CD4+ T CELLS EXPRESSING IL-10 AND CHIMERIC ANTIGEN RECEPTORS AND USES THEREOF

Abstract

The present disclosure relates to a method for the production of CD4 + CD4 produced by genetically modifying T cells IL-10 / CAR Provide cell populations (autologous or allogeneic single donor and allogeneic polydonor). In addition, CD4 IL-10 / CAR Methods for generating CD4 cells and for immune tolerance IL-10 / CAR Methods of using the cells to treat GvHD, cell and organ transplantation, cancer, and other autoimmune and inflammatory disorders are provided.

Description

[Technical Field]

[0001] 1. CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 63 / 295,491, filed December 30, 2021, which is incorporated herein by reference in its entirety.

[0002] 2. Sequence Listing This application has been submitted in XML format and contains a Sequence Listing, which is incorporated herein by reference in its entirety. The XML copy was created on December 24, 2022, is named 37104-50735-Sequence-Listing.xml, and is 95.2 kilobytes (KB) in size. [Background technology]

[0003] 3.Background Regulatory T cells (TCR) are a small but important subset of T cells that maintain immune tolerance to self- and non-pathogenic antigens and maintain homeostasis of the immune system. There are two major populations of TCR: CD4 + FOXP3 + CD25 + T cells (FOXP3 + ) cells and type 1 regulatory T (Tr1) cells. + Both Tr1 and Tr1 cells downregulate pathogenic T cell responses in various preclinical organ and islet transplant models, graft-versus-host disease (GvHD), and various autoimmune and inflammatory diseases.

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

[0005] Despite these encouraging results, the production of donor-derived or autologous Tr1 cells for large-scale treatment of patients with high unmet medical need is not always feasible and is very laborious, and it is not possible to generate large quantities of pure Tr1 cells.

[0006] Recently, Locafaro and coworkers reported purified CD4 + We circumvented some of these problems by transducing T cells with a bidirectional lentiviral vector containing the human IL-10 gene. IL-10 The population shared key features of naturally occurring Tr1 cells. Like Tr1 cells, single-donor CD4 IL-10 The cells produce high levels of IL-10 and express allogeneic CD4 + T cells and allogeneic CD8 + In addition, they are directly cytotoxic to both normal myeloid cells (including antigen-presenting cells, APCs) and myeloid leukemia cells. In a humanized xenograft-versus-host disease (GvHD) model, these single-donor CD4 IL-10 The cells have been shown to be effective in reducing graft-versus-leukemia (GvHD) while retaining GvL activity. See Locafaro et al. Mol Ther. 2017;25(10):2254-2269 and WO 2016 / 146,542.

[0007] While simultaneously preserving the immunosuppressive and homeostatic activities of these cells, CD4 IL-10There is a need for a means to redirect the cytotoxic potential of cells to non-myeloid cells. Summary of the Invention

[0008] 4. Overview The present disclosure describes a novel type of immune cell, CD4, that is engineered to express both exogenous IL-10 and a chimeric antigen receptor (CAR). IL-10 / CAR The present inventors have surprisingly discovered that expression of a CAR in addition to IL-10 redirects the cytotoxic properties of these Tr1-like cells toward therapeutically useful targets other than myeloid targets, without affecting the immunoregulatory properties of the cells. IL-10 / CAR Methods of treatment using cells, CD4 for treatment IL-10 / CAR The use of cells and CD4 in the manufacture of medicines to treat a variety of disorders, where CARs direct the cytotoxic properties of these Tr1-like cells to therapeutically useful targets other than myeloid cells, without affecting the immunomodulatory properties of the cells. IL-10 / CAR Uses of the cells are also described.

[0009] The present disclosure provides a genetically modified CD4+ antibody comprising: (a) a first exogenous polynucleotide segment encoding a chimeric antigen receptor (CAR); and (b) a second exogenous polynucleotide segment encoding interleukin-10 (IL-10). + T cells (CD4 IL10 / CAR ) is provided.

[0010] In some embodiments, the first exogenous polynucleotide segment comprises a regulatory element operably linked to the coding sequence of the CAR. In some embodiments, the regulatory element drives constitutive expression of the CAR.

[0011] In some embodiments, the CAR comprises an antigen-binding domain, a hinge region, a transmembrane domain, and an intracellular signaling domain.

[0012] In some embodiments, the antigen-binding domain comprises a single-chain antibody fragment. In some embodiments, the single-chain antibody fragment comprises a single-chain Fv (scFv).

[0013] In some embodiments, the antigen binding domain targets an antigen associated with an autoimmune disease, an inflammatory disorder, or cancer.

[0014] In some embodiments, the antigen is, in the context of the associated MHC molecule, CD19, CD20, CD22, BCMA, CD27, CD38, B7-H3, CD23, Lyml, Lym2, CLEC5A, CDH179b, FLT3, GCC, Muc, CSF2RA, GFRa4, CD32, CD33, CEA, IL1lRa, IL13Ra, NYBRI, SLea, CD200R, TGFbetaR2, CD276, TROP2, LAMP1, PTK7, DLL3, CDH1, CDH6, CDH17, CDH19, TSHR, tyrosinase, HLA-A * 02. HLA-A * 24, or citrullinated peptides, insulin, MOG, GAD65, IA2, gliadin, and desmoglein.

[0015] In some embodiments, the antigen binding domain comprises an anti-CD19 antigen binding domain. In some embodiments, the anti-CD19 antigen binding domain comprises the sequence of SEQ ID NO: 11.

[0016] In some embodiments, the antigen binding domain is an anti-CD20 antigen binding domain. In some embodiments, the anti-CD20 antigen binding domain comprises the sequence of SEQ ID NO: 18.

[0017] In some embodiments, the antigen-binding domain is an anti-BCMA antigen-binding domain. In some embodiments, the anti-BCMA antigen-binding domain comprises the sequence of SEQ ID NOs: 50-53.

[0018] In some embodiments, the hinge region is selected from a human CD8 hinge region, a human CD28 hinge region, an IgG1 hinge region, or an IgG4 hinge region. In some embodiments, the hinge region is derived from human CD8.

[0019] In some embodiments, the transmembrane domain is selected from the group consisting of a TNFRSF19 transmembrane domain, a CD3 zeta transmembrane domain, a CD8α transmembrane domain, a CD4 transmembrane domain, a CD28 transmembrane domain, and a B7 family-induced costimulatory (ICOS) transmembrane domain. In some embodiments, the transmembrane domain is derived from a CD8α transmembrane domain.

[0020] In some embodiments, the CAR further comprises one or more costimulatory domains, hi some embodiments, the CAR comprises two costimulatory domains.

[0021] In some embodiments, the one or more costimulatory domains are selected from the group consisting of 4-1BB, CD28, OX40, ICOS, CD27, MYD88-CD40, and KIR2DS2.

[0022] In some embodiments, one of the one or more costimulatory domains is derived from CD28.

[0023] In some embodiments, the second costimulatory domain is derived from 4-1BB.

[0024] In some embodiments, the intracellular signaling domain comprises an immunoreceptor tyrosine-based activation motif (ITAM). In some embodiments, the immunoreceptor tyrosine-based activation motif (ITAM) is derived from CD3 zeta.

[0025] In some embodiments, the CAR comprises an anti-CD19 antigen binding domain, an anti-BCMA antigen binding domain, or an anti-CD20 antigen binding domain; a human CD8 hinge region; a TNFRSF19 transmembrane region; a 4-1BB costimulatory domain; and a CD3 zeta chain intracellular signaling domain. In some embodiments, the CAR comprises an anti-CD19 antigen binding domain, an anti-BCMA antigen binding domain, or an anti-CD20 antigen binding domain; a human CD8 hinge region; a CD8α transmembrane region; a 4-1BB costimulatory domain; and a CD3 zeta chain intracellular signaling domain.

[0026] In some embodiments, the CAR comprises the sequence of SEQ ID NO: 9, 16, 22, 34, 41-49, or 54.

[0027] In some embodiments, the first exogenous polynucleotide segment comprises the sequence of SEQ ID NO: 10, 17, 23, 35, or 55.

[0028] In some embodiments, the first exogenous polynucleotide segment is integrated into the T cell nuclear genome.

[0029] In some embodiments, the first exogenous polynucleotide segment is not integrated into the T cell nuclear genome.

[0030] In some embodiments, the first exogenous polynucleotide segment is present in a vector.

[0031] In some embodiments, the second exogenous polynucleotide segment comprises a regulatory element operably linked to a coding sequence for IL-10. In some embodiments, the IL-10 is human IL-10. In some embodiments, the IL-10 is viral IL-10.

[0032] In some embodiments, the IL-10 is a protein having the sequence of SEQ ID NO: 1. In some embodiments, the second exogenous polynucleotide segment comprises the sequence of SEQ ID NO:2.

[0033] In some embodiments, the regulatory element drives constitutive expression of IL-10.

[0034] In some embodiments, the second exogenous polynucleotide segment is integrated into the T cell nuclear genome.

[0035] In some embodiments, the second exogenous polynucleotide segment is not integrated into the T cell nuclear genome.

[0036] In some embodiments, the second exogenous polynucleotide segment is present in a vector.

[0037] In some embodiments, the first exogenous polynucleotide segment and the second exogenous polynucleotide segment are present in the same vector.In some embodiments, the vector is a viral vector.In some embodiments, the vector is a lentiviral vector.

[0038] In some embodiments, CD4 + T cells are CD4 + T cells 10 6 Constitutively express at least 100 pg of IL-10 per cell / mL culture medium.

[0039] In some embodiments, CD4 + T cells are CD4 + T cells 10 6 Constitutively express at least 100 pg, 200 pg, 500 pg, 1 ng, 5 ng, 10 ng, or 50 ng of IL-10 per cell / mL.

[0040] In some embodiments, CD4 + T cells express CD4 after activation with anti-CD3 and anti-CD28 antibodies. + T cells 10 6 Express at least 1 ng of IL-10 per cell / mL.

[0041] In some embodiments, CD4 +T cells express CD4 after activation with anti-CD3 and anti-CD28 antibodies. + T cells 10 6 Express at least 2 ng, 5 ng, 10 ng, 100 ng, 200 ng, or 500 ng of IL-10 per cell / mL.

[0042] In some embodiments, CD4 + T cells are unmodified CD4 + In some embodiments, CD4 + T cells are unmodified CD4 + They express IL-10 at levels at least 10-fold higher than T cells.

[0043] In some embodiments, CD4 + T cells are CD4 + T cells 10 6 In some embodiments, the CD4 + T cells are CD4 + T cells 10 6 Express at least 100 pg, 200 pg, 500 pg, 1 ng, 5 ng, 10 ng, or 50 ng of IL-5 per cell / mL.

[0044] In some embodiments, CD4 + T cells express CD4 after activation with anti-CD3 and anti-CD28 antibodies. + T cells 10 6 Express at least 1 ng of IL-5 per cell / mL.

[0045] In some embodiments, CD4 + T cells express CD4 after activation with anti-CD3 and anti-CD28 antibodies. + T cells 10 6 Express at least 2 ng, 5 ng, 10 ng, 100 ng, 200 ng, or 500 ng of IL-5 per cell / mL.

[0046] In some embodiments, CD4 + T cells are CD4 + T cells 106 Express at least 100 pg of IFN-γ per cell / mL.

[0047] In some embodiments, CD4 + T cells are CD4 + T cells 10 6 Express at least 100 pg, 200 pg, 500 pg, 1 ng, 5 ng, 10 ng, or 50 ng of IFN-γ per cell / mL.

[0048] In some embodiments, CD4 + T cells express CD4 after activation with anti-CD3 and anti-CD28 antibodies. + T cells 10 6 Express at least 1 ng of IFN-γ per cell / mL.

[0049] In some embodiments, CD4 + T cells express CD4 after activation with anti-CD3 and anti-CD28 antibodies. + T cells 10 6 Express at least 2 ng, 5 ng, 10 ng, 100 ng, 200 ng, or 500 ng of IFN-γ per cell / mL.

[0050] In some embodiments, CD4 + T cells are CD4 + T cells 10 6 Express at least 25 pg of IL-4 per cell / mL.

[0051] In some embodiments, CD4 + T cells are CD4 + T cells 10 6 Express at least 25 pg, 50 pg, 75 pg, 100 pg, 200 pg, 500 pg, 1 ng, 5 ng, 10 ng, or 50 ng of IL-4 per cell / mL.

[0052] In some embodiments, CD4 + T cells express CD4 after activation with anti-CD3 and anti-CD28 antibodies. + T cells 10 6Express at least 100 pg of IL-4 per cell / mL.

[0053] In some embodiments, CD4 + T cells express CD4 after activation with anti-CD3 and anti-CD28 antibodies. + T cells 10 6 Express at least 100pg, 200pg, 300pg, 400pg, 500pg, 600pg, 700pg, 800pg, 900pg, 1000pg, 2ng, 5ng, 10ng, 100ng, 200ng, or 500ng of IL-4 per cell / mL.

[0054] In some embodiments, expression of one or more of IL-10, IL-4, IFN-γ, and IL-5 is stable after one or more restimulations.

[0055] In some embodiments, the first exogenous polynucleotide segment or the second exogenous polynucleotide segment further comprises a sequence encoding a selectable marker.

[0056] In some embodiments, the selectable marker is ΔNGFR. In some embodiments, ΔNGFR has the sequence of SEQ ID NO: 3. In some embodiments, the second exogenous polynucleotide segment comprises the sequence of SEQ ID NO: 4.

[0057] In some embodiments, the selectable marker is a truncated form of an EGFR polypeptide.

[0058] In some embodiments, CD4 + T cells express CD19 + In vitro cytotoxicity against target cells is possible.

[0059] In some embodiments, CD4 + T cells express CD19 + In vivo cytotoxicity against target cells is possible.

[0060] In some embodiments, CD19 +The target cells are autoantibody-producing B cells.

