Multiple donor CD4 + t cells expressing il-10 and uses thereof

Abstract

To provide a new Tr1-based therapy using a population of multiple donor CD4IL-10 cells.SOLUTION: The present disclosure provides a population of multi-donor CD4IL-10 cells generated by genetically modifying CD4 + T cells from at least three different T-cell donors. Further provided are methods of making multiple donor CD4L-10 cells and methods of using multiple donor CD4L-10 cells for immunotolerization, GvHD, transplantation of cells and organs, cancer, and other immune disorders.SELECTED DRAWING: None

Description

[Technical Field]

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

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

[0003] 3.Background Regulatory T cells maintain immune tolerance to self-antigens and non-pathogenic antigens and regulate immune homeostasis. Regulatory T cells belong to a small but important subset of T cells that maintain the immune system. -CD4 + , Foxp3 + CD25 + T cells (Foxp3 + cell) and type 1 regulatory T (T r1) cells are present. Foxp3 + Both Tr1 and Tr2 cells have been shown to be effective in organ and islet transplantation, Gv Pathogenic T in various preclinical models of HD and various autoimmune and inflammatory diseases Downregulates cellular responses.

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

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

[0006] Recently, Locafaro et al. developed a bidirectional lentivirus containing the human IL-10 gene. Purified CD4 from a single donor using vector + By transducing T cells circumvented some of these problems. IL-10 The group is heaven These cells shared key functions with naturally occurring Tr1 cells. CD4 IL-10 The cells produce high levels of IL-10 and express allogeneic CD4 + T cells and CD8 + Furthermore, they downregulate the proliferation of both normal myeloid cells (antigen-presenting These single molecules are cytotoxic to both leukemia cells and myeloid leukemia cells. One donor CD4 IL-10 The cells are capable of reproducing human leukemia while retaining graft-versus-leukemia (GvL) activity. It has been shown to be effective in reducing GvHD in a xenogeneic GvHD model. Cafaro et al. Mol Ther. 2017;25(10):2254-2269 and International Publication No. 2016 / 146,54 Please refer to issue 2.

[0007] Highly purified single-donor CD4 for therapeutic use IL-10 Producing cells Although this is possible, it remains significant due to qualitative and quantitative differences between various individual batches. There are significant limitations, due to the inherent differences between different donors as well as variability in buffy coat quality. Most likely related. Summary of the Invention

[0008] 4. Overview The present disclosure provides a multi-donor CD4 IL-10 A new Tr1-based therapy using a population of cells Provide treatment. Multiple donor CD4 IL-10 Cells were collected from at least three different T cell donors. and then genetically modified to contain an exogenous polynucleotide encoding IL-10. Child-modified CD4 + T cells. T cell donors are multi-donor CD4 IL-10 cell A third-party donor who is neither the host treated with the HSC or organ transplant donor. Donor CD4 IL-10 The cells are not alloantigen specific, in other words, host-derived, prior to administration. They have not been primed or stimulated with previous cells.

[0009] The applicant has developed a multi-donor CD4 IL-10 Cells are single-donor CD4 IL-10 Cell have comparable cytokine production profiles, immunosuppressive and cytotoxic capabilities Furthermore, in vivo, they demonstrated that single-donor CD4 IL-10 C than cells D4 + Although they are more effective in preventing T cell-mediated xeno-GvHD, does not induce GvHD alone. Overall, these multiple-donor CD4 IL-10 Thin The functional properties of single-donor CD4 IL-10 cell The functional properties were equal to or better than those of

[0010] Based on these results, the applicants have developed a multi-donor allogeneic CD4 IL-10 The cells are Gv Used for therapeutic purposes in HD, cell and organ transplantation, autoimmune and inflammatory diseases We argue that this can be done.

[0011] Furthermore, by using third-party T cells and eliminating the need for autologous specificity, multiple Donor CD4 IL-10 The cells are intended to be used to administer Tr1-based cell therapy to patients with various genetic backgrounds. make it available to a larger group of people.

[0012] Thus, the present disclosure is intended to include exogenous polynucleotides encoding IL-10. Genetically modified CD4 + A population of T cells, the CD4 + T cells at least 3 Different T cell donors (multiple donor CD4 IL-10 cells obtained from Provide a group.

[0013] In some embodiments, CD4 + T cells are 3, 4, 5, 6, 7, 8, 9, or 10 In some embodiments, the T cells in the population are obtained from different T cell donors. D4 + T cells may collectively express 6, 7, 8, 9, 10, 11, 12, or more different H Possesses the LA haplotype.

[0014] In some embodiments, all CD4 + T cells are HLA-A, HLA- HLA-B, HLA-C, HLA-DRB1, and HLA-DQB1 loci are less likely to be involved in the At least 5 / 10, 6 / 10, 7 / 10, 8 / 10, or 9 / 10 agreement. In some embodiments, all CD4 + T cells are HLA-A, HLA-B, At least 4 / 8 and 5 / 8 of the HLA-C and HLA-DRB1 loci are present in each other. In some embodiments, all of the individuals in the population have 8, 6 / 8, 7 / 8, or 8 / 8 matches. All CD4 + T cells have a 2 / 2 match with each other at the HLA-A locus. In some embodiments, all CD4 + T cells express the HLA-B locus In some embodiments, all CD4 + T The cells have a 2 / 2 match with each other at the HLA-C locus. So, all the CD4 + T cells are genetically linked to HLA-DRB1 and HLA-DQB1 genes. In some embodiments, the chromosomes have at least a 3 / 4 or 4 / 4 match with each other at the chromosomes. is the total number of CD4 + T cells are A * Possess the 02 allele.

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

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

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

[0018] In some embodiments, the exogenous polynucleotide is integrated into the T cell nuclear genome. In some embodiments, the exogenous polynucleotide does not integrate into the T cell nuclear genome. In some embodiments, the exogenous polynucleotide comprises a lentiviral vector sequence. In some embodiments, the exogenous polynucleotide is integrated into the T cell nuclear genome. It won't fit.

[0019] In some embodiments, the CD4 + At least 90% of T cells express IL-10 In some embodiments, the CD4 + At least 95% of T cells are IL-1 In some embodiments, the population expresses CD4 + At least 98% of T cells % express IL-10.

[0020] In some embodiments, the genetically modified CD4 + T cells are CD4 + T cells 10 6 individual / cultivation Constitutively express at least 100 pg of IL-10 per ml of culture medium. In an embodiment, the genetically modified CD4 + T cells are CD4 + T cells 10 6 per ml, At least 200 pg, 500 pg, 1 ng, 5 ng, 10 ng, or 50 ng of IL- 10. In some embodiments, the genetically modified CD4 + T cells express anti-C After activation with anti-CD3 and anti-CD28 antibodies, CD4 + T cells 10 6 per ml, few In some embodiments, the genetically modified C D4 + After activation with anti-CD3 and anti-CD28 antibodies, T cells expressed CD4 + T cells 10 6 At least 2ng, 5ng, 10ng, 100ng, 200ng, or In some embodiments, the genetically modified CD4 + T The cells express IL-10 at levels at least 5-fold higher than unmodified CD4+ T cells. In some embodiments, the genetically modified CD4 + T cells are unmodified CD4 + than T cells They express at least 10-fold higher levels of IL-10.

[0021] In some embodiments, the CD4 + At least 90% of T cells express exogenous polynucleotides In some embodiments, the population expresses a selectable marker from the nucleotide. + T At least 95% of the cells express the selectable marker from the exogenous polynucleotide. In some embodiments, the CD4 + At least 98% of T cells express exogenous polynucleotides The selectable marker is expressed from the

[0022] In some embodiments, the genetically modified CD4 + T cells express CD49b. In some embodiments, the genetically modified CD4 + T cells express LAG-3. In terms of morphology, genetically modified CD4 + The T cells express TGF-β. In some embodiments, , genetically modified CD4 + The T cells express IFNγ. In some embodiments, the genetically modified Weird CD4 + The T cells express GzB. In some embodiments, the genetically modified CD4 + T The cells express perforin. In some embodiments, the genetically modified CD4 + T cells In some embodiments, the gene expresses 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.

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

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

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

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

[0027] In one aspect, the present disclosure provides a multi-donor CD4 IL-10 1. A method of producing a cell, comprising: (i) Primary CD4 T cells from at least three different T cell donors + Obtaining T cells; (ii) transfecting each dome by introducing an exogenous polynucleotide encoding IL-10; Nah's CD4 + Separately modifying the T cells, and then (iii) Genetically modified CD4 + T cells are pooled, thereby genetically modifying CD4 + T Obtaining cells; The present invention provides a method comprising:

[0028] In some embodiments, the method comprises the steps of: after step (i) and before step (ii), or After step (ii), or after step (ii) and before step (iii), or ), followed by First generation CD4 + T cells were treated with anti-CD3 and anti-CD28 antibodies, or anti-CD3 and anti-CD 28 antibody-coated beads.

[0029] In some embodiments, the method further comprises culturing primary CD4 + T cells In some embodiments, the exogenous polynucleotide is , primary CD4 +In some embodiments, the T cells are introduced into In some embodiments, the viral vector is a lentiviral vector. The polynucleotide comprises a segment encoding IL-10 having the sequence of SEQ ID NO: 1. In some embodiments, the polynucleotide segment encoding IL-10 comprises , having the sequence of SEQ ID NO:2.

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

[0031] In some embodiments, the method further comprises, after step (ii), Genetically modified CD4 expressing a selectable marker + Isolating T cells and thereby genetically modifying C D4 + The method further includes generating an enriched population of T cells.

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

[0033] In some embodiments, the method comprises genetically modifying CD4 + Enriched populations of T cells In some embodiments, the genetically modified CD4 + T thin The step of incubating the enriched population of cells comprises incubating the enriched population of cells with an anti-CD3 antibody in the presence of IL-2 and In the presence of beads coated with anti-CD28 antibody or CD3 and CD28 antibodies It is held at.

[0034] In some embodiments, the method comprises genetically modifying CD4 + Subsequent steps to freeze T cells In some embodiments, in step (i), primary CD4 + T cells , 3, 4, 5, 6, 7, 8, 9, or 10 different T cell donors. In some embodiments, the at least three T cell donors are HLA-A, HLA-B, HLA-C, HLA-D, HLA-E, HLA-F ... -C, HLA-DRB1, and HLA-DQB1 loci, at least one of each Some have a 5 / 10, 6 / 10, 7 / 10, 8 / 10, or 9 / 10 agreement. In an embodiment, the at least three T cell donors are HLA-A, HLA-B, HLA-C, and at least 4 / 8, 5 / 8, 6 / 8, In some embodiments, the T cells of at least three individuals have a 7 / 8 or 8 / 8 match. The donors have a 2 / 2 match with each other at the HLA-A locus. In this state, at least three T cell donors are 2 / 2 identical to each other at the HLA-B locus. In some embodiments, at least three T cell donors have a matched HLA-C In some embodiments, the loci have a 2 / 2 match with each other. Human T cell donors share a common genetic identity at the HLA-DRB1 and HLA-DQB1 loci. At least 3 / 4 or 4 / 4 matched. In some embodiments, at least 3 T cells Each donor is A * Possess the 02 allele.

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

[0036] In some embodiments, the method comprises isolating CD4 + Isolating T cells In some embodiments, the peripheral blood mononuclear cells are isolated from buffy coat or apheresis. Obtained from rhesus.

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

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

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

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

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

[0042] In some embodiments, the HSC donor is partially HLA-mismatched to the patient. In some embodiments, the HSC donor is HLA-A, HLA-B, or <5 / 10 at HLA-C, HLA-DRB1 and HLA-DQB1 loci; Matches less than 6 / 10, less than 7 / 10, less than 8 / 10, less than 9 / 10 or less than 10 / 10 In some embodiments, the HSC donor has HLA-A, HLA-B, HLA-C, HLA-D, HLA-E, HLA-F ... -B, HLA-C and HLA-DRB1 loci, less than 4 / 8, less than 5 / 8, 6 In some embodiments, the HSC The donor must have 2 HLA-A, HLA-B, or HLA-C loci with respect to the patient. In some embodiments, the HSC donor has a match of less than 1 / 2 to the patient. Less than 3 / 4 or less than 4 / 4 of the LA-DRB1 and HLA-DQB1 loci It has coincidence.

[0043] In some embodiments, one or more T cell donors are HLA-incompatible with the patient. In some embodiments, one or more T cells are HLA-matched or partially HLA-mismatched. The cell donor must be HLA-A, HLA-B, HLA-C, HLA-DRB1, and at the HLA-DQB1 locus, 5 / 10, 6 / 10, 7 / 10, 8 / 10, and 9 In some embodiments, one or more The T cell donor will have HLA-A, HLA-B, HLA-C and HLA-DR for the patient. At the B1 locus, less than 4 / 8, less than 5 / 8, less than 6 / 8, less than 7 / 8, or less than 8 / 8 In some embodiments, one or more T cell donors have a perfect match to the patient. In contrast, those with less than a 2 / 2 match at the HLA-A, HLA-B, or HLA-C loci In some embodiments, one or more T cell donors have At the LA-DRB1 and HLA-DQB1 loci, less than 2 / 4, less than 3 / 4, or 4 In some embodiments, one or more T cell donors have a match of less than 1 / 4. In some embodiments, the HSC donor is HLA-mismatched or partially HLA-mismatched. One or more T cell donors may be HLA-A, HLA-B, or 5 / 10, 6 at the HLA-C, HLA-DRB1, and HLA-DQB1 loci 10, 7 / 10, 8 / 10, 9 / 10 or less than 10 / 10 agreement. In embodiments, one or more T cell donors are HLA-A, HLA-B, HLA-C, HLA-D, HLA-E, HLA-F ... At the HLA-B, HLA-C, and HLA-DRB1 loci, 4 / 8, 5 / 8, and 6 In some embodiments, 1 or more The T cell donor is HLA-A, HLA-B, or HLA-C relative to the HSC donor. In some embodiments, one or more The T cell donor was genetically identical to the HSC donor with HLA-DRB1 and HLA-DQB1 genes. There is a 3 / 4 or less than 4 / 4 match at the locus.

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

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

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

[0047] In some embodiments, the patient has cancer. In some embodiments, the cancer is a solid or In some embodiments, the patient has adrenal cancer, anal cancer, bile duct cancer, bladder cancer, or hematologic neoplasm. Cancer, bone cancer, adult brain / CNS tumors, pediatric brain / CNS tumors, breast cancer, male breast cancer, unknown primary Cancer of the rectum, Castleman's disease, cervical cancer, colon / rectum cancer, endometrial cancer, esophageal cancer, Ewing's tumor Cancer, eye cancer, gallbladder cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor (GIST), gestational trophoblastic disease Hodgkin's disease, Kaposi's sarcoma, kidney cancer, laryngeal and hypopharyngeal cancer, leukemia, acute lymphocytic ( ALL), acute myeloid (AML including myeloid sarcoma and leukemia cutis), chronic lymphocytic (CL) L), chronic myeloid leukemia (CML), chronic myelomonocytic leukemia (CMML), childhood leukemia, Liver cancer, lung cancer, non-small cell lung cancer, small cell lung cancer, pulmonary carcinoid tumor, lymphoma, skin lymphoma Cancer, malignant mesothelioma, multiple myeloma, myelodysplastic syndrome, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, Lymphoblastoma, Non-Hodgkin's lymphoma, Childhood non-Hodgkin's lymphoma, Oral and oropharyngeal cancer, Bone Sarcoma, ovarian cancer, pancreatic cancer, penile cancer, pituitary tumor, prostate cancer, retinoblastoma, rhabdomyosarcoma, salivary Adenocarcinoma, sarcoma - adult soft tissue cancer, skin cancer, skin cancer - basal cell and squamous cell, skin cancer - melanoma , Skin cancer - Merkel cell, Small intestine cancer, Gastric cancer, Testicular cancer, Thymic cancer, Thyroid cancer, Uterine sarcoma, Vaginal cancer, From the group consisting of vulvar cancer, Waldenstrom's macroglobulinemia, and Wilms' tumor The patient has a cancer selected from the following:

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

[0049] In some embodiments, the patient has an inflammatory disease or an autoimmune disease. In embodiments, the inflammatory or autoimmune disease is type 1 diabetes, autoimmune uveitis, arthritis, Rheumatism, psoriasis, psoriatic arthritis, multiple sclerosis, systemic lupus, inflammatory bowel disease, Addison's disease , Graves' disease, Sjögren's syndrome, Hashimoto's thyroiditis, myasthenia gravis, autoimmune vasculitis , pernicious anemia, ulcerative colitis, bullous disease, scleroderma, and celiac disease. will be done.

