate to severe Crohn's disease resulted in objective, temporary remission (Desreumaux et al., Gastroenterology. 2012; 143(5): 1207-1217.e2.). In addition, after allogeneic hematopoietic stem cell transplantation (allo-HSCT), donor-derived allogeneic specific CD4 + Adoptive transfer of T cell populations into leukemia patients results in rapid reconstitution of the immune system and protection against microbial and viral infections without severe GvHD. In responding patients, long-term remission and tolerance (>7 years) are achieved, leading to cure (Bacchetta et al., Front Immunol. 2014; 5:16).

[0007] Despite these encouraging results, the production of donor-derived or autologous Tr1 cells for large-scale treatment of patients with high unmet medical needs is not always feasible and is cumbersome, and the generation of large quantities of pure Tr1 cells is not achievable.

[0008] Recently, Locafaro and colleagues used a bidirectional lentiviral vector containing the human IL-10 gene to transduce purified CD4 + T cells circumvent some of these problems. The resulting single-donor CD4 IL-10 The cell population has the main functions of naturally occurring Tr1 cells. Like Tr1 cells, single donor CD4 IL-10 Cells produce high levels of IL-10 and downregulate 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 the humanized xeno-GvHD model, these single donor CD4 IL-10 The cells were shown to effectively reduce GvHD in a humanized xeno-GvHD model while retaining graft-versus-leukemia (GvL) activity. See Locafaro et al. Mol Ther. 2017; 25(10): 2254-2269 and WO2016 / 146,542.

[0009] Although it is possible to generate highly purified single-donor CD4 IL-10 The use of cells for therapeutic purposes remains extremely limited due to qualitative and quantitative variability between batches, which is likely related to intrinsic differences between individual donors and variations in the quality of the buffy coat. 4. Overview

[0011] The present disclosure provides for the use of multiple donor CD4 IL-10 Novel Tr1-based therapeutics based on cell populations. Multi-donor CD4 IL-10 The cells are CD4 T cells obtained from at least two different T cell donors and then genetically modified to contain an exogenous polynucleotide encoding IL-10. + T cells. The T cell donor is a third party donor who is neither a recipient of multiple donor CD4 IL-10 The host of cell therapy is not a donor of HSC or organ transplantation. IL-10 The cells are not alloantigen specific, ie, they have not been primed or stimulated with cells from the host prior to administration.

[0012] The applicant proves that multiple donor CD4 IL-10 Cells have single donor CD4 IL-10 The cells have comparable cytokine production characteristics, immunosuppressive and cytotoxic abilities. In addition, in vivo, they are effective in preventing +T cell-mediated xenogeneic GvHD compared with single-donor CD4 IL-10 The cells were more effective, while they themselves did not induce GvHD. Overall, these multi-donor CD4 IL-10 Functional properties of cells in vitro and in vivo compared with single-donor CD4 IL-10 Cells are equal to or better than them.

[0013] Based on these results, the applicant claims that multi-donor allogeneic CD4 IL-10 The cells can be used for therapeutic purposes in GvHD, cell and organ transplantation, autoimmune and inflammatory diseases.

[0014] Furthermore, by using third-party T cells and eliminating the requirement for allogeneic specificity, multiple donor CD4 IL-10 The cells make Tr1-based cell therapies accessible to a larger population of patients with various genetic backgrounds.

[0015] Thus, in a first aspect, the present disclosure provides a CD4 + A population of T cells that has been genetically modified to contain an exogenous polynucleotide encoding IL-10, wherein the CD4 + T cells were obtained from at least two different T cell donors (multiple donor CD4 IL-10 cell).

[0016] In some embodiments, the CD4 + T cells are obtained from two, three, four, five, six, seven, eight, nine, or ten different T cell donors. In some embodiments, the CD4 + The T cells have a total of six, seven, eight, nine, ten, eleven, twelve or more different HLA haplotypes.

[0017] In some embodiments, all CD4+ T cells in the population are at least 1 / 10, 2 / 10, 3 / 10, 4 / 10, 5 / 10, 6 / 10, 7 / 10, 8 / 10, or 9 / 10 matched to each other at the HLA-A, HLA-B, HLA-C, HLA-DRB1, and HLA-DQB1 loci. In some embodiments, all CD4+ T cells in the population are at least 1 / 8, 2 / 8, 3 / 8, 4 / 8, 5 / 8, 6 / 8, 7 / 8, or 8 / 8 matched to each other at the HLA-A, HLA-B, HLA-C, and HLA-DRB1 loci. In some embodiments, all CD4+ T cells in the population are at least 1 / 8, 2 / 8, 3 / 8, 4 / 8, 5 / 8, 6 / 8, 7 / 8, or 8 / 8 matched to each other at the HLA-A, HLA-B, HLA-C, and HLA-DRB1 loci. + T cells have a 2 / 2 match with each other at the HLA-A locus. In some embodiments, all CD4 + T cells have a 2 / 2 match with each other at the HLA-B locus. In some embodiments, all CD4+ T cells have a 2 / 2 match with each other at the HLA-C locus. In some embodiments, all CD4 + The T cells have at least a 3 / 4 or 4 / 4 match with each other at the HLA-DRB1 and HLA-DQB1 loci. In some embodiments, all CD4 + T cells have the A*02 or A*24 allele.

[0018] In some embodiments, all CD4 + T cells 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, all CD4 + T cells 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, all CD4 + T cells have less than a 2 / 2 match with each other at the HLA-A locus. In some embodiments, all CD4 + T cells have less than a 2 / 2 match with each other at the HLA-B locus. In some embodiments, all CD4 + T cells have less than a 2 / 2 match with each other at the HLA-C locus. In some embodiments, all CD4 + The T cells have less than 3 / 4 or 4 / 4 matches with each other at the HLA-DRB1 and HLA-DQB1 loci.

[0019] In some embodiments, all CD4 + T cells do not match each other at the HLA-A, HLA-B, HLA-C, HLA-DRB1, and HLA-DQB1 loci. In some embodiments, all CD4 + T cells do not match each other at the HLA-A, HLA-B, HLA-C, and HLA-DRB1 loci. In some embodiments, all CD4 + T cells are mismatched with each other at the HLA-A locus. In some embodiments, all CD4 + The T cells do not match each other at the HLA-B locus. In some embodiments, all CD4 + T cells are mismatched with each other at the HLA-C locus. In some embodiments, all CD4 +T cells are mismatched at the HLA-DRB1 and HLA-DQB1 loci.

[0020] In some embodiments, none of the CD4 + 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, IL-10 is human IL-10. In some embodiments, IL-10 is a viral IL-10. In some embodiments, IL-10 is a variant of human IL-10, which has a human IL-10 sequence with one, two, three, four, five, six, seven, eight, nine or ten amino acid modifications. In some embodiments, one, two, three, four, five, six, seven, eight, nine or ten amino acid modifications are substituted with the amino acids of viral IL-10 at the corresponding amino acid positions. In some embodiments, the variant of human IL-10 has a sequence of SEQ ID NO:8 or 9.

[0021] In some embodiments, the IL-10 encoding polynucleotide segment encodes a protein 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. In some embodiments, the expression control element drives constitutive expression of the encoded IL-10. In some embodiments, the expression control element drives IL-10 in activated CD4 + In some embodiments, the expression control element drives tissue-specific expression or CD4 + T cell-specific expression.

[0022] 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 a sequence of SEQ ID NO: 3. In some embodiments, the exogenous polynucleotide comprises a sequence of SEQ ID NO: 4. In some embodiments, the exogenous polynucleotide has a sequence of SEQ ID NO: 5.

[0023] In some embodiments, the selection marker is a truncated EGFR polypeptide.In some embodiments, the selection marker is a truncated human EGFR polypeptide.

[0024] In some embodiments, the exogenous polynucleotide is integrated into the T cell nuclear genome. In some embodiments, the exogenous polynucleotide is not integrated into the T cell nuclear genome. In some embodiments, the exogenous polynucleotide further comprises a lentiviral vector sequence. In some embodiments, the exogenous polynucleotide is not integrated into the T cell nuclear genome.

[0025] In some embodiments, at least 70% of the CD4 + In some embodiments, at least 90% of the CD4+ T cells in the population express IL-10. In some embodiments, at least 95%, 98%, or 99% of the CD4+ T cells in the population express IL-10. + 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 this case, the IL-10 expression level can be determined by the expression level of the selection marker.

[0026] In some embodiments, the genetically modified CD4 + T cells constitutively express at least 100 pg IL-10 / 10 6 CD4 + T cells / mL culture medium. In some embodiments, the genetically modified CD4 + T cells constitutively express at least 200pg, 500pg, 1ng, 5ng, 10ng or 50ng IL-10 / 10 6 CD4 + T cells / mL. In some embodiments, the genetically modified CD4 + T cells expressed at least 1 or 2 ng IL-10 / 10 after activation with anti-CD3 and anti-CD28 antibodies 6 CD4 + T cells / mL. In some embodiments, the genetically modified CD4 + T cells expressing at least 2ng, 5ng, 10ng, 100ng, 200ng or 500ng IL-10 / 10 after activation with anti-CD3 and anti-CD28 antibodies 6 CD4 + T cells / mL. In some embodiments, the genetically modified CD4 + T cells were more expressed than unmodified CD4 + T cells express IL-10 at least 5-fold higher levels. In some embodiments, the genetically modified CD4 + T cells were more expressed than unmodified CD4 + T cells expressed IL-10 at least 10-fold higher levels.

[0027] In some embodiments, at least 70% of the CD4 +The T cells express a selection marker from an exogenous polynucleotide. In some embodiments, at least 90% of the CD4+ T cells in the population express a selection marker from an exogenous polynucleotide. In some embodiments, at least 95%, 98%, or 99% of the CD4+ T cells in the population express a selection marker from an exogenous polynucleotide. + The T cells express a selectable marker from an exogenous polynucleotide.

[0028] In some embodiments, the genetically modified CD4 + T cells express CD49b. In some embodiments, the genetically modified CD4 + T cells express LAG-3. In some embodiments, the genetically modified CD4 + T cells express TGF-β. In some embodiments, the genetically modified CD4 + T cells express IFN-γ. In some embodiments, the genetically modified CD4 + T cells express granzyme B (GzB). In some embodiments, the genetically modified CD4 + T cells express perforin. In some embodiments, the genetically modified CD4 + T cells express CD18. In some embodiments, the genetically modified CD4 + T cells express CD2. In some embodiments, the genetically modified CD4 + T cells express CD226. In some embodiments, the genetically modified CD4 + T cells express IL-22.

[0029] In some embodiments, CD4 + The T cells do not lose reactivity in the presence of peripheral blood mononuclear cells (PBMCs) from the host. In some embodiments, the CD4 + T cells did not lose their responsiveness in the presence of recombinant IL-10 protein, wherein the recombinant IL-10 protein was not expressed by the CD4 + T cell expression. In some embodiments, CD4 + T cells did not lose their responsiveness in the presence of DC10 cells from the host.

[0030] In some embodiments, CD4 + T cells are in frozen suspension. In some embodiments, CD4 + The T cells are in a liquid suspension. In some embodiments, the liquid suspension has been previously frozen.

[0031] Another aspect of the present disclosure provides a pharmaceutical composition comprising:

[0032] (i) CD4+ T cell populations suspended in

[0033] (ii) a pharmaceutically acceptable carrier.

[0034] In yet another aspect, the present disclosure provides for preparing multiple donor CD4 IL-10 A method for cells comprising the steps of:

[0035] (i) combining primary CD4+ T cells obtained from at least two different T cell donors; and

[0036] (ii) Modification of the incorporated CD4 + T cells,

[0037] Genetically modified CD4 + T cells.

[0038] In one aspect, the present disclosure provides for preparing multiple donor CD4 IL-10 A method for cells comprising the steps of:

[0039] (i) Obtain primary CD4 T cells from at least two different T cell donors + T cells; and

[0040] (ii) Modifying the CD4 of each donor by introducing an exogenous polynucleotide encoding IL-10 + T cells, then

[0041] (iii) Incorporation of genetically modified CD4 + T cells,

[0042] Genetically modified CD4 + T cells.

[0043] In some embodiments, after step (i) and before step (ii), after step (ii), after step (ii) and before step (iii), or after step (iii), the method further comprises the following steps:

[0044] Primary CD4 + In some embodiments, multiple donor CD4 IL-10 T cells have been used in Miltenyi Biotec's T CellTransAct TM In some embodiments, multiple donor CD4 IL-10T cells have been used in STEMCELL Technologies' ImmunoCult Human T Cell Activator TM cultured in the presence of .

[0045] In some embodiments, the method comprises further incubating primary CD4 + In some embodiments, a viral vector is used to introduce exogenous polynucleotides into primary CD4 + In some embodiments, the viral vector is a lentiviral vector. In some embodiments, the viral vector is a chimeric viral vector. In some embodiments, the viral vector is an adeno-associated viral vector. In some embodiments, the exogenous polynucleotide comprises a segment encoding IL-10 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.

[0046] 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 a 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.

[0047] In some embodiments, after step (ii), the method further comprises the step of isolating the genetically modified CD4 + T cells, thereby generating enriched genetically modified CD4 + T cell populations.

[0048] In some embodiments, at least 70% of the genetically modified CD4 + T cells express IL-10. In some embodiments, at least 90%, 95%, or 98% of the genetically modified CD4 + T cells express IL-10. In some embodiments, at least 70% of the genetically modified CD4 + T cells express a selectable marker. In some embodiments, at least 90%, 95%, or 98% of the genetically modified CD4 + T cells express a selectable marker.

[0049] In some embodiments, the method further comprises incubating the enriched genetically modified CD4 +In some embodiments, the enriched genetically modified CD4 T cells are incubated in the presence of IL-2, in the presence of anti-CD3 antibody and anti-CD28 antibody, or beads coated with anti-CD3 antibody and CD28 antibody. + Steps for T cell populations.

[0050] In some embodiments, the method further comprises freezing the genetically modified CD4 + In some embodiments, in step (i), primary CD4 + T cells are obtained from two, three, four, five, six, seven, eight, nine or ten 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 at 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 at HLA-A, HLA-B, HLA-C and HLA-DRB1 loci. In some embodiments, at least two T cell donors have 2 / 2 matches at HLA-A loci. In some embodiments, at least two T cell donors have 2 / 2 matches at HLA-B loci. 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 at least two T cell donors has an A*02 or A*24 allele.

[0051] 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 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 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 are less than 3 / 4 or 4 / 4 matched to each other at the HLA-DRB1 and HLA-DQB1 loci.

[0052] In some embodiments, in step (i), primary CD4 + T cells are obtained from one or more frozen stocks. In some embodiments, in step (i), primary CD4 + T cells were obtained from unfrozen peripheral blood mononuclear cells from at least two different T cell donors.

[0053] In some embodiments, the method further comprises isolating CD4 + In some embodiments, peripheral blood mononuclear cells are obtained from the buffy coat or apheresis.

[0054] In another aspect, the present disclosure provides a method of treating a patient comprising the steps of:

[0055] Administration of the multi-donor CD4 IL-10 Cells or pharmaceutical compositions.

[0056] In some embodiments, the method further comprises thawing multiple donor CD4 IL-10 A prior step of freezing the suspension of cells.

[0057] In some embodiments, multiple donor CD4 IL-10 The cell or pharmaceutical composition prevents or reduces the severity of a pathogenic T cell response in a patient.

[0058] In some embodiments, multiple donor CD4 IL-10 The cell or pharmaceutical composition prevents or reduces the severity of an inflammatory or autoimmune response.

[0059] In some embodiments, the method further comprises the step of administering to the patient mononuclear cells from a hematopoietic stem cell (HSC) donor. IL-10 Cells or pharmaceutical compositions and mononuclear cells from HSC donors are administered simultaneously. In some embodiments, multiple donor CD4 IL-10 In some embodiments, the mononuclear cells are in PBMCs. In some embodiments, the mononuclear cells are in bone marrow. In some embodiments, the mononuclear cells are in cord blood. In some embodiments, the mononuclear cells have been isolated from PBMCs, bone marrow, or cord blood.

[0060] In some embodiments, the method further comprises the following steps:

[0061] In the administration of multiple donor CD4 IL-10 Before or after administering the cells or pharmaceutical composition, a hematopoietic stem cell (HSC) from a HSC donor is administered to the patient.

[0062] In some embodiments, the HSC donor is partially HLA-mismatched with the patient. In some embodiments, the HSC donor has less than 5 / 10, 6 / 10, 7 / 10, 8 / 10, 9 / 10 or 10 / 10 matching with the patient at 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 matching with the patient at HLA-A, HLA-B, HLA-C and HLA-DRB1 loci. In some embodiments, the HSC donor has less than 2 / 2 matching with the patient at HLA-A, HLA-B or HLA-C loci. In some embodiments, the HSC donor has less than 3 / 4 or 4 / 4 matching with the patient at HLA-DRB1 and HLA-DQB1 loci.

[0063] In some embodiments, one or more of the T cell donors are HLA mismatched or partially HLA mismatched with 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 with the patient at 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 with the patient at 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 with the patient at 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 with the patient at HLA-DRB1 and HLA-DQB1 loci. In some embodiments, one or more of the T cell donors are HLA mismatched or partially HLA mismatched with 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 with 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 with 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 with the HSC donor at the HLA-A, HLA-B or HLA-C loci. In some embodiments, one or more of the T cell donors is less than 3 / 4 or 4 / 4 matched to the HSC donor at the HLA-DRB1 and HLA-DQB1 loci.

[0064] In some embodiments, multiple donor CD4 IL-10 The cells or pharmaceutical compositions prevent GvHD or reduce the severity of GvHD by transplanted hematopoietic stem cells.

[0065] In some embodiments, multiple donor CD4 IL-10 The cells or pharmaceutical compositions prevent or reduce the severity of a pathogenic response by lymphoid cells present in a transplanted hematopoietic stem cell population.

[0066] In some embodiments, the patient has cancer. In some embodiments, the patient has neoplastic cells. In some embodiments, the neoplastic cells express CD13, HLA-I class, and CD54. In some embodiments, the neoplastic cells express CD112, CD58, or CD155.

[0067] In some embodiments, the patient has cancer. In some embodiments, the cancer is a solid or blood neoplasm. In some embodiments, the patient has cancer selected from the group consisting of adrenal cancer, anal cancer, bile duct cancer, bladder cancer, bone cancer, adult brain / CNS tumors, childhood brain / CNS tumors, breast cancer, male breast cancer, cancer of unknown primary, Castleman disease, cervical cancer, colon / rectum cancer, endometrial cancer, esophageal cancer, Ewing family tumors, eye cancer, gallbladder cancer, gastrointestinal carcinoid tumors, gastrointestinal stromal tumors (GIST), gestational trophoblastic disease, Hodgkin disease, Kaposi's sarcoma, kidney cancer, laryngeal and hypopharyngeal cancer, leukemia, acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML, including myeloid sarcoma and skin leukemia), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia ( =CML), chronic myelomonocytic leukemia (CMML), childhood leukemia, liver cancer, lung cancer, non-small cell lung cancer, small cell lung cancer, lung carcinoid tumor, lymphoma, cutaneous lymphoma, malignant mesothelioma, multiple myeloma, myelodysplastic syndrome, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, non-Hodgkin lymphoma, childhood non-Hodgkin lymphoma, oral cavity and oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, penile cancer, pituitary tumor, prostate cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma - adult soft tissue cancer, skin cancer, skin cancer - basal cell and squamous cell, skin cancer - melanoma, skin cancer - Merkel cell, small intestine cancer, stomach cancer, testicular cancer, thymic cancer, thyroid cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Waldenstrom's macroglobulinemia, and Wilms' tumor.

[0068] In some embodiments, the cancer is medullary carcinoma.In some embodiments, the cancer is AML or CML.

[0069] In some embodiments, the patient suffers from an inflammatory or autoimmune disease. In some embodiments, the inflammatory or autoimmune disease is selected from the group consisting of type 1 diabetes, autoimmune uveitis, autoimmune hepatitis, vitiligo, alopecia areata, rheumatoid arthritis, psoriasis, psoriatic arthritis, multiple sclerosis, systemic lupus erythematosus, inflammatory bowel disease, Addison's disease, Graves' disease, Sjögren's syndrome, Hashimoto's thyroiditis, myasthenia gravis, autoimmune vasculitis, pernicious anemia, ulcerative colitis, bullous diseases, scleroderma, Crohn's disease, celiac disease, and celiac disease.

[0070] 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.

[0071] In some embodiments, the patient has a disease or disorder involving overactivity of the NLPR3 inflammasome. In some embodiments, the patient has type 2 diabetes, a neurodegenerative disease, a cardiovascular disease, or an inflammatory bowel disease.

[0072] In some embodiments, the patient has a disease or disorder involving increased production of IL-1 β by activated monocytes, macrophages, or dendritic cells.

[0073] In some embodiments, the patient has a disease or disorder involving increased production of IL-18 by activated monocytes, macrophages, or dendritic cells.

[0074] In some embodiments, the patient has a disease or disorder involving increased production of mature caspase 1 by activated monocytes, macrophages, or dendritic cells.

[0075] In some embodiments, the patient suffers from an allergy or atopic disease. In some embodiments, the allergy or atopic disease is selected from the group consisting of asthma, atopic dermatitis, and rhinitis. In some embodiments, the patient suffers from a food allergy.

[0076] In some embodiments, the method further comprises administering a CD4 + T cell populations or pharmaceutical compositions before or after the patient undergoes cell and organ transplantation. In some embodiments, multiple donor CD4 IL-10 The cells or pharmaceutical compositions prevent or reduce the severity of host rejection of cell and organ transplants.

[0077] In some embodiments, the method further comprises administering a CD4 + The step of transplanting iPS cell-derived cells or tissues into a patient before or after the T cell population or pharmaceutical composition.

[0078] In some embodiments, multiple donor CD4 IL-10 The cells or pharmaceutical compositions prevent or reduce the severity of host rejection of a transplant.

[0079] In some embodiments, the method further comprises administering multiple donor CD4 IL-10The step of 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 before or after the cell or pharmaceutical composition is produced. In some embodiments, multiple donor CD4 IL-10 The cell or pharmaceutical composition reduces the 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.

[0080] In some embodiments, the patient has an excessive 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.

[0081] In some embodiments, the method further comprises administering multiple donor CD4 IL-10 The step of administering an immunogenic therapeutic protein to a patient before or after the cell population or pharmaceutical composition. IL-10 The cell population or pharmaceutical composition reduces the immune response to the immunogenic therapeutic protein.In some embodiments, the immunogenic therapeutic protein is selected from the group consisting of a therapeutic antibody, a Factor VIII replacement, a cytokine, and a cytokine mutein.

[0082] In some embodiments, the method further comprises the step of detecting a selectable marker in a biological sample obtained from the patient to detect the presence or absence of multiple donor CD4 IL-10 T cells. In some embodiments, the biological sample is a biopsy or blood from a patient.