[0061] In some embodiments, CD19 + The target cells are CD19 + They are cancer cells.

[0062] In some embodiments, CD4 + T cells are capable of in vitro cytotoxicity against myeloid target cells.

[0063] In some embodiments, CD4 + T cells are capable of in vivo cytotoxicity against myeloid target cells.

[0064] In some embodiments, CD4 + T cells express CD19 + Cytotoxicity against target cells and myeloid target cells is possible.

[0065] In some embodiments, the myeloid target cells express one or more of class I MHC, CD13, CD54, and CD112.

[0066] In some embodiments, CD19 + Cytotoxicity against target cells is maintained after one or more rounds of in vitro restimulation. + Cytotoxicity against target cells is maintained after one or more rounds of in vitro expansion.

[0067] In some embodiments, the cytotoxicity against myeloid target cells is maintained after one or more rounds of in vitro restimulation, hi some embodiments, the cytotoxicity against myeloid target cells is maintained after one or more rounds of in vitro expansion.

[0068] In some embodiments, CD4 + T cells are allogeneic CD4 + It is possible to suppress T cell proliferation.

[0069] In some embodiments, CD4 + T cells are allogeneic CD8 + It is possible to suppress T cell proliferation.

[0070] In some embodiments, CD4 + T cells are allogeneic CD4 + T cell proliferation, allogeneic CD8 + It is possible to suppress T cell proliferation and PBMC proliferation.

[0071] In some embodiments, the suppressive properties are maintained after one or more restimulations.

[0072] In another aspect, the present disclosure provides a genetically modified CD4 + CD4 containing cells + Characterized by T cell populations.

[0073] In some embodiments, the CD4 + T cells are obtained and pooled from 2, 3, 4, 5, 6, 7, 8, 9, or 10 different T cell donors.

[0074] In some embodiments, the CD4 + Collectively, T cells have 6, 7, 8, 9, 10, 11, 12, or more different HLA haplotypes.

[0075] In some embodiments, all CD4 + The T cells have at least a 1 / 10, 2 / 10, 3 / 10, 4 / 10, 5 / 10, 6 / 10, 7 / 10, 8 / 10, or 9 / 10 match to each other at the HLA-A, HLA-B, HLA-C, HLA-DRB1, and HLA-DQB1 loci.

[0076] In some embodiments, all CD4 + The T cells have at least a 1 / 8, 2 / 8, 3 / 8, 4 / 8, 5 / 8, 6 / 8, 7 / 8, or 8 / 8 match to each other at the HLA-A, HLA-B, HLA-C, and HLA-DRB1 loci.

[0077] In some embodiments, all CD4 + T cells have a 2 / 2 match to each other at the HLA-A locus.

[0078] In some embodiments, all CD4 + T cells have a 2 / 2 match with each other at the HLA-B locus.

[0079] In some embodiments, all CD4 + T cells have a 2 / 2 match to each other at the HLA-C locus.

[0080] In some embodiments, all CD4 + The T cells have at least a 3 / 4 or 4 / 4 match to each other at the HLA-DRB1 and HLA-DQB1 loci.

[0081] In some embodiments, all CD4 + The T cells have less than a 5 / 10, 6 / 10, 7 / 10, 8 / 10, or 9 / 10 match to each other at the HLA-A, HLA-B, HLA-C, HLA-DRB1, and HLA-DQB1 loci.

[0082] In some embodiments, all CD4 + The T cells have a 4 / 8, 5 / 8, 6 / 8, 7 / 8, or less than 8 / 8 match to each other at the HLA-A, HLA-B, HLA-C, and HLA DRB1 loci.

[0083] In some embodiments, all CD4 + The T cells have less than a 2 / 2 match with each other at the HLA-A locus.

[0084] In some embodiments, all CD4 + The T cells have less than a 2 / 2 match with each other at the HLA-B locus.

[0085] In some embodiments, all CD4 + The T cells have less than a 2 / 2 match with each other at the HLA-C locus.

[0086] In some embodiments, all CD4 + The T cells have a 4 / 2, 3 / 4, or less than 4 / 4 match to each other at the HLA-DRB1 and HLA-DQB1 loci.

[0087] In some embodiments, all CD4 + T cells are A * 02 allele or A * 02 is negative.

[0088] In some embodiments, all CD4 + T cells are A * 24 alleles or A * 24 is negative.

[0089] In some embodiments, NGFR + CD4 in cell populations + At least 30% of the T cells express the CAR.

[0090] In some embodiments, NGFR + CD4 in cell populations + At least 60% of the T cells express the CAR.

[0091] In some embodiments, NGFR + CD4 in cell populations + At least 90% of the T cells express the CAR.

[0092] In some embodiments, CD4 + The T cells are present in frozen suspension.

[0093] In some embodiments, CD4 + The T cells are present in a liquid suspension.

[0094] In some embodiments, the liquid suspension is pre-frozen.

[0095] In another aspect, the present disclosure provides a method for detecting a CD4 + any of the T cells or CD4 + The present invention features any of the pharmaceutical compositions provided herein comprising any of the T cell populations.

[0096] In another aspect, the present disclosure provides a method for detecting CD4 IL10 / CAR A method for producing primary CD4 T cells, comprising: (a) culturing primary CD4 T cells from one or more T cell donors; + and (b) obtaining CD4 T cells by introducing (i) a first exogenous polynucleotide segment encoding a chimeric antigen receptor (CAR), and (ii) a second exogenous polynucleotide segment encoding IL-10. + The present invention features a method including modifying a T cell.

[0097] In some embodiments, in step (a), primary cultured CD4 + The T cells are obtained from 2, 3, 4, 5, 6, 7, 8, 9, or 10 different T cell donors.

[0098] In some embodiments, the method further comprises the step of: + The method further comprises pooling the T cells.

[0099] In some embodiments, in step (a), primary cultured CD4 + The T cells are autologous to the patient.

[0100] In some embodiments, the method comprises, after step (a) and before step (b), or after step (b), injecting CD4+ into the cells in the presence of an anti-CD3 antibody and an anti-CD28 antibody, or anti-CD3 antibody and CD28 antibody coated beads.+ The method further comprises the step of incubating the T cells.

[0101] In some embodiments, the method further comprises administering CD4 + Further comprising incubating the T cells.

[0102] In some embodiments, the first exogenous polynucleotide segment, the second exogenous polynucleotide segment, or both are transfected into primary cultured CD4 + In some embodiments, the viral vector is a lentiviral vector.

[0103] In some embodiments, the first exogenous polynucleotide segment comprises a coding sequence for a CAR.

[0104] In some embodiments, the CAR is specific for a target antigen associated with an autoimmune disease, an inflammatory disorder, or cancer.

[0105] In some embodiments, the target antigen is associated with an autoimmune disease, an inflammatory disorder, or cancer.

[0106] In some embodiments, the CAR is an anti-CD19 CAR, an anti-CD20 CAR, an anti-CD22 CAR, an anti-BCMA CAR, an anti-B7-H3 CAR, an anti-CD27 CAR, or an anti-CD38 CAR.

[0107] In some embodiments, the CAR comprises the sequence of SEQ ID NO: 9, 16, 22, 34, 41-49, or 54.

[0108] In some embodiments, the first exogenous polynucleotide segment comprises the sequence of SEQ ID NO: 10, 17, 23, 35, or 55.

[0109] In some embodiments, the IL-10 comprises the sequence of SEQ ID NO: 1. In some embodiments, the second exogenous polynucleotide segment comprises the sequence of SEQ ID NO:2.

[0110] In some embodiments, the first exogenous polynucleotide segment or the second exogenous polynucleotide segment further comprises a segment encoding a selectable marker.

[0111] In some embodiments, the encoded selectable marker is ΔNGFR. In some embodiments, the encoded selectable marker has the sequence of SEQ ID NO:3.

[0112] In some embodiments, the selectable marker is a truncated form of an EGFR polypeptide.

[0113] In some embodiments, the method further comprises, after step (b), providing a genetically modified CD4 + Isolating T cells and thereby genetically modifying CD4 + Further comprising generating an enriched population of T cells.

[0114] In some embodiments, the genetically modified CD4 + At least 30% or at least 60% of the T cells express IL-10 and CAR.

[0115] In some embodiments, the genetically modified CD4 + At least 90% of the T cells express IL-10 and CAR.

[0116] In some embodiments, the genetically modified CD4 + At least 40% of the T cells express the selectable marker.

[0117] In some embodiments, the genetically modified CD4 + At least 75% of the T cells express the selectable marker.

[0118] In some embodiments, the genetically modified CD4 + At least 90% of the T cells express the selectable marker.

[0119] In some embodiments, the method comprises genetically modifying CD4 + The method further comprises the step of incubating the enriched population of T cells.

[0120] In some embodiments, the genetically modified CD4 + The step of incubating the enriched population of T cells is carried out in the presence of IL-2, anti-CD3 antibody and anti-CD28 antibody, or CD3 antibody and CD28 antibody coated beads.

[0121] In some embodiments, the method comprises genetically modifying CD4 + Further included is a subsequent step of freezing the T cells.

[0122] In some embodiments, at least two T cell donors have at least a 1 / 10, 2 / 10, 3 / 10, 4 / 10, 5 / 10, 6 / 10, 7 / 10, 8 / 10, or 9 / 10 match to each other at the HLA-A, HLA-B, HLA-C, HLA-DRB1, and HLA-DQB1 loci.

[0123] In some embodiments, at least two T cell donors have at least a 1 / 8, 2 / 8, 3 / 8, 4 / 8, 5 / 8, 6 / 8, 7 / 8, or 8 / 8 match to each other at the HLA-A, HLA-B, HLA-C, and HLA-DRB1 loci.

[0124] In some embodiments, at least two T cell donors have a 2 / 2 match to each other at the HLA-A locus. In some embodiments, at least two T cell donors have a 2 / 2 match to each other at the HLA-B locus. In some embodiments, at least two T cell donors have a 2 / 2 match to each other at the HLA-C locus.

[0125] In some embodiments, at least two T cell donors have at least a 3 / 4 or 4 / 4 match to each other at the HLA-DRB1 and HLA-DQB1 loci.

[0126] In some embodiments, at least two T cell donors have less than a 5 / 10, 6 / 10, 7 / 10, 8 / 10, or 9 / 10 match to each other at the HLA-A, HLA-B, HLA C, HLA-DRB1, and HLA-DQB1 loci.

[0127] In some embodiments, at least two T cell donors have a 4 / 8, 5 / 8, 6 / 8, 7 / 8, or less than 8 / 8 match to each other at the HLA-A, HLA-B, HLA C, and HLA DRB1 loci.

[0128] In some embodiments, at least two T cell donors have less than a 2 / 2 match to each other at the HLA-A locus. In some embodiments, at least two T cell donors have less than a 2 / 2 match to each other at the HLA-B locus. In some embodiments, at least two T cell donors have less than a 2 / 2 match to each other at the HLA-C locus.

[0129] In some embodiments, at least two T cell donors have a 4 / 2, 3 / 4, or less than 4 / 4 match to each other at the HLA-DRB1 and HLA DQB1 loci.

[0130] In some embodiments, each of the at least two T cell donors is * 02 allele or A * In some embodiments, each of the at least two T cell donors is A02 negative. * 02 allele or A * 02 is negative.

[0131] In some embodiments, in step (a), primary cultured CD4 + The T cells are obtained from one or more frozen stocks.

[0132] In some embodiments, in step (a), primary cultured CD4 + T cells are obtained from unfrozen peripheral blood mononuclear cells of at least two different T cell donors.

[0133] In some embodiments, the method comprises isolating CD4 + The method further comprises the step of isolating the T cells.

[0134] In another aspect, the disclosure provides a method of treating hematological cancer, comprising providing CD4 IL-10 / CAR Any of the cells provided herein, including CD4 IL-10 / CAR The present invention also features methods comprising administering to a patient with hematological cancer a therapeutically effective amount of any of the cell populations or any of the pharmaceutical compositions provided herein.

[0135] In some embodiments, the method comprises administering to a subject a subject, the subject being a patient, a subject ... IL-10 / CAR The method further comprises administering to the patient an allo-HSCT graft before or after administration of the method.

[0136] In another aspect, the present disclosure provides a method of treating a patient with a malignant disease, comprising administering to the patient an allo-HSCT graft and providing CD4 IL-10 / CAR Any of the cells provided herein, including CD4 IL-10 / CAR The present invention also features methods comprising administering a therapeutically effective amount of any of the cell populations or any of the pharmaceutical compositions provided herein.

[0137] In some embodiments, CD4 IL-10 / CAR The amount of cells is still sufficient to suppress or prevent graft-versus-host disease (GvHD) without suppressing the graft-versus-leukemia (GvL) or graft-versus-tumor (GvT) efficacy of allo-HSCT.

[0138] In some embodiments, the malignant disease or hematological cancer is myeloid leukemia.

[0139] In some embodiments, CD4 IL-10 / CAR The cells target and kill cancer cells that express CD13.

[0140] In some embodiments, CD4IL-10 / CAR The cells target and kill cancer cells that express HLA-class I.

[0141] In some embodiments, the myeloid leukemia is acute myeloid leukemia (AML).

[0142] In some embodiments, the malignancy or hematological cancer is CD19 + , CD20 + , CD22 + , CD27 + , CD38 + , BCMA+, or B7-H3 + It is blood cancer.

[0143] In some embodiments, CD19 + , CD20 + , CD22 + , CD27 + , CD38 + , BCMA+, or B7-H3 + The hematological cancer is selected from chronic lymphocytic leukemia, acute lymphoblastic leukemia (ALL), and non-Hodgkin's lymphoma.

[0144] In some embodiments, the allo-HSCT graft is obtained from a related or unrelated donor to the patient.

[0145] In some embodiments, CD4 IL-10 / CAR The cells are non-autologous to the patient.

[0146] In some embodiments, CD4 IL-10 / CAR The cells are autologous to the patient.