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

[0051] In some embodiments, the patient has a disease or disorder associated with hyperactivity of the NLPR3 inflammasome. In some embodiments, the patient has a disorder. have a chronic bowel disease.

[0052] In some embodiments, the patient is treated with activated monocytes, macrophages, or dendritic cells. have a disease or disorder associated with increased L-1β production.

[0053] In some embodiments, the patient is treated with activated monocytes, macrophages, or dendritic cells. Have a disease or disorder associated with increased L-18 production.

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

[0055] In some embodiments, the patient has an allergic or atopic disorder. In some embodiments, the allergic or atopic disease is asthma, atopic dermatitis, and nasal congestion. In some embodiments, the patient has a food allergy. .

[0056] In some embodiments, the method comprises administering to a subject a subject a CD4 + Prior to administration of the population of T cells or pharmaceutical composition The method further comprises transplanting the cells and organ into a patient either at or after the transplantation. In terms of morphology, multiple donor CD4 IL-10 The cells or pharmaceutical compositions may be administered to the host for cell and organ transplantation. Prevent or reduce the severity of rejection.

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

[0058] In some embodiments, multiple donor CD4 IL-10 The cells or pharmaceutical composition may be used for transplantation. Preventing or reducing the severity of host rejection.

[0059] In some embodiments, the method comprises administering a multi-donor CD4 IL-10 Cells or pharmaceutical compositions The method further comprises administering a recombinant AAV to the patient before or after administration of the recombinant AAV. In embodiments, multiple donor CD4 IL-10 The cells or pharmaceutical composition may be directed against recombinant AAV. Decreases immune response.

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

[0061] In some embodiments, the method comprises detecting the selection marker in a biological sample obtained from the patient. Detecting multiple donor CD4 IL-10 The process of detecting the presence or absence of T cells In some embodiments, the biological sample is a biopsy or blood from a patient. do.

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

[0063] In some embodiments, multiple donor CD4 IL-10 Cellular CD4 IL-10 Cell None of the donors were donors of HSCT grafts.

[0064] In another aspect, the disclosure provides a method of treating hematological cancer, comprising administering to a subject a therapeutically effective amount of a compound sufficient to induce an anti-cancer effect. Adequate amounts of multiple donor CD4 IL-10 The method comprises administering cells to a patient with blood cancer, -CD4 IL-10 Cells were obtained from at least three different T cell donors and then constructed Vector-mediated gene transfer of the coding sequence for human IL-10 under the control of a constitutive promoter Genetically modified CD4 + The method includes administering a T cell to a subject.

[0065] In some embodiments, the method comprises administering a multi-donor CD4 IL-10 Prior to administration of cells The method further comprises administering an allogeneic HSCT graft to the patient, either before or after the procedure. In embodiments, multiple donor CD4 IL-10 The amount of cells is related to the graft-versus-leukemia (GVL) of allogeneic HSCT. Prevents graft-versus-host disease (GvH) without suppressing the efficacy of GvL or GvT. D) is still sufficient to inhibit

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

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

[0068] In some embodiments, the allogeneic HSCT graft is obtained from a related or unrelated donor of the recipient. In some embodiments, multiple donor CD4 IL-10 The cells are In some embodiments, multiple donor CD4 IL- 10 The cells are allogeneic to the recipient. In some embodiments, multiple donor C D4 IL-10 The cells must be anergized to the host's alloantigen before administration to the host. stomach.

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

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

[0071] In some embodiments, multiple donor CD4 IL-10 Cells were collected from at least three donors. In some embodiments, the gene is isolated from at least three donors and then genetically modified. In some embodiments, none of the donors is the same donor as the allogeneic HSCT donor. Allogeneic HSCT grafts are obtained from matched or mismatched donors to the recipient. can be done.

[0072] In some embodiments, multiple donor CD4 IL-10 The cells were CD54-expressing cells. In some embodiments, multiple donor CD4 IL-10 cell targets and kills cancer cells that express HLA-class I and CD54. In an embodiment, multiple donor CD4 IL-10 The cells were targeted to cancer cells expressing CD112. In some embodiments, multiple donor CD4 IL-10 The cells are C In some embodiments, multiple donor CD4 IL-10 The cells target and kill cancer cells in the host.

[0073] One aspect of the present disclosure is a method for treating hematological cancers by allogeneic hematopoietic stem cell transplantation (allo-HSCT). It is a law, administering the allogeneic HSCT graft to the subject (host); To evaluate the graft-versus-leukemia (GvL) or graft-versus-tumor (GvT) efficacy of allogeneic HSCT grafts. Multiple donor CD in sufficient doses to suppress graft-versus-host disease (GvHD) without suppression 4 IL-10 administering the cells to a recipient (host) of an allogeneic HSCT; Multiple donor CD4IL-10 Cells were obtained from at least three different T cell donors. Vector-mediated gene transfer of the coding sequence of human IL-10 under the control of a constitutive promoter Transgenic genetically modified CD4 + Contains T cells, Multiple donor CD4 IL-10 The cells are non-autologous to the recipient and are allogeneic HSCs non-self to the T donor, Multiple donor CD4 IL-10 The cells are agonized against the host's alloantigen before administration to the host. It is not energyized, Multiple donor CD4 IL-10 The cells are polyclonal and Tr1-like. do.

[0074] Another aspect of the present disclosure is the treatment of hematological cancers by allogeneic hematopoietic stem cell transplantation (allo-HSCT). 1. A method comprising: administering the allogeneic HSCT graft to the subject (host); To evaluate the graft-versus-leukemia (GvL) or graft-versus-tumor (GvT) efficacy of allogeneic HSCT grafts. Multiple donor CD in sufficient doses to suppress graft-versus-host disease (GvHD) without suppression 4 IL-10 administering the cells to a recipient (host) of an allogeneic HSCT; Multiple donor CD4 IL-10 Cells were obtained from at least three different T cell donors. Vector-mediated gene transfer of the coding sequence of human IL-10 under the control of a constitutive promoter Transgenic genetically modified CD4 + Contains T cells, Multiple donor CD4 IL-10 The cells target and kill cancer cells in the host, Multiple donor CD4 IL-10 The cells are agonized against the host's alloantigen before administration to the host. It is not energyized, Multiple donor CD4 IL-10 The cells are non-autologous to the recipient and polyclonal. The present invention provides a method for the treatment of Tr1-like leukemia. [Brief explanation of the drawings]

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

[0076] These drawings depict various embodiments of the present invention for purposes of illustration only. As will be seen from the following description, it is possible to implement the invention as set forth herein without departing from the principles of the invention as described herein. It will be readily understood that alternative embodiments of the structures and methods described may be used.

[0077] 6. Detailed Description 6.1.Definition Unless otherwise defined, all technical and scientific terms used herein are defined by the principles of the present invention. As used herein, the term "term" has the meaning commonly understood by a person skilled in the art to which it pertains. When used herein, the following terms have the meanings ascribed to them:

[0078] "Graft-versus-leukemia" or "GvL" is a condition that occurs after allogeneic hematopoietic stem cell transplantation (HSCT) or bone marrow transplantation. This refers to the effect that occurs after bone marrow transplantation (BMT). T lymphocytes in the allograft are able to migrate to the tumor-producing tissues and produce residual malignant tumors. Eliminates primary leukemia cells.

[0079] "Graft-versus-tumor effect" or "GvT" is a condition that occurs after allogeneic hematopoietic stem cell transplantation (HSCT) or bone marrow transplantation. This refers to the effects that appear after bone marrow transplantation (BMT). Allograft T lymphocytes are able to inhibit malignant residual host cancer cells, e.g., myeloma and lymphoma. Eliminates myeloid and myeloid leukemia, lymphoma, multiple myeloma, and possibly breast cancer cells. The term vT is a generic term for GvL.

[0080] The terms "treatment," "treating," and the like are used herein as understood in the medical arts. In particular, these terms generally refer to any compound having the desired pharmacological and / or It means to obtain a physiological effect, which effect is to completely or partially alleviate a disease, condition, or its symptoms. may be prophylactic in that it partially prevents and / or reduces the severity of a disease or condition and and / or adverse effects, e.g., partial or complete cure of symptoms attributable to the disease or condition. As used herein, "treatment" refers to the treatment of a disease in a mammal, particularly a human. or condition, and (a) may be predisposed to that disease or condition. from occurring in a subject who has not yet been diagnosed with the disease or condition. (b) inhibiting the disease or condition (e.g., preventing its onset). or (c) alleviating the disease or condition (e.g., causing regression of the disease or condition). resulting in the improvement of one or more symptoms. Improvement in any condition is readily assessed according to standard methods and techniques known in the art. The population of subjects treated by the disease method may include those with an undesirable condition or disease. This includes subjects who are suffering from the disease as well as subjects who are at risk of developing the condition or disease.

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

[0082] For a given locus, a pair of individuals are such that each of the two alleles in one individual is identical to the other. If two alleles of two individuals match, then they have a 2 / 2 match. Only one of the two alleles in one individual matches one of the two alleles in the other individual If a pair of individuals has 10 alleles (HLA) of one individual, they have a 1 / 2 match. each of the HLA-A, HLA-B, HLA-C, HLA-DRB1, and HLA-DQB1 loci If all of the 10 alleles of the other individual match, then H at the HLA-A, HLA-B, HLA-C, HLA-DRB1 and HLA-DQB1 loci There is a 10 / 10 agreement on

[0083] In a preferred embodiment, allele-level typing is used to determine HLA compatibility. Allele-level typing is used, for example, in * Like 02:01:01:01 H is defined by using all the numbers in the first, second, third and fourth sections It corresponds to the unique nucleotide sequence of the LA gene. Functionally, silent substitutions in coding sequences and substitutions in non-coding sequences The third and fourth regions, which characterize different alleles, are regions where substitutions affect HLA allele inheritance. except when it prevents expression of the offspring (e.g., the null allele B*15:01:01:02N). The absence of the null allele is irrelevant and is recognized by alloreactive T cells. This is highly likely and often results in a mismatch that has adverse clinical consequences. Substitutions in the code sequence can affect expression levels (e.g., the A24low allele). Such variability may also contribute to anti-HLA non-self recognition. It can have an impact.

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

[0085] As used herein, the term "partial HLA mismatch" refers to HLA class I (HLA -A, HLA-B and HLA-C) and class II (HLA-DRB1 and HLA-DQ B1) Individuals with mismatched HLA alleles at loci and to an acceptable degree of immunity to each other Refers to pairs of individuals that are genetically incompatible. Some studies have identified tolerated mismatches. Some HLA class I incompatibilities are thought to be better tolerated.

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

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

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

[0089] 6.2. Other Interpretation Rules The ranges set forth herein are shorthand notations for all values ​​within the range, inclusive of the stated endpoints. For example, the range 1 to 50 is understood to be 1, 2, 3, 4, 5, 6, 7, 8, 9 , 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 3 6, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 , 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, do.

[0090] 6.3. Summary of experimental observations The present disclosure provides a method for the production of human IL-10 containing the human IL-10 gene and a truncated, non-signaling form of human NGFR. Highly purified allogeneic CD4 transduced with bidirectional lentiviral vectors + T thin Methods for the production and use of successfully transduced CD4 + T cells, >95% pure IL-10 purified using NGFR-specific monoclonal antibodies CD4 that produces and expresses NGFR + T cells (CD4 IL-10 Three people CD4 from different allogeneic HLA-mismatched donors IL-10 Pool cells at a 1:1:1 ratio did.

[0091] As used herein, multiple donor CD4 IL-10 These pooled populations, also called cells, are Donor CD4 IL-10 Cytokines of cellular and naturally occurring type 1 regulatory T (Tr1) cells These cells had cytokine production profiles comparable to those of the high Produces high levels of IL-10, IL-22, IFN-γ, IL-5, and low levels of IL-4 Multiple donor CD4 IL-10 The cells are polyclonal (have multiple antigen specificities). ), in vitro allogeneic CD4 + Cells and CD8 + The proliferation of both T cells was suppressed. Furthermore, they specifically killed myeloid leukemia cells in vitro. Multiple donor CD4 in a humanized mouse model of GvHD IL-10 Adoptive transfer of cells showed that these cells efficiently homed to the spleen. Multiple donor CD4 in the model IL-10 Adoptive transfer of cells using human CD4 + T cells Importantly, even at high concentrations, it inhibited severe xenograft-vs-host disease induced by multiple Donor CD4IL-10 cells alone did not induce GvHD. Number of donor CD4 IL-10 The cells are used for the treatment and / or prevention of GvHD. and complement allogeneic hematopoietic stem cell transplantation (HSCT) for the treatment of leukemia and other malignancies. Used as an adjunct to reduce GvHD while maintaining the therapeutic effects of GvL or GvT after HSCT to treat cell and organ rejection and autoimmune and inflammatory diseases This indicates that it can be used for

[0092] 6.4. Multiple donor CD4 IL-10 cell In a first embodiment, the gene is modified to include an exogenous polynucleotide encoding IL-10. Modified CD4 + A population of T cells (CD4 IL-10 This population is provided by: At least three different T cell donors (multiple donor CD4 IL-10 cells) CD4 + Contains T cells.

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

[0094] In some embodiments, CD4 + T cells were obtained from pre-frozen stocks of blood or from endogenous T cells. Peripheral blood mononuclear cells (PBMCs) are isolated from previously frozen stocks. In terms of morphology, CD4 + T cells were isolated from peripheral blood or from non-pre-frozen PBMCs. In some embodiments, CD4 + T cells were collected from blood samples obtained from multiple donors. or isolated separately from PBMCs and then pooled. 4 + T cells are isolated from initially pooled blood or PBMCs from multiple donors.

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

[0096] In some embodiments, the at least three different T cell donors are genotypically unrelated. In some embodiments, at least three different T cell donors are selected from genetically engineered individuals. Selection is based on the child type.

[0097] In certain embodiments, at least three different T cell donors have identical HLA haplotypes. Selected based on type.

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

[0099] In some embodiments, all CD4 + T cells are HLA-A, HLA- HLA-B, HLA-C, HLA-DRB1, and HLA-DQB1 loci are less likely to be involved in the At least 5 / 10, 6 / 10, 7 / 10, 8 / 10, or 9 / 10 agreement. In some embodiments, all CD4 +T cells are HLA-A, HLA-B, At least 4 / 8 and 5 / 8 of the HLA-C and HLA-DRB1 loci are present in each other. In some embodiments, all of the individuals in the population have 8, 6 / 8, 7 / 8, or 8 / 8 matches. All CD4 + T cells have a 2 / 2 match with each other at the HLA-A locus. In some embodiments, all CD4 + T cells express the HLA-B locus In some embodiments, all CD4 + T The cells have a 2 / 2 match with each other at the HLA-C locus. So, all the CD4 + T cells are genetically linked to HLA-DRB1 and HLA-DQB1 genes. In some embodiments, the chromosomes have at least a 3 / 4 or 4 / 4 match with each other at the chromosomes. is the total number of CD4 + T cells are A * Possess the 02 allele.

[0100] In a preferred embodiment, at least three different T cell donors each contain CD4 IL -10 In a preferred embodiment, the cells are not treated with the host. Any of the T cell donors may be CD4 T cells in the treatment methods described herein. IL-10 With cells are not donors of stem cells (e.g., HSCs), tissues, or organs used in

[0101] In some embodiments, one or more T cell donors are selected from the patient (host) being treated. In some embodiments, the antibody is HLA-mismatched or partially HLA-mismatched to 1 or more T cell donors to patients with HLA-A, HLA-B, HLA-C, At the HLA-DRB1 and HLA-DQB1 loci, 5 / 10, 6 / 10, and 7 / In some embodiments, the match is less than 10, 8 / 10, 9 / 10, or 10 / 10. One or more T cell donors may be HLA-A, HLA-B, HLA- At the C and HLA-DRB1 loci, <4 / 8, <5 / 8, <6 / 8, and <7 / In some embodiments, one or more T-cells have a match of less than 8 or less than 8 / 8. The cell donor must be mutated to the patient at the HLA-A, HLA-B, or HLA-C locus. In some embodiments, one or more T cell donors have a match of less than 2 / 2. , for patients with less than 2 / 4 at the HLA-DRB1 and HLA-DQB1 loci , having less than 3 / 4 or less than 4 / 4 agreement.