[0083] In one aspect, the present disclosure provides a method for treating a patient with a malignant tumor, comprising: administering to the patient allo-HSCT, and administering a therapeutically effective amount of multiple donor CD4 IL-10 In some embodiments, multiple donor CD4 IL-10 In some embodiments, multiple donor CD4 IL-10 The cells were then administered allo-HSCT.

[0084] In some embodiments, multiple donor CD4 IL-10 CD4 in cells IL-10 None of the cell donors were HSCT donors.

[0085] In another aspect, the present disclosure provides a method for treating a hematological cancer, comprising: administering to a hematological cancer patient an amount of multiple donor CD4 IL-10 cells, wherein the multiple donor CD4 IL-10 The cells include CD4 T cells obtained from at least two different T cell donors + T cells are then genetically modified by vector-mediated gene transfer of the coding sequence for human IL-10 under the control of a constitutive or inducible promoter.

[0086] In some embodiments, the method of treating a hematological cancer comprises the following steps, wherein a sufficient amount of multiple donor CD4 + T cells have been obtained from individual donors and genetically modified individually by vector-mediated gene transfer of the coding sequence for human IL-10 under the control of a constitutive or inducible promoter and then pooled.

[0087] In some embodiments, the method of treating a blood cancer comprises the following steps, wherein the multiple donor CD4+ T cells obtained from the respective donors sufficient to induce an anti-cancer effect to be administered are first combined. + T cells, and this pool is then genetically modified by vector-mediated gene transfer of the coding sequence for human IL-10 under the control of a constitutive or inducible promoter.

[0088] In some embodiments, the method further comprises administering multiple donor CD4 IL-10 In some embodiments, multiple donor CD4 IL-10 The amount of cells is also sufficient to inhibit or prevent graft-versus-host disease (GvHD) without inhibiting the graft-versus-leukemia (GvL) or graft-versus-tumor (GvT) efficacy of allo HSCT.

[0089] In some embodiments, the hematological cancer is a myeloid leukemia.

[0090] In some embodiments, multiple donor CD4 IL-10 Cells target and kill cancer cells expressing CD13. In some embodiments, multiple donor CD4 IL-10 The cells target and kill cancer cells expressing HLA class I. In some embodiments, the myeloid leukemia is acute myeloid leukemia (AML).

[0091] In some embodiments, 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, multiple donor CD4 IL-10The cells are allogeneic to the recipient. In some embodiments, multiple donor CD4 IL-10 The cells do not lose reactivity to host alloantigens prior to administration to the host.

[0092] In some embodiments, multiple donor CD4 IL-10 The cells are Tr1-like cells.

[0093] In some embodiments, multiple donor CD4 IL-10 The cells are polyclonal. In some embodiments, multiple donor CD4 IL-10 The cells are polyclonal and non-autologous to the recipient.

[0094] In some embodiments, multiple donor CD4 IL-10 The cells are isolated from at least two donors and then genetically modified. 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 the recipient.

[0095] In some embodiments, multiple donor CD4 IL-10 Cells are targeted and killed by CD54 expressing cells. In some embodiments, multiple donor CD4 IL-10 Cells target and kill cancer cells expressing HLA class I and CD54. In some embodiments, multiple donor CD4 IL-10 Cells target and kill cancer cells expressing CD112. In some embodiments, multiple donor CD4 IL-10 Cells target and kill cancer cells expressing CD58. In some embodiments, multiple donor CD4 IL-10 The cells target and kill cancer cells in the host.

[0096] One aspect of the present disclosure provides a method of treating a blood cancer by allogeneic hematopoietic stem cell transplantation (allo-HSCT), comprising:

[0097] administering allo-HSCT to a subject (host);

[0098] Administration of multiple donor CD4+ T cells to a host in an amount sufficient to inhibit or prevent graft-versus-host disease (GvHD) without inhibiting graft-versus-leukemia (GvL) or graft-versus-tumor (GvT) efficacy of an allo-HSCT transplant IL-10 cell;

[0099] The multiple donor CD4 IL-10 The cells contain CD4 T cells obtained from at least two different T cell donors. + T cells, and the CD4 +T cells were genetically modified by vector-mediated gene transfer of the coding sequence for human IL-10 under the control of a constitutive or inducible promoter;

[0100] The multiple donor CD4 IL-10 The cells are non-autologous to the host and non-autologous to the allo-HSCT donor;

[0101] The multiple donor CD4 IL-10 The cells do not lose reactivity to host alloantigens prior to administration to the host; and

[0102] The multiple donor CD4 IL-10 The cells are polyclonal and Tr1-like.

[0103] In some embodiments, multiple donor CD4 IL-10 In some embodiments, multiple donor CD4 IL-10 The cells were then administered allo-HSCT.

[0104] Another aspect of the present disclosure provides a method of treating a blood cancer by allogeneic hematopoietic stem cell transplantation (allo-HSCT), comprising:

[0105] administering allo-HSCT to a subject (host);

[0106] Administration of multiple donor CD4+ cells to a host in an amount sufficient to inhibit or prevent graft-versus-host disease (GvHD) without inhibiting the graft-versus-leukemia (GvL) or graft-versus-tumor (GvT) efficacy of allo-HSCT IL-10 cell;

[0107] The multiple donor CD4 IL-10 The cells contain CD4 T cells obtained from at least two different T cell donors. + T cells, and the CD4 + T cells were genetically modified by vector-mediated gene transfer of the coding sequence of human IL-10 under the control of a constitutive promoter;

[0108] The multiple donor CD4 IL-10 The cells target and kill cancer cells in the host;

[0109] The multiple donor CD4 IL-10 The cells do not lose reactivity to host alloantigens prior to administration to the host; and

[0110] All multi-donor CD4 IL-10 The cells are non-autologous to the host, polyclonal and Tr1-like.

[0111] Another aspect of the present disclosure provides CD4 IL-10 cells, wherein the IL-10 is a viral IL-10. The viral IL-10 has a sequence of SEQ ID NO: 6, 19, 20, or 21. In some embodiments, the viral IL-10 is encoded by a polynucleotide having a sequence of SEQ ID NO: 7. In some embodiments, the IL-10 is a human IL-10, wherein one, two, three, four, five, six, seven, eight, nine, or ten amino acids from human IL-10 are replaced by the corresponding amino acid sequence from the viral IL-10. In some embodiments, the CD4 + T cells are transduced with exogenous viral IL-10 under the control of a constitutive promoter. In some embodiments, the expression control element drives viral IL-10 expression in activated CD4 + In some embodiments, the exogenous polynucleotide encoding viral IL-10 is integrated into the T cell nuclear genome. In some embodiments, the exogenous polynucleotide encoding viral IL-10 is not integrated into the T cell nuclear genome. In some embodiments, the exogenous polynucleotide encoding viral IL-10 has a sequence of SEQ ID NO: 7.

[0112] Another aspect of the present disclosure provides CD4 IL-10 cells, wherein the IL-10 is IL-10 from mouse (SEQ ID NO: 10), rat (SEQ ID NO: 11), rhesus monkey (MACMU) (SEQ ID NO: 12), gorilla (SEQ ID NO: 13), cynomolgus monkey (CYNO) (SEQ ID NO: 14), olive baboon (SEQ ID NO: 15), bonobo (SEQ ID NO: 16), chimpanzee (SEQ ID NO: 17) or EBVB9 (SEQ ID NO: 18). In some embodiments, 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 (MACMU) (SEQ ID NO: 12), gorilla (SEQ ID NO: 13), cynomolgus monkey (CYNO) (SEQ ID NO: 14), olive baboon (SEQ ID NO: 15), bonobo (SEQ ID NO: 16), chimpanzee (SEQ ID NO: 17), or EBVB9 (SEQ ID NO: 18).

[0113] Another aspect of the present disclosure provides CD4IL-10 Cells, wherein IL-10 is a variant of human IL-10. The human IL-10 variant has a sequence of SEQ ID NO: 19 or SEQ ID NO: 20. In some embodiments, IL-10 is human IL-10, wherein one, two, three, four, five, six, seven, eight, nine or ten amino acids from human IL-10 are replaced by the corresponding amino acid sequence of IL-10 from another species (e.g., mouse (SEQ ID NO: 10), rat (SEQ ID NO: 11), rhesus monkey (MACMU) (SEQ ID NO: 12), gorilla (SEQ ID NO: 13), cynomolgus monkey (CYNO) (SEQ ID NO: 14), olive baboon (SEQ ID NO: 15), bonobo (SEQ ID NO: 16), chimpanzee (SEQ ID NO: 17) or EBVB9 (SEQ ID NO: 18)). In some embodiments, CD4 + T cells are transduced with exogenous IL-10 variants under the control of a constitutive promoter. In some embodiments, the expression control element drives the IL-10 variant to express in activated CD4 + Expression 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.

[0114] In another aspect, the present disclosure provides for preparing viral IL-10CD4 IL-10 The method comprises the following steps:

[0115] (i) Obtaining primary CD4 T cells from a single donor + T cells; and

[0116] (ii) Modification of donor CD4 by introduction of exogenous polynucleotide encoding viral IL-10 + T cells,

[0117] Genetically modified CD4 + T cells.

[0118] In some embodiments, the method further comprises the following steps after step (i) or after step (ii): incubating primary CD4 + T cells.

[0119] In some embodiments, the method comprises further incubating primary CD4 +In some embodiments, the exogenous polynucleotide encoding viral IL-10 uses a vector.

[0120] 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 a 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.

[0121] In some embodiments, the method further comprises the following steps after step (ii): isolating the genetically modified CD4 + T cells, thereby generating enriched genetically modified CD4 + T cell populations.

[0122] In some embodiments, the method further comprises incubating the enriched genetically modified CD4 + In some embodiments, the enriched genetically modified CD4 T cells are incubated in the presence of IL-2 in the presence of beads coated with anti-CD3 antibody and anti-CD28 antibody or CD3 antibody and CD28 antibody. + Steps for T cell populations.

[0123] In some embodiments, in step (i), primary CD4 + T cells are obtained from frozen stocks. In some embodiments, in step (i), primary CD4 + T cells were obtained from unfrozen peripheral blood mononuclear cells from a single T cell donor.

[0124] In some embodiments, the method further comprises isolating CD4 + In some embodiments, peripheral blood mononuclear cells are obtained from the buffy coat or apheresis. 5. Brief description of the accompanying drawings

[0126] Figure 1 is a non-limiting illustration of a bidirectional lentiviral vector structure for delivering human IL-10 and ΔNGFR coding sequences to CD4 T cells from multiple donors. + Multidonor CD4 T cells IL-10 cell.

[0127] Figure 2The complete circular structure of the bidirectional lentiviral vector is shown to generate a lentiviral vector that delivers human IL-10 and ΔNGFR coding sequences to CD4 T cells from multiple donors. + T cells to generate multiple donor CD4 IL-10 cell.

[0128] Figure 3 shows the method for generating CD4 IL-10 Exemplary scheme for cells.

[0129] Figure 4A Human CD4 T cells transduced with LV-IL-10 / ΔNGFR (a bidirectional lentiviral vector encoding human IL-10 and a truncated form of the human NGF receptor) are shown. + CD4 + ΔNGFR + Percentage of cells (mean ± SD, n = 10, left grey bars) and vector copy number (VCN, mean ± SD, n = 10, right grey bars). Figure 4B Human CD4 T cells transduced with LV-IL-10 / ΔNGFR and purified using anti-CD271 microbeads from two representative donors (Donor B and Donor C) are shown. + FACS analysis of CD4 and ΔNGFR expression in T cells.

[0130] Figure 5 Single donor CD4 IL-10 Cytokine production profiles of cells after the second (TF2) and third (TF3) restimulation. TF2 (left panel) and TF3 (right panel) CD4 IL-10 Cells were not stimulated (as indicated by arrows) or stimulated for 48 hours with fixed CD3 (10 μg / mL) and soluble CD28 mAb (1 μg / mL). Culture supernatants were collected and the 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 are shown, n = 8 tested donors.

[0131] Fig. 6A CD4 expressing granzyme B (GzB) after the second round of stimulation (TF2) analyzed by FACS IL-10 Percentage of cells. Box and whiskers of n=7 different single donors are shown. Figure 6B Shows when CD4 IL -10 Cells (10 5 cells / well) and K562 and ALL-CM cells (10 5The box-and-whisker plots represent data from n=4 donors, and the dots represent data from a single donor.

[0132] Fig. 7A and 7B Single donor CD4 IL-10 Allogeneic CD4 + Proliferation of T cells. Allogeneic PBMC cells were used 670(5×10 4 cells / well) and in the absence or presence of CD4 IL-10 Cells (5×10 4 Allogeneic mature dendritic cells (5 × 10 3 cells / well) and soluble anti-CD3 mAb. After 3 days of culture, flow cytometry was used to analyze the CD4 + ΔNGFR - After T cells were gated, The percentage of proliferative responding cells was determined by dilution at 670. Fig. 7A Results for Donor-C, Donor-E, and Donor-F are shown, and Figure 7B Results for Donor-H, Donor-I, and Donor-L are shown. Percentages of proliferation and inhibition are indicated. CD4 IL-10 Cell-mediated inhibition was calculated as follows: 100-([in CD4 IL-10 proliferation of responders in the presence of cells / proliferation of responders alone] × 100).

[0133] Fig. 8A and 8B Single donor CD4 IL-10 Cells can suppress allogeneic CD8 + Proliferation of T cells. Allogeneic PBMC cells were used 670(5×10 4 cells / well) and in the absence or presence of CD4 IL-10 Cells (5×10 4 Allogeneic mature dendritic cells (5 × 10 3 cells / well) and soluble anti-CD3 mAb. After 3 days of culture, flow cytometry was used to analyze the CD8 + ΔNGFR - T cells were gated by The percentage of proliferative responding cells was determined by dilution at 670. Fig. 8AResults for Donor-C, Donor-E, and Donor-F are shown, and Figure 8B Results for Donor-H, Donor-I, and Donor-L are shown. Percentages of proliferation and inhibition are indicated. CD4 IL-10 Cell-mediated inhibition was calculated as follows: 100-([in CD4 IL-10 proliferation of responders in the presence of cells / proliferation of responders alone] × 100).

[0134] Fig. 9 CD4 IL-10 The average level (+ / -SD) of cells produced compared to multiple donor CD4 IL-10 Cytokine production profile of cells after the third (TF3) restimulation. TF3CD4 IL-10 Cells were pooled at a 1:1:1 ratio and stimulated with immobilized CD3 (10 μg / mL) and soluble CD28 mAb (1 μg / mL) for 48 hours. Culture supernatants were collected and the levels of IL-10, IL-4, IL-5, IFN-γ, and IL-22 were determined by ELISA. IL-10 Results for cells; grey bars represent mean ± SD, n = 8 single donors.

[0135] Fig. 10A CD4 IL-10 The mean % (+ / -SD) of cells expressing granzyme B compared to CD4 expressing granzyme B (GzB) from multiple donors IL-10 The percentage of cells after the third round of stimulation (TF3) was analyzed by FACS. Fig. 10B Shows that when multiple donor CD4 IL-10 Cells (10 5 cells / well) and K562 and ALL-CM cells (10 5 The percentage of dead cells after 3 days of co-culture at a ratio of 1:1 was calculated by FACS counting the residual leukemic cells (CD45+, CD3-) of each target cell. IL-10 Results are shown in Figure 2 and grey bars represent mean ± SD of n = 3 individual donors used to generate the combined pool.

[0136] Fig.11A and 11B Multiple donor CD4 IL-10 Allogeneic CD4 + T cells and CD8 + Proliferation of T cells. Allogeneic PBMC cells were used 670(5×10 4cells / well) and in the absence or presence of multiple donor CD4 IL-10 Cells (5×10 4 Allogeneic mature dendritic cells (1×10 4 cells / well) and soluble anti-CD3 mAb. After 3 days of culture, flow cytometry was used to analyze the CD4 + ΔNGFR – T cells and CD8 + ΔNGFR – After T cells were gated, The percentage of proliferative responding cells was determined by dilution at 670. Fig.11A The CD4 + Multiple donor CD4 IL-10 Cell results. Fig. 11B The CD4 + Multiple donor CD4 IL-10 Results of CD4 cells. IL-10 Cell-mediated inhibition was calculated as follows: 100-([in CD4 IL-10 proliferation of responders in the presence of cells / proliferation of responders alone] × 100).

[0137] Fig.12 Figure 2 shows the use of human PBMCs and / or multiple donor CD4 IL-10 (BC-C / E / F) cell-induced GvHD regimen.

[0138] Fig.13 It shows that after injection of PBMC (5×10 6 cells / mouse), multiple donors (three donors; BC-C / E / F) CD4 IL-10 Cells (5×10 6 cells / mouse), or PBMC (5×10 6 cells / mouse) and multiple donor CD4 IL-10 Cells (three donors; BC-C / E / F) (5×10 6 cells / mouse) every day after combination with GvHD in % of NSG mice.

[0139] Fig.14 It shows that after injection of PBMC (5×10 6 cells / mouse), multiple donors (three donors; (BC-C / E / F)) CD4 IL-10 Cells (5×106 cells / mouse), or PBMC (5×106 cells / mouse) and multiple donor CD4 IL-10 Cells (three donors; (BC-C / E / F)) (5×10 6 cells / mouse) in NSG mice IL-10 Migration of cells to the spleen (left panel) and bone marrow (right panel). Box and whisker plots of n=8 tested animals are shown.

[0140] Fig.15 The results show that CD4+ T cells injected on day 3 after irradiation and CD4+ T cells from multiple donors (BC-H / I / L) or single donors (BC-H) IL-10 Cell-induced GvHD regimen.

[0141] Fig.16 The % of NSG mice free of GvHD for each day following injection is shown.

[0142] Figures 17A-17C The graft-versus-leukemia (GvL) effect based on the reduction of circulating leukemic cells and long-term leukemia-free survival was shown. Leukemia was measured as described previously (Locafaro G. et al Molecular Therapy 2017). On day 0, NSG mice were sublethally irradiated and injected intravenously with myeloid leukemia cells (ALL-CM) (2.5×10 6 indivual). Fig.17A It is a diagram of the experiment. Fig. 17B The results show that PBMC (2.5×10 6 CD4 IL-10 Cells (2.5×10 6 Leukemia-free survival of animals. Fig. 17C The results show that PBMC (2.5×10 6 or multiple donor CD4 IL-10 Cells (from donor BC-I and donor BC-H) (2.5×10 6 Leukemia-free survival of animals.

[0143] Figures 18A-18C The results show that ALL-CM cells (2.5 × 10 6 Long-term leukemia-free survival was measured in NSG mice with 40% paraformaldehyde (P < 0.05). Fig.18A It is a diagram of the experiment. Fig.18B Shown is the injection of PBMCs alone (2.5×10 6 ) or mononuclear cells (PBMC) (2.5×10 6+ single donor (from donor BC-H and donor BC-I) CD4 IL-10 Cells (2.5×10 6 Data of animals of each species. Fig.18C Shown is the injection of PBMCs alone (2.5×10 6 ) or mononuclear cells (PBMC) (2.5×10 6 + multiple donor CD4 IL-10 Cells (BC-I / H) (2.5×10 6 Data of animals of each species.

[0144] Figures 19A-19G CD4 IL-10 Cellular inhibition of NLPR3 inflammasome activation. Fig.19A CD4 from a single donor (#1) is shown IL-10 Effect of cell supernatants on IL-1β production by LPS-activated monocytes. Fig.19B CD4 from another single donor (#2) is shown IL-10 Effect of cell supernatants on IL-1β production by LPS-activated monocytes. Fig.19C CD4 from a single donor (#1) is shown IL-10 Effect of cell supernatants on the enhancement of LPS-induced IL-1β production by nigericine (NIG), an inhibitor of NLPR3 inflammasome activator. Fig.19D CD4 from a single donor (#2) is shown IL -10 Effects of cell supernatants on the enhancement of LPS-induced IL-1β production by nigericin (NIG), an inhibitor of the NLPR3 inflammasome activator. Fig.19E Figure 2 shows CD4 T cells from a single donor (BC-E) in the presence or absence of anti-IL-10 receptor (anti-IL-10R) mAb. IL-10 Bar graph showing the effect of cell supernatants and pooled cells from two different donors (BC-C / E) on LPS-induced IL-1β production by monocytes. Fig.19F The results show that multiple donor CD4 IL-10 Effect of cell (BC-T / U / V) supernatant on IL-1β production by monocytes. Figure 19G The results show that multiple donor CD4 IL-10 Effect of cell (BC-T / U / V) supernatants on IL-18 production induced by the combination of LPS and nigericin.

[0145] Fig. 20 The experimental scheme for testing graft-versus-myeloid leukemia and xenogeneic GvHD effects is shown. On day 0, NSG mice were injected intravenously with ALL-CM cells (2.5×10 6 On day 3, mice were divided into five groups, and each group received the following treatments: (i) no treatment as control, (ii) allogeneic PBMCs; (iii) allogeneic PBMCs and multiple donor CD4 IL-10 cells (BC-V / T / E, 1:1:1 pool); (iv) allogeneic PBMC and single donor CD4 IL-10 cells (BC-E); or (v) multiple donor CD4 IL-10 Cells (BC-V / T / E), concentration as Fig. 20 shown.

[0146] Fig.21 Single donor CD4 IL-10 cells (BC-V, BC-T, BC-V and BC-E) and multiple donor CD4 IL-10 Histogram of cytokine secretion profile of cells (pooled: BC-E, BC-V and BC-T at 1:1:1).

[0147] Fig. 22 Single donor CD4 IL-10 cells (BC-V and BC-E) and multiple donor CD4 IL-10 Cells (BC-V / T / E combined) to allogeneic CD4 + and CD8 + Inhibitory effect on T cell proliferation in vitro. 670(5×10 4 cells / well) and in the absence or presence of CD4 IL-10 Cells (5×10 4 Allogeneic mature dendritic cells (1×10 4 cells / well) and soluble anti-CD3 mAb. After 3 days of culture, flow cytometry was used to analyze the CD4 + ΔNGFR - (above) or CD8 + After gating on ΔNGFR- (lower panel) T cells, The percentage of proliferative responding cells was determined by dilution at 670. Fig. 22 Results for single donors BC-V and BC-E and pooled cells from donors BC-V / T / E are shown. Percentages of proliferation and inhibition are indicated. CD4 IL-10Cell-mediated inhibition was calculated as follows: 100-([in CD4 IL-10 proliferation of responders in the presence of cells / proliferation of responders alone] × 100).

[0148] Fig.23 Single donor CD4 IL-10 cells (BC-E and BC-V) or multiple donor CD4 IL-10 The results showed that the live cells (% of BC-V / T / E) were co-cultured with ALL-CM myeloid tumor cells or K562 cells. IL-10 The cells were selectively cytotoxic to ALL-CM myeloid neoplasms but not to K562 cells that lack MHC class I expression.

[0149] Fig.24 The results show that ALL-CM cells (2.5×10 6 Leukemia-free survival was measured in NSG mice with 100% leukemia-free cells (NSGs). On day 3, mice were divided into five groups, and each group received the following treatments: (i) no treatment as control; (ii) allogeneic mononuclear cells (PBMCs); (iii) allogeneic PBMCs and multiple donor CD4 IL-10 cells (BC-V / T / ET); (iv) allogeneic PBMC and single donor CD4 IL-10 Cells (BC-E) or (v) multiple donor CD4 IL-10 Cells (BC-V / T / E). The graph shows the leukemia-free survival rate of animals in each group.