[0147] In some embodiments, CD4 IL-10 / CAR The cells are allogeneic to the patient.

[0148] In some embodiments, CD4 IL-10 / CAR The cells are not anergized to host alloantigens prior to administration to the patient.

[0149] In some embodiments, CD4 IL-10 / CAR The cells are Tr1-like cells.

[0150] In some embodiments, CD4 IL-10 / CAR The cells are polyclonal.

[0151] In some embodiments, CD4 IL-10 / CAR The cells are polyclonal and non-autologous to the patient.

[0152] In some embodiments, CD4 IL-10 / CAR The cells are polyclonal and autologous to the patient.

[0153] In some embodiments, CD4 IL-10 / CAR Cells are isolated from at least two donors and then genetically modified.

[0154] In some embodiments, none of the at least two donors is the same donor as the allo-HSCT donor.

[0155] In some embodiments, the allo-HSCT graft is obtained from a donor that is matched or mismatched for the patient.

[0156] In some embodiments, CD4 IL-10 / CAR The cells target and kill cells that express CD19, CD20, or BCMA. IL-10 / CAR The cells target and kill cells that express CD54. In some embodiments, CD4 IL-10 / CAR The cells target and kill cancer cells that express HLA-class I and CD54. In some embodiments, CD4 IL-10 / CAR The cells target and kill cancer cells that express CD112 and CD155. In some embodiments, CD4 IL-10 / CAR The cells target and kill cancer cells that express CD58.

[0157] In some embodiments, CD4IL-10 / CAR The cells target and kill cancer cells in the patient.

[0158] In some embodiments, CD4 IL-10 / CAR The cells target and kill solid tumor cells in the patient.

[0159] In another aspect, the present disclosure provides a method of treating hematological cancer by allogeneic hematopoietic stem cell transplantation (allo-HSCT), comprising: administering an allo-HSCT graft to the patient; A quantity of CD4 sufficient to suppress or prevent graft-versus-host disease (GvHD) without suppressing the graft-versus-leukemia (GvL) or graft-versus-tumor (GvT) efficacy of the allo-HSCT graft. IL-10 / CAR administering the cells to the patient Including, CD4 IL-10 / CAR The cells are genetically modified CD4+ cells by vector-mediated gene transfer of one or more vectors containing a coding sequence for human IL-10 under the control of a constitutive or inducible promoter and a coding sequence for CAR under the control of a constitutive or inducible promoter. + Contains T cells; CD4 IL-10 / CAR the cells target and kill cancer cells in the patient; CD4 IL-10 / CAR the cells are not anergized to host alloantigens prior to administration to the patient; and CD4 IL-10 / CAR the cells are non-autologous to the patient and Tr1-like; The method is characterized.

[0160] In another aspect, the present disclosure provides a method of treating hematological cancer by allogeneic hematopoietic stem cell transplantation (allo-HSCT), comprising: administering an allo-HSCT graft to the patient; A certain amount of CD4 IL-10 / CARadministering the cells to the patient, optionally in an amount sufficient to suppress or prevent graft-versus-host disease (GvHD) without suppressing graft-versus-leukemia (GvL) or graft-versus-tumor (GvT) efficacy of the allo-HSCT graft. Including, CD4 IL-10 / CAR The cells are genetically modified CD4+ cells by vector-mediated gene transfer of one or more vectors containing a coding sequence for human IL-10 under the control of a constitutive or inducible promoter and a coding sequence for CAR under the control of a constitutive or inducible promoter. + Contains T cells; CD4 IL-10 / CAR the cells target and kill cancer cells in the patient; CD4 IL-10 / CAR the cells are not anergized to host alloantigens prior to administration to the patient; and CD4 IL-10 / CAR The cells are autologous to the patient and Tr1-like, The method is characterized.

[0161] In another aspect, the present disclosure provides a method for detecting CD19 in a patient. + , CD20 + , CD22 + , CD27 + , CD38 + , BCMA + , or B7-H3 + A method for preventing recurrence of blood cancer, comprising: + , CD20 + , CD22 + , BCMA+, or B7-H3 + Patients identified as having hematologic cancer or CD19 + , CD20 + , CD22 + , BCMA + , or B7-H3 + Provide sufficient CD4 to induce an anti-cancer effect in patients identified as at risk for recurrence of hematologic cancer. IL-10 / CAR Any of the cells provided herein, including CD4 IL-10 / CARIn some embodiments, the method includes administering a therapeutically effective amount of any of the cell populations or any of the pharmaceutical compositions provided herein. IL-10 / CAR The cells are administered after administration of HSCT.

[0162] In another aspect, the present disclosure provides a method for detecting B7-H3 in a patient. + A method for preventing cancer recurrence, comprising: + Patients identified as having cancer are provided with CD4 sufficient to induce an anti-cancer effect. IL-10 / CAR Any of the cells provided herein, including CD4 IL-10 / CAR The present invention also features methods comprising administering a therapeutically effective amount of any of the cell populations or any of the pharmaceutical compositions provided herein.

[0163] In some embodiments, B7-H3 + Cancer is a solid tumor.

[0164] In some embodiments, the solid tumor is selected from the group consisting of breast cancer, brain cancer, lung cancer, liver cancer, stomach cancer, spleen cancer, colon cancer, kidney cancer, pancreatic cancer, prostate cancer, uterine cancer, skin cancer, head cancer, neck cancer, sarcoma, neuroblastoma, and ovarian cancer.

[0165] In another aspect, the disclosure provides a method of treating a patient with minimal residual disease, comprising providing CD4+ sufficient to induce an anti-cancer effect in a patient identified as having or at risk for having minimal residual disease. IL-10 / CAR Any of the cells provided herein, including CD4 IL-10 / CAR The present invention also features methods comprising administering a therapeutically effective amount of any of the cell populations or any of the pharmaceutical compositions provided herein.

[0166] In another aspect, the present disclosure provides a method of treating a patient in need of immune tolerance, comprising administering to the patient a CD4 IL-10 / CAR Any of the cells provided herein, including CD4 IL-10 / CARThe present invention also features methods comprising administering a therapeutically effective amount of any of the cell populations or any of the pharmaceutical compositions provided herein.

[0167] In some embodiments, the method comprises administering to a subject a subject, the subject being a patient, a subject ... IL-10 / CAR cells or CD4 IL-10 / CAR It further includes a prior step of thawing a frozen suspension of the cell population.

[0168] In some embodiments, the patient has an inflammatory or autoimmune disease.

[0169] In some embodiments, the inflammatory or autoimmune disease is selected from the group consisting of autoimmune uveitis, psoriasis, vitiligo, alopecia areata, psoriatic arthritis, inflammatory bowel disease, Hashimoto's thyroiditis, autoimmune vasculitis, ulcerative colitis, bullous diseases, scleroderma, celiac disease, Graves' disease, systemic sclerosis, myasthenia gravis, anti-NMDA encephalitis, pemphigus disease (both vulgaris and foliaceus), epidermolysis bullosa acquisita, thrombotic thrombocytopenic purpura, idiopathic thrombocytopenic purpura, autoantibody-induced vascular inflammation, autoantibody-induced carditis, rheumatoid arthritis, The disease is selected from the group consisting of autoantibody-induced rheumatoid arthritis, neuromyelitis optica spectrum disorder, systemic lupus erythematosus (SLE), multiple sclerosis (MS), Sjogren's syndrome, autoimmune myopathy, type I diabetes, Addison's disease, pernicious anemia, autoimmune hepatitis, primary biliary cholangitis (PBC), autoimmune pancreatitis, Goodpasture's disease, primary membranous nephropathy, ovarian failure, autoimmune orchitis, dry eye disease, aplastic anemia, autoimmune neutropenia, and idiopathic interstitial pneumonia.

[0170] In some embodiments, the inflammatory or autoimmune disease is Crohn's disease, ulcerative colitis, celiac disease, type 1 diabetes, lupus, psoriasis, psoriatic arthritis, ankylosing spondylitis, or rheumatoid arthritis.

[0171] In some embodiments, the patient has a disease or disorder associated with hyperactivity of the NLPR3 inflammasome.

[0172] In some embodiments, the patient has type 2 diabetes, a neurodegenerative disease, a cardiovascular disease, or an inflammatory bowel disease.

[0173] In some embodiments, the patient has a disease or disorder associated with increased IL-1β production by activated monocytes, macrophages, or dendritic cells.

[0174] In some embodiments, the patient has a disease or disorder associated with increased IL-18 production by activated monocytes, macrophages, or dendritic cells.

[0175] In some embodiments, the patient has a disease or disorder associated with increased production of mature caspase-1 by activated monocytes, macrophages, or dendritic cells.

[0176] In some embodiments, the patient has an allergic or atopic disease.

[0177] In some embodiments, the allergic or atopic disease is selected from the group consisting of asthma, atopic dermatitis, and rhinitis.

[0178] In some embodiments, the patient has a food allergy.

[0179] In some embodiments, the patient has a solid tumor.

[0180] In some embodiments, the solid tumor is selected from the group consisting of breast cancer, brain cancer, lung cancer, liver cancer, stomach cancer, spleen cancer, colon cancer, kidney cancer, pancreatic cancer, prostate cancer, uterine cancer, skin cancer, head cancer, neck cancer, sarcoma, neuroblastoma, and ovarian cancer.

[0181] In some embodiments, the method comprises administering to a subject a subject, the subject being a patient, a subject ... IL-10 / CAR cells, CD4 IL-10 / CAR In some embodiments, the method further comprises administering the cell population or pharmaceutical composition to the patient, either before or after administering the cell population or pharmaceutical composition to the patient. IL-10 / CAR cells, CD4 IL-10 / CARThe cell population, or pharmaceutical composition, prevents or reduces the severity of host rejection of an organ transplant.

[0182] In some embodiments, the method comprises administering to a subject a subject, the subject being a patient, a subject ... IL-10 / CAR cells, CD4 IL-10 / CAR In some embodiments, the method further comprises the step of transplanting the iPS cell-derived cells or tissue into the patient either before or after administering the cell population or pharmaceutical composition. IL-10 / CAR cells, CD4 IL-10 / CAR The cell population, or pharmaceutical composition, prevents or reduces the severity of host rejection of the cell transplant.

[0183] In some embodiments, the method comprises administering to a subject a subject, the subject being a patient, a subject ... IL-10 / CAR cells, CD4 IL-10 / CAR In some embodiments, the method further comprises administering a recombinant AAV to the patient either before or after administering the cell population or pharmaceutical composition. IL-10 / CAR cells, CD4 IL-10 / CAR The cell population, or pharmaceutical composition, reduces the immune response to the recombinant AAV.

[0184] In some embodiments, the method comprises administering to a subject a subject, the subject being a patient, a subject ... IL-10 / CAR cells, CD4 IL-10 / CAR In some embodiments, the method further comprises administering to the patient a recombinant viral vector other than AAV, either before or after administering the cell population or pharmaceutical composition. IL-10 / CAR cells, CD4 IL-10 / CAR The cell population, or pharmaceutical composition, reduces the immune response to recombinant viral vectors other than AAV.

[0185] In some embodiments, the method comprises administering to a subject a subject, the subject being a patient, a subject ... IL-10 / CAR cells, CD4 IL-10 / CAR In some embodiments, the method further comprises administering an immunogenic therapeutic protein to the patient either before or after administering the cell population or pharmaceutical composition. IL-10 / CAR cells, CD4 IL-10 / CARThe cell population, or pharmaceutical composition, reduces an immune response to an immunogenic therapeutic protein. In some embodiments, the immunogenic therapeutic protein is selected from a therapeutic antibody, a Factor VIII replacement, a cytokine, and a cytokine mutein.

[0186] In some embodiments, the patient has an exaggerated immune response to a viral or bacterial infection. In some embodiments, the patient has a coronavirus infection. In some embodiments, the patient has organ and / or tissue damage.

[0187] In some embodiments, the method detects a selectable marker in a biological sample obtained from a patient, thereby detecting CD4 IL-10 / CAR Further comprising detecting the presence or absence of the cells.

[0188] In some embodiments, the biological sample is a biopsy or blood from the patient.

[0189] In another aspect, the disclosure provides a method of treating or inhibiting an autoimmune, allergic, or inflammatory disease in a patient, comprising providing to a patient identified as having an autoimmune, allergic, or inflammatory disease, CD4+ antibodies sufficient to treat or inhibit the autoimmune, allergic, or inflammatory disease. IL-10 / CAR Any of the cells provided herein, including CD4 IL-10 / CAR The present invention also features methods comprising administering a therapeutically effective amount of any of the cell populations or any of the pharmaceutical compositions provided herein.

[0190] In another aspect, the present disclosure provides a method for reducing graft rejection in a patient who has received a hematopoietic stem cell, bone marrow cell, umbilical cord blood cell, tissue stem cell, or solid organ transplant, comprising providing to a patient identified as having rejection of the transplanted hematopoietic stem cell, bone marrow cell, tissue stem cell, or solid organ, CD4+ cells sufficient to reduce rejection of the graft. IL-10 / CAR Any of the cells provided herein, including CD4 IL-10 / CARThe present invention also features methods comprising administering a therapeutically effective amount of any of the cell populations or any of the pharmaceutical compositions provided herein.

[0191] In another aspect, the disclosure provides a method of treating graft-versus-host disease (GvHD) in a patient, comprising administering to a patient identified as having or at risk for having graft-versus-host disease (GvHD) a CD4+ antibody sufficient to inhibit or prevent GvHD. IL-10 / CAR Any of the cells provided herein, including CD4 IL-10 / CAR The present invention also features methods comprising administering a therapeutically effective amount of any of the cell populations or any of the pharmaceutical compositions provided herein.

[0192] In some embodiments, the graft-versus-host disease (GvHD) comprises acute GvHD.

[0193] In some embodiments, the graft-versus-host disease (GvHD) comprises chronic GvHD.