[0102] In some embodiments, one or more T cell donors are HLA-indifferent to the HSC donor. In some embodiments, one or more T cells are HLA-matched or partially HLA-mismatched. The cell donor is HLA-A, HLA-B, HLA-C, and HLA-D relative to the HSC donor. RB1 and HLA-DQB1 loci, 5 / 10, 6 / 10, 7 / 10, 8 / In some embodiments, 1 or more More than 100 T cell donors were identified as HLA-A, HLA-B, and HLA-C donors relative to the HSC donor. , and 4 / 8, 5 / 8, 6 / 8, 7 / 8, or 8 / at the HLA-DRB1 locus In some embodiments, one or more T cell donors have a match of less than 8. For SC donors, 2 / 2 at the HLA-A, HLA-B, or HLA-C locus In some embodiments, one or more T cell donors have a match of less than HS. For C donors, 3 / 4 or have less than 4 / 4 agreement.

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

[0104] 6.4.2. Exogenous Polynucleotides Encoding IL-10 The present disclosure of multiple donor CD4 IL-10 The cells were transfected with exogenous polynucleotides encoding IL-10. CD4 that has been genetically modified to contain leutidine + T cells. The peptide comprises a polynucleotide encoding IL-10 operably linked to an expression control element. Contains an octide segment.

[0105] The polynucleotide segment encoding IL-10 is derived from a human, bonobo, or rhesus monkey. In some embodiments, the polynucleotide encoding IL-10 may encode IL-10. The nucleotide segment encodes human IL-10 having the sequence of SEQ ID NO: 1. In some embodiments, the polynucleotide segment encoding IL-10 is set forth in SEQ ID NO: Human IL-10 having at least 90%, 95%, 98% or 99% sequence identity with IL-1. In some embodiments, the polynucleotide encoding IL-10 encodes a variant of The tid segment has the nucleotide sequence of SEQ ID NO: 2. In some embodiments, The polynucleotide segment encoding IL-10 has at least 90% identical sequence to SEQ ID NO:2. , 95%, 98% or 99% sequence identity.

[0106] In some embodiments, the exogenous polynucleotide encodes viral-IL-10. In various embodiments, the exogenous polypeptide is an exogenous polypeptide selected from the group consisting of HCMV, GMCMV, RhCMV, BaCMV, MOCMV, SMCMV, EBV, Bonobo-HV, BaLCV, Ov HV-2, EHV-2, CyHV-3, AngHV-1, ORFV, BPSV, PCPV , LSDV, SPV, GPV, or CNPV-derived IL-10. In embodiments, the exogenous polypeptide comprises viral IL-10 derived from EBV or ORFV. Code.

[0107] The exogenous polynucleotide is transduced into CD4 + Encoded IL in T cells It further comprises expression control elements that direct expression of -10.

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

[0109] In some embodiments, an inducible promoter is used to express IL- In some embodiments, the IL-10 promoter is used. In some embodiments, a tissue-specific promoter is used. In some embodiments, lineage-specific promoters are used. A promoter that is compatible with the promoter sequence is used.

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

[0111] In some embodiments, synthetic promoters are used. The CMV core promoter is used. In certain embodiments, both inducible and constitutive In certain embodiments, a promoter is used. The synthetic bidirectional promoter disclosed in J. Med. Chem. Soc., 23(1):108-116 (2005) is used. The promoter may mediate the coordinate transcription of two mRNAs in a ubiquitous or tissue-specific manner. In certain embodiments, the bidirectional promoter encodes IL-10 and a selectable marker. Induce the expression of

[0112] In some embodiments, the exogenous polynucleotide is a polynucleotide that is capable of transducing CD4 + It further comprises a segment encoding a selectable marker that allows for the selection of T cells. In some embodiments, the selectable marker is ΔNGFR. The selectable marker is a polypeptide having the sequence of SEQ ID NO: 3. In certain embodiments, the selectable marker has at least 90%, 95%, 98% or 99% sequence identity with SEQ ID NO:3 In certain embodiments, the nucleic acid encoding the ΔNGFR selectable marker is a polypeptide. The nucleotide sequence has the sequence of SEQ ID NO: 4. In some embodiments, the ΔNGFR selection matrix The nucleotide sequence encoding the marker has at least 90%, 95%, 98% or more identical sequences to SEQ ID NO:4. % or 99% sequence identity.

[0113] In some embodiments, the selectable marker is a truncated EGFR polypeptide. In some embodiments, the selectable marker is selected from the group described in Wang et al., which is incorporated herein in its entirety. al. “A transgene-encoded cell surface polypeptide for selection, in vivo tracking , and ablation of engineered cells,” Blood, vol. 118, no. 5 (2011) A truncated form of the GFR polypeptide, optionally huEGFRt.

[0114] In some embodiments, the exogenous polynucleotide encodes an antibiotic resistance gene. In some embodiments, the exogenous polynucleotide further comprises a sequence comprising an ampicillin-containing In some embodiments, the exogenous polynucleotide comprises a sequence encoding an agonist resistance gene. The gene contains a sequence encoding a kanamycin resistance gene.

[0115] In an exemplary embodiment, the exogenous polynucleotide is delivered to CD4+ T cells using a vector. In some embodiments, the vector is a plasmid vector. In some embodiments, the vector is a viral vector.

[0116] In certain embodiments, the exogenous polynucleotide is expressed using a lentiviral vector. The exogenous polynucleotide is delivered to CD4+ T cells and contains a lentiviral vector sequence. In certain embodiments, the method of Matrai et al., Molecula The lentivirus disclosed in r Therapy 18(3):477-490(2010) (“Matrai”) A vector is used.

[0117] In some embodiments, the lentiviral vector integrates into the T cell nuclear genome. In some embodiments, the lentiviral vector can be integrated into the T cell nuclear genome. In some embodiments, integration-deficient lentiviral vectors are used. It is used. For example, in some embodiments, the integration-deficient or other vectors disclosed in Matrai may be used. Lentiviral vectors are used. In some embodiments, integrase-deficient lentiviral vectors are used. Antiviruses are used, e.g., containing an inactivating mutation in integrase (D64V). The integrase-deficient lentivirus is described in Matrai et al., which is incorporated herein by reference. et al., Hepatology 53:1696-1707 (2011).

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

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

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

[0121] In some embodiments, multiple donor CD4 IL-10 T cells are CD4 + T cells 10 6 They constitutively express at least 100 pg of IL-10 per ml of culture medium. In some embodiments, multiple donor CD4 IL-10 T cells are CD4 + T cells 10 6 At least 200 pg, 500 pg, 1 ng, 5 ng, 10 per ml of culture medium ng, or 50 ng of IL-10 constitutively.

[0122] In some embodiments, multiple donor CD4 IL-10 T cells express anti-CD3 and anti-C Beads coated with a combination of anti-CD3 and anti-CD28 antibodies or anti-CD3 and anti-CD28 antibodies After activation with CD4 + T cells 10 6 At least 1 ng or more per ml of culture medium In some embodiments, multiple donor CD4 IL-1 0 T cells were coated with anti-CD3 and anti-CD28 antibodies or CD3 and CD28 antibodies. After activation with coated beads, CD4 + T cells 10 6per 1 ml of culture medium , at least 5 ng, 10 ng, 100 ng, 200 ng or 500 ng of IL-10 To manifest.

[0123] In various embodiments, IL-10 production can be measured using a variety of assays for protein detection and measurement. Methods, e.g., ELISA, spectroscopic procedures, colorimetric methods, amino acid analysis, radiolabeling, edema After 12 hours of activation, 2 In a preferred embodiment, the amount of IL-10 produced is measured after 4 hours or 48 hours. Measured by ELISA 48 hours after activation with CD3 and anti-CD28 antibodies.

[0124] In some embodiments, multiple donor CD4 IL-10 T cells are unmodified CD4 + T thin In some embodiments, the cells express IL-10 at levels at least 5-fold higher than the cells. Multiple donor CD4 IL-10 T cells are unmodified CD4 + At least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, or 50 times higher They express IL-10 in the IL-10 subunit.

[0125] In some embodiments, multiple donor CD4 IL-10 T cells express a selection marker In some embodiments, multiple donor CD4 IL-10 T cells typically expresses a protein that is expressed in Tr1 cells. Donor CD4 IL-10 T cells express marker proteins characteristic of Tr1 cells .

[0126] In some embodiments, multiple donor CD4 IL-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 GzB. In some embodiments, multiple donor CD4 IL-10 T cells are myeloid antigen-presenting cells Upon activation, it releases GzB. In some embodiments, multi-donor CD4 IL-1 0 The T cells express perforin. In some embodiments, multi-donor CD4 IL -10 T cells release perforin when activated by myeloid antigen-presenting cells. In some embodiments, multiple donor CD4 IL-10 T cells express CD18. In some embodiments, multiple donor CD4 IL-10 T cells express CD2. In an embodiment, 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.

[0127] In some embodiments, multiple donor CD4 IL-10 T cells are less likely to be Tr1 cells. In various embodiments, the function is the secretion of IL-10, T Myeloid GF-β secretion and release of granzyme B (GzB) and perforin This is due to the specific killing of antigen-presenting cells.

[0128] 6.4.4. Process Products In an exemplary embodiment, multiple donor CD4 IL-10 T cells express IL-10 Exogenous polynucleotides that bind to CD4 + It is obtained by modifying T cells.

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

[0130] In some embodiments, multiple donor CD4 IL-10 T cells are (i) at least 3 Primary CD4 T cells from different human T cell donors + (ii) pooling the T cells; Pooled C by introducing an exogenous polynucleotide encoding IL-10 D4 + and modifying T cells. In some embodiments, NaCD4 IL-10 T cells were (i) primary C cells from at least three different T cell donors; D4 + (ii) obtaining T cells; and (iii) administering an exogenous polynucleotide encoding IL-10. By introducing CD4 + Separately modifying the T cells, and then ii) Genetically modified CD4 + and pooling T cells.

[0131] In some embodiments, multiple donor CD4 IL-10 T cells are CD4 + Activating T cells In some embodiments, the cells are cultured in the presence of a protein that can activate the cells. So, multiple donor CD4 IL-10 T cells are expressed by anti-CD3 and anti-CD28 antibodies, or anti- Cultured in the presence of beads coated with anti-CD3 and anti-CD28 antibodies. In some embodiments, multiple donor CD4 IL-10 T cells respond to anti-CD3 antibodies, Coated with anti-CD28 antibody and IL-2, or anti-CD3 antibody and anti-CD28 antibody In some embodiments, the cells are cultured in the presence of multi-donor beads and IL-2. CD4 IL-10 T cells were analyzed using T Cell Trap (Miltenyi Biotec). In some embodiments, multi-donor CD4 I L-10 T cells were treated with ImmunoCu (STEMCELL Technologies) The cells were cultured in the presence of Human T Cell Activator™.

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

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

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

[0135] In some embodiments, the pharmaceutically acceptable carrier or diluent is saline, lactate In various embodiments, the pharmaceutical composition is a solution of 100 mg of ethanol, 100 mg of ethanol, or other physiologically compatible solution. The solution contains 2 to 20%, preferably 5%, of human serum albumin.

[0136] In some embodiments, the pharmaceutical composition is adapted for administration by systemic administration, particularly intravenous administration. A unit dosage form of the pharmaceutical composition is provided.

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

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

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

[0140] In some embodiments, the method comprises: (i) collecting T cells from at least three different T cell donors; Obtained primary CD4 + (ii) pooling T cells; and (iii) administering to the T cells an exogenous gene encoding IL-10. Pooled CD4 by introducing a specific polynucleotide + Modifying T cells In another embodiment, the method comprises: (i) obtaining T cell samples from at least three different T cell donors; From the first CD4 + (ii) obtaining T cells; and (iii) transfecting the T cells with an exogenous polynucleotide encoding IL-10. By introducing leiotide, each donor's CD4 + separately modifying the T cells; (iii) genetically modified CD4 + T cells are pooled, thereby multi-donor CD4 IL-10 and obtaining the cells. IL-1 cells can be obtained using various methods known in the art. An exogenous polynucleotide encoding 0 was introduced into primary CD4 + It can be introduced into T cells.

[0141] In some embodiments, the method comprises administering an anti-CD3 antibody and an anti-CD28 antibody, or an anti-CD3 Primary CD4 in the presence of antibodies and anti-CD28 antibody-coated beads + T cells is a genetically modified CD4+ The method further includes incubating the T cells. In some embodiments, the method includes combining an anti-CD3 antibody, an anti-CD28 antibody and IL-2, or an anti-CD3 antibody and Primary CD4 cells were cultured in the presence of anti-CD28 antibody-coated beads and IL-2. + T thin Cells or genetically modified CD4 + The method further includes incubating the T cells. In an embodiment, the method comprises culturing primary CD4 + T cells or Genetically modified CD4 + In some embodiments, the method further comprises incubating the T cells. The method involves the incubation of primary CD4+ cells in the presence of a nanopreparation of anti-CD3 and anti-CD28 antibodies. + T cells or genetically modified CD4 + Further comprising incubating the T cells. In this embodiment, the incubation is carried out using T Ce from Miltenyi Biotec. In some embodiments, the ink is The innovation is based on ImmunoCul, manufactured by STEMCELL Technologies. t in the presence of Human T Cell Activator™.

[0142] In some embodiments, the incubation step comprises incubating an exogenous gene encoding IL-10. In some embodiments, incubation is performed before introducing the polynucleotide. The screening process involves (i) primary CD4 T cells obtained from at least three different T cell donors; + T cells (ii) the exogenous polynucleotide encoding IL-10 is added to the IL-10-encoding DNA; By introducing pooled CD4 +This is done before the step of modifying the T cells. In some embodiments, the incubation step comprises: (i) incubation of at least three different T Primary CD4 cells from cell donors + After the step of obtaining T cells, (ii) IL-10 is administered. Each donor's CD4 + T cells separately This is done before the process of modifying the

[0143] In some embodiments, an incubation step is performed after step (ii). In other words, in some embodiments, the incubation step comprises: (ii) incubating IL-10; by introducing an exogenous polynucleotide encoding pooled CD4 + T cells In some embodiments, the incubation step is performed after the step of modifying (ii) introducing an exogenous polynucleotide encoding IL-10 into each donor; -CD4 + (iii) genetically modified CD4 + T cells are pooled, thereby genetically modifying CD4 + This is carried out before the step of obtaining T cells. In some embodiments, the incubation step comprises (iii) genetically modified CD4 + T Cells are pooled, thereby multi-donor CD4 IL-10 This is done after the cell extraction process. do.

[0144] In some embodiments, the incubation step is performed more than once. In an embodiment, the incubation step comprises: + Both before and after genetic modification of T cells It will be held.

[0145] In some embodiments, the exogenous polynucleotide is initially expressed using a viral vector. CD4 + In some embodiments, the viral vector is a lentiviral vector. In some embodiments, the exogenous polynucleotide is a virus vector. In some embodiments, the segment encoding IL-10 has the sequence of The exogenous polynucleotide may be at least 90%, 95%, 98%, or 99% identical to SEQ ID NO:1. In some embodiments, the IL-10 encoding segment has sequence identity to The polynucleotide segment encoding IL-10 has the sequence of SEQ ID NO:2. In some embodiments, the polynucleotide segment encoding IL-10 is It has at least 90%, 95%, 98% or 99% sequence identity with sequence number 2. In some embodiments, the exogenous polynucleotide is a CD4 + T cells In some embodiments, the segment further comprises a segment encoding a marker that allows for selection of the In certain embodiments, the encoded selectable marker is ΔNGFR. The selected selectable marker has the sequence of SEQ ID NO: 3. In certain embodiments, the exogenous polynucleotide The nucleotide comprises the sequence of SEQ ID NO: 4. In some embodiments, the encoded selection vector The marker is a truncated human EGFR polypeptide.

[0146] In some embodiments, the method comprises genetically modifying CD4 + T thin The cells were isolated and genetically modified to express CD4 IL-10 1. Generating Enriched Populations of Cells It further includes the process.

[0147] In some embodiments, the genetically modified CD4 + At least one T cell 90% express the selectable marker. In some embodiments, the gene in the enriched population Modified CD4 + At least 95% of the T cells express the selectable marker. In this study, we investigated the genetically modified CD4 + At least 96, 97, 98, or 99% express the selectable marker.