[0150] Fig.25 It shows that after injection of ALL-CM cells (2.5×10 6 ) and then on day 3 the following treatments were administered: (i) no treatment as control; (ii) allogeneic mononuclear cells (PBMCs); (iii) allogeneic PBMCs and multiple donor CD4 IL-10 cells (BC-V / T / E); (iv) allogeneic PBMC and single donor CD4 IL-10 Cells (BC-E) or (v) multiple donor CD4 IL-10 cell.

[0151] Fig.26 It is shown that NSG mice dosed with 2.5E+06 PBMCs (allogeneic to donors C, E, F, and H) all died of acute and lethal xenogeneic GvHD on day 22. Single donor CD4 IL-10 cells (Batch C) or multiple donor CD4 IL-10Administration of cells (batch CEFH) in combination with PBMCs prevented lethal xenogeneic GvHD in 75% (3 / 4 mice) and 80% (4 / 5 mice), respectively. IL-10 Transfer of cells did not induce any signs of GvHD. Taken together, these results suggest that multidonor CD4 IL-10 Cells suppress pathogenic human T cell responses, with single donor CD4 IL-10 cells, or slightly more effectively. PBMC: peripheral mononuclear cells; GvHD: graft-versus-host disease.

[0152] Fig.27A 10; SEQ ID NO: 11 ; EBVB9 (SEQ ID NO: 12 ); EBVB9 (SEQ ID NO: 13 ); EBVB9 (SEQ ID NO: 14 ); EBVB9 (SEQ ID NO: 15 ); EBVB9 (SEQ ID NO: 16 ); EBVB9 (SEQ ID NO: 17 ); EBVB9 (SEQ ID NO: 18 ); EBVB9 (SEQ ID NO: 19 ); EBVB9 (SEQ ID NO: 20 ); EBVB9 (SEQ ID NO: 21 ); EBVB9 (SEQ ID NO: 22 ); EBVB9 (SEQ ID NO: 23 ); EBVB9 (SEQ ID NO: 24 ); EBVB9 (SEQ ID NO: 25 ); EBVB9 (SEQ ID NO: 26 ); EBVB9 (SEQ ID NO: 27 ); EBVB9 (SEQ ID NO: 28 ); EBVB9 (SEQ ID NO: 29 ); EBVB9 (SEQ ID NO: 30 ); EBVB9 (SEQ ID NO: 31 ); EBVB9 (SEQ ID NO: 32 ); NO:18).

[0153] Fig.27B Sequences of IL-10 variants generated by substituting one or more amino acids of human IL-10 with amino acids of viral IL-10 (EBVB9) at the corresponding positions are provided. Sequences of exemplary variants, possible huIL-10 hybrid #1 (SEQ ID NO: 19) and possible huIL-10 hybrid #2 (SEQ ID NO: 20) are also provided. "*" indicates one or more amino acid positions that are substituted. "#" indicates a preferred I105 to A105 amino acid substitution of IL-10 hybrid #2 (SEQ ID NO: 20).

[0154] Fig.27CAn alignment of human IL-10 (SEQ ID NO: 1) and IL10 EBVB9 (SEQ ID NO: 18) is shown. "*" indicates one or more amino acid positions that were substituted in IL-10 hybrid #1. "#" indicates a preferred I105 to A105 amino acid substitution for IL-10 hybrid #2.

[0155] The accompanying drawings depict various embodiments of the present invention for illustration purposes only. One skilled in the art will readily recognize from the following discussion that alternative embodiments of the structures and methods shown herein may be employed without departing from the principles of the invention described herein. 6. Specific implementation methods

[0156] 6.1. Definitions

[0157] 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 the invention belongs. As used herein, the following terms have the meanings given below.

[0158] "Graft-versus-leukemia effect" or "GvL" refers to the effect that occurs after allogeneic hematopoietic stem cell transplantation (HSCT) or bone marrow transplantation (BMT). T lymphocytes in the allogeneic transplant eliminate malignant residual host leukemia cells.

[0159] The "graft-versus-tumor effect" or "GvT" refers to the effect that occurs after allogeneic hematopoietic stem cell transplantation (HSCT) or bone marrow transplantation (BMT). T lymphocytes in the allogeneic transplant eliminate malignant residual host cancer cells, such as myeloma, lymphocytic and myeloid leukemias, lymphomas, multiple myeloma, and possibly breast cancer cells. The term GvT is a general term for GvL.

[0160] The terms "treatment", "treating", etc. are used herein in the broadest sense as understood in the medical field. Specifically, these terms generally mean obtaining a desired pharmacological and / or physiological effect. As used herein, "treatment" covers any treatment of a disease or condition in a mammal (particularly a human), and includes: (a) preventing a disease or condition from occurring in a subject who may be susceptible to the disease or condition but has not yet been diagnosed with the disease or condition; (b) inhibiting the disease or condition (e.g., preventing its development); or (c) alleviating the disease or condition (e.g., causing the disease or condition to subside, providing one or more symptom improvements). The improvement of any condition can be easily assessed according to standard methods and techniques known in the art. The subject population treated by the disease method includes subjects with an undesirable condition or disease, and subjects at risk of developing the condition or disease.

[0161] As used herein, "HLA-matched" refers to a pair of individuals having matching HLA alleles in HLA class I (HLA-A, HLA-B, and HLA-C) and class II (HLA-DRB1 and HLA-DQB1) loci, thereby making the individuals immunologically compatible with each other. HLA compatibility can be determined using any method available in the art, for example, as described in Tiervy, Haematologica 2016 Volume 101 (6): 680-687, which is incorporated herein by reference.

[0162] For a given locus, when each of the two alleles of one individual matches the two alleles of another individual, the pair has a 2 / 2 match. When only one of the two alleles of one individual matches one of the two alleles of another individual, the pair has a 1 / 2 match. When all ten alleles of one individual (two each of the HLA-A, HLA-B, HLA-C, HLA-DRB1, and HLA-DQB1 loci) match all ten alleles of another individual, the pair has a 10 / 10 match at the HLA-A, HLA-B, HLA-C, HLA-DRB1, and HLA-DQB1 loci.

[0163] In a preferred embodiment, allele level typing is used to determine HLA compatibility. Allele level typing corresponds to the unique nucleotide sequence of HLA genes, defined by using all the numbers in the first, second, third and fourth fields, such as A*02:01:01:01. Functionally, the third and fourth fields characterize alleles that are different by the silent replacement in the coding sequence and the replacement in the non-coding sequence, respectively, and these replacements are irrelevant unless they prevent the expression of HLA alleles (e.g., invalid allele B*15:01:01:02N). Deletion of invalid alleles will lead to mismatching, which is likely to be recognized by alloreactive T cells and produce harmful clinical effects. Replacement in non-coding sequences may affect expression levels (e.g., A24low allele A*24:02:01:02L). This variability may also have an impact on anti-HLA allorecognition.

[0164] As used herein, the term "HLA-mismatched" refers to a pair of individuals having 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.

[0165] As used herein, the term "HLA partially mismatched" refers to a pair of individuals having mismatched HLA alleles in the HLA class I (HLA-A, HLA-B, HLA-C) and HLA class II (HLA-DRB1, HLA-DQB1) loci, such that the individuals are immunologically incompatible with each other to an acceptable degree. Some studies have identified acceptable mismatches. Some HLA class I incompatibilities are considered more acceptable.

[0166] "HLA haplotype" refers to a series of HLA loci alleles of a chromosome, one from the mother and one from the father. The genotypes of 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.

[0167] The term "therapeutically effective amount" refers to an amount effective to treat a disease and thereby ameliorate symptoms of the disease.

[0168] The term "prophylactically effective amount" is an amount effective in preventing a disease, condition, or symptoms thereof, in whole or in part. The term "amelioration" refers to any therapeutically beneficial outcome in treating a disease state (e.g., a neurodegenerative disease state), including prevention, lessening of severity or progression, remission, or cure thereof.

[0169] 6.2. Other interpretation conventions

[0170] 41, 42, 43, 44, 45, 46, 47, 48, 49, and 50.

[0171] 6.3. Multiple Donor CD4 IL-10 cell

[0172] In a first aspect, a CD4 + T cell populations that have been genetically modified to contain an exogenous polynucleotide encoding IL-10 (CD4 IL-10 cells). The population contains T cells from at least two different T cell donors (multiple donor CD4 IL-10 CD4 + T cells.

[0173] 6.3.1.CD4+ T cells and T cell donors

[0174] Multiple donor CD4 T cells can be isolated from peripheral blood, umbilical cord blood, or other blood samples from donors using methods available in the art. IL-10 CD4 used in the group + In typical embodiments, CD4 + T cells are isolated from peripheral blood (preferably human donors). In certain embodiments, CD4 + T cells are isolated from peripheral blood by leukapheresis. In certain embodiments, CD4 + T cells are obtained from a third party blood bank. In certain embodiments, CD4 + T cells were obtained from the buffy coat after centrifugation of whole blood.

[0175] In some embodiments, CD4 + T cells are isolated from a previously frozen stock of blood or a previously frozen stock of peripheral blood mononuclear cells (PBMC). + T cells are isolated from peripheral blood or PBMCs that have not been previously frozen. In some embodiments, CD4 + T cells are isolated individually from blood or PBMC obtained from multiple donors and then pooled. In some embodiments, CD4 + T cells are first isolated from blood or PBMCs pooled from multiple donors.

[0176] In some embodiments, CD4 + The T cells are obtained from three, four, five, six, seven, eight, nine or ten different T cell donors.

[0177] In some embodiments, at least two different T cell donors are selected without regard to genotype. In some embodiments, at least two different T cell donors are selected based on genotype.

[0178] In certain embodiments, at least two different T cell donors are selected based on their HLA haplotypes.

[0179] 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.

[0180] In some embodiments, all CD4 +The T cells are at least 1 / 10, 2 / 10, 3 / 10, 4 / 10, 5 / 10, 6 / 10, 7 / 10, 8 / 10, or 9 / 10 matched to each other at the HLA-A, HLA-B, HLA-C, HLA-DRB1, and HLA-DQB1 loci. In some embodiments, all CD4 + The T cells are at least 1 / 8, 2 / 8, 3 / 8, 4 / 8, 5 / 8, 6 / 8, 7 / 8, or 8 / 8 matched to each other at the HLA-A, HLA-B, HLA-C, and HLA-DRB1 loci. In some embodiments, all CD4 + T cells have a 2 / 2 match with each other at the HLA-A locus. In some embodiments, all CD4 + T cells have a 2 / 2 match with each other at the HLA-B locus. In some embodiments, all CD4 + T cells have a 2 / 2 match with each other at the HLA-C locus. In some embodiments, all CD4 + The T cells have at least a 3 / 4 or 4 / 4 match with each other at the HLA-DRB1 and HLADQB1 loci. In some embodiments, all CD4 + T cells have the A*02 or A*24 allele.

[0181] In some embodiments, all CD4 + T cells 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, all CD4 + T cells 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, all CD4 + T cells have less than a 2 / 2 match with each other at the HLA-A locus. In some embodiments, all CD4 + T cells have less than a 2 / 2 match with each other at the HLA-B locus. In some embodiments, all CD4 + T cells have less than a 2 / 2 match with each other at the HLA-C locus. In some embodiments, all CD4 + The T cells have less than 3 / 4 or 4 / 4 matches with each other at the HLA-DRB1 and HLA DQB1 loci.

[0182] In some embodiments, all CD4+ T cells 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, all CD4 + T cells 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, all CD4 + T cells have less than a 2 / 2 match with each other at the HLA-A locus. In some embodiments, all CD4 + T cells have less than a 2 / 2 match with each other at the HLA-B locus. In some embodiments, all CD4 + T cells have less than a 2 / 2 match with each other at the HLA-C locus. In some embodiments, all CD4 + The T cells have less than 3 / 4 or 4 / 4 matches with each other at the HLA-DRB1 and HLA DQB1 loci.

[0183] In a preferred embodiment, none of the at least two different T cell donors is a CD4 IL-10 In a preferred embodiment, none of the at least two different T cell donors is a CD4 IL-10 The cells are used together with donors of stem cells (eg, HSCs), tissues or organs for the therapeutic methods described herein.

[0184] In some embodiments, one or more of the T cell donors are HLA mismatched or partially HLA mismatched with 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 with the patient at 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 with the patient at 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 with the patient at HLA-A, HLA-B or HLA-C loci. In some embodiments, one or more of the T cell donors has less than a 2 / 4, 3 / 4, or 4 / 4 match to the patient at the HLA-DRB1 and HLA-DQB1 loci.

[0185] In some embodiments, one or more of the T cell donors are HLA mismatched or partially HLA mismatched with 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 with 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 with 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 with the HSC donor at the HLA-A, HLA-B or HLA-C loci. In some embodiments, one or more of the T cell donors is less than 3 / 4 or 4 / 4 matched to the HSC donor at the HLA-DRB1 and HLA-DQB1 loci.

[0186] In a preferred embodiment, none of the CD4 + T cells are immortalized.

[0187] 6.3.2. Exogenous polynucleotide encoding IL-10

[0188] Multiple donor CD4 IL-10 The cells are CD4 + T cells. The exogenous polynucleotide comprises an IL-10 encoding polynucleotide segment operably linked to an expression control element.

[0189] The IL-10 encoding polynucleotide segment may encode IL-10 of a human, bonobo, or rhesus monkey. In some embodiments, the IL-10 encoding polynucleotide segment encodes a human IL-10 having a sequence of SEQ ID NO: 1. In some embodiments, the IL-10 encoding polynucleotide segment encodes a human IL-10 variant 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 a 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.

[0190] In some embodiments, the IL-10 encoding polynucleotide segment encodes IL-10 of Mus musculus, "MOUSE" (SEQ ID NO: 10); Rattus norvegicus, "RAT" (SEQ ID NO: 11); Rhesus macaque, "MACMU" (SEQ ID NO: 12); gorilla, "GORILLA" (SEQ ID NO: 13); cynomolgus macaque, "CYNO" (SEQ ID NO: 14); Polonium, "OLIVE BABOON" (SEQ ID NO: 15); bonobo, "BONOBO" (SEQ ID NO: 16); chimpanzee, "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 Mus musculus, "MOUSE" (SEQ ID NO: 10); Rattus norvegicus, "RAT" (SEQ ID NO: 11); Rhesus macaque, "MACMU" (SEQ ID NO: 12); gorilla, "GORILLA" (SEQ ID NO: 13); cynomolgus macaque, "CYNO" (SEQ ID NO: 14); Polonium, "OLIVE BABOON" (SEQ ID NO: 15); bonobo, "BONOBO" (SEQ ID NO: 16); chimpanzee, "CHIMP" (SEQ ID NO: 17); and EBVB9 (SEQ ID NO: 18).

[0191] In some embodiments, the exogenous polynucleotide encodes a viral IL-10. In various embodiments, the exogenous polypeptide encodes an 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 a viral IL-10 from EBV or ORFV.

[0192] In some embodiments, the IL-10 encoding polynucleotide segment encodes a variant of human IL-10, which has one, two, three, four, five, six, seven, eight, nine, ten or more amino acid substitutions compared to human IL-10 (e.g., SEQ ID NO: 1). In some embodiments, one, two, three, four, five, six, seven, eight, nine or ten amino acid substitutions are substitutions with amino acids of viral IL-10 at the corresponding amino acid positions. In some embodiments, the IL-10 encoding polynucleotide segment encodes a variant of human IL-10, which has one, two, three, four, five, six, seven, eight, nine, ten 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 a sequence of SEQ ID NO: 8 or 9.

[0193] In some embodiments, the IL-10 encoding polynucleotide segment encodes a variant of human IL-10 having one, two, three, four, five, six, seven, eight, nine, ten 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 an amino acid substitution, insertion and / or deletion at the corresponding position with Mus musculus, "mouse" (SEQ ID NO: 10); Rattus norvegicus, "rat (RAT)" (SEQ ID NO: 11); Rhesus macaque, "MACMU" (SEQ ID NO: 12); gorilla, "gorilla (GORILLA)" (SEQ ID NO: 13); cynomolgus monkey, "CYNO" (SEQ ID NO: 14); Baboon, "olive baboon (OLIVE BABOON)" (SEQ ID NO: 15); bonobo, "BONOBO" (SEQ ID NO: 16); chimpanzee, "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.

[0194] In some embodiments, the IL-10 encoding polynucleotide segment encodes a human IL-10 variant having reduced immunostimulatory activity compared to human IL-10. In some embodiments, the variant of human IL-10 includes an I105A substitution. In some embodiments, the variant of human IL-10 is prepared 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.

[0195] The exogenous polynucleotide further comprises an expression control element that directs the expression of the encoded IL-10 in the transduced CD4 + Expression in T cells.

[0196] In some embodiments, the expression control element comprises a protein capable of directing IL-10 to express in CD4 + In some embodiments, the promoter drives IL-10 expression in CD4 + In some embodiments, the promoter drives IL-10 in activated CD4 T cells. + Expression in T cells.

[0197] In some embodiments, when treatment is appropriate, an inducible promoter is used to induce the expression of IL-10. In some embodiments, an 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.

[0198] In some embodiments, a native human promoter is used. In some embodiments, the human elongation factor (EF) 1α promoter is used. In some embodiments, the human phosphoglycerate kinase promoter (PGK) is used. In some embodiments, the human ubiquitin C promoter (UBI-C) is used.

[0199] In some embodiments, a synthetic promoter is used. In certain embodiments, a minimal CMV core promoter is used. In a specific embodiment, an inducible or constitutive bidirectional promoter is used. In a specific embodiment, a synthetic bidirectional promoter disclosed in Amendola et al., Nature Biotechnology, 23(1): 108-116 (2005) is used. The promoter can mediate the coordinated transcription of two mRNAs in a universal or tissue-specific manner. In certain embodiments, the bidirectional promoter induces the expression of IL-10 and a selection marker.

[0200] In some embodiments, the exogenous polynucleotide further comprises a segment encoding a selectable marker that allows for the selection of successfully transduced CD4 + T cells. 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 with SEQ ID NO:3. In a specific embodiment, 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 with SEQ ID NO:4.

[0201] 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. Therefore, in some embodiments, the expression of the selection marker is measured to infer the expression of IL-10 from the exogenous polynucleotide.

[0202] 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, optionally a huEGFR disclosed in Wang et al. "A transgene-encoded cellsurface polypeptide for selection, in vivo tracking, and ablation of engineered cells", Blood, v.118, n.5 (2011), which is incorporated herein by reference in its entirety.

[0203] 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.

[0204] In typical embodiments, a vector is used to deliver the exogenous polynucleotide into CD4+ T cells. In some embodiments, the vector is a plasmid vector. In some embodiments, the vector is a viral vector.

[0205] In certain embodiments, a lentiviral vector is used to deliver the exogenous polynucleotide to CD4+ T cells, and the exogenous polynucleotide comprises a lentiviral vector sequence. In certain embodiments, a lentiviral vector disclosed in Mátrai et al., Molecular Therapy 18(3):477–490 (2010) (“Mátrai”) is used, which is incorporated herein by reference.

[0206] In some embodiments, the lentiviral vector is capable of integrating into the T cell nuclear genome. In some embodiments, the lentiviral vector cannot be integrated 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 Mátrai is used. In some embodiments, an integrase-deficient lentivirus is used. For example, an integrase-deficient lentivirus containing an inactivating mutation (D64V) in the integrase can be used, as described in Mátrai et al., Hepatology 53: 1696-1707 (2011), which is incorporated herein by reference.

[0207] 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 T cell cytoplasm.

[0208] 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.

[0209] 6.3.3. Multiple Donor CD4 IL-10 Gene expression in T cells

[0210] Multiple donor CD4 IL-10 T cells express IL-10. In some embodiments, multiple donor CD4 IL-10 T cells constitutively express IL-10. In some embodiments, multiple donor CD4 IL-10 T cells express IL-10 upon activation.

[0211] In some embodiments, multiple donor CD4 IL-10 T cells constitutively express at least 100 pg of IL-10 / 10 6 CD4 + T cells / mL culture. In some embodiments, multiple donor CD4 IL-10T cells constitutively express at least 200 pg, 500 pg, 1 ng, 5 ng, 10 ng or 50 ng of IL-10 / 10 6 CD4 + T cells / mL culture.

[0212] In some embodiments, after activation with a combination of anti-CD3 antibody and anti-CD28 antibody, or beads coated with anti-CD3 antibody and anti-CD28 antibody, multiple donor CD4 IL-10 T cells expressing at least 1ng or 2ng IL-10 / 10 6 CD4 + T cells / mL culture. In some embodiments, after activation with beads coated with anti-CD3 and anti-CD28 antibodies or CD3 and CD28 antibodies, multiple donor CD4 IL-10 T cells expressing at least 5ng, 10ng, 100ng, 200ng or 500ng IL-10 / 10 6 CD4 + T cells / mL culture.

[0213] In various embodiments, the amount of IL-10 produced is determined 12 hours, 24 hours or 48 hours after activation using various protein detection and measurement methods (e.g., ELISA, spectroscopic procedures, colorimetry, amino acid analysis, radiolabeling, Edman degradation, HPLC, Western blot, etc.). In a preferred embodiment, the amount of IL-10 produced is determined by ELISA 48 hours after activation with anti-CD3 antibodies and anti-CD28 antibodies.

[0214] In some embodiments, multiple donor CD4 IL-10 T cells were more expressed than unmodified CD4 + T cells express IL-10 at least 5-fold higher levels. IL-10 T cells were more expressed than unmodified CD4 + T cells express IL-10 at levels at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, or 50 fold higher.

[0215] In some embodiments, multiple donor CD4 IL-10 T cells further express a selectable marker. In some embodiments, multiple donor CD4 IL-10 T cells express proteins normally expressed in Tr1 cells. In some embodiments, multiple donor CD4 IL-10 T cells express characteristic marker proteins of Tr1 cells.

[0216] In some embodiments, multiple donor CD4IL-10 T cells express CD49b. In some embodiments, multiple donor CD4 IL-10 T cells express LAG-3. In some embodiments, multiple donor CD4 IL-10 T cells express TGF-β. In some embodiments, multiple donor CD4 IL-10 T cells express IFNγ. In some embodiments, multiple donor CD4 IL-10 T cells express granzyme B (GzB). In some embodiments, multiple donor CD4 IL-10 T cells release granzyme B (GzB) when activated with myeloid antigen presenting cells or myeloid tumor cells. In some embodiments, multiple donor CD4 IL-10 T cells express perforin. In some embodiments, multiple donor CD4 IL-10 T cells release perforin when activated with myeloid antigen presenting cells or myeloid tumor cells. IL-10 T cells express CD18. In some embodiments, multiple donor CD4 IL-10 T cells express CD2. In some embodiments, multiple donor CD4 IL-10 T cells express CD226. In some embodiments, multiple donor CD4 IL-10 T cells express IL-22. In some embodiments, multiple donor CD4 IL-10 T cells express IL-10.