[0194] In another aspect, the present disclosure provides a method of treating tissue or organ damage in a patient, comprising administering to a patient identified as having or at risk of having tissue or organ damage, a CD4 antibody as provided herein sufficient to induce repair of the tissue or organ damage. IL-10 / CAR Any of the cells provided herein, including CD4 IL-10 / CAR The present invention also features methods comprising administering a therapeutically effective amount of any of the cell populations or pharmaceutical compositions provided herein.

[0195] In another aspect, the disclosure features a polynucleotide construct that includes: (a) a first polynucleotide segment encoding a chimeric antigen receptor (CAR); and (b) a second polynucleotide segment encoding interleukin-10 (IL-10).

[0196] In some embodiments, the first polynucleotide segment comprises a regulatory element operably linked to the coding sequence of the CAR. In some embodiments, the regulatory element drives constitutive expression of the CAR.

[0197] In some embodiments, the second polynucleotide segment comprises a regulatory element operably linked to a coding sequence for IL-10. In some embodiments, the regulatory element drives constitutive expression of IL-10.

[0198] In some embodiments, the method further comprises an internal ribosome entry site (IRES) or a self-cleaving peptide between the first polynucleotide segment and the second polynucleotide segment. In some embodiments, the self-cleaving peptide is selected from the group consisting of F2A, P2A, T2A, and E2A.

[0199] In some embodiments, the CAR comprises an antigen-binding domain, a hinge region, a transmembrane domain, and an intracellular signaling domain.

[0200] In some embodiments, the antigen-binding domain comprises a single-chain antibody fragment. In some embodiments, the single-chain antibody fragment comprises a single-chain Fv (scFv).

[0201] In some embodiments, the antigen binding domain targets an antigen associated with an autoimmune disease, an inflammatory disorder, or cancer.

[0202] In some embodiments, the antigen is selected from the group consisting of CD19, CD20, CD22, BCMA, B7-H3, CD27, CD38, CEA, BCMA, CD23, Lyml, Lym2, CLEC5A, CDH179b, FLT3, GCC, Muc, CSF2RA, GFRa4, CD32, CD33, IL1lRa, IL13Ra, NYBRI, SLea, CD200R, TGFbetaR2, CD276, TROP2, LAMP1, PTK7, DLL3, CDH1, CDH6, CDH17, CDH19, TSHR, tyrosinase, HLA-A2, citrullinated peptides, insulin, GAD65, IA2, gliadin, and desmoglein.

[0203] In some embodiments, the antigen-binding domain is an anti-CD19 antigen-binding domain. In some embodiments, the anti-CD19 antigen-binding domain comprises the sequence of SEQ ID NO: 11.

[0204] In some embodiments, the antigen binding domain is an anti-CD20 antigen binding domain. In some embodiments, the anti-CD20 antigen binding domain comprises the sequence of SEQ ID NO: 18.

[0205] In some embodiments, the antigen-binding domain is an anti-BCMA antigen-binding domain. In some embodiments, the anti-BCMA antigen-binding domain comprises the sequence of SEQ ID NOs: 50-53.

[0206] In some embodiments, the hinge region is selected from a human CD8 hinge region, a human CD28 hinge region, an IgG1 hinge region, or an IgG4 hinge region. In some embodiments, the hinge region is derived from human CD8.

[0207] In some embodiments, the transmembrane domain is selected from the group consisting of a TNFRSF19 transmembrane domain, a CD3 zeta transmembrane domain, a CD8α transmembrane domain, a CD4 transmembrane domain, a CD28 transmembrane domain, and a B7 family-induced costimulatory (ICOS) transmembrane domain. In some embodiments, the transmembrane domain is derived from CD8α.

[0208] In some embodiments, the CAR further comprises one or more costimulatory domains.

[0209] In some embodiments, the CAR comprises two costimulatory domains.

[0210] In some embodiments, the one or more costimulatory domains are selected from the group consisting of 4-1BB, CD28, OX40, ICOS, CD27, MYD88-CD40, and KIR2DS2.

[0211] In some embodiments, one of the one or more costimulatory domains is derived from CD28.

[0212] In some embodiments, the second costimulatory domain is derived from 4-1BB.

[0213] In some embodiments, the intracellular signaling domain comprises an immunoreceptor tyrosine-based activation motif (ITAM). In some embodiments, the immunoreceptor tyrosine-based activation motif (ITAM) is derived from CD3 zeta.

[0214] In some embodiments, the CAR comprises an anti-CD19 antigen-binding domain, a BCMA, or an anti-CD20 antigen-binding domain; a human CD8 hinge region; a CD8 transmembrane region; a CD28 costimulatory domain; and a CD3 zeta chain intracellular signaling domain.

[0215] In some embodiments, the CAR comprises the sequence of SEQ ID NO: 9, 16, 22, or 34.

[0216] In some embodiments, the first polynucleotide segment comprises the sequence of SEQ ID NO: 10, 17, 23, 35, or 55.

[0217] In some embodiments, the IL-10 is human IL-10. In some embodiments, the IL-10 is viral IL-10.

[0218] In some embodiments, the IL-10 is a protein having the sequence of SEQ ID NO: 1. In some embodiments, the second polynucleotide segment comprises the sequence of SEQ ID NO:2.

[0219] In some embodiments, the first polynucleotide segment or the second polynucleotide segment further comprises a sequence encoding a selectable marker.

[0220] In some embodiments, the selectable marker is ΔNGFR. In some embodiments, ΔNGFR has the sequence of SEQ ID NO: 3. In some embodiments, the polynucleotide construct comprises the sequence of SEQ ID NO: 4.

[0221] In some embodiments, the selectable marker is a truncated form of an EGFR polypeptide.

[0222] In some embodiments, the construct is a vector. In some embodiments, the vector is a viral vector. In some embodiments, the vector is a lentiviral vector.

[0223] In another aspect, the disclosure features a polynucleotide construct including a first polynucleotide segment having the sequence of SEQ ID NO: 10, 17, 23, 35, or 55; and a second polynucleotide segment having the sequence of SEQ ID NO: 2.

[0224] In another aspect, the disclosure features a polynucleotide construct including: a first polynucleotide segment having the sequence of SEQ ID NO: 10, 17, 23, 35, or 55; a second polynucleotide segment having the sequence of SEQ ID NO: 2; and, between the first and second polynucleotide segments, a third polynucleotide segment having the sequence of SEQ ID NO: 33.

[0225] In another aspect, the disclosure features a polynucleotide construct including: a first polynucleotide segment having the sequence of SEQ ID NO: 10, 17, 23, 35, or 55; a second polynucleotide segment having the sequence of SEQ ID NO: 2; a third polynucleotide segment having the sequence of SEQ ID NO: 27, between the first and second polynucleotide segments; and a fourth polynucleotide segment having the sequence of SEQ ID NO: 4.

[0226] In another aspect, the present disclosure provides CD4 IL-10 CD4 cells, wherein the IL-10 is viral IL-10. IL-10 The viral IL-10 has the sequence of SEQ ID NO: 6 or 18. In some embodiments, the viral IL-10 is encoded by a polynucleotide having the sequence of SEQ ID NO: 7. In some embodiments, the IL-10 is human IL-10 in which 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids from human IL-10 have been replaced by the corresponding amino acid sequence from the viral IL-10. In some embodiments, the viral IL-10 is human IL-10 in which 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids from human IL-10 have been replaced by the corresponding amino acid sequence from the viral IL-10. + T cells are transduced with exogenous viral IL-10 under the control of a constitutive promoter. In some embodiments, the expression control element is activated CD4 + The exogenous polynucleotide encoding viral IL-10 is driven to express viral IL-10 in T cells. In some embodiments, the exogenous polynucleotide encoding viral IL-10 is integrated into the T cell nuclear genome. In some embodiments, the exogenous polynucleotide encoding viral IL-10 is not integrated into the T cell nuclear genome. In some embodiments, the exogenous polynucleotide encoding viral IL-10 has the sequence of SEQ ID NO: 7.

[0227] In another aspect, the present disclosure provides CD4 IL-10CD4 cells, wherein IL-10 is IL-10 of house mouse (Mus musculus), "MOUSE" (SEQ ID NO: 58); brown rat (Rattus norvegicus), "RAT" (SEQ ID NO: 59); rhesus monkey (Macaca mulatta), "MACMU" (SEQ ID NO: 60); western gorilla (Gorilla gorilla), "GORILLA" (SEQ ID NO: 61); cynomolgus monkey (Macaca fascicularis), "CYNO" (SEQ ID NO: 62); olive baboon (Papio anubis), "OLIVE BABOON" (SEQ ID NO: 63); bonobo (Pan paniscus), "BONOBO" (SEQ ID NO: 64); chimpanzee (Pan troglodytes), "CHIMP" (SEQ ID NO: 65); and EBVB9 (SEQ ID NO: 66). IL-10 In some embodiments, the IL-10 is a protein having at least 90%, 95%, 98%, or 99% sequence identity to IL-10 from house mouse (Mus musculus), "MOUSE" (SEQ ID NO: 58); brown rat (Rattus norvegicus), "RAT" (SEQ ID NO: 59); rhesus monkey (Macaca mulatta), "MACMU" (SEQ ID NO: 60); western gorilla (Gorilla gorilla), "GORILLA" (SEQ ID NO: 61); cynomolgus monkey (Macaca fascicularis), "CYNO" (SEQ ID NO: 62); olive baboon (Papio anubis), "OLIVE BABOON" (SEQ ID NO: 63); bonobo (Pan paniscus), "BONOBO" (SEQ ID NO: 64); chimpanzee (Pan troglodytes), "CHIMP" (SEQ ID NO: 65); and EBVB9 (SEQ ID NO: 66).

[0228] In another aspect, the present disclosure provides CD4 IL-10 CD4 cells, wherein the IL-10 is a variant of human IL-10. IL-10The cells are characterized in that the variants of human IL-10 have the sequence of SEQ ID NO: 67 or SEQ ID NO: 68. In some embodiments, the IL-10 is human IL-10 in which 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids from human IL-10 have been replaced by the corresponding amino acid sequence from IL-10 of another species (e.g., IL-10 from house mouse (Mus musculus), "MOUSE" (SEQ ID NO: 58); brown rat (Rattus norvegicus), "RAT" (SEQ ID NO: 59); rhesus monkey (Macaca mulatta), "MACMU" (SEQ ID NO: 60); western gorilla (Gorilla gorilla), "GORILLA" (SEQ ID NO: 61); cynomolgus monkey (Macaca fascicularis), "CYNO" (SEQ ID NO: 62); bonobo (Pan paniscus), "BONOBO" (SEQ ID NO: 64); chimpanzee (Pan troglodytes), "CHIMP" (SEQ ID NO: 65); and EBVB9 (SEQ ID NO: 66)). In some embodiments, CD4 + The T cells are transduced with an exogenous IL-10 variant under the control of a constitutive promoter. In some embodiments, the expression control element is a constitutive promoter for activating CD4 + In some embodiments, the exogenous polynucleotide encoding the IL-10 variant is integrated into the T cell nuclear genome. In some embodiments, the exogenous polynucleotide encoding the IL-10 variant is not integrated into the T cell nuclear genome.

[0229] In yet another aspect, the present disclosure provides a method for the treatment of viral IL-10 CD 4IL-10 1. A method of making a polymerizable composition comprising the steps of: (i) Primary CD4 T cells from a single T cell donor + Obtaining T cells; and (ii) transfecting donor CD4 by introducing an exogenous polynucleotide encoding viral IL-10 + T cells, thereby genetically modifying CD4+ Steps for obtaining T cells The present invention provides a method comprising:

[0230] In some embodiments, the method further comprises, after step (i) or after step (ii), culturing primary CD4 in the presence of anti-CD3 and anti-CD28 antibodies, or anti-CD3 and CD28 antibody coated beads. + The method further comprises the step of incubating the T cells.

[0231] In some embodiments, the method further comprises culturing primary CD4 + In some embodiments, the T cells are incubated with an exogenous polynucleotide encoding viral IL-10 using a vector.

[0232] In some embodiments, the exogenous polynucleotide encoding viral IL-10 comprises a segment encoding a selectable marker. In some embodiments, the encoded selectable marker is ΔNGFR. In some embodiments, the encoded selectable marker has the sequence of SEQ ID NO: 3. In some embodiments, the encoded selectable marker is a truncated EGFR polypeptide. In some embodiments, the encoded selectable marker is a truncated human EGFR polypeptide.

[0233] In some embodiments, the method further comprises, after step (ii), providing a genetically modified CD4 + Isolating T cells and thereby genetically modifying CD4 + Further comprising generating an enriched population of T cells.

[0234] In some embodiments, the method comprises genetically modifying CD4 + In some embodiments, the enriched population of T cells further comprises incubating the enriched population of T cells. + The step of incubating the enriched population of T cells is carried out in the presence of IL-2, anti-CD3 antibody and anti-CD28 antibody, or CD3 antibody and CD28 antibody coated beads.

[0235] In some embodiments, in step (i), primary cultured CD4 + In some embodiments, the T cells are obtained from a cryopreservation. In some embodiments, in step (i), primary cultured CD4 + T cells are obtained from unfrozen peripheral blood mononuclear cells of a single T cell donor.