[0148] In some embodiments, the genetically modified CD4 + At least one T cell 90% express IL-10. In some embodiments, the genetically modified cells in the enriched population Weird CD4 + At least 95% of the T cells express IL-10. , genetically modified CD4 in enriched populations + At least 96, 97, 98, or 9 of T cells 9% express IL-10.

[0149] In some embodiments, the method comprises genetically modifying CD4 + Enriched populations of T cells In some embodiments, the incubation further comprises: Coating with anti-CD3 and anti-CD28 antibodies, or anti-CD3 and anti-CD28 antibodies In some embodiments, the incubation is carried out in the presence of beads. In some embodiments, the incubation is further carried out in the presence of IL-2. In some embodiments, the incubation is carried out in the presence of an antibody. In some embodiments, the nanopreparation is performed in the presence of a CD3 antibody and an anti-CD28 antibody. The incubation was performed using T Cell Trap (Miltenyi Biotec). In some embodiments, the incubation is carried out in the presence of nsAct™. is the ImmunoCult Huma manufactured by STEMCELL Technologies n in the presence of T Cell Activator™.

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

[0151] In some embodiments, primary CD4 + T cells are selected based on HLA haplotype In some embodiments, the method involves analyzing the genetic information of the donor. In some embodiments, the method further comprises selecting a T cell donor by: The method includes analyzing the genetic information or HLA haplotype of a candidate T cell donor. include.

[0152] In some embodiments, primary CD4 + T cells are primary CD4 + T cells or modified T cells derived from a donor who is at least partially HLA-matched to the host being treated with In one embodiment, primary CD4 + T cells are derived from stem cells (HSCs), tissue or organ donors and In some embodiments, the primary donor is an at least partially HLA matched donor. CD4 + The T cells are obtained from a third-party donor who is not biologically related to the host. In some embodiments, primary CD4 + T cells are biologically related to stem cell, tissue, or organ donors. obtained from third-party donors who are not connected to the organization.

[0153] In some embodiments, in step (i), primary CD4 + T cells, 3, 4, 5, In some embodiments, the T cells are obtained from 6, 7, 8, 9, or 10 different T cell donors. At least three T cell donors were HLA-A, HLA-B, HLA-C, and HLA- At least 5 / 10, 6 / 10, and 1 / 2 at the DRB1 and HLA-DQB1 loci, respectively. In some embodiments, the match is at least 10, 7 / 10, 8 / 10, or 9 / 10. At least three T cell donors were HLA-A, HLA-B, HLA-C, and HLA-D At least 4 / 8, 5 / 8, 6 / 8, 7 / 8, or 8 of each other at the RB1 locus In some embodiments, at least three T cell donors have a HL / 8 match. In some embodiments, the AA locus has a 2 / 2 match with each other. All three T cell donors have a 2 / 2 match with each other at the HLA-B locus. In some embodiments, at least three T cell donors are heterozygous for at the HLA-C locus. In some embodiments, at least three T cell donors have a 2 / 2 match with each other. - at least 3 / 4 of each other at the HLA-DRB1 and HLA-DQB1 loci or 4 / 4 matched. In some embodiments, each of the at least three T cell donors , A * Possess the 02 allele.

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

[0155] In some embodiments, donor-derived CD4 + T cells are transformed into antigen-presenting cells (monocytes, dendritic cells, or DC-10 cells), to generate allospecific CD4 + Generate T cells, This was followed by high levels of IL-10 (allo-CD4 IL-10 cells) do.

[0156] In some embodiments, the method comprises the step of: CD4 + In some embodiments, the method does not include anergizing the T cells. CD4 in the presence of recombinant IL-10 protein + It includes a step of anergizing T cells. First, recombinant IL-10 protein binds to CD4 + Not expressed by T cells. In one embodiment, the method comprises transfecting a host with CD4 + Anergizing T cells This does not include a step of

[0157] 6.7. Multiple donor CD4 IL-10 How to use the cells In yet another aspect, the disclosure provides a method of treating a patient in need of immune tolerization, comprising administering to said patient a therapeutically effective amount of ... and administering to a patient a multi-donor CD4IL-10 Administering cells or pharmaceutical compositions The present invention provides a method comprising the steps of:

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

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

[0160] In some embodiments, the treatment method comprises administering a multi-donor CD4 IL-10 Thin In some embodiments, the method further comprises monitoring the cells from the patient. Detecting a selection marker in the obtained biological sample, thereby detecting multiple donor CD4 I L-10 In some embodiments, the selectable marker is , patient multiple donor CD4 IL-10 Multiple assays were performed to track changes in the presence of cells. In some embodiments, the biological sample is a biopsy or blood sample from the patient. It is a liquid sample.

[0161] Multiple donor CD4 IL-10 The T cells are administered in a therapeutically effective amount, which is determined by body weight and The determination may be based on other clinical factors. 3 cell / kg to 10 9 In some embodiments, 10 cells / kg are administered. 3 cells / kg From 10 8 In some embodiments, 10 cells / kg are administered. 3 cells / kg to 1 0 7 In some embodiments, 10 cells / kg are administered. 3 cells / kg to 10 6 Thin In some embodiments, 10 cells / kg are administered. 3 cells / kg to 10 5 cells / k In some embodiments, 10 g is administered. 3 cells / kg to 10 4 Cells / kg injected It is given.

[0162] In various embodiments, multiple donor CD4 IL-10 T cells are effective in the treatment schedule In some embodiments, multiple donor CD4 IL-10 A single dose of T cells In some embodiments, multiple donor CD4 IL-10 Cells were cultured daily, every 3 days, and every 7 days. It is administered daily, every 14 days, every 21 days, or monthly.

[0163] Multiple donor CD4 IL-10 T cells can be administered according to different routes, e.g., systemically, subcutaneously In some embodiments, the cells may be administered intraperitoneally or intraperitoneally. Preferably, the drug is administered in saline or a physiological solution which may contain 5% human serum albumin. can be.

[0164] 6.7.1. Methods for reducing or preventing GvHD In some embodiments, multiple donor CD4 IL-10 Cell or multi-donor CD4 IL -10 The pharmaceutical composition comprising the cells may be administered to a patient in need of hematopoietic stem cell (HSC) transplantation (HSCT) prior to HSC transplantation. It is used to treat patients concomitantly with T or after HSCT.

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

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

[0167] In some embodiments, the T cell donor is a multi-donor CD4 IL-10 Cells and HS C. The genetic information of the patient to be treated and / or the genetic information of the HSC donor are selected. In some embodiments, the T cell donor is a multi-donor CD4 IL-10 Cells and H HLA haplotype of the patient treated with SC and / or the HLA haplotype of the HSC donor In some embodiments, the method comprises selecting a CD4 IL-10 Thin Before administering the cells, a step of analyzing the genetic information or HLA haplotype of the T cell donor may be further included. In some embodiments, the method includes: In some embodiments, the method further comprises analyzing the genetic makeup of the HSC donor. The method further includes analyzing the information or HLA haplotype.

[0168] In some embodiments, the T cell donor, the host, and the HSC donor are biologically connected. In some embodiments, the T cell donor, the host, and the HSC donor are from different HL In some embodiments, the T cell donor, the host, and the HSC donor have the A haplotype. The patient has at least a partial mismatch in HLA haplotype. In this case, T cell donors are identified as HLA haplotype donors with an HLA match above a threshold. It is selected if it has a

[0169] In some embodiments, the HSC donor is partially HLA-mismatched to the patient. In some embodiments, the HSC donor is HLA-A, HLA-B, or <5 / 10 at HLA-C, HLA-DRB1 and HLA-DQB1 loci; Matches less than 6 / 10, less than 7 / 10, less than 8 / 10, less than 9 / 10 or less than 10 / 10 In some embodiments, the HSC donor has HLA-A, HLA-B, HLA-C, HLA-D, HLA-E, HLA-F ... -B, HLA-C and HLA-DRB1 loci, less than 4 / 8, less than 5 / 8, 6 In some embodiments, the HSC The donor must have 2 HLA-A, HLA-B, or HLA-C loci with respect to the patient. In some embodiments, the HSC donor has a match of less than 1 / 2 to the patient. Less than 3 / 4 or less than 4 / 4 of the LA-DRB1 and HLA-DQB1 loci It has coincidence.

[0170] In some embodiments, one or more T cell donors are HLA-incompatible with the patient. In some embodiments, one or more T cells are HLA-matched or partially HLA-mismatched. The cell donor must be HLA-A, HLA-B, HLA-C, HLA-DRB1, and at the HLA-DQB1 locus, 5 / 10, 6 / 10, 7 / 10, 8 / 10, and 9 In some embodiments, one or more The T cell donor will have HLA-A, HLA-B, HLA-C and HLA-DR for the patient. At the B1 locus, less than 4 / 8, less than 5 / 8, less than 6 / 8, less than 7 / 8, or less than 8 / 8 In some embodiments, one or more T cell donors have a perfect match to the patient. In contrast, those with less than a 2 / 2 match at the HLA-A, HLA-B, or HLA-C loci In some embodiments, one or more T cell donors have At the LA-DRB1 and HLA-DQB1 loci, less than 2 / 4, less than 3 / 4, or 4 have a match of less than / 4.

[0171] In some embodiments, one or more T cell donors are HLA-indifferent to the HSC donor. In some embodiments, one or more T cells are HLA-matched or partially HLA-mismatched. The cell donor is HLA-A, HLA-B, HLA-C, and HLA-D relative to the HSC donor. RB1 and HLA-DQB1 loci, 5 / 10, 6 / 10, 7 / 10, 8 / In some embodiments, 1 or more More than 100 T cell donors were identified as HLA-A, HLA-B, and HLA-C donors relative to the HSC donor. , and 4 / 8, 5 / 8, 6 / 8, 7 / 8, or 8 / at the HLA-DRB1 locus In some embodiments, one or more T cell donors have a match of less than 8. For SC donors, 2 / 2 at the HLA-A, HLA-B, or HLA-C locus In some embodiments, one or more T cell donors have a match of less than HS. For C donors, 3 / 4 or In some embodiments, multiple donor CD4 IL-10 cell Alternatively, the pharmaceutical composition prevents or reduces the severity of GvHD caused by transplanted hematopoietic stem cells.

[0172] In some embodiments, multiple donor CD4 IL-10 The cells or pharmaceutical composition are transplanted In certain embodiments, the method prevents or reduces the severity of a pathological T cell response by the induced hematopoietic cells. Multiple donor CD4 IL-10 The cells prevent or reduce GvHD.

[0173] 6.7.2. Cancer Treatment Methods In some embodiments, multiple donor CD4 IL-10 Cells used for cancer treatment In a preferred embodiment, multiple donor CD4 IL-10 The cells have anti-tumor effects (transplantation directly mediates graft-versus-tumor (GvT) and in certain embodiments, anti-leukemia effects (graft-versus-leukemia, GvL) directly mediates

[0174] In some embodiments, multiple donor CD4 IL-10 The cells are allogeneic for the treatment of cancer. In some embodiments, the multi-donor CD4 IL-10 The cells may be administered before or after administration of the PBMCs. In embodiments, multiple donor CD4 IL-10 Cells and allogeneic mononuclear cells or PBMCs were simultaneously It is administered.

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

[0176] In some embodiments, the neoplastic cells express CD13. In some embodiments, the neoplastic cells express HLA-class I. In some embodiments, the neoplastic cells express HLA-class I. In some embodiments, the neoplastic cells express CD13, HLA-class D54. In some embodiments, the neoplastic cells express CD112 and CD54. In some embodiments, the neoplastic cells express CD58. In some embodiments, the neoplastic cells express CD155. In some embodiments, the tumor expresses CD112. , CD58, or CD155. In various embodiments, the tumor is a solid tumor or a blood tumor. It's a tumor.

[0177] In some embodiments, the patient has adrenal cancer, anal cancer, bile duct cancer, bladder cancer, bone cancer, adult brain cancer, / CNS tumors, pediatric brain / CNS tumors, breast cancer, male breast cancer, cancer of unknown primary, Castlema Cancer of the cervix, colon / rectum, endometrial cancer, esophagus, Ewing's tumor, eye cancer, gallbladder cancer, Gastrointestinal carcinoid tumors, gastrointestinal stromal tumors (GIST), gestational trophoblastic disease, Hodgkin's disease, Posi's sarcoma, kidney cancer, laryngeal and hypopharyngeal cancer, leukemia, acute lymphocytic (ALL), acute myeloid leukemia (AML, including myeloid sarcoma and leukemia cutis), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (C ML) leukemia, chronic myelomonocytic leukemia (CMML), childhood leukemia, liver cancer, lung cancer, non-small cell lung cancer cell lung cancer, small cell lung cancer, pulmonary carcinoid tumor, lymphoma, cutaneous lymphoma, malignant mesothelioma, Multiple myeloma, myelodysplastic syndrome, nasal cavity and paranasal sinus cancer, nasopharyngeal carcinoma, neuroblastoma, non-Hodgkin's disease Hodgkin's lymphoma, childhood non-Hodgkin's lymphoma, oral and oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer , Penile cancer, Pituitary tumor, Prostate cancer, Retinoblastoma, Rhabdomyosarcoma, Salivary gland cancer, Sarcoma - Adult soft tissue Skin Cancer, Skin Cancer, Skin Cancer - Basal Cell and Squamous Cell, Skin Cancer - Melanoma, Skin Cancer - Merkel Cell, small intestine cancer, stomach cancer, testicular cancer, thymus cancer, thyroid cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Waldenstrom and Wilms' tumor. do.

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

[0179] In some embodiments, the methods include treating hematological cancers affecting the blood, bone marrow, and lymph nodes. In various embodiments, the hematological cancer is a lymphoma (e.g., Hodgkin's disease). In various embodiments, the hematological cancer is acute myeloid leukemia, acute myeloma ... or chronic myeloid (bone marrow) leukemia (AML, CML), or myelodysplastic syndrome.

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

[0181] In some embodiments, multiple donor CD4 IL-10 Cells can be combined with therapeutic interventions The combination may be performed simultaneously or at different times. Preferably, the therapeutic intervention is chemotherapy, radiotherapy, allogeneic HSCT, immunosuppression, transfusion. The therapeutic agent is selected from the group consisting of blood, bone marrow transplant, growth factors, and biological agents.

[0182] In some embodiments, multiple donor CD4 IL-10 The cells were tumor-infiltrating myeloid cells ( Induces cell death in cells such as monocytes, macrophages, and neutrophils.

[0183] 6.7.3. Methods for treating other disorders In some embodiments, multiple donor CD4IL-10 Cells can treat autoimmune diseases It is administered to

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

[0185] In some embodiments, multiple donor CD4 IL-10 The cells were transplanted other than HSCT. Administered to prevent or reduce the severity of pathogenic T cell responses to transplants and organ transplants In some embodiments, the method comprises administering a multi-donor CD4 IL-10 T cells or pharmaceutical compositions The method further includes administering the organ to the patient either before or after administration of the agent. In a preferred embodiment, the organ is a kidney, a heart, or pancreatic islet cells. CD4 IL-10 The cells or pharmaceutical compositions are useful for preventing or reducing the severity of host rejection of organ transplants. do.

[0186] In some embodiments, multiple donor CD4 IL-10The cells may be used in gene therapy, e.g. It is administered to prevent or reduce the immune response associated with administration of recombinant AAV (rAAV). In these embodiments, the method comprises administering a multi-donor CD4 IL-10 Administration of cells or pharmaceutical compositions The method further comprises administering recombinant AAV to the patient before or after the administration.

[0187] In some embodiments, multiple donor CD4 IL-10 The cells may be iPS-derived tissue or It is administered to prevent or reduce the immune response associated with cell transplantation. Examples of cells include cardiomyocytes, hepatocytes, epithelial cells, cartilage, bone and muscle cells, and neurons. Examples include, but are not limited to:

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

[0189] In some embodiments, multiple donor CDs 4IL-10 Cells express NLPR3 inflammasome In some embodiments, the compound is administered to treat a disease or disorder associated with increased activity of the serotonin receptor. So, multiple donor CD 4IL-10 The cells may be activated monocytes, macrophages, or dendritic cells. It is administered to treat diseases or disorders associated with increased IL-1β production by some In embodiments, multiple donor CDs 4IL-10 The cells may be activated monocytes, macrophages, or dendritic cells. The antibody is administered to treat a disease or disorder associated with increased IL-18 production by endothelial cells. In some embodiments, multiple donor CD 4IL-10 The cells are activated monocytes, macrophages, for treating diseases or disorders associated with increased production of mature caspase-1 by mammalian or dendritic cells It is administered.