[0217] In some embodiments, multiple donor CD4 IL-10 The T cells exhibit at least one phenotypic function of Tr1 cells. In various embodiments, the function is secretion of IL-10, secretion of TGF-β, and specific killing of myeloid antigen presenting cells by releasing granzyme B (GzB) and perforin.

[0218] 6.3.4. Products produced by the method

[0219] In typical embodiments, multiple donor CD4 IL-10 T cells are activated by modifying CD4 T cells with exogenous polynucleotides encoding IL-10 + T cell acquisition.

[0220] In some embodiments, the exogenous polynucleotide is introduced into the CD4 + In a specific embodiment, CD4 T cells are transduced with a lentivirus containing a coding sequence for IL-10. + T cells.

[0221] In some embodiments, multiple donor CD4 IL-10T cells were generated by: (i) combining primary CD4 + T cells; and (ii) modifying the combined CD4 T cells by introducing an exogenous polynucleotide encoding IL-10 + In some embodiments, multiple donor CD4 IL-10 T cells were generated by: (i) obtaining primary CD4 T cells from at least two different T cell donors; + (ii) modifying each donor's CD4 T cells by introducing an exogenous polynucleotide encoding IL-10 + T cells; then (iii) combining genetically modified CD4 + T cells.

[0222] In some embodiments, multiple donor CD4 IL-10 T cells can activate CD4 + In some embodiments, multiple donor CD4 IL-10 T cells are cultured in the presence of anti-CD3 antibody and anti-CD28 antibody or beads coated with anti-CD3 antibody and anti-CD28 antibody. IL-10 T cells are cultured in the presence of anti-CD3 antibody, anti-CD28 antibody and IL-2, or beads coated with anti-CD3 antibody and anti-CD28 antibody and IL-2. IL-10 T Cell TransAct from Miltenyi Biotec TM In some embodiments, multiple donor CD4 IL-10 T cells in ImmunoCult Human TCell Activator from STEMCELL Technologies TM cultured in the presence of .

[0223] In some embodiments, multiple donor CD4 IL-10 T cells are in frozen stock.

[0224] 6.4. Pharmaceutical Compositions

[0225] In another aspect, a pharmaceutical composition is provided. The pharmaceutical composition comprises multiple donor CD4 IL-10 T cells and a pharmaceutically acceptable carrier or diluent.

[0226] The pharmaceutical composition can be formulated for use by any suitable human or veterinary route of administration. In typical embodiments, 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 an acceptable aqueous solution for parenteral administration, which is pyrogen-free and has suitable pH value, isotonicity and stability.

[0227] In some embodiments, the pharmaceutically acceptable carrier or diluent is saline, lactated Ringer's solution or other physiologically compatible solutions. In various embodiments, the pharmaceutical composition solution comprises 2-20%, preferably 5% human serum albumin.

[0228] In some embodiments, unit dosage forms of pharmaceutical compositions are provided that are suitable for administering the pharmaceutical compositions by systemic administration, particularly intravenous administration.

[0229] In some embodiments, the unit dosage form contains 10 4 Up to 10 11 Multiple donor CD4 IL-10 T cells, 10 4 Up to 10 10 Multiple donor CD4 IL-10 T cells, 10 4 Up to 10 9 Multiple donor CD4 IL-10 T cells, 10 5 Up to 10 10 Multiple donor CD4 IL-10 T cells, 10 5 Up to 10 9 Multiple donor CD4 IL-10 T cells, 10 5 Up to 10 8 Multiple donor CD4 IL-10 T cells or 10 5 Up to 10 7 Multiple donor CD4 IL-10 T cells.

[0230] In typical embodiments, the pharmaceutical composition in unit dosage form is in liquid form.

[0231] 6.5. Preparation of multiple donor CD4 IL-10 Cell Methods

[0232] In another aspect, the present disclosure provides for preparing multiple donor CD4 IL-10 Cellular methods.

[0233] In some embodiments, the method comprises the steps of: (i) combining primary CD4 T cell lines obtained from at least two different T cell donors; + T cells; and (ii) modifying the combined CD4 T cells by introducing an exogenous polynucleotide encoding IL-10 + In other embodiments, the method comprises the following steps: (i) obtaining primary CD4 T cells from at least two different T cell donors; + (ii) modifying each donor's CD4 T cells by introducing an exogenous polynucleotide encoding IL-10 + T cells; then (iii) incorporating genetically modified CD4 + T cells, thereby obtaining multiple donor CD4 IL-10 Cells. Various methods known in the art can be used to introduce exogenous polynucleotides encoding IL-10 into primary CD4 + T cells.

[0234] In some embodiments, the method further comprises incubating primary CD4 + T cells or genetically modified CD4 + In some embodiments, the method further comprises incubating primary CD4 T cells in the presence of anti-CD3 antibody, anti-CD28 antibody and IL-2 or beads coated with anti-CD3 antibody and anti-CD28 antibody and IL-2. + T cells or genetically modified CD4 + In some embodiments, the method further comprises incubating primary CD4 T cells in the presence of a feeder cell mixture. + T cells or genetically modified CD4 + In some embodiments, the method further comprises incubating primary CD4 T cells in the presence of the nanoformulation of anti-CD3 antibody and anti-CD28 antibody. + T cells or genetically modified CD4 + In some embodiments, in a T Cell TransAct from Miltenyi Biotec TM In some embodiments, the incubation is carried out in the presence of STEMCELL Technologies' ImmunoCult Human T Cell Activator TM Incubate in the presence of .

[0235] In some embodiments, the incubation step is performed prior to introducing the exogenous polynucleotide encoding IL-10. In some embodiments, in (i) combining primary CD4 T cells obtained from at least two different T cell donors + T cells; but after (ii) modifying the combined CD4 + In some embodiments, in (i) obtaining primary CD4 T cells from at least two different T cell donors, + T cells; but after (ii) modifying each donor's CD4 + T cells were previously incubated.

[0236] In some embodiments, an incubation step is performed after step (ii). In other words, in some embodiments, after (ii) the combined CD4 + In some embodiments, in (ii) each donor's CD4 T cells are modified by introducing an exogenous polynucleotide encoding IL-10. + T cells, but in combination with genetically modified CD4 + T cells were previously cultured to obtain genetically modified CD4 + In some embodiments, in (iii) combining genetically modified CD4 + T cells were then incubated to obtain multi-donor CD4 IL-10 cell.

[0237] In some embodiments, the incubation step is performed more than once. In some embodiments, in the CD4 + The incubation steps were performed before and after the genetic modification of the T cells.

[0238] In some embodiments, viral vectors are used to introduce exogenous polynucleotides into primary CD4 +T cells. In some embodiments, the viral vector is a lentiviral vector. In some embodiments, the exogenous polynucleotide comprises a segment encoding IL-10 having a 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 with SEQ ID NO: 1. In some embodiments, the IL-10 encoding polynucleotide segment has a 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 with SEQ ID NO: 2. In some embodiments, the exogenous polynucleotide further comprises a segment encoding a marker, thereby allowing selection of successfully transduced CD4+ T cells. In some embodiments, the encoded selection marker is ΔNGFR. In certain embodiments, the encoded selection marker has a sequence of SEQ ID NO: 3. In specific embodiments, the exogenous polynucleotide comprises a sequence of SEQ ID NO: 4. In some embodiments, the encoded selection marker is a truncated form of a human EGFR polypeptide.

[0239] In some embodiments, the method further comprises isolating a genetically modified CD4 + Steps to generate enriched genetically modified CD4 IL-10 Cell population.

[0240] In some embodiments, at least 70% of the genetically modified CD4 + In some embodiments, at least 95% of the genetically modified CD4+ T cells in the enriched population express the selectable marker. In some embodiments, at least 96%, 97%, 98%, or 99% of the genetically modified CD4+ T cells in the enriched population express the selectable marker. + T cells express a selectable marker.

[0241] In some embodiments, the method further comprises incubating the enriched genetically modified CD4 + In some embodiments, the incubation is performed in the presence of anti-CD3 antibody and anti-CD28 antibody, or beads coated with anti-CD3 antibody and anti-CD28 antibody. In some embodiments, the incubation is 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 nanoformulations of anti-CD3 antibody and anti-CD28 antibody. In some embodiments, the incubation is performed in the presence of T Cell TransAct from Miltenyi Biotec. TMIn some embodiments, the incubation is carried out in the presence of ImmunoCult Human TCell Activator from STEMCELL Technologies. TM Incubate in the presence of .

[0242] In some embodiments, the method further comprises freezing the genetically modified CD4 + T cell steps.

[0243] In some embodiments, primary CD4 + The T cells are from a donor selected based on its HLA haplotype. In some embodiments, the method further comprises the step of selecting a T cell donor by analyzing its genetic information. In some embodiments, the method comprises the step of analyzing the genetic information or HLA haplotype of a potential T cell donor.

[0244] In some embodiments, primary CD4 + T cells are derived from primary CD4 + The host treated with T cells or their modifications has a donor that is at least partially HLA-matched. In some embodiments, the primary CD4 + T cells are from a donor that is at least partially HLA matched to a stem cell (HSC), tissue, or organ donor. In some embodiments, primary CD4 + T cells are obtained from a third party donor who is not biologically related to the host. In some embodiments, primary CD4 + The T cells are obtained from a third party donor who is not biologically related to the stem cell, tissue or organ donor.

[0245] In some embodiments, in step (i), primary CD4 +T cells are obtained from two, three, four, five, six, seven, eight, nine or ten 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 at 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 at HLA-A, HLA-B, HLA-C and HLA-DRB1 loci. In some embodiments, at least two T cell donors have 2 / 2 matches at HLA-A loci. In some embodiments, at least two T cell donors have 2 / 2 matches at HLA-B loci. 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 at least two T cell donors has an A*02 or A*24 allele.

[0246] 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 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 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 are less than 3 / 4 or 4 / 4 matched to each other at the HLA-DRB1 and HLA-DQB1 loci.

[0247] In some embodiments, in step (i), primary CD4 + T cells are obtained from one or more frozen stocks. In some embodiments, in step (i), primary 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 further comprises isolating CD4+ In some embodiments, in step (i), primary CD4 + The T cells are obtained from a liquid suspension. In some embodiments, the liquid suspension is obtained from a previously frozen stock solution.

[0248] In some embodiments, CD4 + T cells come into contact with the patient's antigen-presenting cells (monocytes, dendritic cells, or DC-10 cells) to generate allospecific CD4 + T cells, which are then modified to produce high levels of IL-10 (allo-CD4 IL-10 cell).

[0249] In some embodiments, the method does not include exposing CD4 + In some embodiments, the method does not include a step of desensitizing the CD4 T cells in the presence of the recombinant IL-10 protein. + The step of T cell apoptosis in which recombinant IL-10 protein is not removed from CD4 + In some embodiments, the method does not include the presence of DC10 cells from the host. + Steps in which T cells lose their responsiveness.

[0250] Use multiple donor CD4 IL-10 Cell Methods

[0251] In another aspect, the present disclosure provides a method of treating a patient, comprising administering to a patient in need of immune tolerance a plurality of donor CD4 IL-10 The step of preparing a cell or pharmaceutical composition.

[0252] In some embodiments, the method further comprises thawing multiple donor CD4 IL-10 Freeze the suspension of cells as in the previous step.

[0253] In some embodiments, multiple donor CD4 IL-10 The cell or pharmaceutical composition prevents or reduces the severity of a pathogenic T cell response in a patient. In some embodiments, multiple donor CD4 IL-10 Cells or pharmaceutical compositions reduce inflammation. In some embodiments, multiple donor CD4 IL-10 Cells or pharmaceutical compositions enhance tissue repair. In some embodiments, multiple donor CD4 IL-10 The cells or pharmaceutical compositions enhance immune tolerance to self and non-pathogenic antigens and maintain immune homeostasis.IL-10 The cells or pharmaceutical compositions downregulate pathogenic T cell responses associated with organ transplantation, GvHD, and various autoimmune and inflammatory diseases. IL-10 Cells or pharmaceutical compositions for treating autoimmune diseases. In some embodiments, multiple donor CD4 IL-10 The cell or pharmaceutical composition reduces the overactivity of the NLPR3 inflammasome or reduces symptoms associated with overactivity of the NLPR3 inflammasome. IL-10 The cell or pharmaceutical composition induces tumor cell death or reduces tumor growth. In some embodiments, multiple donor CD4 IL-10 The cell or pharmaceutical composition increases disease-free survival (e.g., absence of minimal residual disease). In some embodiments, multiple donor CD4 IL-10 The cells or pharmaceutical compositions induce wound healing or tissue repair.

[0254] In some embodiments, multiple donor 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. IL-10 The cells or pharmaceutical compositions are administered in an amount effective to enhance immune tolerance to self and pathogenic antigens and maintain immune homeostasis. 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. IL-10 The cells or pharmaceutical compositions are administered in an amount effective to treat the autoimmune disease. IL-10 The cells or pharmaceutical compositions are administered in an amount effective to reduce overactivity of the NLPR3 inflammasome or to reduce symptoms associated with overactivity of the NLPR3 inflammasome. IL-10 The cells or pharmaceutical compositions are administered in an amount effective to induce tumor cell death or reduce tumor growth. IL-10 The cells or pharmaceutical compositions are administered in an amount effective to increase disease-free survival (eg, the absence of minimal residual disease).

[0255] In some embodiments, the method of treatment further comprises monitoring the multi-donor CD4IL-10 In some embodiments, the method comprises the steps of detecting a selectable marker in a biological sample obtained from a patient to detect the presence or absence of multiple donor CD4 IL-10 In some embodiments, the markers are detected at multiple time points to track multiple donor CD4 IL-10 Changes in the presence of cells. In some embodiments, the biological sample is a biopsy or a blood sample from a patient.

[0256] Multiple donor CD4 IL-10 T cells are administered in a therapeutically effective amount. This amount can be determined based on body weight and other clinical factors. In some embodiments, 10 3 Up to 10 9 In some embodiments, administration of 10 3 Up to 10 8 In some embodiments, administration of 10 3 Up to 10 7 In some embodiments, administration of 10 3 Up to 10 6 In some embodiments, administration of 10 3 Up to 10 5 In some embodiments, administration of 10 3 Up to 10 4 cells / kg.

[0257] In various embodiments, multiple donor CD4 IL-10 T cells are administered on a schedule that is therapeutically effective. In some embodiments, multiple donor CD4 IL-10 T cells are administered once. In some embodiments, multiple donor CD4 IL-10 T cells are administered daily, every 3 days, every 7 days, every 14 days, every 21 days, or monthly.

[0258] Multiple donor CD4 IL-10 T cells can be administered according to different routes of administration (eg, systemic, subcutaneous, or intraperitoneal). In some embodiments, the cells are administered in saline or physiological solution that may contain 2-20%, preferably 5%, human serum albumin.

[0259] In some embodiments, multiple donor CD4 IL-10 Is preventive, meaning it completely or partially prevents a disease, condition, or its symptoms.

[0260] 6.6.1. Methods to reduce or prevent GvHD

[0261] In some embodiments, multiple donor CD4 IL-10Cells may contain multiple donor CD4 IL-10 The pharmaceutical compositions of cells are used to treat patients prior to hematopoietic stem cell (HSC) transplantation (HSCT), concurrently with HSCT, or after HSCT.

[0262] In various embodiments, the HSCT is a matched related HSCT. In various embodiments, the HSCT is a haploidentical HSCT, an unmatched related HSCT, or an unmatched unrelated HSCT.

[0263] In some embodiments, the patient has a hematological malignancy requiring treatment with allo-HSCT. In some embodiments, the hematological malignancy is mediated by abnormal myeloid cells.

[0264] In some embodiments, based on the multiple donor CD4 IL-10 T cell donors are selected based on the genetic information of the patient to be treated with cells and HSCs and / or the genetic information of the HSC donor. In some embodiments, the T cell donors are selected based on the genetic information of the multiple donor CD4 IL-10 The method further comprises administering a CD4 IL-10 In some embodiments, the method further comprises the step of analyzing the genetic information or HLA haplotype of the T cell donor before the cells are grown. In some embodiments, the method further comprises the step of analyzing the genetic information or HLA haplotype of the host. In some embodiments, the method further comprises the step of analyzing the genetic information or HLA haplotype of the HSC donor.

[0265] In some embodiments, the T cell donor, the host, and the HSC donor are not biologically related. 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 are at least partially mismatched in HLA haplotype. In some embodiments, the T cell donor is selected when the T cell donor has an HLA haplotype and the HLA match exceeds a threshold.

[0266] In some embodiments, the HSC donor is partially HLA-mismatched with the patient. In some embodiments, the HSC donor has less than 5 / 10, 6 / 10, 7 / 10, 8 / 10, 9 / 10 or 10 / 10 matching with the patient at 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 matching with the patient at HLA-A, HLA-B, HLA-C and HLA-DRB1 loci. In some embodiments, the HSC donor has less than 2 / 2 matching with the patient at HLA-A, HLA-B or HLA-C loci. In some embodiments, the HSC donor has less than 3 / 4 or 4 / 4 matching with the patient at HLA-DRB1 and HLA-DQB1 loci.

[0267] In some embodiments, one or more of the T cell donors are HLA mismatched or partially HLA mismatched with 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 with the patient at 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 with the patient at 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 with the patient at 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 with the patient at HLA-DRB1 and HLA-DQB1 loci.

[0268] In some embodiments, one or more of the T cell donors are HLA mismatched or partially HLA mismatched with 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 with 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 with 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 with the HSC donor at the HLA-A, HLA-B or HLA-C loci. In some embodiments, one or more of the T cell donors has less than 3 / 4 or 4 / 4 match with the HSC donor at the HLA-DRB1 and HLA-DQB1 loci. IL-10 The cells or pharmaceutical compositions prevent or reduce the severity of GvHD via transplanted hematopoietic stem cells.

[0269] In some embodiments, multiple donor CD4 IL-10 The cell or pharmaceutical composition prevents or reduces the severity of a pathological T cell response caused by transplanted hematopoietic cells. In certain embodiments, multiple donor CD4 IL-10 cells to prevent or reduce GvHD.

[0270] In some embodiments, multiple donor CD4 IL-10 The cell or pharmaceutical composition prevents or reduces the severity of tissue damage induced by pathogenic T cells or inflammation.

[0271] 6.6.2. Methods for treating cancer

[0272] In some embodiments, multiple donor CD4 IL-10 Cells are used to treat cancer. In a preferred embodiment, multiple donor CD4 IL-10 The cells directly mediate anti-tumor effects, and in certain embodiments, anti-leukemic effects.

[0273] In some embodiments, multiple donor CD4 IL-10 Cells are administered in combination with allogeneic monocytes or PBMCs for the treatment of cancer. IL-10 Cells are administered before or after administration of PBMCs. In some embodiments, multiple donor CD4 IL-10Cells and allogeneic monocytes or PBMCs were administered simultaneously.

[0274] In some embodiments, multiple donor CD4 IL-10 Cells and allogeneic monocytes or PBMCs were administered at a ratio of 1:3, 1:2, 1:1, 2:1 or 3:1.

[0275] In some embodiments, the neoplastic cell expresses CD13. In some embodiments, the neoplastic cell expresses HLA class I. In some embodiments, the neoplastic cell expresses CD54. In some embodiments, the neoplastic cell expresses CD13, HLA class I, and CD54. In some embodiments, the neoplastic cell expresses CD112. In some embodiments, the neoplastic cell expresses CD58. In some embodiments, the neoplastic cell expresses CD155. In some embodiments, the tumor expresses CD112, CD58, or CD155. In various embodiments, the tumor is a solid tumor or a hematological tumor.

[0276] In some embodiments, the patient has a cancer selected from the group consisting of adrenal cancer, anal cancer, bile duct cancer, bladder cancer, bone cancer, adult brain / CNS tumors, childhood brain / CNS tumors, breast cancer, male breast cancer, cancer of unknown primary, Castleman's disease, cervical cancer, colon / rectum cancer, endometrial cancer, esophageal cancer, Ewing family tumors, eye cancer, gallbladder cancer, gastrointestinal carcinoid tumors, gastrointestinal stromal tumors (GIST), gestational trophoblastic disease, Hodgkin's disease, Kaposi's sarcoma, kidney cancer, laryngeal and hypopharyngeal cancer, leukemia, acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML, including myeloid sarcoma and cutaneous leukemia), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), chronic myeloid leukemia (CML), Monocytic leukemia (CMML), childhood leukemia, liver cancer, lung cancer, non-small cell lung cancer, small cell lung cancer, lung carcinoid tumor, lymphoma, cutaneous lymphoma, malignant mesothelioma, multiple myeloma, myelodysplastic syndrome, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, non-Hodgkin lymphoma, childhood non-Hodgkin lymphoma, oral cavity and oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, penile cancer, pituitary tumor, prostate cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma - adult soft tissue cancer, skin cancer, skin cancer - basal cell and squamous cell, skin cancer - melanoma, skin cancer - Merkel cell, small intestine cancer, stomach cancer, testicular cancer, thymic cancer, thyroid cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Waldenstrom's macroglobulinemia and Wilms' tumor.

[0277] In some embodiments, the cancer is a myeloid tumor. In specific embodiments, the cancer is AML or CML. In some embodiments, the cancer is a myeloid tumor.

[0278] In some embodiments, the method is used to treat blood cancers involving the blood, bone marrow, and lymph nodes. In various embodiments, the blood cancer is a lymphoma (e.g., Hodgkin's lymphoma), lymphocytic leukemia, myeloma. In various embodiments, the blood cancer is acute or chronic myeloid (myeloid) leukemia (AML, CML) or myelodysplastic syndrome.

[0279] In some embodiments, the cancer is refractory or resistant to therapeutic intervention.

[0280] In some embodiments, multiple donor CD4 IL-10 The cells are used in combination with a 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.

[0281] In some embodiments, multiple donor CD4 IL-10 The cells induce cell death of tumor-infiltrating and tumor-growth-promoting myeloid cells (e.g., monocytes, macrophages, neutrophils).

[0282] 6.6.3. Methods for treating inflammatory or autoimmune diseases

[0283] In some embodiments, multiple donor CD4 IL-10 cells to treat inflammatory or autoimmune diseases. In some embodiments, multiple donor CD4 IL-10 The cells can be used to treat diseases or conditions involving overactivity of the NLPR3 inflammasome.

[0284] The NOD-like receptor family (NLR) protein NLRP3 is an intracellular signaling molecule that senses danger signals from pathogenic, environmental or endogenous sources. Upon activation, NLPR3 interacts with caspase-1 to form a complex called the inflammasome. This leads to caspase-1 activation, which cleaves the proinflammatory cytokines IL-1β and IL-18 into their active forms and mediates an inflammatory cell death called pyroptosis.