[0236] In some embodiments, the method comprises isolating CD4 + Further comprising isolating T cells. In some embodiments, the peripheral blood mononuclear cells are obtained from a buffy coat or apheresis. [Brief explanation of the drawings]

[0237] [Figure 1] FIG. 1 is a non-limiting illustration of various chimeric antigen receptor (CAR) structures provided herein. [Figure 2-A] Figure 2A illustrates the partial structure of the anti-CD19 CAR coding sequence, including the scFv from the fully human anti-CD19 monoclonal antibody FMC63, the CD8α hinge, the CD8α transmembrane domain, the CD28 costimulatory domain, and the CD3ζ activation domain, used in a lentiviral vector to deliver the anti-CD19 CAR to CD4+ T cells to generate CD4IL-10 anti-CD19 CAR cells. [Figure 2-B] Figure 2B illustrates the lentiviral vector (pLV anti-CD19 CAR) used to deliver anti-CD19 CAR to CD4+ T cells to produce CD4IL-10 anti-CD19CAR cells. [Figure 3-A]Figures 3A-3B illustrate the bidirectional plasmid (pLVIL-10) used to generate the lentiviral vector (LVV; LVVIL-10-NGFR) used to deliver human IL-10 and ΔNGFR coding sequences to CD4+ T cells to produce CD4IL-10 cells or CD4IL-10 anti-CD19 CAR cells. Figure 3A illustrates the partial structure of the bidirectional lentiviral vector for delivering human IL-10 and ΔNGFR coding sequences to CD4+ T cells from multiple donors to produce polydonor CD4IL-10 cells. Figure 3B illustrates the complete circular structure of the bidirectional lentiviral vector (hPGK.IL10.WPRE.mhCMV.ΔNGFR.SV40PA) for delivering human IL-10 and ΔNGFR coding sequences to CD4+ T cells from multiple donors to produce polydonor CD4IL-10 cells. [Figure 3-B] Same as Figure 3-A. [Figure 4] FIG. 4 shows, for illustrative purposes, antigen expression during natural B cell differentiation. [Figure 5-A]Figures 5A-5C show schematic diagrams of non-limiting examples of methods for generating CD4IL-10 anti-CD19 CAR cells. In each method, human CD4+ T cells were activated with anti-CD3 / anti-CD28 beads (Miltenyi) (cell:bead ratio of 3:1) in complete culture medium (X-vivo supplemented with 5% human AB serum and rhIL-2 (50 U / mL)). Figure 5A shows that activated CD4+ T cells were transduced with a lentiviral vector encoding an anti-CD19 CAR (LVVCD19-CAR) 48 hours after activation and with a bidirectional lentiviral vector encoding a truncated form of human IL-10 and the human NGF receptor (LVVIL-10-NGFR) 24 hours later (i.e., 72 hours after activation). Both LVVs were used at a multiplicity of infection (MOI) of 20. Figure 5B shows that activated CD4+ T cells were transduced with LVVIL-10-NGFR 48 hours after activation and then transduced with LVVCD19-CAR 24 hours later (i.e., 24 hours after activation). Figure 5C shows that activated CD4+ T cells were simultaneously transduced with LVVIL-10-NGFR and LVVCD19-CAR 48 hours after activation. A control (not expressing CAR) was generated by transducing activated CD4+ T cells with LVVIL-10-NGFR alone 48 hours after activation. For each of Figures 5A-5C, transduction was performed in the presence of polybrene (8 μg / mL). Additionally, for each of Figures 5A-5C, transduced cells (i.e., ΔNGFR+ cells) were purified using anti-CD271 microbeads (Miltenyi) and anti-CD19 CAR microbeads (Miltenyi) 10 to 28 days after transduction. Purified cells were restimulated every 14 days as previously described (Andolfi et al. Mol. Ther. 20(9):1778-1790(2012) and Locafaro et al. Mol. Ther. 25(10):2254-2269(2017)). After the second (TF2) and third (TF3) restimulations, the resulting cells (CD4IL-10 anti-CD19 CAR cells or control CD4IL-10 cells) were characterized in vitro and in vivo. Culture medium was changed every 2-3 days throughout the culture period, as needed. [Figure 5-B] Same as Figure 5-A. [Figure 5-C] Same as Figure 5-A. [Figure 6-A] Figure 6A shows flow cytometry plots (anti-CD19 CAR expression on the x-axis and NGFR expression as a surrogate for IL-10 on the y-axis) for a control (left panel) and a CD4IL-10 anti-CD19 CAR (right panel) produced according to the method described in Figure 5A. Figure 6B shows flow cytometry plots (CD19 on the x-axis and NGFR on the y-axis) for a control (left panel) and a CD4IL-10 anti-CD19 CAR (right panel) produced according to the method described in Figure 5B. Figure 6C shows flow cytometry plots (CD19 on the x-axis and NGFR on the y-axis) for a control (left panel) and a CD4IL-10 anti-CD19 CAR (right panel) produced according to the method described in Figure 5C. [Figure 6-B] Same as Figure 6-A. [Figure 6-C] Same as Figure 6-A. [Figure 7] Figure 7 shows flow cytometry plots (anti-CD19 CAR expression on the x-axis and NGFR expression on the y-axis) for CD4IL-10 T cells (control) and CD4IL-10 anti-CD19 CART cells. The top panel shows CD4IL-10 T cells (control) and CD4IL-10 anti-CD19 CART cells generated from donor 26.1 according to the method shown in Figure 5C. The bottom panel shows CD4IL-10 T cells (control) and CD4IL-10 anti-CD19 CART cells generated from donor 26.2 according to the method shown in Figure 5C. [Figure 8-A] Figures 8A-8D show the cytokine production profiles for CD4IL-10 T cells (control) and CD4IL-10 / CART cells generated from CD4 T cells isolated from three different donors (26.1, 26.2, and 26.3). The cytokine production profiles include IL-10 (Figure 8A), IFN-γ (Figure 8B), IL-4 (Figure 8C), and IL-5 (Figure 8D). [Figure 8-B] Same as Figure 8-A. [Figure 8-C]Same as Figure 8-A. [Figure 8-D] Same as Figure 8-A. [Figure 9] FIG. 9 shows the cytokine production profiles of CD4IL-10 T cells (control) and CD4IL-10 / CART cells generated from CD4+ T cells isolated from donor 24.2. [Figure 10] Figure 10 shows that CD4IL-10 anti-CD19 CAR cells generated from CD4+ T cells isolated from donor 24.2 killed CD19-expressing NALM6 cells in vitro. ALL-CM is a myeloid cell line, and K562, a cell line sensitive to NK killing, was included as a control. [Figure 11-A] Figure 11A shows that CD4IL-10 anti-CD19 CAR cells generated from CD4+ T cells isolated from three different donors (26.1, 26.2, and 26.3) killed CD19+ NALM6 cells in vitro. [Figure 11-B] Figure 11B shows IL-10 levels in the supernatants on day 3 of co-culture of NALM6 cells and CD4IL-10 T cells (control) or NALM6 cells and CD4IL-10 anti-CD19 CAR cells. BLQ: below the level of quantification (31 pg / mL). [Figure 12]Figure 12 shows flow cytometry plots (count vs. eFluor® 670) from a proliferation assay of allogeneic CD4+ T cells after co-culture with CD4IL-10 T cells (control) and CD4IL-10 anti-CD19 CAR cells isolated after a second restimulation (TF2) (see Figure 5C). The top panel shows proliferation of CD4+ T cells after co-culture with CD4IL-10 anti-CD19 CAR cells. The bottom panel shows proliferation of CD4+ T cells after co-culture with CD4IL-10 T cells (control). The flow cytometry plots show "non-dividing" and "dividing" cells, with dividing cells being smaller and containing less eFluor® 670 due to dilution after each division. Suppression mediated by CD4IL-10 T cells (control) and CD4IL-10 anti-CD19 CAR cells was calculated as follows: 100 − ([proliferation of responders in the presence of CD4IL-10 T cells (control) or CD4IL-10 anti-CD19 CAR cells / proliferation of responders] × 100). [Figure 13] Figure 13 shows flow cytometry plots (count vs. eFluor® 670) from a proliferation assay of CD4IL-10 T cells (control) isolated after a second restimulation (TF2) and allogeneic CD8+ T cells after co-culture with CD4IL-10 anti-CD19 CAR cells (see Figure 5C). The top panel shows proliferation of CD8+ T cells after co-culture with CD4IL-10 anti-CD19 CAR cells. The bottom panel shows proliferation of CD8+ T cells after co-culture with CD4IL-10 T cells (control). The flow cytometry plots show "non-dividing" and "dividing" cells, with dividing cells being smaller and containing less eFluor® 670 due to dilution after each division. Suppression mediated by CD4IL-10 T cells (control) and CD4IL-10 anti-CD19 CAR cells was calculated as follows: 100 − ([proliferation of responders in the presence of CD4IL-10 T cells (control) or CD4IL-10 anti-CD19 CAR cells / proliferation of responders] × 100). [Figure 14]Figure 14 shows flow cytometry plots (count vs. eFluor® 670) from a proliferation assay of allogeneic PBMCs after co-culture with CD4IL-10 T cells (control) and CD4IL-10 anti-CD19 CAR cells isolated after the third restimulation (TF3) (see Figure 5C). The flow cytometry plots show "proliferating" cells, with dividing cells being smaller and containing less eFluor® 670 due to dilution after each division. Suppression mediated by CD4IL-10 T cells (control) and CD4IL-10 anti-CD19 CAR cells was calculated as follows: 100 - ([responder proliferation in the presence of CD4IL-10 T cells (control) or CD4IL-10 anti-CD19 CAR cells / responder proliferation] x 100). [Figure 15] Figure 15 shows a schematic diagram of the experimental setup for assessing CD19+ tumor cell growth in mice in vivo. On day 0, NSG mice were injected with (i) NALM6-luciferase (1 x 105 cells / mouse), (ii) NALM6-luciferase (1 x 105 cells / mouse) + PBMCs (2.5 x 106 cells / mouse), (iii) NALM6-luciferase (1 x 105 cells / mouse) + CD4IL-10 anti-CD19 CAR cells (2.5 x 106 cells / mouse), or (iv) NALM6-luciferase (1 x 105 cells / mouse) + PBMCs (2.5 x 106 cells / mouse) + CD4IL-10 anti-CD19 CAR cells (2.5 x 106 cells / mouse). [Figure 16-A] Figure 16A shows bioluminescence from NALM6-luciferase cells in NSG mice on days 3, 6, 9, and 12 after injection under conditions (i) to (iv) described in Figure 15. [Figure 16-B] Figure 16B shows a line graph quantifying bioluminescence from the images in Figure 16A. Total luminous flux [p / s] over time (days after NALM6-luciferase injection) for each of conditions (i)-(iv) described in Figure 15. P values ​​were determined on day 12 using a Mann-Whitney test comparing conditions (ii)-(iv) with condition (i) (NALM6-luciferase alone). [Figure 17]Figure 17 shows a line graph of weight loss over time for the mice described in Figure 15 for conditions (i), (ii), and (iii). Weight loss is used as an indicator of xenograft-versus-host disease (GvHD). [Figure 18-A] Figures 18A-18C show single-donor and poly-donor CD4IL-10 anti-CD19 CAR cells in a humanized xeno-GvHD mouse model. Figure 18A shows single-donor CD4IL-10 anti-CD19 CAR cells tested in an allogeneic PBMC-induced xeno-GvHD humanized mouse model in which irradiated NSG mice were intravenously (iv) injected on day 3 with (i) allogeneic PBMCs (2.5E6 cells / mouse), (ii) single-donor CD4IL-10 anti-CD19 CAR cells (2.5E6 cells / mouse), (iii) poly-donor CD4IL-10 anti-CD19 CAR cells (2.5E6 cells / mouse), (iv) PBMCs + single-donor CD4IL-10 anti-CD19 CAR cells, or (v) PBMCs + poly-donor CD4IL-10 anti-CD19 CAR cells (2.5E6 cells / mouse). Figure 18B shows CD4IL-10 anti-CD19 CAR cells tested in an allogeneic PBMC-induced xeno-GvHD humanized mouse model, in which irradiated NSG mice were intravenously (iv) injected with allogeneic PBMCs (2.5E6 cells / mouse) and / or single-donor CD4IL-10 anti-CD19 CAR cells on day 3. Figure 18C shows CD4IL-10 anti-CD19 CAR cells tested in an allogeneic PBMC-induced xeno-GvHD humanized mouse model, in which irradiated NSG mice were intravenously (iv) injected with allogeneic PBMCs (2.5E6 cells / mouse) and / or poly-donor CD4IL-10 anti-CD19 CAR cells (2.5E6 cells / mouse) on day 3. PBMC: peripheral blood mononuclear cells; GvHD: graft-versus-host disease. [Figure 18-B] Same as Figure 18-A. [Figure 18-C] Same as Figure 18-A. [Figure 19] FIG. 19 illustrates an exemplary protocol for generating CD4IL-10 cells. [Figure 20-A] Figure 20A shows the percentage of CD4+ΔNGFR+ cells (mean ± SD, n = 10) and vector copy number (VCN, mean ± SD, n = 10) among human CD4+ T cells transduced with LV-IL-10 / ΔNGFR (a bidirectional lentiviral vector encoding truncated forms of human IL-10 and the human NGF receptor). Figure 20B 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 20-B] Same as Figure 20-A. [Figure 21] Figure 21 shows the cytokine production profile of single-donor CD4IL-10 cells after a second (TF2) and third (TF3) restimulation. TF2 and TF3 CD4IL-10 cells were left unstimulated (arrows indicate spontaneous / unstimulated samples) or stimulated with fixed CD3 (10 μg / mL) and soluble CD28 mAb (1 μg / mL) for 48 hours. Culture supernatants were collected, and levels of IL-10, IL-4, IL-5, IFN-γ, and IL-22 were determined by ELISA. All samples were tested in triplicate. Mean ± SD of n=8 donors tested is represented. [Figure 22] Figure 22A shows the percentage of CD4IL-10 cells expressing granzyme B (GzB) after a second round of stimulation (TF2), as analyzed by FACS. Boxplots are shown for n=7 donors and single donors. Figure 22B shows the % dead cells when CD4IL-10 cells (10 cells / well) were cocultured with K562 and ALL-CM cells (10 cells / well) at a 1:1 ratio for 3 days. Boxplots represent data from n=4 donors, and dots represent data from a single donor. [Figure 23-A]Figures 23A and 23B 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, the percentage of proliferating responder cells was determined by flow cytometry by dilution of eFluor® 670 after gating on CD4+ΔNGFR- T cells. Figure 23A shows results from donors C, E, and F, and Figure 23B shows results from donors H, I, and L. Percentages of proliferation and suppression are shown. Suppression mediated by CD4IL-10 cells was calculated as follows: 100-([proliferation of responders in the presence of CD4IL-10 cells / proliferation of responders alone] x 100). [Figure 23-B] Same as Figure 23-A. [Figure 24-A] Figures 24A and 24B 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, the percentage of proliferating responder cells was determined by flow cytometry by dilution of eFluor® 670 after gating on CD4+ΔNGFR- T cells. Figure 24A shows results from donors C, E, and F, and Figure 24B shows results from donors H, I, and L. Percentages of proliferation and suppression are shown. Suppression mediated by CD4IL-10 cells was calculated as follows: 100-([proliferation of responders in the presence of CD4IL-10 cells / proliferation of responders alone] x 100). [Figure 24-B] Same as Figure 24-A. [Figure 25] Figure 25 shows the cytokine production profile of polydonor CD4IL-10 cells after the third restimulation (TF3) compared with the mean levels (±SD) produced by CD4IL-10 cells from eight individual donors. TF3 CD4IL-10 cells from three donors were pooled at a 1:1:1 ratio and stimulated with fixed CD3 (10 μg / mL) and soluble CD28 mAb (1 μg / mL) for 48 hours. Culture supernatants were collected, and IL-10, IL-4, IL-5, IFN-gamma, and IL-22 levels were determined by ELISA. Dots represent the results for polydonor CD4IL-10 cells; gray bars represent the mean ±SD for n=8 single donors. [Figure 26] Figure 26A shows the percentage of polydonor CD4IL-10 cells expressing granzyme B (GzB) compared to the mean % level (±SD) of granzyme B expression by CD4IL-10 cells from n=3 single donors used to generate the pools. Cells were analyzed by FACS after three rounds of stimulation (TF3). Figure 26B shows the % dead cells when polydonor CD4IL-10 cells (10 cells / well) were cocultured with K562 and ALL-CM cells (10 cells / well) at a 1:1 ratio for 3 days. Residual leukemia cells (CD45+CD33+) were counted by FACS for each target cell type. Dots represent polydonor CD4IL-10 results, and gray bars represent the mean ±SD of n=3 single donors used to generate the pools. [Figure 27-A]Figures 27A and 27B show that polydonor CD4IL-10 cells can suppress the proliferation of allogeneic CD4+ and CD8+ T cells. Allogeneic PBMC cells were labeled with eFluor® 670 (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 polydonor CD4IL-10 cells (10 cells / well). After 4 days of culture, the percentage of proliferating responder cells was determined by flow cytometry using eFluor® 670 dilution after gating on CD4+ and CD8+ ΔNGFR- T cells. Figure 27A shows results from polydonor CD4IL-10 cells containing pooled CD4+ cells from donors C, E, and F. Figure 27B shows results from a polydonor CD4IL-10 cell line containing pooled CD4+ cells from donor H, donor I, and donor L. Suppression mediated by CD4IL-10 cells was calculated as follows: 100 - ([proliferation of responder in the presence of CD4IL-10 cells / proliferation of responder alone] x 100). [Figure 27-B] Same as Figure 27-A. [Figure 28] FIG. 28 illustrates a protocol for testing the induction of GvHD by human PBMC and / or polydonor CD4IL-10 cells injected on day 0 after radiation. [Figure 29] Figure 29 shows the % of NSG mice demonstrating GvHD on each day after injection of PBMCs (5x106 / mouse), polydonor (3 donors) CD4IL-10 cells (5x106 / mouse), or PBMCs (5x106 / mouse) combined with polydonor CD4IL-10 cells (3 donors) (5x106 / mouse). [Figure 30]Figure 30 shows migration of CD4IL-10 cells to the spleen (left panel) and bone marrow (right panel) in NSG mice injected with PBMCs (5x106 / mouse), polydonor (3 donors) CD4IL-10 cells (5x106 / mouse), or PBMCs (5x106 / mouse) combined with polydonor CD4IL-10 cells (3 donors) (5x106 / mouse). Boxplots for n=8 donors and single donors are shown. [Figure 31] FIG. 31 illustrates the protocol for testing the induction of GvHD by CD4+ T cells and polydonor or single donor (BC-H) CD4IL-10 cells injected on day 0 after radiation. [Figure 32] FIG. 32 shows the % of NSG mice demonstrating GvHD on each day post-injection. [Figure 33-A] Figures 33A-33C show the graft-versus-leukemia (GvL) effect, assessed by reduction of circulating leukemia cells and long-term leukemia-free survival. Leukemia was measured as previously described (Locafaro G. et al. Molecular Therapy 2017). NSG mice were sublethally irradiated and intravenously injected with myeloid leukemia cells (ALL-CM) (5 × 10 cells) on day 0. Figure 33A is illustrative of the experiment. Figure 33B 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. FIG. 33C shows leukemia-free survival in animals injected on day 3 with PBMCs (5×10 6 ) or polydonor CD4IL-10 cells (from donors BC-I and BC-H) (2.5×10 6 ). [Figure 33-B] Same as Figure 33-A. [Figure 33-C] Same as Figure 33-A. [Figure 34-A]Figures 34A-34C show long-term leukemia-free survival measured in sublethally irradiated NSG mice intravenously injected with ALL-CM cells (5x10) on day 0. Figure 34A shows an illustration of the experiment. Figure 34B shows data from animals injected on day 3 with mononuclear cells (PBMC) (5x10) alone or with mononuclear cells (PBMC) (5x10) plus single-donor CD4IL-10 cells (2.5x10) from donors BC-H and BC-I. Figure 34C shows data from animals injected on day 3 with mononuclear cells (PBMC) (5x10) alone or with mononuclear cells (PBMC) (5x10) plus polydonor CD4IL-10 cells (BC-I / H) (2.5x10). [Figure 34-B] Same as Figure 34-A. [Figure 34-C] Same as Figure 34-A. [Figure 35-A]Figures 35A-35G show the inhibition of NLPR3 inflammasome activation by CD4IL-10 cells. Figure 35A shows the effect of CD4IL-10 cell supernatant from a single donor (#1) on IL-1β production by LPS-activated monocytes. Figure 35B shows the effect of CD4IL-10 cell supernatant from another single donor (#2) on IL-1β production by LPS-activated monocytes. Figure 35C shows the effect of CD4IL-10 cell supernatant from a single donor (#1) on the inhibition of LPS-induced IL-1β production enhanced by the NLPR3 inflammasome activator nigericin (NIG). Figure 35D shows the effect of CD4IL-10 cell supernatant from a single donor (#2) on the inhibition of LPS-induced IL-1β production enhanced by the NLPR3 inflammasome activator nigericin (NIG). Figure 35E is a bar graph showing the effect of CD4IL-10 cell supernatants from a single donor and from cells pooled from two different donors on LPS-induced IL-1β production by monocytes in the presence or absence of anti-IL-10 receptor (anti-IL-10R) mAb. Figure 35F shows the effect of polydonor CD4IL-10 cell supernatants on IL-1β production by monocytes in the presence or absence of anti-IL-10 receptor (anti-IL-10R) mAb. Figure 35G shows the effect of polydonor CD4IL-10 cell supernatants on IL-18 production induced by LPS in combination with nigericin in the presence or absence of anti-IL-10R mAb. [Figure 35-B] Same as Figure 35-A. [Figure 35-C] Same as Figure 35-A. [Figure 35-D] Same as Figure 35-A. [Figure 35-E] Same as Figure 35-A. [Figure 35-F] Same as Figure 35-A. [Figure 35-G] Same as Figure 35-A. [Figure 36]Figure 36 illustrates the experimental protocol for testing graft-versus-myeloid leukemia and xeno-GvHD effects. NSG mice were intravenously injected with ALL-CM cells (2.5 x 10 cells) on day 0. On day 3, mice were divided into five groups and treated with (i) no cells as a control, (ii) allogeneic mononuclear cells (PBMCs), (iii) allogeneic PBMCs and polydonor CD4IL-10 cells (a 1:1:1 pool of BC-E, BC-V, and BC-T), (iv) allogeneic PBMCs and single-donor CD4IL-10 cells (BC-E), or (v) polydonor CD4IL-10 cells at the concentrations indicated in Figure 36. [Figure 37] Figure 37 is a bar graph depicting the cytokine secretion profile of single donor (BC-T, BC-V, and BC-E) and polydonor CD4IL-10 cells (POOL: BC-E, BC-V, and BC-T pooled 1:1:1). [Figure 38] Figure 38 shows the suppressive effect of single-donor (BC-V and BC-E) and polydonor CD4IL-10 cells (pool of BC-V / E / T) on the in vitro proliferation of allogeneic CD4+ and CD8+ T cells. Allogeneic PBMC cells were labeled with eFluor® 670 (5 × 104 cells / well) and stimulated with allogeneic mature dendritic (DC) cells (1 × 104 cells / well) and soluble anti-CD3 mAb at a 1:1 responder:suppressor ratio in the absence or presence of CD4IL-10 cells (5 × 104 cells / well). After 3 days of culture, the percentage of proliferating responder cells was determined by flow cytometry using eFluor® 670 dilution after gating on CD4+-ΔNGFR- (top) or CD8+ΔNGFR- (bottom) T cells. Figure 38 shows the results for cells pooled from single donors BC-V and BC-E, and donors BC-V, BC-E, and BC-T. Percentages of proliferation and suppression are shown. Suppression mediated by CD4IL-10 cells was calculated as follows: 100 - ([proliferation of responders in the presence of CD4IL-10 cells / proliferation of responders alone] x 100). [Figure 39]Figure 39 shows the % of viable cells in cocultures of single (BC-E and BC-V) or polydonor CD4IL-10 cells (BC-E + BC-V + BC-T) with ALL-CM myeloid tumor cells or K562 cells. The results demonstrate the selective cytotoxic effect of single-donor and polydonor CD4IL-10 cells against ALL-CM myeloid tumor cells, but not against K562 cells, which lack class I MHC expression. [Figure 40] Figure 40 shows leukemia-free survival measured in NSG mice intravenously injected with ALL-CM cells (2.5 x 106) on day 0. On day 3, mice were divided into five groups, and each group was treated with (i) no cells as a control, (ii) allogeneic mononuclear cells (PBMCs); (iii) allogeneic PBMCs and polydonor CD4IL-10 cells (B BC-V / T / ET); (iv) allogeneic PBMCs and single-donor CD4IL-10 cells (BC-E); or (v) polydonor CD4IL-10 cells. The graph shows the leukemia-free survival of animals in each group. [Figure 41] Figure 41 shows the percentage of NSG mice free of GvHD on each day following injection of ALL-CM cells (2.5 x 106), followed by administration of (i) no cells as a control; (ii) allogeneic mononuclear cells (PBMCs); (iii) allogeneic PBMCs and polydonor CD4IL-10 cells (BC-E, BC-V, BC-T); (iv) allogeneic PBMCs and single-donor CD4IL-10 cells (BC-E), or (v) polydonor CD4IL-10 cells on day 3. [Figure 42]Figure 42 shows that all NSG mice administered 2.5E+06 PBMCs (allogeneic to donors C, E, F, and H) died on day 22 due to acute, lethal xeno-GvHD. Administration of single-donor CD4IL-10 cells (lot C) or polydonor CD4IL-10 cells (lot CEFH) in combination with PBMCs prevented the development of lethal xeno-GvHD in 75% (3 / 4 mice) and 80% (4 / 5 mice), respectively. In contrast, introduction of 2.5E+06 polydonor CD4IL-10 cells did not induce any signs of GvHD. Collectively, these results demonstrate that polydonor CD4IL-10 cells from four different donors suppress pathogenic human T cell responses more potently, or slightly more potently, than single-donor CD4IL-10 cells. PBMC: peripheral blood mononuclear cells; GvHD: graft-versus-host disease. [Figure 43-A] Figure 43A shows an alignment of IL-10 protein sequences from various species, including human (SEQ ID NO: 1), house mouse (Mus musculus), "MOUSE" (SEQ ID NO: 58); brown rat (Rattus norvegicus), "RAT" (SEQ ID NO: 59); rhesus monkey (Macaca mulatta), "MACMU" (SEQ ID NO: 60); western gorilla (Gorilla gorilla), "GORILLA" (SEQ ID NO: 61); cynomolgus monkey (Macaca fascicularis), "CYNO" (SEQ ID NO: 62); olive baboon (Papio Anubis), "OLIVE BABOON" (SEQ ID NO: 63); bonobo (Pan paniscus), "BONOBO" (SEQ ID NO: 64); chimpanzee (Pan troglodytes), "CHIMP" (SEQ ID NO: 65); and EBVB9 (SEQ ID NO: 66). [Figure 43-B]Figure 43B provides the sequences of IL-10 variants generated by substituting one or more amino acids of human IL-10 with amino acids of viral IL-10 (EBVB9) at the corresponding positions. Also provided are the sequences of exemplary variants, potential huIL-10 hybrid #1 (SEQ ID NO: 67) and potential huIL-10 hybrid #2 (SEQ ID NO: 68). A "*" indicates one or more substituted amino acid positions. A "#" indicates a preferred I105 to A105 amino acid substitution for IL-10 hybrid #2 (SEQ ID NO: 68). [Figure 43-C] Figure 43C shows an alignment of human IL-10 (SEQ ID NO: 1) with IL10 EBVB9 (SEQ ID NO: 66). "*" indicates one or more amino acid positions that are substituted in IL-10 Hybrid #1. "#" indicates the preferred I105 to A105 amino acid substitution for IL-10 Hybrid #2. DETAILED DESCRIPTION OF THE INVENTION