[0190] In some embodiments, multiple donor CDs 4IL-10 The cells are activated monocytes, macrophages, It is administered to reduce IL-1β production by phage or dendritic cells. In an embodiment, multiple donor CDs 4IL-10 The cells may be activated monocytes, macrophages, or dendritic cells. In some embodiments, the compound is administered to reduce IL-18 production by the cells. Number of donor CDs 4IL-10 The cells are matured by activated monocytes, macrophages or dendritic cells. It is administered to reduce caspase-1 production.

[0191] In some embodiments, multiple donor CD4 IL-10 Cells are resistant to viral infection In some embodiments, the virus is administered to reduce an overactive immune response in a patient. In some embodiments, the virus is SARS-CoV-2. IL- 10 Cells are involved in overactive immune responses to bacterial infections, e.g., toxic shock and cytotoxicity. It is administered to reduce kinestroke. [Example]

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

[0193] 6.8.1. Example 1: Multiple Donor CD4 IL-10 Cell preparation and vector production No. WO 2016 / 146542, which is incorporated herein by reference in its entirety. As described, human IL-10 and a truncated form of NGFR (ΔNGFR) (Figure 1 Transduction with a lentiviral vector containing the coding sequences for both α- and β-actin (Figure 2) and multiple donor CD4 IL-10 The vector sequence is shown as SEQ ID NO: 5. Briefly, lentiviral vectors were transfected into the pH15C (ATCC 68192) The coding sequence of human IL-10 from a 549 bp fragment was cloned into plasmid #1074.1071. Ligated to hPGK.GFP.WPRE.mhCMV.dNGFR.SV40PA The bidirectional promoter (human PGK promoter + The presence of the minimal core elements of the CMV promoter allows for simultaneous expression of two transgenes. The plasmid contains an antibiotic resistance gene (e.g., ampicillin or kanamycin). It further comprises a code sequence.

[0194] Lentiviral vectors were co-transfected into 293T cells with Ca3PO4 transient transfection of four plasmids. The resulting solution was prepared by transfection, concentrated by ultracentrifugation, and diluted with 1 μM sodium butyrate. The vector was added to the culture for vector recovery. The titer on 293T cells was determined by limiting dilution. The vector particles were then transferred to HIV-1 Gag p24 antigen immunocapture (NEN Life Science Products, Waltham, MA) The vector infectious titer was calculated as the ratio of titer to particles. For concentrated vectors, the titer was 5 × 1. 0 8 From 6 x 10 9 transducing units / ml, with an infectious titer of 5 x 10 4 From 5 x 10 5 The transducing units / ng range.

[0195] CD4 IL-10 Cell production Figure 3 shows the CD4 IL-10 Schematic diagram of the cell production process. 4 + Purified human CD4 T cells + Soluble anti-CD3 mAb, soluble anti-CD28 T cells After activation with mAb and rhIL-2 (50 U / mL) for 48 h, the cells were cultured at a multiplicity of infection (MOI) of 20 ( Human IL-10 and truncated human NGF receptor (LV-IL-10 / ΔNGF) were incubated at a MOI of 10. Transduction was performed with a bidirectional lentiviral vector encoding R).

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

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

[0198] ΔNGFR +T cells were purified using anti-CD271 mAb-coated microbeads. Made with over 95% pure CD4 IL-10 After purification, the cells were separated into CD4 and Δ The cells were stained with a marker for NGFR and analyzed by FACS. Figure 4B shows that the purity of the purified IgG from two of the ten donors tested was greater than 98%. FACS data obtained from two representative human donors (Donor B and Donor C) are shown. CD4 IL-10 Cells were restimulated three times at 14-day intervals, with the second restimulation (TF2) and / or the first After restimulation of TF3, their in vitro and in vivo functions were tested.

[0199] Resting CD4 IL-10 The cells constitutively produced IL-10. Upon activation, production The levels of IL-10 induced by the treatment were strongly enhanced.

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

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

[0202] CD4 IL-10 The cells express high levels of granzyme B and select for myeloid leukemia cells. to kill CD4 IL-10 Cells were then re-stimulated with granzyme B (GzB) after a second restimulation (TF2). The data in Figure 6A show that most CD4 IL-10 Cells Total CD4 from seven different donors was shown to express GzB. IL-10 Cell Over 95% expressed high levels of granzyme B.

[0203] CD4 obtained from the second (TF2) restimulationIL-10 The cells were then treated with myeloid leukemia cells ( Their cytotoxic effects on ALL-CM and erythroleukemia cell lines (K562) were investigated. Further analysis was performed using CD4 IL-10 cells (10 5 cells / well) and K562 cells LL-CM cells (10 5 Residual leukemia cells were co-cultured with 1:1 cells / well for 3 days. strain (CD45 low CD33 + ) were counted by FACS for each target cell.

[0204] CD4 IL-10 As shown in Figure 6B, myeloid leukemia cells (ALL-CM) were selected. The percentage of killed ALL-CM cells varied between 62% and 100%. erythroleukemia cell line K562 (highly sensitive to nonspecific cytotoxic activity) Mortality varied between 0 and 27% (tested in four different donors). The data are CD4 IL-10 Cells express granzyme B and efficiently kill myeloid leukemia cells. As expected, individual donor-derived CD4 IL- 10 Some variability in cell killing ability was observed.

[0205] CD4 IL-10 The cells were allogeneic CD4 + T cells and CD8 + Both T cell proliferation responses suppress CD4 IL-10 The cells were then transfected with allogeneic CD4 + T cells or CD8 + Their effects on T cells Specifically, allogeneic PBMC cells were treated with eFluor® 67 0(10 5cells / well) and labeled with CD4 IL-10 In the absence or presence of cells (10 5 cells / well), allogeneic mature dendritic (DC) cells at a 1:1 responder:suppressor ratio (5×10 4 After 4 days of culture, the cells were stimulated with soluble anti-CD3 mAb (1000 cells / well). The percentage of proliferative responder cells was determined by CD4 + ΔNGFR - T cells or CD8 + ΔNGFR - T cell After plating, eFluor® 670 dilutions were analyzed using flow cytometry. Figures 7A and 7B show the results of six different unpooled donors (Figure 7 Donors C, E, and F in Figure 7A and Donors H, I, and L in Figure 7B) CD4 derived IL-10 Cellular CD4 + The effect on T cells is expressed as the rate of proliferation and suppression Figures 8A and 8B show six different unpooled donors (donor C in Figure 8A, donor D in Figure 8B). CD4 from donors E and F and donors H, I, and L in Figure 8B IL -10 Cellular CD8 + Shows the effect on T cells.

[0206] These results are not pooled and are based on C from six different donors tested separately. D4 IL-10 The cells are allogeneic CD4 + T cells and CD8 + Downregulation of both T cell proliferation responses It was shown that CD4 + The suppressive effect on T cells varied between 51% and 96%. However, CD8 + The inhibitory effect on T cells varied between 62% and 73%.

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

[0208] Multiple donor CD4 IL-10 The cells were CD4 Il-10 have a cytokine production profile comparable to that of cells . The culture supernatant was collected and the levels of IL-10, IL-4, IL-5, IFN-γ, and IL-22 were analyzed. The levels were measured by ELISA. The results presented in Figure 9 show the results of the IgG antibody titers from three different allogeneic donors. Pooled (1:1:1 pooled) multi-donor CD4 IL-10 Cellular cytokines CD4 production (red dots) from individual donors (n=8) IL-10 Cell-derived CD4 IL-10 The cytokine production of the cells (gray bar) was comparable to that of the control. Donor CD4 IL-10 The cells express high levels of IL-10, IL-5, IFN-γ and IL- -22 and produced low levels of IL-4 and undetectable levels of IL-2 (not shown). These data are consistent with the CD4 IL-10 It is feasible to pool cells, and these Multiple donor CD4 IL-10 The cells were derived from a single donor.IL-10 Cells and Tr1 Importantly, pooled IL-16 / ... The homogeneous cell population contained over 95% viable cells and did not kill each other. This shows that.

[0209] Multiple donor CD4 IL-10 The cells express high levels of granzyme B and are associated with myeloid leukemia. Kill the cell line. Multiple donor CD4 IL-10 Cells were analyzed a third time for granzyme B (GzB) expression. The data in Figure 10A show that most multiple donors were CD4 IL-10 Showing that the cells express GzB. Multi-donor CD4 IL-10 cell Over 95% of CD4+ cells express granzyme B and are derived from a single donor. IL-10 GzB expression in cells It was comparable to the current situation.

[0210] CD4 obtained from the third (TF3) restimulation IL-10 The cells were then treated with myeloid leukemia cells ( Their cytotoxic effects on ALL-CM cell line or K562 were further analyzed. Multiple donor CD4 IL-10 cells (10 5 cells / well) were added to K562 cells and ALL- CM cells (10 5 The cells were co-cultured with the residual leukemia cell line (C D45 low CD33 + ) was counted for each target cell by FACS. The results shown demonstrate that certain cytotoxicity events were observed against K562 cells, which are highly sensitive to nonspecific cytotoxicity. However, it shows a cytotoxic effect against myeloid leukemia cells (ALL-CM). This provides a level of selectivity for CD4 IL-10 Cell selection It is comparable to selectivity.

[0211] Multiple donor CD4 IL-10 The cells were expressed as both allogeneic CD4+ and CD8+ T cells. Suppresses proliferative responses. Multiple donor CD4 IL-10 The cells were then transfected with allogeneic CD4 + T cells or CD8 + against T cells These effects were also analyzed. Specifically, allogeneic PBMC cells were treated with eFluor (registered trademark) Trademark)670(10 5 cells / well) and labeled with multiple donor CD4 IL-10 Absence of cells in existence or existence (10 5 cells / well), homogenous at a 1:1 responder:suppressor ratio Mature dendritic (DC) cells (5 × 10 4 cells / well) and stimulated with soluble anti-CD3 mAb After 4 days of culture, the percentage of proliferative responder cells was determined by CD4 + ΔNGFR - T cells and CD8 + Δ NGFR - Gating on T cells and then using flow cytometry to identify eFluorescence Figure 11A shows the results of donors C, E, and F. CD4 derived IL-10 Multiple donor CD4 containing cells IL-10 The results for cells are shown. FIG. 11B shows the results of donors H, I, and L, which were frozen, stored, and thawed prior to testing. CD4 derived IL-10 Multiple donor CD4 containing cells IL-10 The results for cells are shown.

[0212] Figure 11A shows a multi-donor CD4 IL-10Cells (from three different donors) were CD4 + and CD8 + The results show that T cell responses are suppressed by 96% and 74%, respectively. A second, different batch of multi-donor CD4 cells was frozen, stored, thawed, and then tested. IL -10 Comparable results were obtained using CD4 cells (Fig. 11B). + and CD8 + T cell proliferation The suppression of CD4+ T cells was 68% and 75%, respectively. These data are consistent with the results of a multi-donor CD4+ T cell study. I L-10 This demonstrates that cells can be frozen and preserved without loss of function.

[0213] In summary, multiple donor CD4 IL10 Data obtained using cells are It has been shown that preparations can be pooled without problems. They contain >95% viable cells. Single-donor CD4 IL-10 Related functions of cells (cytokine production, cell maintains all of the immune responses (cytotoxicity, and suppression of allogeneic T cell responses). IL-10 Thin The use of a larger pool of cells allows for the identification of CD4 IL-10 Cell This should reduce the natural variability observed between batches, allowing for the development of large-scale formulations for human therapy. Unilateral CD4 IL-10 The cells should be provided.

[0214] Multiple donor CD4 IL-10 Cellular products have the significant advantage of being more homogeneous products. This allows for well-defined efficacy and release criteria with low lot-to-lot variability. Furthermore, it will be possible to determine the standard for continuous large-scale cell manufacturing processes. This enables the development of

[0215] Multiple donor CD4 IL-10 Other methods of producing cells Prior to lentiviral transduction, buffy coats from a minimum of 3–5 different donors were collected. Pool CD4 + Cells were isolated from buffy coats by positive selection using anti-CD4 antibodies. Isolated from pooled CD4 + The purity of the cells is confirmed by FACS. Frozen human CD4 + Cells are obtained from a minimum of 3-5 normal healthy donors. Human CD4 + Thaw cells before use. CD4 cells from buffy coat or frozen stocks + The cells were incubated with a combination of CD3 antibody and CD28 antibody or a combination of CD3 antibody and CD28 antibody in the presence of IL-2. and CD28 antibody-coated beads for 24-48 hours. Therefore, CD4 from buffy coat or frozen stocks + Cells were treated with soluble anti-CD3, soluble Activated with anti-CD28 mAb and rhIL-2 (50 U / mL) for 48 hours, CD4 IL- 10 Bidirectional lentiviral vector encoding human IL-10 for the production of cells Transduce with.

[0216] In some cases, the T cell donor (or CD4 + Cell) HL The A haplotype is first determined, and CD4 + Select cells The funds will be pooled and used.

[0217] Multiple donor CD4 IL-10 The cells were activated with CD4 +The cells were cultured in the same manner as described above. Transduction with lentiviral vectors containing the 10 and ΔNGFR coding sequences This is how it is made.

[0218] On days 7-11, 5-9 days after transduction, cells were harvested and successfully transduced T cells were analyzed. The cells were purified using anti-NGFR antibodies. This process typically yields 95% pure multi-drug NaCD4 IL-10 resulting in a cell population.

[0219] Purified multi-donor CD4 IL-10 Cells were counted and stained with CD3 antibody in the presence of IL-2. and a mixture of CD28 antibodies, on beads coated with CD3 and CD28 antibodies. Therefore, the cells are optionally restimulated for another 8-10 days in the presence of feeder cells. Therefore, purified multiple donor CD4 IL-10 Re-stimulating cells in the presence of feeder cells .

[0220] After a total culture period of 14–18 days, CD4 IL-10 Cells were harvested, counted, and spontaneously their ability to produce IL-10, or CD3 and CD28 antibodies or CD3 antibodies IL-10 production after activation with antibodies and CD28-coated beads These abilities are tested. In addition, the levels of GrzB and perforin are measured. In addition, human T cells (PBMC) and purified CD4 + and CD8 + Suppresses T cell proliferation Their ability to do so will also be tested.

[0221] In addition, IL-22 production is constitutive and dependent on other cytokines, e.g., IFNγ. As previously described, the production of IL-10, IL-4, and IL-5 was stimulated by CD3 antibodies and Using a combination of CD28 and CD28 antibodies, 200,000 cells were collected in a volume of 200 microliters. 0 CD4 IL-10 The level of IL-22 production is measured after cell activation. As described for other cytokines in WO 2016 / 146542 , as measured by IL-22-specific ELISA. Prior to storage, pooled CD4 IL- 10 Freeze the cells.

[0222] 6.8.2. Example 2: Multiple Donor CD4 IL-10 Treatment of GvHD using cells or prevention In vivo multiple donor CD4 IL-10 Cellular effects Multiple donor CD4 IL-10 The cell population was cultured with human PBMCs as shown in Figure 12. Their effects on GvHD induced by xenografts were investigated using humanized xenografts of GvHD disease models. The study was conducted in the NSG mouse model, which is a cancer-specific model. NSG mice were irradiated with sublethal doses of radiation, and Human PBMCs (5 × 10 6 cells / mouse), multiple donor (3 donors) CD4 IL-1 0 cells (5×10 6 cells / mouse), or multiple donor CD4 IL-10 Cells (3 donors) -)(5×10 6 cells / mouse) combined with human PBMCs (5 × 10 6 Cells / Mouse ) was intravenously injected. GvHD was induced as previously reported (Bondanza et al., Blood 2006), weight loss (greater than 20% weight loss), skin lesions, coat condition, activity and posture. and evaluated it.

[0223] Figure 13 shows the percentage of NSG mice exhibiting GvHD on each day after injection. 5 x 10 to NSG mice 6 Administration of human PBMCs unexpectedly resulted in an abnormally excitable G All mice died on day 10, which was a highly lethal GvH event. Reflects D. 5 x 10 6 Multiple donor CD4 IL-10 Co-administration of cells Although fulminant GvHD was delayed, the mice were at 20% of the pre-designated human body mass index for sacrifice. The animals were sacrificed on day 14 after reaching the weight loss criterion (Figure 13). Multiple donor CD4 IL-10 have shown that it can delay very severe GvHD. Importantly, multi-donor CD4 IL-10 cell (5×10 6 cells) did not induce any signs of GvHD.