[0285] In some embodiments, multiple donor CD4 IL-10 Cells are used to treat an inflammatory disease selected from the group consisting of Muckle-Wells syndrome (MWS), familial cold autoinflammatory syndrome (FCAS), and neonatal-onset multisystem inflammatory disease (NOMID). In some embodiments, multiple donor CD4 IL-10Cells are used to treat a chronic disease selected from metabolic syndrome, type 2 diabetes, atherosclerosis, Alzheimer's disease, Parkinson's disease, ALS, nonalcoholic steatohepatitis, osteoarthritis, silicosis, asbestosis, gout, and pulmonary fibrosis. In some embodiments, multiple donor CD4 IL-10 cells 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.

[0286] In some embodiments, multiple donor CD4 IL-10 Cells are used to treat diseases associated with NLRP3. The disease may be selected from the group consisting of CAPS, NASH, Alzheimer's disease, Parkinson's disease, cardiovascular disease, osteoarthritis, gout, pseudogout, nephrocalcinosis, type II diabetes, Sjögren's syndrome, sickle cell disease (SCD), AMD, infection, cerebral malaria, asbestosis, contact hypersensitivity, sunburn, silicosis, cystic fibrosis, inflammatory bowel disease, renal calcification, ALS, myelodysplastic syndrome and uveitis.

[0287] 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, cerebral hemorrhage, sepsis-related encephalopathy, cerebral ischemia, subarachnoid hemorrhage, epilepsy, acrylamide poisoning, opioid-induced neuroinflammation, chronic migraine, perioperative neurocognitive disorder, post-stroke cognitive impairment, post-cardiac arrest cognitive impairment, social isolation-induced cognitive impairment, anxiety, and post-traumatic stress disorder.

[0288] 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.

[0289] In some embodiments, the disease is a cardiac disorder selected from atherosclerosis, heart failure, hypertension, myocardial infarction, atrial fibrillation, cardiac damage caused by metabolic dysfunction, and endothelial dysfunction.

[0290] 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, liver injury caused by idiosyncratic drugs, and liver fibrosis. In some embodiments, the disease is a pancreatic or renal disorder selected from diabetic encephalopathy, diabetic-related atherosclerosis, insulin resistance, islet transplant rejection, chronic crystalline nephropathy, renal fibrosis, I / R renal injury, obesity-related nephropathy, and renal hypertension. In some embodiments, the disease is a skin or eye disorder selected from psoriasis and retinal angiogenesis. 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 diseases. In some embodiments, the disease is a pain condition selected from multiple sclerosis-related neuropathic pain, chronic prostatitis / chronic pelvic pain, bone pain caused by cancer, and hyperalgesia. In some embodiments, the disease is cancer, such as human head and neck squamous cell carcinoma. In some embodiments, the disease is an infectious condition, such as a bacterial, viral, or parasitic infection.

[0291] In some embodiments, multiple donor CD4 IL-10 The cells were used in combination with currently available treatments for NLRP3-related diseases, such as biologics targeting IL-1, including the recombinant IL-1 receptor antagonist anakinra, the IL-1β neutralizing antibody canakinumab, and the soluble decoy IL-1 receptor rilonacept.

[0292] In some embodiments, multi-donor CD4IL-10 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.

[0293] In some embodiments, multiple donor CD4 IL-10 Cells to treat inflammation. Inflammation may be associated with, but is not limited to, coronary artery disease (CAD), type 2 diabetes, neurodegenerative diseases, or inflammatory bowel disease.

[0294] In some embodiments, multiple donor CD4 IL-10Cells are used to treat diseases or conditions involving increased production of IL-1β by activated monocytes, macrophages, or dendritic cells. In some embodiments, administration of multiple donor CD4 IL-10 Cells are used to treat diseases or conditions involving increased production of IL-18 by activated monocytes, macrophages, or dendritic cells. In some embodiments, administration of multiple donor CD4 IL-10 The cells are used to treat diseases or disorders involving increased production of mature caspase 1 by activated monocytes, macrophages or dendritic cells.

[0295] In some embodiments, multiple donor CD4 IL-10 cells to reduce the production of IL-1β by activated monocytes, macrophages or dendritic cells. In some embodiments, administration of multiple donor CD4 IL-10 cells to reduce the production of IL-18 by activated monocytes, macrophages or dendritic cells. In some embodiments, administration of multiple donor CD4 IL-10 cells to reduce the production of mature caspase 1 by activated monocytes, macrophages or dendritic cells.

[0296] 6.6.4. Methods of treating other diseases

[0297] In some embodiments, multiple donor CD4 IL-10 cells to treat autoimmune diseases.

[0298] 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, Sjögren's syndrome, Hashimoto's thyroiditis, myasthenia gravis, autoimmune vasculitis, pernicious anemia, ulcerative colitis, bullous diseases, 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 suffers from allergies or atopic diseases. Allergies or atopic diseases can be selected from the group consisting of: asthma, atopic dermatitis and rhinitis. In some embodiments, the patient suffers from food allergies.

[0299] In some embodiments, multiple donor CD4 IL-10 cells to prevent or reduce the severity of pathogenic T cell responses to cell and organ transplantation other than HSCT. In some embodiments, the method comprises administering multiple donor CD4 IL-10T cells or pharmaceutical compositions before or after the patient undergoes an organ transplant. In certain embodiments, the organ is a kidney, heart, or pancreatic islet cells. In a preferred embodiment, multiple donor CD4 IL-10 The cells or pharmaceutical compositions prevent or reduce the severity of host rejection of an organ transplant.

[0300] In some embodiments, multiple donor CD4 IL-10 cells to prevent or reduce immune responses associated with gene therapy (e.g., administration of recombinant AAV (rAAV)). In these embodiments, the method further comprises administering multiple donor CD4 IL-10 The step of administering the recombinant AAV to the patient before or after the cell or pharmaceutical composition.

[0301] In some embodiments, multiple donor CD4 IL-10 The cells are used to prevent or reduce the immune response associated with transplantation of iPS-derived tissues or cells. iPS-derived tissues and cells include, but are not limited to, cardiomyocytes, hepatocytes, epithelial cells, cartilage, bone and muscle cells, and neurons.

[0302] In some embodiments, multiple donor CD4 IL-10 In some embodiments, multiple donor CD4 IL-10 cells to reduce excessive immune responses to bacterial infections, such as toxic shock and cytokine storm.

[0303] In some embodiments, the method further comprises administering multiple donor CD4 IL-10 The step of administering an immunogenic therapeutic protein to a patient before or after the cell population or pharmaceutical composition. IL-10 The cell population or pharmaceutical composition reduces the immune response to the immunogenic therapeutic protein.In some embodiments, the immunogenic therapeutic protein is selected from the group consisting of a therapeutic antibody, a Factor VIII replacement, a cytokine, and a cytokine mutein.

[0304] 6.7. Example

[0305] The following examples are offered for illustrative purposes only and are not intended to be limiting.

[0306] 6.7.1. Experimental observations summary

[0307] The present disclosure provides methods for producing and using highly purified allogeneic CD4 +T cells that have been transduced with a bidirectional lentiviral vector containing the human IL-10 gene and a truncated, non-signaling form of human NGFR. Successfully transduced CD4 + T cells with purity >95% producing IL-10 and expressing NGFR CD4 + T cells (designated as CD4 IL-10 CD4 cells from 3 different allogeneic HLA-mismatched donors IL-10 The cells were combined at a 1:1:1 ratio.

[0308] These combined populations, also referred to herein as multi-donor CD4 IL-10 cells, with a single donor CD4 IL-10 The cells have a cytokine production profile comparable to naturally derived type 1 regulatory T (Tr1) cells. They produce high levels of IL-10 and IL-22, varying levels of IFN-γ and IL-5, and low levels of IL-4. IL-10 The cells are polyclonal (have multiple antigen specificities) and suppress allogeneic CD4 + and CD8 + T cells. In addition, they specifically kill myeloid leukemia cells in vitro. In addition, multiple donor CD4 IL-10 The cells inhibited NLPR3 inflammasome activation and pro-inflammatory IL-1β and IL-18 production by human monocytes in vitro.

[0309] Adoptive transfer of multiple donor CD4 IL-10 In a humanized mouse model of GvHD, adoptive transfer of multiple donor CD4 IL-10 Human CD4 + Importantly, even at high concentrations, multiple donor CD4 IL-10 The cells themselves do not induce GvHD. In addition, multiple donor CD4 IL-10 The cells had a cytotoxic effect on cancer cells in NSG mice injected intravenously with ALL-CM cells. Three days after administration of ALL-CM cells (when these cells had already expanded to a large extent), single-donor and multi-donor CD4 IL-10 These results suggest that multiple donor CD4 IL-10 The cells have direct anti-myeloid leukemia therapeutic effects in vivo. When single-donor and multi-donor CD4 IL-10 When cells were administered together with PBMC, CD4 IL-10The cells further downregulated allogeneic PBMC-induced xeno-GvHD.

[0310] These results suggest that multiple donor CD4 IL-10 The cells can be used to treat and / or prevent 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 preserving the GvL or GvT therapeutic effects of HSCT; and can be used to treat cellular and organ rejection and autoimmune and inflammatory diseases.

[0311] 6.7.2. Example 1: Multiple Donor CD4 IL-10 Cell generation

[0312] Vector production

[0313] Multiple donor CD4 IL-10 Cells were generated by transduction with a lentiviral vector (LV-IL-10 / ΔNGFR) containing the coding sequences for human IL-10 and a truncated form of NGFR (ΔNGFR) ( Figure 1 and 2 ), as described in WO2016 / 146542, which is incorporated herein by reference in its entirety. The plasmid sequence encoding human IL-10 and ΔNGFR (pLVIL-10) for producing LV-IL-10 / ΔNGFR is provided as SEQ ID NO:5. In brief, pLVIL-10 is generated by connecting a 549bp fragment of the human IL-10 sequence encoding pH15C (ATCC 68192) to plasmid #1074.1071.hPGK.GFP.WPRE.mhCMV.dNGFR.SV40PA. The presence of a bidirectional promoter (human PGK promoter plus the minimal core element of the reverse CMV promoter) enables the co-expression of two transgenes. The plasmid further contains the coding sequence of an antibiotic resistance gene (e.g., ampicillin or kanamycin).

[0314] Lentiviral vectors were produced by transient four-plasmid co-transfection into 293T cells with Ca3PO4 and concentrated by ultracentrifugation: 1 μM sodium butyrate was added to the culture for vector collection. Titers in 293T cells were estimated by limiting dilution, and vector particles were measured by HIV-1 Gag p24 antigen immunocapture (NEN Life Science Products; Waltham, MA). Vector infectivity was calculated as the ratio between titer and particles. For concentrated vectors, the titer range was 5 × 10 8 Up to 6×10 9 transduction units / mL, and the infectivity range is 5×10 4 Up to 5×10 5Transduction units / ng.

[0315] CD4 IL-10 Cell production

[0316] Figure 3 It is CD4 IL-10 Schematic diagram of the cell generation process. Purification of CD4 from healthy donors + Human CD4 T cells were activated with soluble anti-CD3, soluble anti-CD28 mAb and rhIL-2 (50 U / mL) + T cells were incubated 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.

[0317] After 9-11 days, transduced cells were analyzed for ΔNGFR expression by FACS, and vector copy number (VCN) was quantified by digital droplet PCR (ddPCR).

[0318] CD4 from 10 different donors + The average transduction efficiency was 45 ± 17% for T cells and 2.7 ± 0.6% for VCN. Figure 4A Human CD4 T cells transduced with LV-IL-10 / ΔNGFR (a bidirectional lentiviral vector encoding human IL-10 and a truncated form of the human NGF receptor) are shown. + CD4 + ΔNGFR + Percentage of cells (mean ± SD, n = 10 left column) and vector copy number (VCN, mean ± SD, n = 10 right column). Quantification of CD4 by digital droplet PCR (ddPCR) IL-10 CD4 + ΔNGFR + Frequency of cells and vector copy number.

[0319] Purification of ΔNGFR using anti-CD271 mAb-coated beads + T cells with a purity of >95% CD4 IL-10 Cell population. After purification, cells were stained with markers for CD4 and ΔNGFR and analyzed by FACS. The data showed that the purification step resulted in a purity of over 98%. Figure 4B FACS data of two representative donors (Donor B and Donor C) out of 10 tested donors are shown. The CD4 IL-10 The cell purity was 98.3% and 99.2%, respectively. IL-10Cells were restimulated three times at 14-day intervals and their in vitro and in vivo functions were tested after the second (TF2) and third (TF3) functional restimulations.

[0320] Resting CD4 IL-10 Cells constitutively produce IL-10. Upon activation, the levels of IL-10 produced are strongly increased.

[0321] CD4 IL-10 The cells have a cytokine production profile comparable to naturally derived Tr1 cells.

[0322] Analysis of single donor CD4 after the second (TF2) and third (TF3) restimulation IL-10 The cytokine production characteristics of the cells and the results in Figure 5 Specifically, CD4 IL-10 Cells (2×10 in 200 μl) 5 Cells) were restimulated as previously described (Andolfi et al. Mol Ther. 2012; 20(9): 1778-1790 and Locafaro et al. Mol Ther. 2017; 25(10): 2254-2269). On day 14, after the second (TF2) and third (TF3) rounds of restimulation, CD4 IL-10 Cells were not re-stimulated or activated with immobilized CD3 (10 μg / mL) and soluble CD28 mAb (1 μg / mL) for 48 hours. Culture supernatants were collected and the 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 are shown, n = 8 tested donors. Figure 5 The results presented in the present study showed that CD4 IL-10 The cells displayed cytokine production characteristic of Tr1 cells.

[0323] Although differences between different donors were observed, the second (TF2) ( Figure 5 Left thumbnail) or the third time (TF3) ( Figure 5 The overall cytokine production profile after restimulation (right panel) is comparable and reflects the cytokine production profile of Tr1 cells (Roncarolo et al., Immunity, 2018). Like Tr1 cells, CD4 IL-10 The cells produced high levels of IL-10 and IL-22, varying levels of IL-5 and IFN-γ, but relatively low levels of IL-4, and undetectable levels of IL-2 (not shown).

[0324] CD4 IL-10 Cells express high levels of granzyme B and selectively kill myeloid leukemia cells

[0325] After the second round of restimulation (TF2), CD4 IL-10 Granzyme B (GzB) expression in cells. Fig. 6A The data in Figure 2 show that all CD4 IL-10 More than 95% of the cells expressed high levels of granzyme B.

[0326] Further analysis of CD4 IL-10 The cells showed cytotoxic effects on human myeloid leukemia cell line (ALL-CM) and erythroid leukemia cell line (K562). IL-10 Cells (10 5 cells / well) and K562 and ALL-CM cells (10 5 The remaining leukemia cell lines (CD45 低 , CD3-).

[0327] CD4 IL-10 Cells selectively kill myeloid leukemia cells (ALL-CM), such as Figure 6B The % killing of ALL-CM cells ranged from 62% to 100%, whereas the % killing of the erythroid leukemia cell line K562 (which is highly sensitive to nonspecific cytotoxicity and natural killer (NK) cell activity) varied from 0 to 27% (4 different donors were tested). Taken together, these data confirm that CD4 IL-10 The cells expressed granzyme B and effectively killed myeloid leukemia cells. As expected, CD4 IL-10 There is some variation in the killing ability of the cells.

[0328] CD4 IL-10 Allogeneic CD4 + and CD8 + T cell proliferative response

[0329] CD4 IL-10 Allogeneic CD4 + T cells or CD8 + Specifically, allogeneic PBMC cells were used 670(5×10 4 cells / well) and in the absence or presence of CD4 IL-10 Cells (5×10 4 Allogeneic mature dendritic cells (mDCs) (5 × 10 3 cells / well) and soluble anti-CD3 mAb. After 3 days of culture, CD4 + ΔNGFR –T cells or CD8 + ΔNGFR – After T cells were gated, The percentage of proliferative responding cells was determined by dilution at 670. Fig. 7A and 7B Shown are the results from six different unpooled donors ( Fig. 7A Donor-C, Donor-E and Donor-F, and Figure 7B CD4 IL-10 CD4 + T cells affected, as well as the percentage of proliferation and suppression. Fig. 8A and 8B Shown are the results from six different single donors ( Fig. 8A Donor-C, Donor-E and Donor-F, and Figure 8B CD4 IL-10 CD8 + Effects on T cell proliferation.

[0330] The results showed that CD4 IL-10 Cells (unpooled and tested individually) down-regulate allogeneic CD4 + and CD8 + T cell proliferation response. + The inhibitory effect on T cells ranged from 51% to 96%, while the inhibitory effect on CD8 + The inhibitory effect of T cells ranged from 62% to 73%.

[0331] Multiple donor CD4 IL-10 Cell generation and characterization

[0332] Using CD4 from multiple donors + Cells were cultured as described above and Figure 3 The generation of CD4 IL-10 CD4 cells per donor IL-10 The cells were stimulated by a second (TF2) and third (TF3) restimulation. After the third stimulation, CD4 IL-10 Cells were combined at a 1:1:1 ratio and stimulated with immobilized CD3 (10 μg / mL) and soluble CD28 mAb (1 μg / mL) for 48 hours.

[0333] Multiple donor CD4 IL-10 The cells have the same CD4 IL-10 Cytokine production of cells comparable to that of Tr1 cells Life characteristics.

[0334] The culture supernatants were collected and the levels of IL-10, IL-4, IL-5, IFN-γ, and IL-22 were determined by ELISA. Fig. 9The results presented in the study showed that multiple donor CD4 IL-10 Cytokine production of cells (black dots) and CD4 IL-10 CD4 IL-10 Cells are equivalent. Multiple donor CD4 IL-10 The cells produced high levels of IL-10 and IL-22, varying levels of IL-5, IFN-γ, and low levels of IL-4, as well as undetectable levels of IL-2 (not shown). These data suggest that combined CD4 IL-10 The cells are viable and these multiple donor CD4 IL-10 The cells retain the CD4 IL-10 Importantly, the pooled allogeneic cell populations contained >95% viable cells, indicating that they do not kill each other.

[0335] Multiple donor CD4 IL-10 The cells expressed high levels of granzyme B and killed myeloid leukemia cell lines.

[0336] After restimulation in round 3 (TF3), multiple donor CD4 IL-10 Granzyme B (GzB) expression in cells. Fig. 10A The data showed that most multi-donor CD4 IL-10 Cells express GzB. More than 95% of multi-donor CD4 IL-10 Cells expressing granzyme B, with CD4 IL-10 The GzB expression of cells was comparable ( Fig. 10A ).

[0337] Further analysis of CD4 IL-10 Cytotoxicity of cells against myeloid leukemia cells (ALL-CM cell line) or K562. Multiple donor CD4 IL-10 Cells (10 5 cells / well) and K562 and ALL-CM cells (10 5 The residual leukemia cell lines (CD45 低 , CD3 - ). Fig. 10B The results presented in showed some degree of cytotoxicity against K562 cells, which were highly sensitive to nonspecific cytotoxicity. IL-10 The selective killing level of cells (black dots) against myeloid leukemia cells (ALL-CM) is related to the CD4 IL-10 Cells (open columns) are equivalent.

[0338] Multiple donor CD4 IL-10 The cells inhibit the proliferative response of allogeneic CD4+ and CD8+ T cells.

[0339] We also analyzed multiple donor CD4 IL-10 Allogeneic CD4 + T cells or CD8 + Specifically, allogeneic PBMC cells were used 670(5×10 4 cells / well) and in the absence or presence of multiple donor CD4 IL-10 Cells (5×10 4 Allogeneic mature dendritic cells (5 × 10 3 cells / well) and soluble anti-CD3 mAb. After 3 days of culture, CD4 + ΔNGFR – T cells or CD8 + ΔNGFR – After T cells were gated, The percentage of proliferative responding cells was determined by dilution at 670. Fig.11A CD4 from donor-C, donor-E and donor-F (CEF) are shown. IL-10 Multiple donor CD4 IL-10 Cell results. Fig. 11B CD4 IL-10 Multiple donor CD4 IL-10 Results for cells that were frozen, stored, and thawed prior to testing.

[0340] Fig.11A Shown, multiple donor CD4 IL-10 cells (from 3 different donors) suppressed CD4 + and CD8 + T cell response. Using a second different batch of multiple donor CD4 IL-10 Similar results were obtained with cells that were frozen, stored, and thawed prior to testing ( Fig. 11B ). CD4 + and CD8 + The inhibition rates of T cell proliferation were 68% and 75%, respectively. These data suggest that multiple donor CD4 IL-10 Cells can be frozen, stored, and thawed without loss of function.

[0341] In summary, using multiple donor CD4 IL-10The data obtained with the cells showed that these cell preparations could be pooled without problems. They contained >95% viable cells and maintained single donor CD4 IL-10 All relevant functions of cells (cytokine production, cytotoxic capacity and suppression of allogeneic T cell responses). Use more pooled multi-donor CD4 IL-10 Cells should be reduced CD4 from different individual donors IL-10 The natural variation observed between cell batches should provide a large supply of readily available CD4 IL-10 cell.

[0342] Multiple donor CD4 IL-10 Cell products will have the significant advantage of more homogeneous products, which will allow for clear batch-to-batch variability, potency and release criteria to be defined. In addition, it will facilitate the development of continuous large-scale cell production methods.

[0343] For the generation of multiple donor CD4 IL-10 Other cell methods

[0344] Before lentiviral transduction, pool buffy coats from at least 3-5 different donors. Isolate CD4 from buffy coats by positive selection using anti-CD4 antibody + Cells. FACS was used to examine the presence of incorporated CD4 + Cell purity. Optionally, obtain frozen human CD4 + Cells. Frozen human CD4 + Cells were thawed before use. CD4 + Cells were activated for 24-48 hours with a combination of CD3 and CD28 antibodies or a combination of CD3 and CD28 antibody-coated beads. In some cases, CD4 from buffy coat or frozen stocks were activated with soluble anti-CD3 mAb, soluble anti-CD28 mAb, and rhIL-2 (50 U / mL). + Cells were then transduced with a bidirectional lentiviral vector encoding human IL-10 for 48 h as described above to generate CD4 IL-10 cell.

[0345] In some cases, the T cell donor (or CD4 + cells) and selectively combine and use CD4 + cell.

[0346] The activated CD4 + Cell generation from multiple donor CD4 IL-10 cell.

[0347] On days 7-11, 5-9 days after transduction, cells were harvested and transduced T cells were successfully purified using anti-NGFR antibodies. This process typically yielded 95% pure multi-donor CD4 IL-10 Cell population.

[0348] Purified multi-donor CD4 IL-10 The cells are counted and restimulated for 8-10 days in the presence of IL-2, optionally in the presence of feeder cells, by a mixture of CD3 and CD28 antibodies, beads coated with CD3 and CD28 antibodies. In some cases, purified multi-donor CD4 IL-10 Cell stimulation.

[0349] After a total culture period of 5 weeks, CD4 IL-10 Cells were cultured and tested for their ability to produce IL-10 spontaneously or after activation with CD3 and CD28 antibodies or beads coated with CD3 and CD28 antibodies. In addition, levels of GrzB and perforin were measured. They were also tested for their ability to inhibit the proliferation of human T cells (PBMCs) and purified CD4 + and CD8 + The ability of T cells to proliferate.