[0238] The drawings depict various embodiments of the present invention for purposes of illustration only. Those skilled in the art will readily recognize from the following discussion that alternative embodiments of the structures and methods illustrated herein may be used without departing from the principles of the present invention as described herein.

[0239] 6. Detailed Description 6.1.Definition Unless otherwise defined, all technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this invention belongs. As used herein, the following terms have the meanings ascribed below.

[0240] The "graft-versus-leukemia effect" or "GvL" refers to the effect that occurs after allogeneic hematopoietic stem cell transplantation (HSCT) or bone marrow transplantation (BMT): T lymphocytes in the allograft eliminate residual malignant host leukemia cells.

[0241] The "graft-versus-tumor effect" or "GvT" refers to an effect that occurs after allogeneic hematopoietic stem cell transplantation (HSCT) or bone marrow transplantation (BMT). T lymphocytes in the allograft eliminate malignant residual host cancer cells, such as myeloma and lymphoid and myeloid leukemias, lymphomas, multiple myeloma, and possibly breast cancer cells. The term GvT is a general form of GvL.

[0242] The terms "treatment," "treating," and the like are used herein in the broadest sense understood in the medical arts. In particular, the term generally refers to obtaining a desired pharmacological and / or physiological effect. "Treatment," as used herein, encompasses any treatment of a mammalian, particularly a human, disease or condition, including (a) preventing a disease or condition from occurring in a subject who may be predisposed to the disease or condition but has not yet been diagnosed with it; (b) inhibiting a disease or condition (e.g., halting its development); or (c) alleviating a disease or condition (e.g., causing regression of a disease or condition, providing improvement in one or more symptoms). Improvement of any condition can be readily assessed according to standard methods and techniques known in the art. The subject population treated by the disease method includes subjects suffering from an undesirable condition or disease, as well as subjects at risk of developing a condition or disease.

[0243] "HLA match" as used herein refers to a pair of individuals with matching HLA alleles at the HLA class I (HLA-A, HLA-B, and HLA-C) and class II (HLA-DRB1 and HLA-DQB1) loci, making the individuals immunologically compatible with each other. HLA compatibility can be determined using any method available in the art, for example, the method described in Tiervy, Haematologica 2016 Volume 101(6):680-687, which is incorporated herein by reference.

[0244] For a given locus, a pair of individuals has a 2 / 2 match if each of two alleles in one individual matches two alleles in the other individual. A pair of individuals has a 1 / 2 match if only one of two alleles in one individual matches one of two alleles in the other individual. A pair of individuals has a 10 / 10 match at the HLA-A, HLA-B, HLA-C, HLA-DRB1, and HLA-DQB1 loci if all 10 alleles in one individual (two at each of the HLA-A, HLA-B, HLA-C, HLA-DRB1, and HLA-DQB1 loci) match all 10 alleles in the other individual.

[0245] In a preferred embodiment, allele-level typing is used to determine HLA compatibility. Allele-level typing involves typing all digits in the first, second, third, and fourth regions, e.g., A * 02:01:01:01. Functionally, the third and fourth regions, which characterize alleles that differ by silent substitutions in coding sequences and substitutions in non-coding sequences, respectively, are used to characterize alleles where the substitution prevents expression of the HLA allele (e.g., null allele B). * The deletion of the invalid allele results in a mismatch that is highly likely to be recognized by alloreactive T cells and has adverse clinical consequences. Substitutions in non-coding sequences can affect expression levels (e.g., the A24low allele A * 24:02:01:02L). Such diversity may also affect anti-HLA allorecognition.

[0246] The term "HLA mismatched," as used herein, refers to a pair of individuals who have mismatched HLA alleles at the HLA class I (HLA-A, HLA-B, and HLA-C) and class II (HLA-DRB1 and HLA-DQB1) loci, making the individuals immunologically incompatible with each other.

[0247] The term "partial HLA mismatch" as used herein refers to a pair of individuals with mismatched HLA alleles at the HLA class I (HLA-A, HLA-B, and HLA-C) and class II (HLA-DRB1 and HLA-DQB1) loci, making the individuals immunologically incompatible to an acceptable degree. Some studies have identified acceptable mismatches. Some HLA class I mismatches are considered to be more acceptable.

[0248] "HLA haplotype" refers to the set of HLA locus alleles, one inherited from the mother and one inherited from the father. Genotypes at the HLA class I (HLA-A, HLA-B, and HLA-C) and class II (HLA-DRB1 and HLA-DQB1) loci can be used to determine the HLA haplotype.

[0249] The term "therapeutically effective amount" is an amount that is effective to treat and thus ameliorate the symptoms of a disease.

[0250] The term "prophylactically effective amount" is an amount effective with respect to the complete or partial prevention of a disease, condition, or symptom thereof.

[0251] The term "ameliorate" refers to any therapeutically beneficial outcome in the treatment of a disease state, eg, a neurodegenerative disease state, including prevention, attenuation of the severity or progression, remission, or cure thereof.

[0252] The term "CD4 IL-10 / CAR " refers to any CD4 that has been genetically modified to express a chimeric antigen receptor. IL-10 Refers to cells. CD4 IL-10 / CAR When referring to a specific chimeric antigen receptor, the name should be based on the specificity of the CAR, e.g., "CD4 IL-10 / 抗[抗gen] CAR " cells. For example, CD4 IL-10 / CAR and CD4 IL-10 / CD19 CAR may be used interchangeably.

[0253] The term "autologous," as used herein, refers to cells derived from the same individual (eg, patient) into which the cells are reintroduced.

[0254] The term "allogenic," as used herein, refers to cells taken from two or more different individuals of the same species who are not genetically identical.

[0255] 6.2. Other Interpretive Conventions Ranges recited herein are understood to be shorthand for all values ​​within the range, including the recited endpoints. For example, the range 1 to 50 is understood to include any number, combination of numbers, or subrange from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, and 50.

[0256] CD4 IL-10 / CAR cell In a first aspect, a CD4+ cell line is provided that has been genetically modified to express a chimeric antigen receptor (CAR) from a first exogenous polynucleotide encoding the CAR, and to express IL-10 from a second exogenous polynucleotide encoding IL-10. + T cells or CD4 + T cell population (CD4 IL-10 / CAR Cells) are described.

[0257] CD4 + T Cells and T Cell Donors CD4 IL-10 / CAR (autologous or allogeneic single-donor CD4 IL-10 / CAR or allogeneic polydonor CD4 IL-10 / CAR ) used to generate the CD4 population + T cells can be isolated from peripheral blood, umbilical cord blood, or other blood samples from a donor, preferably a human donor, using methods available in the art. In a typical embodiment, CD4 +T cells are isolated from peripheral blood. In certain embodiments, CD4 + T cells are isolated using leukopheresis and buffy coat. In certain embodiments, CD4 + T cells are isolated from peripheral blood obtained from a third party.

[0258] In some embodiments, CD4 + T cells were obtained from pre-frozen blood stocks, or pre-frozen peripheral blood mononuclear cells (PBMCs), or CD4 + In some embodiments, the CD4 T cells are isolated from a previously frozen stockpile of CD4 T cells. + T cells are isolated from peripheral blood or PBMCs that have not been previously frozen. In some embodiments, CD4 + T cells are isolated individually from blood or PBMCs from individual donors and then pooled. + T cells are isolated from blood or PBMCs that are initially pooled from multiple donors.

[0259] In some embodiments, CD4 + The T cells are obtained from a single T cell donor. In some embodiments, the CD4 + The T cells are obtained from 2, 3, 4, 5, 6, 7, 8, 9, or 10 different T cell donors.

[0260] In some embodiments, CD4 + T cells are CD4 IL-10 / CAR The cells are obtained from the patient to be treated.

[0261] In some embodiments, the one or more T cell donors are selected regardless of genotype. In some embodiments, the one or more T cell donors are selected based on genotype.

[0262] In certain embodiments, one or more T cell donors are selected based on their HLA haplotype.

[0263] In some embodiments, some or all of the at least two different T cell donors have matching HLA haplotypes. In some embodiments, some or all of the at least two different T cell donors have mismatched HLA haplotypes.

[0264] In some embodiments, the CD4 + The T cells all have at least a 1 / 10, 2 / 10, 3 / 10, 4 / 10, 5 / 10, 6 / 10, 7 / 10, 8 / 10, or 9 / 10 match to each other at the HLA-A, HLA-B, HLA-C, HLA-DRB1, and HLA-DQB1 loci. + The T cells all have at least a 1 / 8, 2 / 8, 3 / 8, 4 / 8, 5 / 8, 6 / 8, 7 / 8, or 8 / 8 match to each other at the HLA-A, HLA-B, HLA-C, and HLA-DRB1 loci. + All T cells have a 2 / 2 match to each other at the HLA-A locus. In some embodiments, the CD4 + The T cells all have a 2 / 2 match with each other at the HLA-B locus. + The T cells all have a 2 / 2 match to each other at the HLA-C locus. In some embodiments, the CD4 + All of the T cells have at least a 3 / 4 or 4 / 4 match to each other at the HLA-DRB1 and HLA DQB1 loci. In some embodiments, the CD4 + All T cells are A * 02 allele, or A * There are 24 alleles.

[0265] In some embodiments, any of the one or more T cell donors is CD4 IL-10 / CAR In some embodiments, none of the one or more T cell donors are the host treated with the CD4 T cells in the methods of treatment described herein. IL-10 / CARIn some embodiments, one or more of the T cell donors are not donors of stem cells (e.g., HSCs), tissues, or organs used with the cells. IL-10 / CAR The host treated by the cells.

[0266] In some embodiments, one or more of the T cell donors are HLA-mismatched or partially HLA-mismatched to the patient (host) being treated. In some embodiments, one or more of the T cell donors have less than a 5 / 10, 6 / 10, 7 / 10, 8 / 10, 9 / 10, or 10 / 10 match to the patient at the HLA-A, HLA-B, HLA-C, HLA-DRB1, and HLA-DQB1 loci. In some embodiments, one or more of the T cell donors have less than a 4 / 8, 5 / 8, 6 / 8, 7 / 8, or 8 / 8 match to the patient at the HLA-A, HLA-B, HLA-C, and HLA-DRB1 loci. In some embodiments, one or more of the T cell donors have less than a 2 / 2 match to the patient at the HLA-A, HLA-B, or HLA-C loci. In some embodiments, one or more of the T cell donors have a 2 / 4, 3 / 4, or less than 4 / 4 match to the patient at the HLA-DRB1 and HLA-DQB1 loci.

[0267] In some embodiments, one or more of the T cell donors are HLA-mismatched or partially HLA-mismatched to the HSC donor. In some embodiments, one or more of the T cell donors have less than a 5 / 10, 6 / 10, 7 / 10, 8 / 10, 9 / 10, or 10 / 10 match to the HSC donor at the HLA-A, HLA-B, HLA-C, HLA-DRB1, and HLA-DQB1 loci. In some embodiments, one or more of the T cell donors have less than a 4 / 8, 5 / 8, 6 / 8, 7 / 8, or 8 / 8 match to the HSC donor at the HLA-A, HLA-B, HLA-C, and HLA-DRB1 loci. In some embodiments, one or more of the T cell donors have less than a 2 / 2 match to the HSC donor at the HLA-A, HLA-B, or HLA-C loci. In some embodiments, one or more of the T cell donors have a 3 / 4 or less than 4 / 4 match to the HSC donor at the HLA-DRB1 and HLA-DQB1 loci.

[0268] In a preferred embodiment, CD4 + None of the T cells are immortalized.

[0269] 6.3.2. Exogenous Polynucleotides Encoding CAR CD4 of the present disclosure IL10 / CAR The cells are genetically modified to express a first exogenous polynucleotide segment encoding a chimeric antigen receptor (CAR). + T cells. CAR has a modular design with four main components: antigen binding domain, hinge, transmembrane domain and intracellular signaling domain.Each of these elements has a distinct function, and the optimal molecular design of CAR can be achieved through the diversity of component protein domains, as described in Rafiq et al. (Nat. Rev. Clin. Onco., 17: 147-167 (2020), the entirety of which is incorporated herein by reference).

[0270] In some embodiments, the CAR-encoding polynucleotide segment encodes a first-generation CAR, a second-generation CAR, or a third-generation CAR. In some embodiments, the first-generation CAR comprises an antigen binding domain, a hinge region, a transmembrane domain, and an intracellular signaling domain. In some embodiments, the second-generation CAR comprises an antigen binding domain, a hinge region, a transmembrane domain, a costimulatory domain, and an intracellular signaling domain. Non-limiting examples of second-generation CARs include those described in U.S. Patent Application Publication Nos. 2004 / 0043401, 2013 / 0287748, 2014 / 0227237, 2014 / 0099309, and 2014 / 0050708; International Publication No. WO2012 / 079000; and International Publication No. WO2015 / 157252, the entire contents of which are incorporated herein by reference.

[0271] In some embodiments, second-generation CAR is pCAR.In some embodiments, pCAR comprises second-generation CAR, which comprises (i) (a) signal transduction region; (b) costimulatory signal transduction region; (c) transmembrane domain; and (d) first binding element that specifically interacts with the first epitope on target antigen; and (ii) (e) (b) different costimulatory signal transduction region; (f) transmembrane domain; and (g) second binding element that specifically interacts with the second epitope on second target antigen.Non-limiting examples of pCAR are those described in US Patent No. 10,703,794, which is incorporated herein by reference in its entirety.

[0272] In some embodiments, the third-generation CAR comprises an antigen binding domain, a hinge region, a transmembrane domain, a first costimulatory domain and a second costimulatory domain, and an intracellular signaling domain.Non-limiting examples of third-generation CAR include those described in US Patent Application Publication No. 2014 / 0322275; No. 2019 / 0345217; No. 2019 / 0112380; and No. 2020 / 0031904, the entirety of which is incorporated herein by reference.

[0273] In some embodiments, the antigen-binding domain may be a single chain antibody fragment (e.g., scFv), a nanobody (e.g., a camelid V H H domains), cytokines, ligands, or peptides (adenectins and DARPins).