[0224] In addition, human PBMCs (5 × 10 6 cells / mouse), multiple donor (3 donors) CD4 IL-10 cells (5×10 6 cells / mouse) or human PBMCs (5 × 10 6 Cells / Mouse ) multiple donor CD4 IL-10 Cells (3 donors) (5 × 10 6 cells / mouse) and Spleen (Figure 14, left panel) and bone marrow (Figure 14, right panel) of NSG mice injected with the combination of Human CD4 in IL-10 The presence of cells was examined 14 days after injection. The results were multi-donor CD4 IL-10 The cells migrated to the spleen and bone marrow. Fourteen days after injection, these cells were found to be present in low proportions. Multiple donor CD4 IL-10 Fulminant GvHD induced by human PBMC cells These results suggest that the IL-16 expression of IL-16 is associated with a delayed response to IL-16, indicating that they do not induce any xeno-GvHD by themselves.

[0225] Multiple donor CD4 IL-10 Cells were purified CD4 + Severe xenogeneic GvHD caused by cells To inhibit. As shown in Figure 15, 2.5 x 10 6 Administration of purified human CD4+ T cells resulted in Gv In a humanized xenogeneic GvHD model in which HD disease was induced, multiple donor CD4 IL-10 NSG mice were sublethally irradiated on day 0 and human CD4+ cells were cultured on day 3. 4 + T cells (2.5×10 6 cells / mouse) single or multiple donor CD4 IL-10 Cells (three different donors) (2.5 × 10 6 in combination with or in pools of cells / mice Single-donor CD4 IL-10 cells (2.5×10 6 cells / mouse) The combined doses were intravenously injected. GvHD was induced as previously reported (Bondanza et al. Food 2006), weight loss (>20% weight loss), skin lesions, coat, activity and posture was evaluated based on

[0226] Figure 16 shows the percentage of NSG mice exhibiting GvHD on each day after injection. The results showed that multiple donor CD4 IL-10 Cells are human allogeneic CD4 + T cell-mediated G In this particular experiment, CD4+ T cells were shown to be able to inhibit vHD. All mice in the control group receiving IFN-γ died by day 20, demonstrating that xenogeneic GvHD was not In contrast, 2.5 × 10 6 Multiple donor CD4 IL-10 Simultaneous throwing of Administration of single-donor CD4 IL-10 Cells were also protective But the effect wasn't that strong.

[0227] Other experiments Multiple donor CD4 IL-10 The therapeutic effect of the cells was evaluated in four different mouse groups, namely, i) CD4 IL-10 The cells were administered to mice receiving human PBMCs from unrelated donors ( GvHD positive control), (ii) multiple donor CD4 IL-10 Mice that received cells (negative gender control), (iii) PBMC and multiple donor CD4 IL-10 Cell combinations were injected at a 1:1 ratio. mice given (iv) PBMC and multiple donor CD4 IL-10 The cell combinations were either in a 2:1 ratio or The study was conducted in mice receiving PBMC and multiple donor CD4 IL-10 cell Among animals receiving the combination, some animals received both PBMC and multi-donor CD4 IL- 10 Some animals were administered PBMCs at the same time as the cells, and some animals were administered PBMCs several days later (e.g., 5 days later). After) multiple donor CD4 IL-10 Some animals will receive cells, and some will receive PBMCs. Multiple donor CD4 counts from several days ago (e.g., 5 days ago) IL-10 The cells are administered.

[0228] PBMC and / or multiple donor CD4 IL-10 1, 2, 3, and 4 weeks after cell administration Mice were monitored for the development of GvHD by measuring body weight at week 5 and, if necessary, week 6. In addition to weight loss, mice are monitored for skin lesions, coat condition, and activity. Mice in the treatment group will be monitored for an additional period to assess long-term survival. Multiple donor CD4 IL-10 Examine the effects on cells.

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

[0230] These results are consistent with multiple donor CD4 IL-10 Cells are effective in reducing and preventing xenogeneic GvHD This indicates that it is effective.

[0231] 6.8.3. Example 3: Inhibition of GvHD and Treatment of Cancer Multiple donor CD4 IL-10 Populations of cells were transplanted with human PBMCs and AML tumor cells. In the NSG mouse model, human PBMC-induced xenogeneic GvHD was The efficacy and anti-tumor effects of these cells will be tested. Administered intravenously as previously described in Patent Publication No. 2016 / 146542. MC or multiple donor CD4 IL-10 The cells or a combination thereof are administered three days later.

[0232] As described in Example 1, multiple donor CD4 IL-10 Obtaining cells from multiple donors D4 IL-10 The therapeutic effect of cells was evaluated by irradiating 5 × 10 cells on day 0. 6 AL Four different groups of mice (AML mice) bearing L-CM cells were further analyzed: (i) AML mice without any treatment; (ii) multiple donor CD4 IL-10 Unrelated to cells 5 x 10 donor-derived 6 AML mice given human PBMCs - PBMCs were severe (iii) 2.5 × 10 6 Multiple donor CD4 IL-1 0 AML mice administered cells, and (iv) PBMCs and multiple donor CD4 IL-10 AML mice administered in combination with AML cells at a ratio of 1:1 or 2:1, or at different ratios. One additional group of mice will be administered ALL-CML cells. However, on the third day after irradiation, 5 × 10 6 The mice are administered human PBMCs.

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

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

[0235] The results were multi-donor CD4 IL-10 The cells are useful for both inhibiting xenogeneic GvHD and treating cancer. This shows that it is effective in

[0236] 6.8.4. Example 4: Multiple Donor CD4 IL-10 Cell-based cancer treatment Multiple donor CD4 IL-10 Populations of cells in NSG mice treated with T cell therapy for ALL -CM leukemia model.

[0237] On day 0, NSG mice were irradiated with sublethal doses of radiation and transduced with myeloid leukemia cells (ALL-C M)(5×10 6 In the first group of animals, PBMCs (5 × 10 6 pieces ) or CD4 from a single donor (donors BC-I and BC-H) IL-10 cell (2.5×10 6 ) were injected on day 3. In the second group of animals, PBMCs (5 × 10 6 ) or multiple donor CD4 IL-10 cells (2.5×10 6 100 mg / kg) was injected on the third day. Graft-versus-leukemia (GvL) effect in animals is demonstrated by reducing blood leukemia cells and achieving long-term leukemia-free survival. Leukemia was measured based on disease survival as previously reported (Locafaro G .et al Molecular Therapy 2017).

[0238] As shown in Figures 17A and 17B, mice injected with ALL-CM myeloid leukemia cells All of the mice had extensive leukemic progression by day 17. 6 PBMCs Administration resulted in a strong inhibition of leukemia progression. Interestingly, a similar degree of leukemia progression was observed. The level of inhibition was found to be lower (2.5 × 106 (units) single donor CD4 IL10 (Figure 17 A) or multiple donor CD4 IL10 (Figure 17B). These data were obtained Single-donor and multiple-donor CD4 IL10 demonstrated a strong direct anti-leukemia effect. is doing.

[0239] Single donor CD4 IL10 and multiple donor CD4 IL10 Graft-versus-leukemia (GvL) The effect was compared in mice injected with ALL-CM myeloid leukemia cells combined with PBMCs. Further testing was carried out. 6 Administration of PBMCs resulted in potent inhibition of leukemia progression , single donor CD4 IL10 (2.5×10 6 5×10 6 PBM Administration of C had a synergistic effect (Figure 18A). 6 Multiple Do NaCD4 IL10 5×10 combined with 6 Administration of PBMCs produced equivalent synergistic Gv These data support the multi-donor CD4 IL10 PBMC These results suggest that IL-17 acts synergistically with IL-17 to mediate potent GvL effects.

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

[0241] NLPR3 inflammasome activation is involved in many chronic inflammatory and autoimmune diseases The NLPR3 inflammasome mediates monocyte / macrophage-mediated pro-inflammatory responses. "Danger signals" leading to caspase-1-mediated production of the kinases IL-1β and IL-18 NLPR3 inflammasome and IL-1β / I by human monocytes. Multiple donor CD4 for L-18 production IL-10 To investigate the effects of the cells, a series of experiments were performed. Conduct in vitro experiments.

[0242] First, human PBMCs were collected by Ficoll / Paque (Sigma-Aldrich) Monocytes are isolated from peripheral blood by standard density centrifugation using the Monocyte Isolation Kit II (Milt). Monocytes were isolated from human PBMCs by negative selection using the ELISA kit (enyi) according to the manufacturer's instructions. Since positive selection or adhesion can lead to unwanted activation of cells, negative selection is The preferred choice is to culture the cells in a 96-well plate in culture medium containing 3% toxin-free human AB serum. In a microtiter plate, 2 x 10 per 200 μL well 5 pieces or 1 x 10 5 Multiple donor CD4 IL-10 Where cells exist, isolated single The ball is 5 x 10 4 Seed at cells / 200 μl.

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

[0244] After treatment as outlined in Table 1, supernatants were collected from six wells per group and analyzed for IL-1β / I. IL-18 production was monitored by IL-1β specific for mature IL-1β or IL-18 (Biolegend). Measured by ISA. 6-way for groups No. 3, 10, 13, 14 and 17. Cells harvested from the cells were analyzed by Western blot to determine the levels of activated caspase-1. Check the rules.

[0245] Data from the experiment showed that multiple donor CD4 IL-10 IL-1 cells are activated by monocytes The present inventors further demonstrated that multiple donor IL-1β and IL-18 production are downregulated. CD4 IL-10 cells downregulate mature caspase-1 production in activated monocytes Furthermore, multiple donor CD4 IL-10 and multiple donor CD4 IL-10 By The IL-10 produced downregulates inflammasomes.

[0246] Similar experiments will be performed using human macrophages or dendritic cells instead of monocytes. The results showed that multiple donor CD4 IL-10 The cells are derived from activated macrophages and dendritic cells. These results suggest that IL-1β, IL-18, and mature caspase-1 production are downregulated. do.

[0247] These results are consistent with multiple donor CD4 IL-10 Cells express the NLPR3 inflammasome It is suggested that it may be used to treat diseases or disorders associated with hyperactivation. In general, multiple donor CD4 IL-10 The cells can be used to treat chronic inflammatory and autoimmune diseases. The NLPR3 inflammasome can treat exogenous or endogenous "danger signals." Signals, such as pathogen-associated molecular patterns (PAMPs), silica, asbestos, and damaged membranes. Danger-associated molecular pattern (DAMP)-like products from mitochondrial, necrotic, and stressed cells It can be activated by substances and uremic acid crystals.

[0248] 6.8.6. Experimental methods and materials Cell preparations and cell lines. Peripheral blood mononuclear cells (PBMCs) were cultured on Ficoll-Hypaque media. Prepared by centrifugation on a gradient. CD4 + T cells were isolated using a CD4 T cell isolation kit (Mi ltenyi Biotec, Bergisch Gladbach, Germany) Mature dendritic cells (DCs) were purified using a CD14 + Micro Beads (Miltenyi Biotech, Germany) were used according to the manufacturer's instructions. Positively selected peripheral blood CD14 + Generated from monocytes and supplemented with 10% fetal bovine serum (FBS) , Lonza, Italy), 100 U / mL penicillin / streptomycin (Lonz a, Italy), RPMI supplemented with 2 mM L-glutamine (Lonza, Italy). 10 ng / mL recombinant human (rh) IL-4 ( R&D Systems, Minneapolis, MN, USA) and 100ng / mL r in the presence of hGM-CSF (Genzyme, Seattle, WA) at 37°C for 5 days The cells were cultured and treated with 1 mg / mL lipopolysaccharide (LPS, Sigma, CA, USA). The seeds were then allowed to mature for two days.

[0249] Plasmid construction: The coding sequence of human IL-10 was inserted into pH15C (ATCC no. 68192) The 549 bp fragment was then multicloned into pBluKSM (Invitrogen). The plasmid pBluKSM-hIL-10 was obtained by cloning into the cloning site of the plasmid pBluKSM-hIL-10. hIL-10 was excised from SM-hIL-10 and expressed as 1074.1071.hPGK.GFP .WPRE.mhCMV.dNGFR.SV40PA (herein referred to as LV-ΔNGFR) ) to obtain a 555 bp fragment, giving LV-IL-10 / ΔNGFR. Bidirectional promoter (minimum of human PGK promoter + CMV promoter in opposite direction) The presence of the nucleotide sequence (nucleotides) allows for simultaneous expression of two transgenes (Locafaro et al., Mol T her.2017;25(10):2254-2269). The sequence of LV-IL-10 / ΔNGFR was determined by pyrosequencing. This was verified by Primm.

[0250] Vector production and titration of VSV-G-pseudotyped third-generation bidirectional lentiviral vectors Transient four-plasmid co-transfection of 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, and HIV-1 Gag p24 antigen immunocapture (NEN Life Science Product, Walsa Vector particles were measured by a ELISA (Immunology, Massachusetts) and the vector infectious titer was calculated as a function of the titer and particle size. The titer was calculated as the ratio of 5 × 10 8 From 6 x 10 9 Transducing units / mL range The infectivity titer is 5 x 10 4 From 10 5 The transducing units / ng of p24 were in the range.

[0251] CD4 IL-10Generation of cell lines using polyclonal CD4 transduced cells as previously reported (Andolfi et al. Mol Ther. 2012;20(9):1778-1790). D4 purified T cells were incubated with soluble anti-CD3 monoclonal antibody (mAb, 30 ng / mL, OKT 3, Janssen-Cilag, Raritan, NJ, USA), anti-C D28mAb (1 μg / mL, BD) and rhIL-2 (50 U / mL, PROLEUK) T cells were activated with HIV-1 vaccine (NIST, Novartis, Italy) for 48 hours. LV-IL-10 / ΔNGFR (CD4 IL-10 ) was transduced. On day 11, CD271 + Microbeads (Miltenyi Biotec, Berg isch Gladbach, Germany) using CD4 + ΔNGFR + Cells are bead-selected 5% human serum (BioWhittaker-Lonza, Washington), 100 U / mL penicillin-streptomycin (BioWhittaker) and 50 U / X-VIV containing mL of rhIL-2 (PROLEUKIN, Novartis, Italy) On days 7 and 10, the medium was supplemented with 50 U / mL of rhIL-2 On day 14, cells were harvested, washed, and incubated for 14 days as previously reported. The cells were restimulated with the allogeneic feeder mixture as described previously (Andolfi et al. Mol Ther. 2012;20(9): After 14 days, the cells were harvested and frozen. IL-10 Regenerate cells After a second and third round of restimulation and expansion, functional characterization was performed in vitro. , was used for in vivo experiments.

[0252] Vector copy number analysis was performed 11 days after transduction to remove non-integrated vector forms. The cells were cultured using the QIAamp DNA Blood Mini Kit (QIAgen Genomic DNA was isolated using a vector construct (Protein-like DNA fragment, 51106) according to the manufacturer's instructions. The mixture was then mixed with the QX200 Droplet Digital PCR System (Bi Quantification was performed by o-Rad according to the manufacturer's instructions.

[0253] Cytokine Measurements To measure cytokine production, the second and third restimulations were performed. After single-donor and multiple-donor CD4 IL-10 Cells were left unstimulated or A final volume of 200 μl of medium (96-well round-bottom plate, 2 × 10 5 Immobilized antibodies in Stimulation was performed with CD3 (10 μg / mL) and soluble anti-CD28 (1 μg / mL) mAbs. After 8 hours of culture, the supernatant was collected and analyzed for IL-10, IL-4, IL-5, IFN-γ, and IL-10. -22 levels were measured by ELISA according to the manufacturer's instructions (BD Biosciences). was measured by.

[0254] Flow cytometry analysis of granzyme B after surface staining with CD4 (clone MHG B04, Invitrogen, USA) for the expression of CD4 IL-10 Fixing cells Permeabilize and permeabilize cells using the BD Cytofix / Cytoperm™ kit according to the manufacturer's instructions. The staining was performed according to the manufacturer's instructions (Cat. No. 554714, Biolegend, USA). The stained cells were washed twice with PBS supplemented with 1% FBS and then incubated with FlowJo10 software. The analysis was performed using a BD LSRFortessa software.