[0350] In addition, 200,000 CD4 IL-10 Constitutive production of IL-22 by cells and measurement of 200,000 CD4 in a 200 μl volume after activation using a combination of CD3 and CD28 antibodies IL-10 IL-22 production by cells. IL-22 production levels were measured in an IL-22-specific ELISA as described for other cytokines in WO2016 / 146542. Pooled CD4 IL-10 Cells were frozen before storage.

[0351] 6.7.3. Example 2: Using multiple donor CD4 IL-10 Cell therapy or prevention of GvHD

[0352] Multiple donor CD4 IL-10 The role of cells in the body.

[0353] Testing multiple donor CD4 in a humanized xenogeneic GvHD disease model (NSG mouse model) IL-10 Effects of cell populations on xenogeneic GvHD induced by human PBMCs, such as Fig.12 NSG mice were sublethally irradiated and intravenously injected with (i) human PBMC (5×10 6cells / mouse), (ii) multiple donors (three donors; BC-C / E / F) CD4 IL-10 Cells (5×10 6 cells / mouse), or (iii) human PBMC (5×10 6 cells / mouse) and multiple donor CD4 IL-10 Cells (BC-C / E / F) (5×10 6 Xeno-GvHD was evaluated based on survival, weight loss (>20% weight loss), skin lesions, coat condition, mobility, and hunched back as previously described (Bondanza et al. Blood 2006).

[0354] Fig.13 The percentage of NSG mice free of xeno-GvHD is shown every day after injection. 6 Administration of personal PBMC to irradiated NSG mice unexpectedly resulted in an exceptionally fulminant xeno-GvHD. All mice died by day 10, reflecting a highly lethal xeno-GvHD. Co-administration of 5 × 10 6 Multiple donor CD4 IL-10 cells delayed this fulminant xeno-GvHD, but mice were sacrificed on day 14 because they reached the pre-specified humane 20% body weight loss criterion ( Fig.13 ). Nevertheless, these results suggest that multiple donor CD4 IL-10 It can delay the development of severe xeno-GvHD. Importantly, PBMC (5×10 6 Multiple donor CD4 IL-10 The cells failed to induce any signs of xeno-GvHD.

[0355] The spleens of NSG mice were also tested 14 days after injection ( Fig.14 , left panel) and bone marrow ( Fig.14 , right small picture) in human CD4 IL-10 These NSG mice were injected with human PBMC (5×10 6 cells / mouse), multiple donors (three donors; BC-C / E / F) CD4 IL-10 Cells (5×10 6 cells / mouse), or human PBMC (5×10 6 cells / mouse) and multiple donor CD4 IL-10 Cells (three donors; BC-C / E / F) (5×10 6 cells / mouse). Fig.14 The results presented in the study showed that multiple donor CD4 IL-10The cells migrated to the spleen and bone marrow. These cells were found at low percentages 14 days after cell infusion. These results suggest that multiple donor CD4 IL-10 The cells delayed fulminant xeno-GvHD induced by human PBMCs and did not induce any xeno-GvHD themselves.

[0356] Multiple donor CD4 IL-10 Cells were purified by CD4 + Cells suppress severe xeno-GvHD.

[0357] Testing multiple donor CD4 in a humanized xeno-GvHD model IL-10 cells, where 2.5×10 6 Purified human CD4 + T cell-induced GvHD disease, such as Fig.15 On day 0, NSG mice were sublethally irradiated and injected intravenously with human CD4 + T cells (2.5×10 6 cells / mouse) or with multiple donor CD4 IL-10 Cells (three different donors; BC-H / I / L) (2.5×10 6 cells / mouse) or with CD4 IL-10 Cells (2.5×10 6 Xeno-GvHD was evaluated based on survival, weight loss (>20% weight loss), skin lesions, coat condition, mobility and hunched back as previously described (Bondanza et al. Blood 2006).

[0358] Fig.16 The percentage of NSG mice free of GvHD is shown for each day after injection. IL-10 (BC-H / I / L) cells can suppress the expression of human allogeneic CD4 + T cell-mediated xeno-GvHD. In this experiment, xeno-GvHD was very severe, as all mice in the control group that received CD4+ T cells died by day 20. In contrast, co-administration of 2.5×10 6 Multiple donor CD4 IL-10 Suppresses GvHD by 75%. Single donor CD4 IL-10 Cells also have a protective effect, but the effect is weaker.

[0359] Other experiments

[0360] Multiple donor CD4 IL-10 Therapeutic effect of cells: (i) Mice received IL-10Human PBMC from unrelated donors (xeno-GvHD positive control); (ii) mice received multiple donor CD4 IL -10 cells (negative control); (iii) mice received PBMC and multiple donor CD4 IL-10 and (iv) mice received PBMC and multiple donor CD4 IL-10 Combination of cells. In the case of receiving PBMC and multiple donor CD4 IL-10 Some animals received both PBMC and multiple donor CD4 IL-10 Some animals received CD4 IL-10 cells, and some animals received CD4 IL-10 cell.

[0361] In the administration of PBMC and / or multiple donor CD4 IL-10 After the cells were added, the mice were monitored for the development of GvHD by measuring body weight at weeks 1, 2, 3, 4, and, if necessary, week 5. In addition to weight loss, the mice were examined for skin lesions, hair condition, and mobility. The mice in the treatment groups were monitored for an additional period of time to determine whether multiple donor CD4 IL-10 Effects of cellular on long-term survival.

[0362] After administration, the peripheral blood and tissue CD4 IL-10 Specifically, the number and location of multi-donor CD4 IL-10 Monitoring of multiple donor CD4 IL-10 Other sites where the cells were present included the lymph nodes and the intestine. The treated mice were monitored for an additional three weeks to determine their long-term survival.

[0363] The results showed that multiple donor CD4 IL-10 The cells effectively reduced and prevented xeno-GvHD.

[0364] 6.7.4. Example 3: Inhibition of GvHD and Treatment of Cancer

[0365] Testing of multi-donor CD4 in the NSG mouse model transplanted with human PBMCs and AML tumor cells IL-10 Effect of cell populations on human PBMC-induced xenogeneic GvHD and antitumor effects. AML cells (ALL-CM) were administered intravenously as previously described in WO 2016 / 146542. PBMC or multi-donor CD4 IL-10 cells or a combination thereof.

[0366] Multiple donor CD4 IL-10 Cells were obtained as described in Example 1. Multiple donor CD4 IL -10 Therapeutic effect of cells. Each group of mice received irradiation and 5×10 6 ALL-CM cells (AML mice): (i) AML mice that did not receive additional treatment; (ii) AML mice that received CD4 IL-10 5×10 cells from unrelated donors 6 AML mice with personal PBMCs – PBMCs induce severe xeno-GvHD; (iii) received 2.5×10 6 Multiple donor CD4 IL-10 and (iv) mice that received PBMC and multiple donor CD4 IL-10 Another group of mice did not receive ALL-CML cells but received 5×10 6 Individual PBMCs.

[0367] Testing of multiple donor CD4 based on weight loss, skin lesions, coat condition, motility, mortality, and long-term survival IL-10 The effect of cells on human PBMC-induced xeno-GvHD was tested based on the reduction of circulating tumor cells and long-term tumor-free survival. IL-10 Anti-tumor or graft-versus-leukemia (GvL) effects of cells.

[0368] Some mice were monitored for up to seven weeks to monitor long-term survival and complete tumor remission.

[0369] The results showed that multiple donor CD4 IL-10 The cells were effective in both suppressing xeno-GvHD and treating cancer.

[0370] 6.7.5. Example 4: Using multiple donor CD4 IL-10 Cell therapy for cancer

[0371] Testing multi-donor CD4 T cell therapy in the ALL-CM leukemia model in NSG mice IL-10 Cell population.

[0372] On day 0, NSG mice were sublethally irradiated and injected intravenously 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 (2.5×106 In the third group of animals, multiple donor CD4 IL-10 Cells (2.5×10 6 In the fourth group of animals, a single donor (from donor BC-1) CD4 IL-10 Cells (2.5×10 6 In the fifth group of animals, a single donor (from donor BC-H) CD4 IL-10 Cells (2.5×10 6 The graft-versus-leukemia (GvL) effect was tested in animals based on the reduction of circulating leukemic cells and long-term leukemia-free survival. Leukemia was measured as previously described (Locafaro G. et al Molecular Therapy 2017). Fig.17A .

[0373] like Fig. 17B and Fig. 17C As shown, all mice injected with ALL-CM myeloid leukemia cells alone developed extensive leukemia progression on day 17. 6 Interestingly, single donor CD4 IL-10 ( Fig. 17B ) or multiple donor CD4 IL-10 ( Fig. 17C ) cells achieved comparable levels of leukemia progression inhibition. These data suggest that single-donor and multi-donor CD4 IL-10 It has a strong direct anti-leukemia effect.

[0374] Single donor CD4 IL-10 and multiple donor CD4 IL-10 Graft versus leukemia (GvL) effect in combination with PBMCs ( Fig.18A ). Injection 2.5×10 6 Administration of 2.5×10 PBMC resulted in strong inhibition of leukemia progression. 6 PBMC and single donor CD4 IL-10 (2.5×10 6 The combination of cells produced stronger inhibition of leukemia progression ( Fig.18B ). Apply 2.5×10 6 PBMC and 2.5×10 6 Multiple donor CD4 IL-10 The combination of cells has single donor CD4 IL-10 (2.5×106) cells equivalent to leukemia synergistic inhibition ( Fig.18C ). These data suggest that multiple donor CD4IL-10 The cells did not interfere with the protective GvL effect of PBMCs but synergized with PBMCs to mediate a strong GvL effect.

[0375] 6.7.6. Example 5: Using multiple donor CD4 IL-10 Cell therapy for chronic inflammatory and autoimmune diseases

[0376] Many chronic inflammatory and autoimmune diseases involve activation of the NLPR3 inflammasome. The NLPR3 inflammasome can be activated by “danger signals,” which leads to caspase 1-mediated production of proinflammatory cytokines IL-1β and IL-18 by monocytes / macrophages. A series of in vitro experiments were performed to investigate the effects of multiple donor CD4 IL-10 Effects of cellular factors on the NLPR3 inflammasome and IL-1β / IL-18 production in human monocytes.

[0377] First, human PBMCs were isolated from peripheral blood by standard density centrifugation over Ficoll / Paque (Sigma-Aldrich). Monocytes were isolated from human PBMCs by negative selection using the Monocyte Isolation Kit II (Miltenyi) according to the manufacturer's instructions. Negative selection is preferred because positive selection or adhesion can lead to undesired cell activation. 2×10 5 or 1×10 5 Multiple donor CD4 IL-10 The isolated mononuclear cells were cultured at 5 × 10 cells / 200 μl. 4 Cells / 200 μl were plated in 96-well microtiter plates in toxin-free medium containing 3% human AB serum.

[0378] Table 1 summarizes the treatment conditions applied to nine groups of monocytes, each group consisting of six wells of cells.

[0379]

[0380] *Z-YVADfmk is a specific inhibitor of caspase 1. 20uM Z-YVADfmk (Biovision, Enzo Life Sciences, or Axxora Life Sciences) dissolved in DMSO was used as indicated. MCC950 is an NLRP3 inhibitor. 10uM MCC950 (Invivogen) was used as indicated.

[0381] **LPS (Sigma-Aldrich) 100 ng / mL plus nigericin (Nig, Invivogen) 10 uM were added during the last 30 min of LPS incubation.

[0382] ***Anti-IL-10R antibody (Biolegend) 30ug / mL.

[0383] **** By adding CD4 IL-10 or CD4 GFP cells at 1×10 6 / mL culture for 3 days and collect the supernatant to obtain CD4 IL-10 The supernatants of CD4 and GFP cultures were collected. IL-10 production levels were measured by IL-10-specific ELISA.

[0384] After treatment as outlined in Table 1, supernatants were collected from 6 wells of each group and IL-1β / IL-18 production was measured by ELISA specific for mature IL-1β or IL-18 (Biolegend). Cells collected from 6 wells of selected groups were analyzed by Western blot to determine the level of activated caspase 1.

[0385] Experimental data show that multiple donor CD4 IL-10 cells downregulated IL-1β and IL-18 produced by activated monocytes. IL-10 cells downregulate mature caspase-1 production in activated monocytes. In addition, multiple donor CD4 IL-10 and CD4 IL-10 The produced IL-10 downregulates the inflammasome.

[0386] Similar experiments were performed using human macrophages or dendritic cells instead of monocytes. IL-10 The cells further downregulated the production of IL-1β, IL-18, and mature caspase-1 by activated macrophages and dendritic cells.

[0387] These data suggest that multiple donor CD4 IL-10 The cells can be used to treat diseases or conditions involving overactivity of the NLRP3 inflammasome. IL-10 The cells can be used to treat chronic inflammatory and autoimmune diseases. The NLPR3 inflammasome can be activated by exogenous or endogenous "danger signals" such as pathogen-associated molecular patterns (PAMPs), silica, asbestos, danger-associated molecular patterns (DAMPs) such as products of damaged mitochondria, necrotic and stressed cells, and uremic acid crystals.

[0388] 6.7.7. Example 6: Multiple Donor CD4 IL-10 Cell supernatants inhibit NLPR3 inflammasome activation and IL-1β and IL-18 production in human monocytes

[0389] CD14 was isolated from PBMC using a pan-monocyte isolation kit (Miltenyi Biotec, Bergisch Gladbach, Germany). + Monocytes were plated at 2 × 10 per well. 5 100 μL / well were plated in 96 flat-bottom microtiter wells and cultured in the presence of LPS. Z-YVADfmk (20 microMol), MMC950 (10 microMol), IL-10 (10 ng / mL) or various concentrations of single or pooled donor CD4 IL-10 The cells were further cultured in the presence of the cell supernatant.

[0390] CD4 T cells from single or pooled donors activated for 72 hours with a combination of anti-CD3 and anti-CD28 antibodies IL-10 Cells were obtained from supernatants as previously described (Andolfi et al. 2012, Mol. Therapy Vol. 20, 1778-1790, Locafaro et al. Mol Ther 2017, 25, 2254). Fig.19C and 19D ), monocytes were incubated with a combination of LPS and the NLPR3 inflammasome activator nigericin ("NIG"), with NIG added during the last 30 min of LPS activation.

[0391] The NLPR3 inflammasome is activated by LPS, leading to the production of mature caspase 1 and biologically active forms of IL-1β and IL- 18. Monocytes plated in the absence of LPS activation did not produce detectable levels of IL-10 during incubation (not shown).

[0392] At concentrations of 50%, 25% and 12.5% ​​( Fig.19A and Fig.19B ) Add CD4 IL-10 The supernatant of the cells (containing 1769pg IL-10 / mL) inhibited the production of IL-1β by LPS-activated monocytes from donors #1 and #2, respectively, in a dose-dependent manner. The supernatant of GFP-transduced CD4+ cells was used as a control. Complete inhibition of IL-1β production was observed at concentrations of 50% and 25%, while the supernatant of GFP-transduced control CD4+ cells at a concentration of 50% was ineffective.

[0393] Different concentrations of CD4 IL -10T cell supernatants (50%, 25%, or 12.5%), Z-YVADfmk, or MCC950. CD4+ T cells from single donors (BC-E) or pooled donors IL-10 The supernatant of the cells contained 5295 or 3532 pg IL-10 / mL, respectively. IL-10 The supernatant of the cells also very effectively inhibited LPS-induced IL-1β production enhanced by the NLPR3 inflammasome activator nigericin ( Fig.19C and Fig.19D ).

[0394] The data showed that a concentration of 50% CD4 IL-10 The cell supernatant was 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 the supernatant containing IL-10 inhibited NLPR3 inflammasome activation and mature caspase 1 production, thereby strongly inhibiting the production of the proinflammatory cytokine IL-1β ( Figures 19A-19D ).

[0395] In a second series of experiments, supernatants from a single donor (BC-E) and pooled CD4 IL-10 Similar results were obtained for 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 These supernatants contained 5295 and 3532 pg IL-10 / mL, respectively, and inhibited LPS-induced IL-1β production by monocytes from donor #3 in a dose-dependent manner ( Fig.19E ). The supernatant at 50% concentration was as effective as Z-YVADfmk and MCC950. Pooled CD4 IL-10 The inhibitory effect of the cell supernatant was completely neutralized by anti-IL-10 receptor antibody. Similarly, the pooled supernatant from 3 different donors contained 2589 pg IL-10 / mL and dose-dependently inhibited the production of IL-1β by monocytes from donor #4 ( Fig.19F The inhibitory effect of the supernatant was completely neutralized by IL-10 receptor antibody, indicating that NLPR3 activation is mediated by IL-10. IL-10IL-10 produced by T cells strongly inhibits the production of the proinflammatory cytokine IL-1β. As expected, anti-IL-10 receptor antibodies had no effect on the inhibition of IL-1β production mediated by Z-VADfmk and MCC950 ( Fig.19E ).

[0396] CD4 was further tested on monocytes from donor #4 activated by LPS and nigericin IL-10 Different concentrations of single donor (BC-V) or multiple donor (three donors; BC-T / U / V) CD4 IL-10 Cell supernatant, ZYVADfmk or MCC950. Figure 19G The data presented in show that pooled supernatants from 3 different donors (BCT-UV) down-regulated IL-18 production induced by the combination of LPS and nigericin.

[0397] Together, these data suggest that single-donor and multi-donor CD4 IL-10 IL-10 produced by the cells strongly downregulates the NLPR3 inflammasome, leading to a strong suppression of the proinflammatory cytokines IL-1β and IL-18.

[0398] 6.7.8. Example 7: Single and Multiple Donor CD4 IL-10 Cells inhibit xeno GvHD and myeloid tumor growth in vivo

[0399] Single-donor (BC-T, BC-V, and BC-E) or multi-donor (BC-V / T / E) CD4 IL-10 Functional properties and quality of cells. Single-donor and multi-donor CD4 IL-10 The cells all produced high levels of IL-10, varying levels of IFN-γ, very low levels of IL-4, and undetectable levels of IL-2 (the latter not shown), reflecting the characteristic cytokine production profile of Tr1 cells ( Fig.21 ).

[0400] In addition, single-donor (BC-T, BC-V, and BC-E) or multi-donor (BC-V / T / E) CD4 IL-10 CD4 + and CD8 + The inhibitory capacity of T cell proliferation. PBMCs were labeled with eFLuor670 (Invitrogen). Labeled PBMCs (1×10 5Single and multiple donor CD4 IL-10 Cells were added to 96-well round-bottom plates at a 1:1 ratio in a final volume of 0.2 mL. After 4 days of co-culture, their inhibitory effects on the proliferation of eFluor670-labeled responder cells were determined by eFluor670 dilution using flow cytometry as described in (Locafaro et al. Mol Ther 2017, 25, 2254). Fig. 22 Flow cytometry results are provided. Single and multiple donor CD4 IL-10 The cells strongly inhibited the proliferation of allogeneic CD4+ and CD8+ T cells in vitro, with an inhibition rate of more than 80% ( Fig. 22 ).

[0401] Further analysis of CD4 IL-10 Cytotoxicity of cells against myeloid leukemia cells (ALL-CM) and erythroid leukemia cell lines (K562). Single donor (BC-E and BC-V) or multiple donor (BC-V / T / E) CD4 IL-10 The cells were co-cultured with ALL-CM or K562 cells at a 1:1 ratio. The cells were harvested 3 days later and the viable CD45 低 CD3-target cells were isolated and analyzed by FACS as described (Locafaro et al. Mol Ther 2017, 25, 2254). Single and multiple donor CD4 IL-10 The cells also have potent direct cytotoxic effects against ALL-CM myeloid neoplasms, but they are unable to kill sensitive K562 cells because K562 cells lack the expression of class I MHC required for their cytotoxic activity ( Fig.23 Single donor (BC-E and BC-V) or multiple donor (BC-V / T / E) CD4 IL-10 The cells had comparable cytotoxic activity against both target cell lines (ALL-CM and K562).

[0402] We also used a humanized xeno GvHD disease model (intravenous injection of ALL-CM cells (2.5 × 10 6 Single donor (BC-E) and multiple donor (BC-V / T / E) CD4 IL-10 Cytotoxic effects on cells. Fig. 20 As shown, their effects on GvHD induced by human PBMCs from allogeneic donors and their effects on the growth of acute myeloid leukemia in the cell line ALL CM in a therapeutic setting were tested.

[0403] Female NOD scid gamma (NSG) mice aged eight to ten weeks were obtained from Charles-River Italia (Calco, Italy). The experimental protocol was approved by the Internal Committee for Animal Research of Ospedale San Raffaele (Institutional Animal Care and Use Committee (IACUC)). On day 0, mice received whole-body irradiation from a linear accelerator. ALL-CM cells (2.5 × 10 6 On day 0, mice in different groups were injected with no injection, allogeneic PBMC (2.5×10 6 ), single donor (BC-E, 2.5×10 6 or multiple donor CD4 IL-10 Cells (from 3 different donors (BC-V / T / E, 2.5×10 6 On day 3, allogeneic PBMC (2.5×10 6 or multiple donor CD4 IL-10 Cells (2.5×10 6 All cells were administered intravenously in a volume of 250 μl of Iscove's modified Dulbecco's medium. Mice were monitored 3-4 times per week.

[0404] NSG mice were divided into five cohorts of 5 mice each, and each group received the following treatments on day 0: (i) no treatment as control; (ii) allogeneic PBMCs; (iii) allogeneic PBMCs and multiple donor CD4 IL-10 cells (BC-V / T / E); (iv) allogeneic PBMC and single donor CD4 IL-10 cells (BC-E); or (v) administration of multiple donor CD4 IL-10 (BC-V / T / E) cells. Myeloid leukemia progression was measured as previously described (Locafaro et al. Mol Ther 2017, 25, 2254).

[0405] Administration of ALL-CM cells to NSG mice resulted in rapid expansion of these cells, and all mice died or had to be sacrificed by day 20. As expected, injection of PBMCs prevented leukemia progression. Single and multiple donor CD4 IL-10 Administration of the cells in combination with allogeneic PBMCs did not interfere with the anti-myeloid leukemia effects of PBMCs.

[0406] Three days after administration of ALL-CM cells (when these cells have already expanded to a large extent), multiple donor CD4 IL-10(BC-V / T / E) cells led to inhibition of tumor growth. These results suggest that multiple donor CD4 IL-10 The cells have direct anti-myeloid leukemia therapeutic effects in vivo ( Fig.24 ).

[0407] However, despite their beneficial anti-myeloid leukemia effects, PBMCs induced a very severe form of xeno-GvHD, and all mice died by day 24 ( Fig.25 In this study, single-donor (BC-E) and multi-donor (BC-V / T / E) CD4 IL-10 The ability of cells to inhibit PBMC-induced xeno-GvHD after administration into NSG mice. On day 0, NSG mice were injected with ALL-CM cells (2.5×10 6 The mice were divided into five groups, and each group received the following treatments: (i) no treatment as control; (ii) allogeneic PBMCs; (iii) allogeneic PBMCs and multi-donor (BC-V / T / E) CD4 IL-10 cells; (iv) injection of allogeneic PBMC and single donor CD4 IL-10 Cells (BC-E) or multiple donors (BC-V / T / E) CD4 IL -10 Cells. In animals, xeno-GvHD was measured by survival and weight loss. In addition, the hunchback, coat condition, and skin integrity were monitored as described by (Bondanza et al, Blood, 2006, 107, 1828). Mice were sacrificed for ethical reasons if the weight loss was greater than 20%. Fig.25 The results show that ALL-CM cells (2.5×10 6 ) and subsequently with PBMC (with or without single-donor or multiple-donor CD4 IL-10 % of NSG mice without GvHD every day after treatment with (cells).