[0255] Killing assay: Single-donor and multiple-donor CD4 counts after second and third restimulations. IL -10 The cytotoxicity of cells was analyzed in co-culture experiments. The target cells were K562 and ALL-CM, which are myeloid leukemia cell lines, respectively. Use CD4 IL-10 Cells were cultured at a 1:1 ratio (10 5 target cells and 10 5 CD4 IL-10 At the end of the co-culture, the cells were harvested and plated in K5 62 and ALL-CM cells were analyzed and counted by FACS.

[0256] Suppression assays single-donor and multi-donor CD4 IL-10 To measure the inhibitory potential of cells Allogeneic PBMCs were cultured in a cell proliferation kit according to the manufacturer's instructions. Dye eFluor® 670 (Invitrogen, CA, USA) After labeling with allogeneic mature DCs (5 × 10 4 cells / well) and soluble anti-CD3 ( PBMCs and suppressor cells were stimulated with mAb (50 ng / mL) at a 1:1 ratio. (10 5 PBMCs and 10 5 CD4 IL-10 After 3 days of culture, CD4 + ΔNGFR - or CD8 + ΔNGFR - eFluor670 dilutions of T cells were analyzed by F The proliferation of responding cells was examined by analysis by ACS.

[0257] Graft-versus-host disease model: 6 / 8-week-old female NSG mice were used in all experiments. On day 0, mice received 175–200 cGy from a linear accelerator depending on the mouse weight. received a single dose of total body irradiation and injected PBMC cells (5 × 10 6 ), or CD4 IL-10 cell (single donor or multiple donor (pool of 3 donors) 5 x 10 6 or 2.5 x 10 6 ), or CD4 IL-10 cells (5×10 6 or 2.5 x 10 6 Combined with PBMCs (5 × 10 6 The survival rate, weight loss, activity, hair, skin and Posture was monitored at least three times per week as previously reported (Bondanza e (t al. Blood. 2006;107(5):1828-1836). When mice lost 20% of their body weight, it was deemed ethically unacceptable. The mice were euthanized for the following reasons.

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

[0259] 7. Incorporation by Reference All publications, patents, patent applications, and other references cited in this application are hereby incorporated by reference in their entirety. Any patent, patent application or other document is expressly incorporated by reference for all purposes. and is hereby incorporated by reference in its entirety for all purposes to the same extent as if separately set forth. To be incorporated.

[0260] 8. Equivalents While various specific embodiments have been illustrated and described, the above specification is not intended to be limiting. Various modifications may be made without departing from the spirit and scope of the present invention. It will be understood that many variations will become apparent to those skilled in the art upon review of this specification. Deaf. 9. Arrays SEQ ID NO: 1 (amino acid sequence of human IL-10) JPEG2026035590000002.jpg23118 SEQ ID NO: 2 (an exemplary nt sequence of human IL-10) JPEG2026035590000003.jpg4199 SEQ ID NO: 3 (amino acid sequence of ΔNGFR) JPEG2026035590000004.jpg31118 SEQ ID NO: 4 (Exemplary nt sequence of ΔNGFR) JPEG2026035590000005.jpg7187 SEQ ID NO: 5 (nucleotide sequence of bd.ΔNGFR.PGK.IL-10) JPEG2026035590000006.jpg87118 JPEG2026035590000007.jpg172119 JPEG2026035590000008.jpg80118

Claims

1. C that has been genetically modified to contain an exogenous polynucleotide encoding IL-10 D4 ＋ a population of T cells, said CD4 ＋ T cells were collected from at least three different T cell donors. -obtained from CD4 ＋ T cell population (multiple donor CD4 ＩＬ－１０ cell).

2. The CD4 ＋ T cells are collected from 3, 4, 5, 6, 7, 8, 9, or 10 different T cell donors. The CD4 according to claim 1, obtained from ＋ A population of T cells.

3. The CD4 ＋ T cells total 6, 7, 8, 9, 10, 11, 12 or 3. The CD4 antibody of claim 1 or claim 2, having more than two different HLA haplotypes. ＋ A population of T cells.

4. All of the CD4 ＋ T cells are HLA-A, HLA-B, HLA-C, At least 5 / 1 at the HLA-DRB1 and HLA-DQB1 loci 4. Any of claims 1 to 3 having a match of 0, 6 / 10, 7 / 10, 8 / 10, or 9 / 10. The CD4 according to any one of claims 1 to 4 ＋ A population of T cells.

5. All of the CD4 ＋ T cells are HLA-A, HLA-B, HLA-C, and at least 4 / 8, 5 / 8, 6 / 8, 7 / 8 or 8 / 8 match of any one of claims 1 to 4. ＋ T A population of cells.

6. All of the CD4 ＋ T cells have two copies of each other at the HLA-A locus.

6. The CD4 according to claim 1, having a match of 1 / 2. ＋ A population of T cells.

7. All of the CD4 ＋ T cells have two copies of each other at the HLA-B locus.

7. The CD4 according to claim 1, wherein the CD4 ＋ A population of T cells.

8. All of the CD4 ＋ T cells have two copies of each other at the HLA-C locus.

8. The CD4 according to claim 1, wherein the CD4 ＋ A population of T cells.

9. All of the CD4 ＋ T cells express HLA-DRB1 and HLA-DQB1 10. The method of claim 1, wherein the loci have at least 3 / 4 or 4 / 4 identity with each other. CD4 according to any one of ＋ A population of T cells.

10. All CD4 ＋ T cells are A ＊ 02 allele of claim 1 9. The CD4 ＋ A population of T cells.

11. Any CD4 ＋ 11. The method of claim 1, wherein the T cells are also not immortalized. CD4 ＋ A population of T cells.

12. The exogenous polynucleotide is operably linked to an expression control element.

12. The method of claim 1, comprising a polynucleotide segment encoding . CD4 as described ＋ A population of T cells.

13. 13. The method according to any one of claims 1 to 12, wherein the IL-10 is human IL-10. D4 ＋ A population of T cells.

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

15. The polynucleotide segment encoding IL-10 has the sequence of SEQ ID NO:

1. The CD4 gene according to any one of claims 1 to 14, which encodes a protein ＋ T cell Group.

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

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

18. 10. The method of claim 1, wherein the exogenous polynucleotide further comprises a sequence encoding a selectable marker. 17 to 19. ＋ A population of T cells.

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

20. 20. The CD4 of claim 19, wherein the ΔNGFR has the sequence of SEQ ID NO:

3. ＋ T cell collection Group.

21. 20. The CD4 of claim 19, wherein the exogenous polynucleotide comprises the sequence of SEQ ID NO:

4. ＋ A population of T cells.

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

23. 23. Any of claims 1 to 22, wherein the exogenous polynucleotide has the sequence of SEQ ID NO:

5. The CD4 ＋ A population of T cells.

24. 23. The method of claim 1, wherein the exogenous polynucleotide is integrated into the T cell nuclear genome. CD4 according to any one of ＋ A population of T cells.

25. 3. The method of claim 1, wherein the exogenous polynucleotide is not integrated into the T cell nuclear genome.

3. The CD4 ＋ A population of T cells.

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

27. 3. The method of claim 1, wherein the exogenous polynucleotide is not integrated into the T cell nuclear genome.

6. The CD4 ＋ A population of T cells.

28. The CD4 ＋ At least 90% of the T cells express IL-10.

28. The CD4 according to any one of claims 1 to 27. ＋ A population of T cells.

29. The CD4 ＋ At least 95% of the T cells express IL-10. CD4 according to 28 ＋ A population of T cells.

30. The CD4 ＋ At least 98% of the T cells express IL-10. CD4 according to 29 ＋ A population of T cells.

31. The genetically modified CD4 ＋ T cells are CD4 ＋ T cell 10 ６ per ml of culture medium 31. A method according to claim 1, wherein the method constitutively expresses at least 100 pg of IL-10. The CD4 ＋ A population of T cells.

32. The genetically modified CD4 ＋ T cells are CD4 ＋ T cell 10 ６ At least 1 per ml 00 pg, 200 pg, 500 pg, 1 ng, 5 ng, 10 ng, or 50 ng of IL- 32. The CD4 of claim 31, which constitutively expresses . ＋ A population of T cells.

33. The genetically modified CD4 ＋ T cells after activation with anti-CD3 and anti-CD28 antibodies , CD4 ＋ T cell 10 ６ expressing at least 1 ng of IL-10 per cell / ml. Item 33. The CD4 according to any one of items 1 to 32. ＋ A population of T cells.

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

35. The genetically modified CD4 ＋ T cells are unmodified CD4 ＋ At least five times higher than T cells IL-10 is expressed in the CD4+ / CD ... ＋ T cell A group of.

36. The genetically modified CD4 ＋ T cells are unmodified CD4 ＋ At least 10 times higher than T cells 36. The CD4 of claim 35, which expresses IL-10 at a level ＋ A population of T cells.

37. The CD4 ＋ At least 90% of T cells express the exogenous polynucleotide.

37. The CD4 vector of claim 1, wherein the vector expresses the selectable marker. ＋ T thin A group of cells.

38. The CD4 ＋ At least 95% of T cells express the exogenous polynucleotide.

38. The CD4 vector of claim 37, which expresses the selectable marker from ＋ A population of T cells.

39. The CD4 ＋ At least 98% of T cells express the exogenous polynucleotide.

39. The CD4 vector of claim 38, wherein the vector expresses the selectable marker. ＋ A population of T cells.

40. The genetically modified CD4 ＋ 40. Any of claims 1 to 39, wherein the T cells express CD49b. The CD4 ＋ A population of T cells.

41. The genetically modified CD4 ＋ Any of claims 1 to 40, wherein the T cells express LAG-3. The CD4 ＋ A population of T cells.

42. The genetically modified CD4 ＋ 42. Any of claims 1 to 41, wherein the T cells express TGF-β. The CD4 ＋ A population of T cells.

43. Genetically modified CD4 ＋ 43. The method of claim 1, wherein the T cells express IFNγ. CD4 as described ＋ A population of T cells.

44. The genetically modified CD4 ＋ 44. Any one of claims 1 to 43, wherein the T cells express GzB. CD4 described in ＋ A population of T cells.

45. The genetically modified CD4 ＋ 45. The method of claim 1, wherein the T cells express perforin. The CD4 ＋ A population of T cells.

46. The genetically modified CD4 ＋ 46. ​​The method of claim 1, wherein the T cells express CD18. The CD4 ＋ A population of T cells.

47. The genetically modified CD4 ＋ 47. Any one of claims 1 to 46, wherein the T cells express CD2. CD4 described in ＋ A population of T cells.

48. The genetically modified CD4 ＋ 48. Any of claims 1 to 47, wherein the T cells express CD226. The CD4 ＋ A population of T cells.

49. The genetically modified CD4 ＋ Any of claims 1 to 48, wherein the T cells express IL-22. The CD4 ＋ A population of T cells.

50. The CD4 ＋ T cells become anergic in the presence of host-derived peripheral blood mononuclear cells (PBMCs).

50. The CD4 antibody of claim 1, wherein the antibody is unmodified. ＋ A population of T cells.

51. The CD4 ＋ T cells were not anergized in the presence of recombinant IL-10 protein. First, the recombinant IL-10 protein is ＋ 10. The method of claim 1, wherein the antibody is not expressed by T cells.

50. The CD4 ＋ A population of T cells.

52. The CD4 ＋ T cells are not anergized in the presence of host-derived DC10 cells; 52. The CD4 of any one of claims 1 to 51. ＋ A population of T cells.

53. The CD4 ＋ 53. The method of any one of claims 1 to 52, wherein the T cells are in a frozen suspension. CD4 ＋ A population of T cells.

54. The CD4 ＋ 53. The method of any one of claims 1 to 52, wherein the T cells are in liquid suspension. CD4 ＋ A population of T cells.

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

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

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

58. Multiple donor CD4 ＩＬ－１０ 1. A method of producing a cell, comprising: (i) Primary CD4 T cells from at least three different T cell donors ＋ Obtaining T cells; (ii) introducing an exogenous polynucleotide encoding IL-10 into each domain; Nurse's CD4 ＋ separately modifying the T cells, and then (iii) the genetically modified CD4 ＋ T cells are pooled, thereby D4 ＩＬ－１０ and obtaining the cells.

59. After step (i) and before step (ii), or after step (ii), or and before step (iii), or after step (iii), The primary CD4 ＋ T cells are treated with anti-CD3 antibody and anti-CD28 antibody, or anti-CD3 antibody and The method further comprises incubating the mixture in the presence of beads coated with CD28 antibody.

59. The method of claim 57 or claim 58.

60. The primary CD4 ＋ 59. The T cells are further incubated in the presence of IL-2. The method described below.

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

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

63. The exogenous polynucleotide is a sequence encoding IL-10 having the sequence of SEQ ID NO:

1.

63. The method of any one of claims 57 to 62, comprising a segment.

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

65. wherein the exogenous polynucleotide further comprises a segment encoding a selectable marker.

65. The method of any one of claims 57 to 64.

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

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

3. 。

68. After step (ii), the genetically modified CD4 expressing the selection marker ＋ T cells alone thereby generating genetically modified CD4 ＋ The method further comprises generating an enriched population of T cells.

68. The method of any one of claims 65 to 67, comprising:

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

70. the genetically modified CD4 ＋ At least 98% of T cells express IL-1 69. The method of claim 68, wherein the gene expresses 0.

71. the genetically modified CD4 ＋ At least 90% or less of the T cells 70. The method of claim 68, wherein at least 95% of the cells express the selectable marker. method.

72. the genetically modified CD4 ＋ At least 98% of the T cells 71. The method of claim 70, wherein the marker is expressed.

73. The genetically modified CD4 ＋ The method further comprises incubating the enriched population of T cells.

73. The method of any one of claims 68 to 72.

74. The genetically modified CD4 ＋ Incubating the enriched population of T cells includes incubating the enriched population of T cells with IL- In the presence of 2, the cells were coated with anti-CD3 antibody and anti-CD28 antibody, or CD3 antibody and CD28 antibody.

74. The method of claim 73, wherein the method is carried out in the presence of coated beads.

75. The genetically modified CD4 ＋ 57 to 59, further comprising the subsequent step of freezing the T cells.

74. The method of any one of claims 74 to 74.

76. In step (i), the primary CD4 ＋ T cells are 3, 4, 5, 6, 7, 8, 9 or 1 76. The method of any one of claims 57 to 75, wherein the T cells are obtained from 0 different T cell donors. Law.

77. The at least three T cell donors are HLA-A, HLA-B, HLA-C, HLA-D, HLA-E, HLA-F, HLA-G, HLA-H, HLA-H, HLA-H, HLA-H, HLA-I ... - At least 5 / 10, 6 / 10, and 6 / 10 alleles at the DRB1 and HLA-DQB1 loci, respectively 77. The method of claim 76, wherein the method has a match of 10, 7 / 10, 8 / 10, or 9 / 10. How to post.

78. The at least three T cell donors are HLA-A, HLA-B, HLA-C and HLA-D. At least 4 / 8, 5 / 8, 6 / 8, 7 / 8 or 78. The method of any one of claims 57 to 77, wherein: has an 8 / 8 agreement.

79. The at least three T cell donors share a 2 / 2 identity with each other at the HLA-A locus.

79. The method of any one of claims 57 to 78, comprising a match.

80. The at least three T cell donors are 2 / 2 identical at the HLA-B locus.

80. The method of any one of claims 57 to 79, comprising a match.

81. The at least three T cell donors are 2 / 2 identical at the HLA-C locus.

81. The method of any one of claims 57 to 80, comprising a match.

82. The at least three T cell donors have HLA-DRB1 and HLA-DQB1 genes.

82. The method of claim 57 to 81, wherein the loci have at least 3 / 4 or 4 / 4 identity with each other.

10. The method according to any one of claims 1 to 9.

83. Each of the at least three T cell donors is A ＊ 57. Having the 02 allele 83. The method of any one of claims 1 to 82.

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

85. In step (i), the primary CD4 ＋ The T cells are T cells from the at least three different individuals.

84. The method of any one of claims 57 to 83, obtained from non-frozen peripheral blood mononuclear cells of a donor. How to do it.

86. CD4 ＋ 86. The method of claim 85, further comprising isolating T cells. How to post.

87. 1. A method of treating a patient, comprising:

56. The multi-donor CD4 of any one of claims 1 to 55. ＩＬ－１０ Cells or claims 57. A method comprising administering the pharmaceutical composition of 56 to a patient in need of immune tolerization.

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

89. The multiple donor CD4 ＩＬ－１０ the cells or the pharmaceutical composition are capable of inhibiting pathogenicity in the patient 89. The method of claim 87 or 88, which prevents or reduces the severity of a T cell response.