[0408] The results showed that multiple donor CD4 IL-10 The cells did not induce xeno-GvHD and downregulated xeno-GvHD induced by allogeneic PBMCs. Taken together, these results suggest that multi-donor CD4 IL-10 The cells downregulated severe xeno-GvHD, had a direct anti-myeloid leukemia effect in a therapeutic setting, and did not interfere with the protective anti-myeloid leukemia effects of PBMCs.

[0409] 6.7.9. Example 8: Adoptive transfer of multi-donor CD4IL-10 cells from four different donors

[0410] Testing of multiple donor CD4 derived from four different donorsIL-10 Adoptive transfer of cells inhibited the transfer capacity of PBMC-induced xeno-GvHD.

[0411] In these experiments, single donor CD4 IL-10 cells (donor C; batch C) and multi-donor CD4 IL-10 In this model, NSG mice were sublethally irradiated on day 0 and injected (slow intravenous bolus) on day 3 with (i) 2.5E+06 allogeneic PBMCs, (ii) 2.5E+06 allogeneic PBMCs combined with 2.5E+06 single donor CD4 IL-10 Combination of cells (Batch C), (iii) 2.5E+06 allogeneic PBMCs and 2.5E+06 cells multi-donor CD4 IL-10 cells (batch CEFH), or (iv) single injection of 2.5E+06 cells multi-donor CD4 IL-10 Cells (batch CEFH). Xeno-GvHD was determined using a composite score of weight loss, hair appearance, skin appearance, hunchback, and mobility (see Bondanza A, et al. Blood 2006; 107: 1828–36). Fig.26 As shown, at the end of the study only multi-donor CD4 IL-10 Only mice that received the cells were 100% free of xeno-GvHD.

[0412] In summary, these data suggest that multidonor CD4 IL-10 Adoptive transfer of cells suppressed PBMC-induced xeno-GvHD and did not induce xeno GvHD.

[0413] 6.7.10. Example 9: Generation of IL-10 variants

[0414] Variants of human IL-10 are generated by introducing amino acid modifications (e.g., substitutions, insertions, deletions) based on IL-10 sequences from other species. The modification sites are identified by Fig.27A Amino acid positions that differ between species are identified from the alignment and modified by introducing amino acid substitutions, insertions or deletions.

[0415] Fig.27BTwo examples of human IL-10 variants are provided. Potential huIL-10 hybrid #1 (SEQ ID NO: 19) was 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) was 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. Fig.27C An alignment of human IL-10 (SEQ ID NO: 1) and IL-10EBVB9 (SEQ ID NO: 18) is shown, wherein "*" indicates one or more amino acid positions that are substituted in IL-10 hybrid #1, and "#" indicates the preferred I105 to A105 amino acid substitution in IL-10 hybrid #2.

[0416] As described above, variants of human IL-10 were cloned into expression vectors and tested for expression and function of the variant proteins. IL-10 Cells. CD4 IL-10 The efficiency of the cells was tested as provided herein.

[0417] 6.7.11. Experimental methods and materials

[0418] Cell preparation and cell lines. Peripheral blood mononuclear cells (PBMCs) were prepared by Ficoll-Hypaque gradient centrifugation. CD4 T cell isolation kit (Miltenyi Biotec, Bergisch Gladbach, Germany) was used to purify CD4 T cells. + T cells, the purity was >95%. Mature dendritic cells (DC) were isolated from peripheral blood CD14 + Monocyte generation using CD14 +Microbeads (Miltenyi Biotech, Germany) were used for positive selection according to the manufacturer's instructions and cultured for 5 days at 37°C in RPMI 1640 (Lonza, Italy) supplemented with 10% fetal bovine serum (FBS; Lonza, Italy), 100 U / mL penicillin / streptomycin (Lonza, Italy), 2 mM L-glutamine (Lonza, Italy) in the presence of 10 ng / mL recombinant human (rh) IL-4 (R&D Systems, Minneapolis MN, USA) and 100 ng / mL rhGM-CSF (Genzyme, Seattle, WA, USA) and matured for an additional two days with 1 mg / mL lipopolysaccharide (LPS, Sigma, CA, USA).

[0419] Plasmid construction. The coding sequence of human IL-10 was excised from pH15C (ATCC n°68192) and the 549 bp fragment was cloned into the multiple cloning site of pBluKSM (Invitrogen) to obtain pBluKSM-hIL-10. hIL-10 was excised from pBluKSM-hIL-10 to obtain a 555 bp fragment and ligated 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 element of the reverse CMV promoter) enables the co-expression of the two transgenes (Locafaro et al. Mol Ther. 2017; 25(10): 2254-2269). The sequence of pLVIL-10 was verified by pyrosequencing (Primm).

[0420] Vector production and titration. VSV-G-pseudotyped third-generation bidirectional lentiviral vectors were produced by transient co-transfection of four plasmids into 293T cells with Ca3PO4 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 ranged from 5 × 10 8 Up to 6×10 9 transduction units / mL, and the infectivity range is 5×10 4 Up to 10 5 transducing units / ng p24.

[0421] CD4 IL-10 Generation of cell lines. Polyclonal CD4 transduced cells were obtained as described previously (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 treated with LV-IL-10 / ΔNGFR (CD4 IL-10 ) with a multiplicity of infection (MOI) of 20. On day 11, CD271 + Microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany) for CD4 + ΔNGFR + Cells were bead selected and expanded in X-VIVO15 medium containing 5% human serum (BioWhittaker-Lonza, Washington), 100 U / mL penicillin-streptomycin (BioWhittaker), and 50 U / mL rhIL-2 (PROLEUKIN, Novartis, Italy). On days 7 and 10, the medium was replaced with fresh medium supplemented with 50 U / mL rhIL-2. On day 14, cells were collected, washed, and restimulated with allogeneic feeder mix as previously described (Locafaro et al. Mol Ther 2017, 25, 2254). After 14 days, cells were collected and frozen. Thawed CD4 IL-10 Cells were restimulated and functionally characterized in vitro after the second and third restimulations and expansions and used for in vivo experiments.

[0422] Vector copy number analysis. Cells were cultured for 11 days after transduction to remove non-integrated vector forms. Genomic DNA was isolated using the QIAamp DNA Blood Mini Kit (QIAGEN, 51106) according to the manufacturer's instructions. Vector integration was quantified by the QX200 Droplet Digital PCR System (Bio-Rad) according to the manufacturer's instructions.

[0423] Cytokine Assays. To measure cytokine production, single-donor and multi-donor CD4IL-10 Cells were either 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 culture medium (96-well round-bottom plates, 2 × 10 5 After 48 hours of culture, the supernatants were harvested and the 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).

[0424] Flow cytometric analysis. For the expression of granzyme B (clone MHGB04, Invitrogen, USA), after surface staining with CD4, BD Cytofix / Cytoperm was used according to the manufacturer's instructions. TM Kit (Cat. No. 554714, Biolegend, USA) for CD4 IL-10 Cells were fixed, permeabilized and stained. Stained cells were washed twice with PBS supplemented with 1% FBS and analyzed with BD LSRFortessa using FlowJo 10 software.

[0425] Killing assay. Analysis of single-donor and multi-donor CD4 IL-10 In brief, non-myeloid leukemia and myeloid leukemia cell lines K562 and ALL-CM were used as target cells, respectively, and the cells were incubated at a 1:1 ratio (10 5 target cells and 10 5 CD4 IL-10 At the end of co-culture, cells were harvested and K562 and ALL-CM cells were analyzed based on CD45+, CD3- expression and counted by FACS.

[0426] Suppression assay. To measure single-donor and multi-donor CD4 IL-10 The inhibitory capacity of cells was measured using cell proliferation dye according to the manufacturer's instructions. Allogeneic PBMCs were labeled with 670 (Invitrogen, CA, USA). In the presence of anti-CD3 mAb (50 ng / mL), GM-CSF and IL-4 were used to label the cells with CD14 +Allogeneic mature dendritic cells (DC) of cells activate labeled cells. Peripheral blood CD14 monocytes were positively selected using CD14 microbeads (Miltenyi Biotec) according to the manufacturer's instructions. In the presence of 10ng / mL recombinant human (rh) IL-4 (R&D Systems) and 100ng / mL recombinant human granulocyte-macrophage colony stimulating factor (rhGM-CSF) (Genzyme), cells were cultured in RPMI 1640 (Lonza) supplemented with 10% fetal bovine serum (FBS), 100U / mL penicillin / streptomycin (Lonza), 2mM L-glutamine (Lonza) at 37°C for 5 days. In order to generate mature dendritic cells (mDC), cells were stimulated for another 2 days with 1mg / mL lipopolysaccharide (LPS; Sigma) on the 5th day. On the 7th day, DCs were collected, phenotyped, and used to stimulate T cells. The purity and maturation status of DCs were examined by flow cytometry to determine the expression of CD1a, CD14, CD86, CD83, and HLA-DR.

[0427] The labeled cells were plated in a 96-well round plate in a final volume of 200 μL and incubated for 3 days as follows: (i) 5×10 labeled PBMCs alone 4 cells / well; (ii) labeled PBMC 5×10 4 cells / well + mature DC 5×10 3 cells / well + anti-CD3 mAb (50 ng / mL); (iii) labeled PBMC 5×10 4 cells / well + single or multiple donor CD4 IL-10 Cells 5×10 4 cells / well + mature DC 5×10 3 cells / well+anti-CD3 mAb (50 ng / mL).

[0428] After 3 days of culture, cells were harvested and transferred to 96-well V-bottom plates for immunofluorescence staining. CD4+eFluor670+ and CD8+eFluor670+ live cells were gated and cells were analyzed by FACS. The percentage of inhibition was calculated by measuring the dilution of the eFluor670 marker as described previously (Locafaro et al. Mol Ther 2017, 25, 2254).

[0429] Graft-versus-host disease model: 6 / 8-week-old female NSG mice were used in all experiments. On day 0, mice received a single dose of whole-body irradiation of 175-200 cGy from a linear accelerator, depending on their body weight. In some experiments, mice received a single dose of 350 cGy. Mice were injected intravenously with PBMC cells (5×10 6 or 2.5×10 6 or CD4 IL-10 Cells (single donor or multiple donors—pooled from three donors—5×10 6 or 2.5×10 6 ) or PBMC (5×10 6 or 2.5×10 6 ) and CD4 IL-10 Cells (5×10 6 or 2.5×10 6 Survival, weight loss, activity, fur, skin, and kyphosis were monitored at least 3 times per week as described previously (Bondanza et al. Blood. 2006; 107(5): 1828-1836). For ethical reasons, mice were euthanized when they lost 20% of their body weight.

[0430] Alternatively, on day 0, mice received whole body irradiation as described above. On day 3, mice were injected with CD4 + T cells (2.5×10 6 single donor and multiple donor (pool of three donors) CD4 IL-10 Cells (2.5×10 6 ), or CD4 + T cells (2.5×10 6 single donor and multiple donor (pool of three donors) CD4 IL-10 Cells (2.5×10 6 GvHD induction was monitored as described above.

[0431] 7. Incorporation by Reference

[0432] All publications, patents, patent applications, and other documents cited in this application are hereby incorporated by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application, or other document was individually indicated to be incorporated by reference for all purposes.

[0433] 8. Equivalent solutions

[0434] Although various specific embodiments have been illustrated and described, the above description is not restrictive. It should be understood that various changes can be made without departing from the spirit and scope of the present invention. After reading this specification, many changes will be apparent to those skilled in the art.

[0435] 9. Sequence

[0436] SEQ ID NO: 1 (human IL-10 amino acid sequence - protein sequence: Ref P22301)

[0437]

[0438] SEQ ID NO: 2 (human IL-10 exemplary nucleic acid sequence)

[0439]

[0440] SEQ ID NO:3 (ΔNGFR amino acid sequence)

[0441]

[0442] SEQ ID NO:4 (ΔNGFR exemplary nucleic acid sequence)

[0443]

[0444] SEQ ID NO:5 (nucleotide sequence of pLVIL-10)

[0445]

[0446]

[0447]

[0448]

[0449] SEQ ID NO:6 (viral interleukin-10 homolog, also known as interleukin-10BCRF1, also known as IL-10H_EBVB9) protein sequence: Ref: P03180

[0450]

[0451] SEQ ID NO:7 (cDNA sequence of viral interleukin-10 homolog)

[0452] Nucleotide sequence (cDNA): Ref: NC_007605.1

[0453]

[0454] SEQ ID NO: 8 (Exemplary human IL-10 variant with amino acid substitutions based on viral IL-10)

[0455]

[0456] SEQ ID NO: 9 (Exemplary human IL-10 variant with amino acid substitutions based on viral IL-10)

[0457]

[0458] SEQ ID NO: 10 (Mus musculus; "mouse")

[0459]

[0460] SEQ ID NO: 11 (Rattus norvegicus; "rat")

[0461]

[0462] SEQ ID NO: 12 (Rhesus monkey; "MACMU")

[0463]

[0464] SEQ ID NO: 13 (Gorilla; "Gorilla")

[0465]

[0466] SEQ ID NO: 14 (Cynomolgus monkey; "CYNO")

[0467]

[0468] SEQ ID NO: 15 (Papio fascicularis; "Olive Baboon")

[0469]

[0470] SEQ ID NO: 16 (Bonobo; "Bonobo")

[0471]

[0472] SEQ ID NO: 17 (Chimpanzee; "CHIMP")

[0473]

[0474] SEQ ID NO:18 (EBVB9)

[0475]

[0476] SEQ ID NO: 19 huIL-10 hybrid #1

[0477]

[0478] SEQ ID NO:20 huIL-10 hybrid #2

[0479]

Claims

1. A CD4 + T cells (multiple donor CD4 IL-10 A cell population that has 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.

2. The CD4 according to claim 1 + T cell population, in which the CD4 + The T cells are obtained from two, three, four, five, six, seven, eight, nine or ten different T cell donors.

3. The CD4 according to claim 1 or claim 2 + T cell population, wherein the CD4 + The T cells have a total of six, seven, eight, nine, ten, eleven, twelve or more different HLA haplotypes.

4. The CD4 according to any one of the preceding claims. + T cell population, wherein all CD4 + The T cells are at least 1 / 10, 2 / 10, 3 / 10, 4 / 10, 5 / 10, 6 / 10, 7 / 10, 8 / 10 or 9 / 10 matched with each other at the HLA-A, HLA-B, HLA-C, HLA-DRBl and HLA-DQBl loci.

5. The CD4 according to any one of the preceding claims. + T cell population, wherein all CD4 + The T cells are at least 1 / 8, 2 / 8, 3 / 8, 4 / 8, 5 / 8, 6 / 8, 7 / 8, or 8 / 8 matched to each other at the HLA-A, HLA-B, HLA-C, and HLA-DRBl loci.

6. The CD4 according to any one of the preceding claims. + T cell population, wherein all CD4 + The T cells have a 2 / 2 match with each other at the HLA-A locus.

7. The CD4 according to any one of the preceding claims. + T cell population, wherein all CD4 + The T cells have a 2 / 2 match with each other at the HLA-B locus.

8. The CD4 according to any one of the preceding claims. + T cell population, wherein all CD4 + The T cells have a 2 / 2 match with each other at the HLA-C locus.

9. The CD4 according to any one of the preceding claims. + T cell population, wherein all CD4 + The T cells have at least a 3 / 4 or 4 / 4 match with each other at the HLA-DRB1 and HLA-DQB1 loci.

10. The CD4 according to any one of claims 1 to 3 + T cell population, wherein all CD4 + The T cells are less than 5 / 10, 6 / 10, 7 / 10, 8 / 10, or 9 / 10 matched with each other at the HLA-A, HLA-B, HLA-C, HLA-DRBl, and HLA-DQBl loci.

11. The CD4 according to claim 10 + T cell population, wherein all CD4 + The T cells 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-DRBl loci.

12. The CD4 according to any one of claims 10-11. + T cell population, wherein all CD4 + T cells have less than a 2 / 2 match with each other at the HLA-A locus.

13. The CD4 according to any one of claims 10-12. + T cell population, wherein all CD4 + T cells have less than a 2 / 2 match with each other at the HLA-B locus.

14. The CD4 according to any one of claims 10 to 13. + T cell population, wherein all CD4 + T cells have less than a 2 / 2 match with each other at the HLA-C locus.

15. The CD4 according to any one of claims 10 to 14. + T cell population, wherein all CD4 + The T cells have less than 2 / 4, 3 / 4, or 4 / 4 matches with each other at the HLA-DRB1 and HLA-DQB1 loci.

16. The CD4 according to any one of the preceding claims. + T cell population, wherein all CD4 + T cells have the A*02 or A*24 allele.

17. The CD4 according to any one of the preceding claims + T cell population, none of which has a CD4 + T cells are immortalized.

18. The CD4 according to any one of the preceding claims + T cell population, wherein the exogenous polynucleotide comprises an IL-10 encoding polynucleotide segment operably linked to an expression control element.

19. The CD4 according to any one of the preceding claims + T cell population, wherein the IL-10 is human IL-10.

20. The CD4 according to any one of claims 1-19. + The invention relates to a population of T cells, wherein the IL-10 is a viral IL-10.

21. The CD4 according to any one of the preceding claims + T cell population, wherein the IL-10 encoding polynucleotide segment encodes a protein having a sequence of SEQ ID NO:

1.

22. The CD4 according to claim 21 + T cell population, wherein the IL-10 encoding polynucleotide segment has the sequence of SEQ ID NO:

2.

23. The CD4 according to claim 20 + T cell population, wherein the IL-10 encoding polynucleotide segment encodes a protein having a sequence of SEQ ID NO:

6.

24. The CD4 according to claim 23 + T cell population, wherein the IL-10 encoding polynucleotide segment has the sequence of SEQ ID NO:

7.

25. The CD4 according to any one of claims 18-24. + A population of T cells, wherein the expression control element drives constitutive expression of the encoded IL-10.

26. The CD4 according to any one of the preceding claims + T cell population, wherein the exogenous polynucleotide further comprises a polynucleotide segment encoding a selection marker.

27. The CD4 according to claim 26 + A population of T cells, wherein the selection marker is ΔNGFR.

28. The CD4 according to claim 27 + The T cell population, wherein the ΔNGFR has the sequence of SEQ ID NO:

3.

29. The CD4 according to claim 27 + T cell population, wherein the polynucleotide segment comprises the sequence of SEQ ID NO:

4.

30. The CD4 according to claim 26 + The invention relates to a population of T cells, wherein the selection marker is a truncated form of an EGFR polypeptide.

31. The CD4 according to any one of the preceding claims + T cell population, wherein the exogenous polynucleotide has the sequence of SEQ ID NO:

5.

32. The CD4 according to any one of claims 1-31. + A population of T cells, wherein the exogenous polynucleotide is integrated into the nuclear genome of the T cells.

33. The CD4 according to any one of claims 1-31. + A population of T cells, wherein the exogenous polynucleotide is not integrated into the T cell nuclear genome.

34. The CD4 according to claim 32 or 33. + T cell population, wherein the exogenous polynucleotide further comprises a lentiviral vector sequence.

35. The CD4+T cell population according to any one of claims 1-34, wherein the exogenous polynucleotide is not integrated into the T cell nuclear genome.

36. The CD4 according to any one of the preceding claims. + A population of T cells wherein at least 70% of the population is CD4 + T cells express IL-10.

37. The CD4 according to claim 36 + A population of T cells wherein at least 90% of the population is CD4 + T cells express IL-10.

38. The CD4 according to claim 37 + A population of T cells wherein at least 95% or 98% of the population is CD4 + T cells express IL-10.

39. The CD4 according to any one of the preceding claims + T cell populations, in which genetically modified CD4 + T cells constitutively express at least 100 pg IL-10 / 10 6 CD4 + T cells / mL culture medium.

40. The CD4 according to claim 39 + T cell population, wherein 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 IL-10 / 10 6 CD4 + T cells / mL.

41. The CD4 according to any one of the preceding claims + T cell populations, in which genetically modified CD4 + T cells expressed at least 1 ng IL-10 / 10 after activation with anti-CD3 and anti-CD28 antibodies 6 CD4 + T cells / mL.

42. The CD4 according to claim 41 + T cell population, wherein the genetically modified CD4 + T cells expressing at least 2ng, 5ng, 10ng, 100ng, 200ng or 500ng IL-10 / 10 after activation with anti-CD3 and anti-CD28 antibodies 6 CD4 + T cells / mL.

43. The CD4 according to any one of the preceding claims + T cell population, wherein the genetically modified CD4 + T cells were more expressed than unmodified CD4 + T cells expressed IL-10 at least 5-fold higher levels.

44. The CD4 according to claim 43 + T cell population, wherein the genetically modified CD4 + T cells were more expressed than unmodified CD4 + T cells expressed IL-10 at least 10-fold higher levels.

45. The CD4 according to any one of the preceding claims. + A population of T cells wherein at least 70% of the population is CD4 + The T cells express the selectable marker from the exogenous polynucleotide.

46. ​​The CD4 according to claim 45 + A population of T cells wherein at least 90% of the population is CD4 + The T cells express the selectable marker from the exogenous polynucleotide.

47. The CD4 according to claim 46 + A population of T cells wherein at least 95% or 98% of the population is CD4 + The T cells express the selectable marker from the exogenous polynucleotide.

48. The CD4 according to any one of the preceding claims + T cell populations, in which genetically modified CD4 + T cells express CD49b.

49. The CD4 according to any one of the preceding claims + T cell populations, in which genetically modified CD4 + T cells express LAG-3.

50. The CD4 according to any one of the preceding claims + T cell populations, in which genetically modified CD4 + T cells express TGF-β.

51. The CD4 according to any one of the preceding claims + T cell populations, in which genetically modified CD4 + T cells express IFN-γ.

52. The CD4 according to any one of the preceding claims + T cell populations, in which genetically modified CD4 + T cells express GzB.

53. The CD4 according to any one of the preceding claims + T cell populations, in which genetically modified CD4 + T cells express perforin.

54. The CD4 according to any one of the preceding claims + T cell populations, in which genetically modified CD4 + T cells express CD18.

55. The CD4 according to any one of the preceding claims + T cell populations, in which genetically modified CD4 + T cells express CD2.

56. The CD4 according to any one of the preceding claims + T cell populations, in which genetically modified CD4 + T cells express CD226.

57. The CD4 according to any one of the preceding claims + T cell populations, in which genetically modified CD4 + T cells express IL-22.

58. The CD4 according to any one of the preceding claims + T cell population, in which the CD4 + T cells did not lose reactivity in the presence of peripheral blood mononuclear cells (PBMCs) from the host.