90. 90. Any one of claims 87 to 89, further comprising administering mononuclear cells to said patient. The method described below.

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

92. The mononuclear cells are ＩＬ－１０ Before administration of the cells or the pharmaceutical composition 91. The method of claim 90, wherein the administration is either at or after the first day of the first trimester.

93. The pooled donor CD4 ＩＬ－１０ Either before or after administration of the cells or pharmaceutical composition or administering hematopoietic stem cells (HSCs) of an HSC donor to said patient.

93. The method of any one of claims 87 to 92, further comprising:

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

95. The HSC donor is HLA-A, HLA-B, HLA-C, HLA-D, HLA-E, HLA-F, HLA-H ... At the A-DRB1 and HLA-DQB1 loci, 5 / 10, 6 / 10, and 7 / 10; 95. The method of claim 94, having an agreement of less than 8 / 10, 9 / 10, or 10 / 10.

96. The HSC donor provides the patient with HLA-A, HLA-B, HLA-C, and HLA-D. At the LA-DRB1 locus, 4 / 8, 5 / 8, 6 / 8, 7 / 8, or less than 8 / 8 mutations 95. The method of claim 94, comprising a match.

97. The HSC donor has HLA-A, HLA-B, or HLA-C genotype for the patient.

95. The method of claim 94, having less than 2 / 2 matches at the locus.

98. The HSC donor has HLA-DRB1 and HLA-DQB1 genes 95. The method of claim 94, having less than 3 / 4 or less than 4 / 4 matches at the locus.

99. One or more of the T cell donors are HLA-mismatched or partially HLA-matched to the patient.

99. The method of any one of claims 87 to 98, wherein the LA mismatch.

100. One or more of the T cell donors may provide HLA-A, HLA-B, At the HLA-C, HLA-DRB1, and HLA-DQB1 loci, 5 / 10, 6 / 100, 7 / 10, 8 / 10, 9 / 10, or 10 / 10 agreement. The method described.

101. One or more of the T cell donors may provide HLA-A, HLA-B, At the HLA-C and HLA-DRB1 loci, 4 / 8, 5 / 8, 6 / 8, 7 / 8 or 100. The method of claim 99, wherein: has less than 8 / 8 agreement.

102. One or more of the T cell donors may be HLA-A, HLA-B or 100. The method of claim 99, wherein said HIV-1 mutated human has less than a 2 / 2 match at the HLA-C locus.

103. One or more of the T cell donors provide HLA-DRB1 and HL-DRB2 to the patient. A-DQB1 locus with less than 2 / 4, 3 / 4 or 4 / 4 matching.

99. The method according to claim 99.

104. One or more of the T cell donors are HLA-mismatched or HLA-mismatched to the HSC donor.

104. The method of any one of claims 87 to 103, wherein there is partial HLA mismatch.

105. One or more of the T cell donors may have HLA-A, HL-A, or IL-1 deficiency with respect to the HSC donor. 5 / 1 at the AB, HLA-C, HLA-DRB1 and HLA-DQB1 loci Claims with 0, 6 / 10, 7 / 10, 8 / 10, 9 / 10 or less than 10 / 10 matches Item 105. The method according to item 104.

106. One or more of the T cell donors may have HLA-A, HL-A, or IL-1 deficiency with respect to the HSC donor. At the AB, HLA-C, and HLA-DRB1 loci, 4 / 8, 5 / 8, 6 / 8, 105. The method of claim 104, having a match of less than 7 / 8 or 8 / 8.

107. One or more of the T cell donors may have HLA-A, HL-A, or IL-1 deficiency with respect to the HSC donor.

105. The method of claim 104, having less than a 2 / 2 match at the A-B or HLA-C locus. How to do it.

108. One or more of the T cell donors have HLA-DRB1 and having a 3 / 4 or less than 4 / 4 match at the HLA-DQB1 locus.

104. The method according to claim 104.

109. The multiple donor CD4 ＩＬ－１０ The cells or the pharmaceutical composition are 109. The method of claim 79, wherein the method prevents or reduces the severity of GvHD caused by the cells. The method described.

110. The multiple donor CD4 ＩＬ－１０ The cells or the pharmaceutical composition are the transplanted hematopoietic cells. Prevent or reduce the severity of a pathogenic response in lymphoid cells derived from the antibody of claim 93 to 109.

10. The method according to any one of the preceding claims.

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

112. 12. The method of claim 11, wherein the neoplastic cells express CD13, HLA-class I, and CD54.

1. The method according to claim 1.

113. 112 or 113, wherein the neoplastic cells express CD112, CD58, or CD155.

113. The method of any one of claims 112 to 113.

114. 111 or 112, wherein the patient has cancer, and optionally the cancer is a solid or hematological neoplasm.

113. The method of any one of claims 113 to 113.

115. The patient is a patient with adrenal cancer, anal cancer, bile duct cancer, bladder cancer, bone cancer, brain / CNS tumors in adults, pediatric Brain / CNS tumors, breast cancer, male breast cancer, cancer of unknown primary, Castleman's disease, cervical cancer, colon / Rectal cancer, endometrial cancer, esophageal cancer, Ewing's tumor, eye cancer, gallbladder cancer, gastrointestinal carcinoid tumor , gastrointestinal stromal tumor (GIST), gestational trophoblastic disease, Hodgkin's disease, Kaposi's sarcoma, kidney cancer, throat Head and hypopharyngeal cancer, leukemia, acute lymphocytic leukemia (ALL), acute myeloid sarcoma and skin cancer leukemia (AML), chronic lymphocytic (CLL), chronic myeloid leukemia (CML), chronic bone marrow leukemia Myelomonocytic leukemia (CMML), childhood leukemia, liver cancer, lung cancer, non-small cell lung cancer, small cell lung cancer , pulmonary carcinoid tumor, lymphoma, cutaneous lymphoma, malignant mesothelioma, multiple myeloma, myelodysplastic syndromes rhinoplasty syndrome, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, non-Hodgkin's lymphoma, pediatric Non-Hodgkin's lymphoma, oral and oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, penile cancer, pituitary tumor , prostate cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma - adult soft tissue cancer, skin cancer, Skin cancer - basal cell and squamous cell, Skin cancer - melanoma, Skin cancer - Merkel cell, Small intestine cancer, Gastric cancer, Testicular cancer, thymic cancer, thyroid cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Waldenstrom's macroglobulinemia 87 to 89, wherein the patient has a cancer selected from the group consisting of erythrocyte sedimentation syndrome (ERS), ... and Wilms' tumor.

114. The method of any one of claims 114 to 114.

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

117. 116. The method of claim 115, wherein the patient has AML or CML.

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

119. The inflammatory or autoimmune disease is type 1 diabetes, autoimmune uveitis, or rheumatoid arthritis. , psoriasis, psoriatic arthritis, multiple sclerosis, systemic lupus, inflammatory bowel disease, Addison's disease, gray Usu disease, Sjogren's syndrome, Hashimoto's thyroiditis, myasthenia gravis, autoimmune vasculitis, pernicious poverty ulcerative colitis, bullous disease, scleroderma, and celiac disease; The method of claim 118.

120. The inflammatory or autoimmune disease is selected from the group consisting of Crohn's disease, ulcerative colitis, celiac disease, type 1 diabetes, and the like.

120. The method of claim 119, wherein the disease is rheumatoid arthritis, lupus, psoriasis, psoriatic arthritis, or rheumatoid arthritis. 。

121. the patient has a disease or disorder associated with hyperactivity of the NLPR3 inflammasome.

121. The method of any one of paragraphs 87 to 113 or 118 to 120.

122. the patient has type 2 diabetes, a neurodegenerative disease, a cardiovascular disease, or an inflammatory bowel disease.

122. The method of any one of paragraphs 87 to 113 or 118 to 121.

123. The patient has increased IL-1β production by activated monocytes, macrophages, or dendritic cells.

122. Any one of claims 87 to 113 or 118 to 121, having an associated disease or disorder The method described below.

124. The patient has increased IL-18 production by activated monocytes, macrophages, or dendritic cells.

122. Any one of claims 87 to 113 or 118 to 121, having an associated disease or disorder The method described below.

125. The patient has a history of suppressing mature caspase-1 production by activated monocytes, macrophages, or dendritic cells.

122. Any of claims 87 to 113 or 118 to 121, wherein the patient has a disease or disorder associated with an increase 1. The method according to claim 1.

126. 114. Any of claims 87 to 113, wherein the patient has an allergic or atopic disorder.

1. The method according to claim 1.

127. The allergic or atopic disease is selected from the group consisting of asthma, atopic dermatitis, and rhinitis.

127. The method of claim 126, wherein the

128. 114. The method of any one of claims 87 to 113, wherein the patient has a food allergy. Law.

129. The CD4 ＋ The patient is administered a population of T cells or the pharmaceutical composition either before or after administration.

114. The method of any one of claims 87 to 113, further comprising the step of transplanting the organ into a subject. 。

130. The multiple donor CD4 ＩＬ－１０ The cells or the pharmaceutical composition are capable of inhibiting host rejection of the organ transplant.

130. The method of claim 129, wherein the method prevents or reduces the severity of

131. The CD4 ＋ iPS cells either before or after administration of the population of T cells or the pharmaceutical composition.

113. The method of claim 87, further comprising transplanting cells or tissue derived from the cells into the patient.

10. The method according to any one of the preceding claims.

132. The multiple donor CD4 ＩＬ－１０ The cells or the pharmaceutical composition prevent host rejection of the cell transplant.

132. The method of claim 131, wherein the method prevents or reduces the severity of

133. The multiple donor CD4 ＩＬ－１０ Before or after administration of the cells or the pharmaceutical composition, 114. Any one of claims 87 to 113, further comprising administering to the patient an AAV. The method described below.

134. The multiple donor CD4 ＩＬ－１０ The cells or the pharmaceutical composition are capable of expressing the recombinant AAV. The method of claim 133, wherein the method reduces an immune response caused by the antibody.

135. 88. The patient has an exaggerated immune response to a viral or bacterial infection.

114. The method of any one of claims 1 to 113.

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

137. detecting a selectable marker in a biological sample obtained from said patient; Thereby, multiple donor CD4 ＩＬ－１０ further comprising detecting the presence or absence of T cells.

137. The method of any one of claims 87 to 136.

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

139. 1. A method of treating a patient having a malignant tumor, comprising: administering an allogeneic HSCT graft to said patient; Therapeutically effective amounts of multiple donor CD4 ＩＬ－１０ and administering the cells.

140. The multiple donor CD4 ＩＬ－１０ The CD4 ＩＬ－１０ None of the cell donors 140. The method of claim 139, which is not the donor of the HSCT graft.

141. 1. A method of treating hematological cancer, comprising: Multiple donor CD4 sufficient to induce anti-cancer effects ＩＬ－１０ The cells are injected into blood cancer patients providing a The multiple donor CD4 ＩＬ－１０ Cells were obtained from at least three different T cell donors. and vector-borne expression of the coding sequence for human IL-10 under the control of a constitutive promoter. Genetically modified CD4 by transgenesis ＋ The method comprises administering to a subject a T cell.

142. The multiple donor CD4 ＩＬ－１０ Before or after administration of the cells, the allogeneic HSCT graft is 142. The method of claim 141, further comprising administering to a patient.

143. The multiple donor CD4 ＩＬ－１０ The amount of cells is related to the graft-versus-leukemia (GvL) of allogeneic HSCT. ) or graft-versus-host disease (GvHD) without suppressing graft-versus-tumor (GvT) efficacy.

143. The method of claim 142, wherein the amount is further sufficient to suppress.

144. 144. The method of any one of claims 141 to 143, wherein the blood cancer is myeloid leukemia. Law.

145. The multiple donor CD4 ＩＬ－１０ The cells target and kill cancer cells that express CD13.

143. The method of any one of claims 141 to 142, wherein

146. The multiple donor CD4 ＩＬ－１０ The cells target cancer cells that express HLA-class I.

146. The method of any one of claims 141 to 145, wherein the bacteria are killed.

147. 147. Any of claims 141 to 146, wherein the myeloid leukemia is acute myeloid leukemia (AML). The method according to any one of claims 1 to 4.

148. The allogeneic HSCT graft may be obtained from a related or unrelated donor to the recipient.

148. The method of any one of claims 141 to 147,

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

150. wherein the multi-donor CD4IL-10 cells are allogeneic to the recipient.

149. The method of any one of paragraphs 141 to 148.

151. The multiple donor CD4 ＩＬ－１０ The cells may be adapted to react with the host's alloantigens prior to administration to said host.

149. The method of any one of claims 141 to 148, wherein the antibody is not anergized.

152. The multiple donor CD4 ＩＬ－１０ Claims 141 to 148, wherein the cell is a Tr1-like cell.

10. The method according to any one of the preceding claims.

153. The multiple donor CD4 ＩＬ－１０ 14. The cells are polyclonal.

9. The method according to any one of claims 8 to 8.

154. The multiple donor CD4 ＩＬ－１０ The cells are polyclonal and 149. The method of any one of claims 141 to 148, wherein the gene is non-autologous.

155. The multiple donor CD4 ＩＬ－１０ After cells were isolated from at least three donors, 149. The method of any one of claims 141 to 148, wherein the method is genetically modified.

156. None of the at least three donors is the same donor as the allogeneic HSCT donor. The method of claim 155.

157. The allogeneic HSCT graft may be obtained from a donor that is matched or mismatched to the recipient.

157. The method of any one of claims 141 to 156,

158. The multiple donor CD4 ＩＬ－１０ The cells target and kill cells expressing CD54.

158. The method of any one of claims 141 to 157,

159. The multiple donor CD4 ＩＬ－１０ Cancer whose cells express HLA-class I and CD54 159. A method according to any one of claims 141 to 158, wherein the method targets and kills cells.

160. The multiple donor CD4 ＩＬ－１０ The cells target and kill cancer cells that express CD112.

160. The method of any one of claims 141 to 159, wherein the method further comprises:

161. The multiple donor CD4 ＩＬ－１０ The cells target and kill cancer cells that express CD58.

161. The method of any one of claims 141 to 160,

162. The multiple donor CD4 ＩＬ－１０ The cells target and kill cancer cells in the host.

162. The method according to any one of claims 141 to 161.

163. 1. A method of treating hematological cancer by allogeneic hematopoietic stem cell transplantation (allo-HSCT), comprising: administering an allogeneic HSCT graft to the subject (host); The graft-versus-leukemia (GvL) or graft-versus-tumor (GvT) efficacy of the allogeneic HSCT graft Multiple donors in sufficient quantities to suppress graft-versus-host disease (GvHD) without suppressing the immune system CD4 ＩＬ－１０ administering the cells to a recipient (host) of the allogeneic HSCT. Including, The multiple donor CD4 ＩＬ－１０ Cells were obtained from at least three different T cell donors. and vector-mediated transfection of the coding sequence of human IL-10 under the control of a constitutive promoter. Genetically modified CD4 by transgenesis ＋ comprising T cells, The multiple donor CD4 ＩＬ－１０ The cells are non-autologous to the recipient and The allogeneic HSCT donor is non-autologous, The multiple donor CD4 ＩＬ－１０ The cells may be adapted to react with the host's alloantigens prior to administration to said host. and not anergized, The multiple donor CD4 ＩＬ－１０ The method wherein the cells are polyclonal and Tr1-like.

164. 1. A method of treating hematological cancer by allogeneic hematopoietic stem cell transplantation (allo-HSCT), comprising: administering an allogeneic HSCT graft to the subject (host); The graft-versus-leukemia (GvL) or graft-versus-tumor (GvT) efficacy of the allogeneic HSCT graft Multiple donors in sufficient quantities to suppress graft-versus-host disease (GvHD) without suppressing the immune system CD4 ＩＬ－１０ administering the cells to a recipient (host) of the allogeneic HSCT. Including, The multiple donor CD4 ＩＬ－１０ Cells were obtained from at least three different T cell donors. and vector-mediated transfection of the coding sequence of human IL-10 under the control of a constitutive promoter. Genetically modified CD4 by transgenesis ＋ comprising T cells, The multiple donor CD4 ＩＬ－１０ the cells target and kill cancer cells in the host; The multiple donor CD4 ＩＬ－１０ The cells may be adapted to react with the host's alloantigens prior to administration to said host. and not anergized, The multiple donor CD4 ＩＬ－１０ The cells are non-autologous to the recipient and A method that is reclonal and Tr1-like.

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