59. The CD4 according to any one of the preceding claims + T cell population, in which the CD4 + T cells do not lose responsiveness in the presence of recombinant IL-10 protein, wherein the recombinant IL-10 protein is not expressed by the CD4 + T cell expression.

60. The CD4 according to any one of the preceding claims + T cell population, in which the CD4 + T cells did not lose their responsiveness in the presence of DC10 cells from the host.

61. The CD4 of any one of claims 1-60. + T cell population, in which the CD4 + T cells are in frozen suspension.

62. The CD4 of any one of claims 1-60. + T cell population, in which the CD4 + The T cells are in liquid suspension.

63. The CD4 according to claim 62 + A population of T cells, wherein the liquid suspension has been previously frozen.

64. A pharmaceutical composition comprising: (i) CD4 as claimed in any one of the preceding claims + T cell populations suspended in (ii) a pharmaceutically acceptable carrier.

65. A method for preparing multiple donor CD4 IL-10 A method for cells comprising the steps of: (i) Pooling of primary CD4 T cell lines obtained from at least two different T cell donors + T cells; and (ii) Modification of the incorporated CD4 + T cells, Thus, the multi-donor CD4 IL-10 cell.

66. A method for preparing multi-donor CD4 IL-10 A method for cells comprising the steps of: (i) Obtain primary CD4 T cells from at least two different T cell donors + T cells; and (ii) Modifying the CD4 of each donor by introducing an exogenous polynucleotide encoding IL-10 + T cells, then (iii) Incorporation of genetically modified CD4 + T cells, Thus, the multi-donor CD4 IL-10 cell.

67. The method of claim 65 or claim 66, further comprising the following steps after step (i) and before step (ii), after step (ii), after step (ii) and before step (iii), or after step (iii): The primary CD4 + T cells.

68. The method of claim 67, wherein the primary CD4 + T cells.

69. The method according to any one of claims 65-68, wherein the exogenous polynucleotide is introduced into the primary CD4 + T cells.

70. The method of claim 69, wherein the viral vector is a lentiviral vector.

71. The method of any one of claims 65-70, wherein the exogenous polynucleotide comprises a segment encoding IL-10 having a sequence of SEQ ID NO:

1.

72. The method of any one of claims 65-70, wherein the IL-10 encoding polynucleotide segment has the sequence of SEQ ID NO: 2 or 7.

73. The method of any one of claims 65-72, wherein the exogenous polynucleotide further comprises a segment encoding a selection marker.

74. The method of claim 73, wherein the encoded selectable marker is ΔNGFR.

75. The method of claim 74, wherein the encoded selection marker has a sequence of SEQ ID NO:

3.

76. The method according to any one of claims 73 to 75, further comprising the following steps after step (ii): Isolate genetically modified CD4 + T cells, thereby generating enriched genetically modified CD4 + T cell populations.

77. The method of claim 76, wherein at least 70% of the genetically modified CD4 + T cells express IL-10.

78. The method of claim 77, wherein at least 90%, 95% or 98% of the genetically modified CD4 + T cells express IL-10.

79. The method of any one of claims 76-69, wherein at least 70% of the genetically modified CD4 + The T cells express the selection marker.

80. The method of claim 70, wherein at least 90%, 95%, or 98% of the genetically modified CD4 + The T cells express the selection marker.

81. The method according to any one of claims 76-80, further comprising incubating the enriched genetically modified CD4 + Steps for T cell populations.

82. The method of claim 81, wherein the enriched genetically modified CD4 + Steps for T cell populations.

83. The method of any one of claims 65-82, further comprising freezing the genetically modified CD4 + Next steps for T cells.

84. The method according to any one of claims 65-83, wherein in step (i), the primary CD4 + The T cells are obtained from two, three, four, five, six, seven, eight, nine or ten different T cell donors.

85. The method of any one of claims 84, wherein at least two T cell donors are at least 1 / 10, 2 / 10, 3 / 10, 4 / 10, 5 / 10, 6 / 10, 7 / 10, 8 / 10 or 9 / 10 matched to each other at the HLA-A, HLA-B, HLA-C, HLA-DRB1 and HLA-DQB1 loci.

86. The method of any one of claims 65-85, wherein at least two T cell donors are at least 1 / 8, 2 / 8, 3 / 8, 4 / 8, 5 / 8, 6 / 8, 7 / 8, or 8 / 8 matched to each other at the HLA-A, HLA-B, HLA-C, and HLA-DRBl loci.

87. The method of any one of claims 65-86, wherein at least two T cell donors have a 2 / 2 match with each other at the HLA-A locus.

88. The method of any one of claims 65-87, wherein at least two T cell donors have a 2 / 2 match to each other at the HLA-B locus.

89. The method of any one of claims 65-88, wherein at least two T cell donors have a 2 / 2 match with each other at the HLA-C locus.

90. The method of any one of claims 65-89, wherein at least two T cell donors are at least 3 / 4 or 4 / 4 matched to each other at the HLA-DRB1 and HLA-DQB1 loci.

91. The method of any one of claims 84, wherein at least two T cell donors are less than 5 / 10, 6 / 10, 7 / 10, 8 / 10 or 9 / 10 matched to each other at the HLA-A, HLA-B, HLA-C, HLA-DRB1 and HLA-DQB1 loci.

92. The method of claim 84 or 91, wherein at least two T cell donors are less than 4 / 8, 5 / 8, 6 / 8, 7 / 8, or 8 / 8 matched to each other at the HLA-A, HLA-B, HLA-C, and HLA-DRBl loci.

93. The method of any one of claims 84 and 91-92, wherein at least two T cell donors have less than a 2 / 2 match with each other at the HLA-A locus.

94. The method of any one of claims 84 and 91-93, wherein at least two T cell donors have less than a 2 / 2 match with each other at the HLA-B locus.

95. The method of any one of claims 84 and 91-94, wherein at least two T cell donors have less than a 2 / 2 match with each other at the HLA-C locus.

96. The method of any one of claims 84 and 91-95, wherein at least two T cell donors have less than 3 / 4 or 4 / 4 matches with each other at the HLA-DRB1 and HLA-DQB1 loci.

97. The method of any one of claims 65-96, wherein each of at least two T cell donors has an A*02 or A*24 allele.

98. The method according to any one of claims 65-97, wherein in step (i), the primary CD4 + T cells are obtained from one or more frozen stocks.

99. The method according to any one of claims 65-97, wherein in step (i), the primary CD4 + T cells were obtained from unfrozen peripheral blood mononuclear cells from at least two different T cell donors.

100. The method of claim 99, further comprising isolating CD4 + T cell steps.

101. A method of treating a patient, comprising: Administering the multiple donor CD4 IL-10 Cells, or the pharmaceutical composition of claim 64.

102. The method of claim 101, further comprising thawing multiple donor CD4 IL-10 A prior step of freezing the suspension of cells.

103. The method of claim 101 or 102, wherein the multiple donor CD4 IL-10 The cell or the pharmaceutical composition prevents a pathogenic T cell response in the patient or reduces the severity of a pathogenic T cell response in the patient.

104. The method of claim 101 or 102, wherein the multiple donor CD4 IL-10 The cells or the pharmaceutical composition reduce inflammation or enhance immune tolerance.

105. The method of claim 101 or 102, wherein the multiple donor CD4 IL-10 The cells or the pharmaceutical composition enhance tissue repair.

106. The method of any one of claims 101-105, further comprising the step of administering monocytes to the patient.

107. The method of claim 106, wherein the multiple donor CD4 IL-10 The cells or the pharmaceutical composition and the monocytes are administered simultaneously.

108. The method of claim 106, wherein the multiple donor CD4 IL-10 The monocytes are administered before or after the cells or the pharmaceutical composition.

109. The method according to any one of claims 101-108, further comprising the steps of: In the administration of the multiple donor CD4 IL-10 Before or after administering the cells or pharmaceutical composition, a hematopoietic stem cell (HSC) from a HSC donor is administered to the patient.

110. The method of claim 109, wherein the HSC donor is partially HLA-mismatched to the patient.

111. The method of claim 110, wherein the HSC donor has less than 5 / 10, 6 / 10, 7 / 10, 8 / 10, 9 / 10, or 10 / 10 match with the patient at the HLA-A, HLA-B, HLA-C, HLA-DRBl, and HLA-DQBl loci.

112. The method of claim 110, wherein the HSC donor has less than a 4 / 8, 5 / 8, 6 / 8, 7 / 8, or 8 / 8 match with the patient at the HLA-A, HLA-B, HLA-C, and HLA-DRBl loci.

113. The method of claim 110, wherein the HSC donor has less than a 2 / 2 match with the patient at the HLA-A, HLA-B or HLA-C loci.

114. The method of claim 110, wherein the HSC donor has less than 3 / 4 or 4 / 4 match with the patient at the HLA-DRB1 and HLA-DQB1 loci.

115. The method of any one of claims 101-114, wherein one or more of the T cell donors is HLA-mismatched or partially HLA-mismatched to the patient.

116. The method of claim 115, wherein one or more of the T cell donors has less than a 5 / 10, 6 / 10, 7 / 10, 8 / 10, 9 / 10, or 10 / 10 match with the patient at the HLA-A, HLA-B, HLA-C, HLA-DRB1, and HLA-DQB1 loci.

117. The method of claim 115, wherein one or more of the T cell donors has less than a 4 / 8, 5 / 8, 6 / 8, 7 / 8, or 8 / 8 match with the patient at the HLA-A, HLA-B, HLA-C, and HLA-DRB1 loci.

118. The method of claim 115, wherein one or more of the T cell donors has less than a 2 / 2 match with the patient at the HLA-A, HLA-B, or HLA-C loci.

119. The method of claim 115, wherein one or more of the T cell donors has less than a 2 / 4, 3 / 4, or 4 / 4 match with the patient at the HLA-DRB1 and HLA-DQB1 loci.

120. The method of any one of claims 101-119, wherein one or more of the T cell donors is HLA-mismatched or partially HLA-mismatched to the HSC donor.

121. The method of claim 120, wherein one or more of the T cell donors is less than 5 / 10, 6 / 10, 7 / 10, 8 / 10, 9 / 10, or 10 / 10 matched to the HSC donor at the HLA-A, HLA-B, HLA-C, HLA-DRB1, and HLA-DQB1 loci.

122. The method of claim 120, wherein one or more of the T cell donors has less than a 4 / 8, 5 / 8, 6 / 8, 7 / 8, or 8 / 8 match with the HSC donor at the HLA-A, HLA-B, HLA-C, and HLA-DRB1 loci.

123. The method of claim 120, wherein one or more of the T cell donors has less than a 2 / 2 match with the HSC donor at the HLA-A, HLA-B, or HLA-C loci.

124. The method of claim 120, wherein one or more of the T cell donors has less than 3 / 4 or 4 / 4 matching with the HSC donor at the HLA-DRB1 and HLA-DQB1 loci.

125. The method of any one of claims 101-124, wherein the multiple donor CD4 IL-10 The cells or the pharmaceutical composition prevents GvHD caused by transplanted hematopoietic stem cells or reduces the severity of GvHD.

126. The method of any one of claims 109-125, wherein the multiple donor CD4 IL-10 The cell or the pharmaceutical composition prevents or reduces the severity of a pathogenic response of lymphoid cells from transplanted hematopoietic cells.

127. The method of any one of claims 101-126, wherein the patient has neoplastic cells.

128. A method according to claim 127, wherein the neoplastic cells express CD13, HLA-I class and CD54.

129. The method of any one of claims 127-128, wherein the neoplastic cells express CD112, CD58, or CD155.

130. The method of any one of claims 127-129, wherein the patient has cancer, optionally wherein the cancer is a solid neoplasm or a hematological neoplasm.

131. The method of any one of claims 101-130, wherein the patient has a cancer selected from the group consisting of adrenal cancer, anal cancer, bile duct cancer, bladder cancer, bone cancer, adult brain / CNS tumors, childhood brain / CNS tumors, breast cancer, male breast cancer, cancer of unknown primary, Castleman's disease, cervical cancer, colon / rectum cancer, endometrial cancer, esophageal cancer, Ewing family tumors, eye cancer, gallbladder cancer, gastrointestinal carcinoid tumors, gastrointestinal stromal tumors (GIST), gestational trophoblastic disease, Hodgkin's disease, Kaposi's sarcoma, kidney cancer, laryngeal and hypopharyngeal cancer, leukemia, acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML, including myeloid sarcoma and cutaneous leukemia), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), chronic myelomonocytic leukemia (CMML), childhood leukemia, liver cancer, lung cancer, non-small cell lung cancer, small cell lung cancer, lung carcinoid tumor, lymphoma, cutaneous lymphoma, malignant mesothelioma, multiple myeloma, myelodysplastic syndrome, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, non-Hodgkin lymphoma, childhood non-Hodgkin lymphoma, oral cavity and oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, penile cancer, pituitary tumor, prostate cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma - adult soft tissue cancer, skin cancer, skin cancer - basal cell and squamous cell, skin cancer - melanoma, skin cancer - Merkel cell, small intestine cancer, stomach cancer, testicular cancer, thymic cancer, thyroid cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Waldenstrom's macroglobulinemia, and Wilms' tumor.

132. The method of claim 131, wherein the patient has medullary carcinoma.

133. The method of claim 131, wherein the patient has AML or CML.

134. The method of any one of claims 101, wherein the patient has an inflammatory or autoimmune disease.

135. The method of claim 134, wherein 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 erythematosus, inflammatory bowel disease, Addison's disease, Graves' disease, Sjögren's syndrome, Hashimoto's thyroiditis, myasthenia gravis, autoimmune vasculitis, pernicious anemia, ulcerative colitis, bullous diseases, scleroderma, and celiac disease.

136. The method of claim 135, wherein the inflammatory or autoimmune disease is Crohn's disease, ulcerative colitis, celiac disease, type 1 diabetes, lupus, psoriasis, psoriatic arthritis, or rheumatoid arthritis.

137. The method of any one of claims 101-129 or 134-136, wherein the patient suffers from a disease or disorder involving overactivity of the NLPR3 inflammasome.

138. The method of any one of claims 101-129 or 134-137, wherein the patient has type 2 diabetes, a neurodegenerative disease, a cardiovascular disease, or an inflammatory bowel disease.

139. The method of any one of claims 101-129 or 134-137, wherein the patient has a disease or disorder involving increased production of IL-1β by activated monocytes, macrophages, or dendritic cells.

140. The method of any one of claims 101-129 or 134-137, wherein the patient has a disease or disorder involving increased production of IL-18 by activated monocytes, macrophages, or dendritic cells.

141. The method of any one of claims 101-129 or 134-137, wherein the patient has a disease or disorder involving increased production of mature caspase 1 by activated monocytes, macrophages, or dendritic cells.

142. The method of any one of claims 101-129, wherein the patient suffers from an allergic disease or an atopic disease.

143. The method of claim 142, wherein the allergic or atopic disease is selected from the group consisting of asthma, atopic dermatitis, and rhinitis.

144. The method of any one of claims 101-129, wherein the patient suffers from a food allergy.

145. The method according to any one of claims 101-129, further comprising administering the CD4 + T cell population or the pharmaceutical composition, before or after performing an organ transplant on the patient.

146. The method of claim 145, wherein the multiple donor CD4 IL-10 The cells or the pharmaceutical composition prevents host rejection of the organ transplant or reduces the severity of host rejection of the organ transplant.

147. The method according to any one of claims 101-129, further comprising administering the CD4 + The step of transplanting iPS cell-derived cells or tissues into the patient before or after the T cell population or the pharmaceutical composition.

148. The method of claim 147, wherein multiple donor CD4 IL-10 The cells or the pharmaceutical composition prevents or reduces the severity of host rejection of the cell transplant.

149. The method of any one of claims 101-129, further comprising administering the multiple donor CD4 IL -10 The step of administering the recombinant AAV to the patient before or after administering the cell or the pharmaceutical composition.

150. The method of claim 149, wherein the multiple donor CD4 IL-10 The cell or the pharmaceutical composition reduces the immune response to the recombinant AAV.

151. The method of claim 150, further comprising administering a CD4 IL-10 / CAR cells, CD4 IL-10 / CAR The immunogenic therapeutic protein is administered to the patient before or after administration of the cell population or the pharmaceutical composition.

152. The method of claim 151, wherein the CD4 IL-10 / CAR cells, the CD4 IL-10 / CAR The cell population or the pharmaceutical composition reduces the immune response to the immunogenic therapeutic protein.

153. The method of claim 151 or 152, wherein the immunogenic therapeutic protein is selected from a therapeutic antibody, a Factor VIII replacement, a cytokine, and a cytokine mutein.

154. The method of any one of claims 101-129, wherein the patient has an exaggerated immune response to a viral or bacterial infection.

155. The method of claim 154, wherein the patient has a coronavirus infection.

156. The method of claim 149 or 155, wherein the patient has organ and / or tissue damage.

157. The method according to any one of claims 101-156, further comprising the steps of: detecting a selectable marker in a biological sample obtained from said patient, Thus detecting the presence or absence of multiple donor CD4 IL-10 T cells.

158. The method of claim 157, wherein the biological sample is a biopsy or blood from the patient.

159. A method for treating a patient with a malignant tumor, comprising: administering allo-HSCT to the patient, and Administration of therapeutically effective amounts of multiple donor CD4 IL-10 cell.

160. The method of claim 159, wherein the multiple donor CD4 IL-10 CD4 in cells IL-10 None of the cell donors were HSCT donors.

161. A method of treating blood cancer, comprising: Administration of multiple donor CD4+ cells to patients with hematological cancers in amounts sufficient to induce anticancer effects IL-10 cell, wherein the multiple donor CD4 IL-10 The cells contain CD4 T cells obtained from at least two different T cell donors. + T cells, and the CD4 IL-10 The cells are genetically modified by vector-mediated gene transfer of the coding sequence for human IL-10 under the control of a constitutive or inducible promoter.

162. The method of claim 161, further comprising administering the multiple donor CD4 IL-10 cells, before or after the step of administering allo HSCT to the patient.

163. The method of claim 162, wherein multiple donor CD4 IL-10 The amount of cells is further sufficient to inhibit or prevent graft-versus-host disease (GvHD) without inhibiting the graft-versus-leukemia (GvL) or graft-versus-tumor (GvT) efficacy of allo HSCT.

164. The method of any one of claims 161-163, wherein the hematological cancer is a myeloid leukemia.

165. The method of any one of claims 161-162, wherein the multiple donor CD4 IL-10 The cells target and kill cancer cells that express CD13.

166. The method of any one of claims 161-165, wherein the multiple donor CD4 IL-10 The cells target and kill cancer cells that express HLA class I.

167. The method of any one of claims 161-166, wherein the myeloid leukemia is acute myeloid leukemia (AML).

168. The method of any one of claims 161-167, wherein the allo-HSCT is obtained from a donor that is related or unrelated to the recipient.

169. The method of any one of claims 161-168, wherein the multiple donor CD4 IL-10 The cells are non-self to the recipient.

170. The method of any one of claims 161-168, wherein the multiple donor CD4IL-10 cells are allogeneic to the recipient.

171. The method of any one of claims 161-168, wherein the multiple donor CD4 IL-10 The cells do not lose reactivity to host alloantigens prior to administration to the host.

172. The method of any one of claims 161-168, wherein the multiple donor CD4 IL-10 The cells are Tr1-like cells.

173. The method of any one of claims 161-168, wherein the multiple donor CD4 IL-10 The cells are polyclonal.

174. The method of any one of claims 161-168, wherein the multiple donor CD4 IL-10 The cells are polyclonal and non-autologous to the receptor.

175. The method of any one of claims 161-168, wherein the multiple donor CD4 IL-10 The cells are isolated from at least two donors and then genetically modified.

176. The method of claim 170, wherein none of the at least two donors is the same donor as the allo-HSCT donor.

177. The method of any one of claims 161-176, wherein the allo-HSCT is obtained from a donor that is matched or mismatched to the recipient.

178. The method of any one of claims 161-177, wherein the multiple donor CD4 IL-10 Cells target and kill cells expressing CD54.

179. The method of any one of claims 161-178, wherein the multiple donor CD4 IL-10 The cells target and kill cancer cells expressing HLA class I and CD54.

180. The method of any one of claims 161-179, wherein the multiple donor CD4 IL-10 The cells target and kill cancer cells that express CD112.

181. The method of any one of claims 161-180, wherein the multiple donor CD4 IL-10 The cells target and kill cancer cells that express CD58.

182. The method of any one of claims 161-181, wherein the multiple donor CD4 IL-10 The cells target and kill cancer cells in the host.

183. A method for treating blood cancer by allogeneic hematopoietic stem cell (allo-HSCT) transplantation, comprising: Allo-HSCT was administered to the subjects; Administering multiple donor CD4 IL-10 cells, the multiple donor CD4 IL-10 The amount of cells is sufficient to inhibit or prevent graft-versus-host disease (GvHD) without inhibiting the graft-versus-leukemia (GvL) or graft-versus-tumor (GvT) efficacy of the allo-HSCT; wherein the multiple donor CD4 IL-10 The cells contain CD4 T cells obtained from at least two different T cell donors. + T cells, and the CD4 + T cells were genetically modified by vector-mediated gene transfer of the coding sequence for human IL-10 under the control of a constitutive or inducible promoter; wherein the multiple donor CD4 IL-10 The cells are non-autologous to the subject and are non-autologous to the allo-HSCT donor; wherein the multiple donor CD4 IL-10 The cells have not lost reactivity to the subject's alloantigens prior to administration to the subject; and wherein the multiple donor CD4 IL-10 The cells are polyclonal and Tr1-like.

184. The method of claim 183, wherein the multiple donor CD4 IL-10 The cells are then administered following allo-HSCT.

185. The method of claim 183, wherein the multiple donor CD4 IL-10 The cells were administered prior to the allo-HSCT.

186. A method for treating blood cancer by allogeneic hematopoietic stem cell (allo-HSCT) transplantation, comprising: Allo-HSCT was administered to the subjects; Administering multiple donor CD4 IL-10 cells, the multiple donor CD4 IL-10 The amount of cells is sufficient to inhibit or prevent graft-versus-host disease (GvHD) without inhibiting the graft-versus-leukemia (GvL) or graft-versus-tumor (GvT) efficacy of the allo-HSCT, wherein the multiple donor CD4 IL-10 The cells contain CD4 T cells obtained from at least two different T cell donors. + T cells, and the CD4 + T cells are genetically modified by vector-mediated gene transfer of the coding sequence for human IL-10 under the control of a constitutive or inducible promoter. wherein the multiple donor CD4 IL-10 cells to target and kill cancer cells in said subject, wherein the multiple donor CD4 IL-10 The cells have not lost reactivity to the subject's alloantigens prior to administration to the subject; and wherein the multiple donor CD4 IL-10 The cells are non-autologous to the subject, and are polyclonal and Tr1-like.

187. The method of claim 186, wherein the multiple donor CD4 IL-10 The cells are then administered following allo-HSCT.

188. The method of claim 186, wherein the multiple donor CD4 IL-10 The cells were administered prior to the allo-HSCT.

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