[0274] Antigen-binding domain In some embodiments, the antigen-binding domain of the CAR comprises a single-chain antibody fragment. In some embodiments, the single-chain antibody fragment comprises a single-chain Fv (scFv).

[0275] In some embodiments, the antigen binding domain targets an antigen associated with an autoimmune disease, an inflammatory disorder, or cancer. In some embodiments, the antigen binding domain targets an autoantigen.

[0276] In some embodiments, the antigen binding domain targets an antigen associated with an autoimmune disease or inflammatory disorder. In some embodiments, the antigen associated with an autoimmune disease or inflammatory disorder is targeted in the context of an associated MHC molecule, such as, but not limited to, an anti-HLA-A * 02. Anti-HLA-A * 24, or citrullinated peptides, insulin, MOG, GAD65, IA2, gliadin, and desmoglein. In some embodiments, the antigen associated with an autoimmune disease or inflammatory disorder is selected from the group consisting of CD19, CD20, CD22, CD27, BCMA, and CD38.

[0277] In some embodiments, the antigen-binding domain targets a cancer-associated antigen, which in some embodiments is selected from the group consisting of CD19, CD20, CD22, BCMA, B7-H3, CEA, BCMA, CD23, Lyml, Lym2, CLEC5A, CDH179b, FLT3, GCC, Muc, CSF2RA, GFRa4, CD32, CD33, IL1lRa, IL13Ra, NYBRI, SLea, CD200R, TGFbetaR2, CD276, TROP2, LAMP1, PTK7, DLL3, CDH1, CDH6, CDH17, CDH19, TSHR, and tyrosinase.

[0278] In some embodiments, the antigen binding domain targets a B cell antigen. In some embodiments, the B cell antigen is selected based in part on its expression during B cell differentiation, e.g., as shown in FIG. 4. Non-limiting examples of antigen binding domains that target B cell antigens are described in WO 2020 / 010235, which is incorporated herein by reference in its entirety. In some embodiments, the antigen binding domain targets CD19. In some embodiments, the antigen binding domain targets CD20. In some embodiments, the antigen binding domain targets CD22. In some embodiments, the antigen binding domain targets BCMA. In some embodiments, the antigen binding domain targets B7-H3. In some embodiments, the antigen binding domain targets CD27. In some embodiments, the antigen binding domain targets CD38. In some embodiments, the antigen binding domain targets B cell maturation antigen (BCMA). In some embodiments, the antigen binding domain targets carcinoembryonic antigen (CEA).

[0279] In some embodiments, the single-chain antibody fragment comprises a single-chain Fv (scFv) that binds to CD19 (eg, cluster of differentiation 19 protein (CD19) (eg, OMIM Accession No. 107265)).

[0280] In some embodiments, the CD19-targeting scFv is derived from the anti-human CD19-specific mAb clone FMC63 (Nicholson et al., Mol. Immunol., 34(16-17): 1157-65(1997), which is incorporated herein by reference in its entirety). In some embodiments, the CD19-targeting scFv is described in U.S. Patent Application Publication No. 2018 / 0355052, or U.S. Patent Application Publication No. 2020 / 0392200, or International Patent Application Publication No. WO2020 / 010235, each of which is incorporated herein by reference in its entirety.

[0281] In some embodiments, the anti-CD19 antigen-binding domain comprises the sequence of SEQ ID NO: 11. In some embodiments, the anti-CD19 antigen-binding domain comprises a sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 11. In some embodiments, the polynucleotide segment encoding the anti-CD19 antigen-binding domain comprises SEQ ID NO: 12. In some embodiments, the polynucleotide segment encoding the anti-CD19 antigen-binding domain comprises a sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 12.

[0282] In some embodiments, the anti-CD19 antigen-binding domain comprises a heavy chain variable domain comprising a sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 13. In some embodiments, the anti-CD19 antigen-binding domain comprises a light chain variable domain comprising a sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 14. In some embodiments, the anti-CD19 antigen-binding domain comprises a heavy chain variable domain comprising a sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 13, and a light chain variable domain comprising a sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 14.

[0283] In some embodiments, the single-chain antibody fragment comprises a single-chain Fv (scFv) that binds to CD20 (e.g., cluster of differentiation 20 protein (CD20) (e.g., OMIM Accession No. 112210)). Non-limiting examples of scFvs that target CD20 include those described in WO 2020 / 010235 and U.S. Patent Application Publication No. 2020 / 0392200, which are incorporated by reference in their entireties.

[0284] In some embodiments, the anti-CD20 antigen-binding domain comprises the sequence of SEQ ID NO: 18. In some embodiments, the anti-CD20 antigen-binding domain comprises a sequence having at least 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 18. In some embodiments, the polynucleotide segment encoding the anti-CD20 antigen-binding domain comprises SEQ ID NO: 19. In some embodiments, the polynucleotide segment encoding the anti-CD20 antigen-binding domain comprises a sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 19.

[0285] In some embodiments, the anti-CD20 antigen-binding domain comprises a heavy chain variable domain comprising a sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 20. In some embodiments, the anti-CD20 antigen-binding domain comprises a light chain variable domain comprising a sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 21. In some embodiments, the anti-CD20 antigen-binding domain comprises a heavy chain variable domain comprising a sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 20, and a light chain variable domain comprising a sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 21.

[0286] In some embodiments, the single-chain antibody fragment comprises a single-chain Fv (scFv) that binds to CD22 (e.g., cluster of differentiation 22 protein (CD22) (e.g., OMIM Accession No. 107266)). Non-limiting examples of scFvs that target CD22 include those described in WO 2020 / 010235 and U.S. Patent Application Publication No. 2020 / 0392200, which are incorporated by reference in their entireties.

[0287] In some embodiments, the anti-CD22 antigen-binding domain comprises the sequence of SEQ ID NO: 24. In some embodiments, the anti-CD22 antigen-binding domain comprises a sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 24. In some embodiments, the polynucleotide segment encoding the anti-CD22 antigen-binding domain comprises SEQ ID NO: 25. In some embodiments, the polynucleotide segment encoding the anti-CD22 antigen-binding domain comprises a sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 25.

[0288] In some embodiments, the anti-CD22 antigen-binding domain comprises a heavy chain variable domain comprising a sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 26. In some embodiments, the anti-CD22 antigen-binding domain comprises a light chain variable domain comprising a sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 27. In some embodiments, the anti-CD22 antigen-binding domain comprises a heavy chain variable domain comprising a sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 26, and a light chain variable domain comprising a sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 27.

[0289] In some embodiments, the single-chain antibody fragment comprises a single-chain Fv (scFv) that binds to B-cell maturation antigen (BCMA), also known as tumor necrosis factor receptor superfamily member 17 (TNFRSF17).

[0290] Non-limiting examples of scFvs that target BCMA include those described in U.S. Pat. No. 9,765,342 or WO 2010 / 104949, the entire contents of which are incorporated by reference herein. For example, as described in WO 2010 / 104949, an anti-BCMA scFv may comprise the antigen-binding domain of the A7D12.2, C11 D5.3, C12A3.2, or C13F12.1 antibody.

[0291] In some embodiments, the anti-BCMA antigen binding domain comprises a heavy chain variable domain comprising a sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 50 or 52. In some embodiments, the anti-BCMA antigen binding domain comprises a light chain variable domain comprising a sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 51 or 53. In some embodiments, the anti-BCMA antigen binding domain comprises a heavy chain variable domain comprising a sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 50 or 52, and a light chain variable domain comprising a sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 51 or 53.

[0292] In some embodiments, the single-chain antibody fragment comprises a single-chain Fv (scFv) that binds to B7-H3, also known as CD276 (e.g., cluster of differentiation 276 protein (CD276) (e.g., OMIM Accession No. 605715)).

[0293] Non-limiting examples of scFvs targeting B7-H3 include those described in U.S. Patent Application Publication Nos. 2016 / 0053017 and 2018 / 0346544, which are incorporated by reference in their entireties. For example, as described in U.S. Patent Application Publication No. 2018 / 0346544, an anti-CD276 scFv can include the antigen-binding domain of the MGA271 (CD276.MG), CD276.N1, CD276.N2, CD276.N3, CD276.N4, or CD276.N5 antibody.

[0294] In some embodiments, the anti-B7-H3 antigen-binding domain comprises the sequence of SEQ ID NO: 36. In some embodiments, the anti-B7-H3 antigen-binding domain comprises a sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 36. In some embodiments, the polynucleotide segment encoding the anti-B7-H3 antigen-binding domain comprises SEQ ID NO: 37. In some embodiments, the polynucleotide segment encoding the anti-B7-H3 antigen-binding domain comprises a sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 37.

[0295] In some embodiments, the anti-B7-H3 antigen-binding domain comprises a heavy chain variable domain comprising a sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 38. In some embodiments, the anti-B7-H3 antigen-binding domain comprises a light chain variable domain comprising a sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 39. In some embodiments, the anti-B7-H3 antigen-binding domain comprises a heavy chain variable domain comprising a sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 38, and a light chain variable domain comprising a sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 39.

[0296] In some embodiments, the single-chain antibody fragment comprises a single-chain Fv (scFv) that binds to CD27 (eg, OMIM Accession No. 186711).

[0297] In some embodiments, the single-chain antibody fragment comprises a single-chain Fv (scFv) that binds to CD38 (eg, OMIM Accession No. 107270).

[0298] In some embodiments, the single-chain antibody fragment comprises a single-chain Fv (scFv) that binds to CEA (eg, carcinoembryonic antigen protein (CEA) (eg, OMIM Accession No. 114890)).

[0299] In some embodiments, the single chain antibody fragment comprises a single chain Fv (scFv) that binds to BCMA (e.g., B cell maturation antigen (BCMA) (e.g., OMIM Accession No. 109545)).

[0300] Hinge Area The hinge region connects the extracellular antigen-binding domain to the intracellular signaling domain (e.g., one or more costimulatory domains and the intracellular signaling domain) through the transmembrane domain. The hinge provides sufficient flexibility to overcome steric hindrance and an appropriate length to facilitate access and binding of the antigen-binding domain to the target antigen. In some embodiments, differences in hinge length and composition can affect antigen binding and signaling through the CAR. For example, a spacer sequence in or added to the hinge region facilitates access and binding of the antigen-binding domain to the target antigen. In some embodiments, the hinge region affects cytokine production.

[0301] In some embodiments, the hinge region is selected from a human CD8 hinge region, a human CD28 hinge region, an IgG1 hinge region, or an IgG4 hinge region. In some embodiments, the hinge region is derived from human CD8. In some embodiments, the hinge region derived from human CD8 comprises the sequence of SEQ ID NO: 28. In some embodiments, the hinge region derived from human CD8 comprises a sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 28.

[0302] 6.3.2.3. Transmembrane Domain The transmembrane region binds the CAR to CD4 IL10 / CAR In some embodiments, the transmembrane domain affects the stability and function of the CAR.

[0303] In some embodiments, the transmembrane domain is selected from the group consisting of a TNFRSF19 transmembrane domain, a CD3 zeta transmembrane domain, a CD8α transmembrane domain, a CD4 transmembrane domain, a CD28 transmembrane domain, or a B7 family-induced costimulatory (ICOS) transmembrane domain. In some embodiments, the transmembrane domain is derived from TNFRSF19. In some embodiments, the transmembrane domain derived from TNFRSF19 comprises the sequence of SEQ ID NO: 29. In some embodiments, the transmembrane domain derived from TNFRSF19 comprises a sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 29.

[0304] 6.3.2.4. Intracellular Signaling and Costimulatory Domains The intracellular signaling domain is CD4 IL10 / CAR Activates T cells. In some embodiments, the intracellular signaling domain is involved in T cell function, metabolism, and survival.

[0305] In some embodiments, the intracellular signaling domain comprises an immunoreceptor tyrosine-based activation motif (ITAM). In some embodiments, the intracellular signaling domain comprises two or more immunoreceptor tyrosine-based activation motifs (ITAMs). In some embodiments, the immunoreceptor tyrosine-based activation motifs (ITAMs) are derived from CD3 zeta. In some embodiments, the intracellular signaling domain derived from CD3 zeta comprises the sequence of SEQ ID NO: 30. In some embodiments, the intracellular signaling domain derived from CD3 zeta comprises a sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 30.

[0306] In some embodiments, the intracellular signaling domain further comprises one or more costimulatory domains. In such cases, the one or more costimulatory domains are selected from the group consisting of CD4, CD4+ ... + Enhances T cell activation, function, metabolism, and survival compared to T cells. CD4 with a CAR containing a costimulatory domain in addition to the activation domain. + (e.g., CD4 IL10 / CAR ) T cells produce IL-2 and can proliferate with repeated antigen exposure.

[0307] In some embodiments, costimulatory domains from different sources (e.g., 4-1BB and CD28) are combined with CD4 + (e.g., CD4 IL10 / CAR ) induce distinct functional and metabolic profiles in T cells. For example, CD4 T cells bearing CARs containing costimulatory domains derived from CD28 + (e.g., CD4 IL10 / CAR ) T cells undergo enhanced differentiation into effector memory T cells. In another example, CD4 T cells bearing a CAR containing a costimulatory domain derived from 4-1BB undergo enhanced differentiation into effector memory T cells. + (e.g., CD4 IL10 / CAR ) T cells undergo enhanced differentiation into central memory T cells.

[0308] In some embodiments, the one or more costimulatory domains are 4-1BB, CD28, OX40, ICOS, CD27, MYD88-CD40, and KIR2DS2.

[0309] In some embodiments, one or more costimulatory domains are derived from 4-1BB. In some embodiments, the costimulatory domain derived from 4-1BB comprises the sequence of SEQ ID NO: 31. In some embodiments, the costimulatory domain derived from 4-1BB comprises a sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 31.

[0310] In some embodiments, the one or more costimulatory domains are derived from CD28. In some embodiments, the one or more costimulatory domains comprise a CD28 costimulatory domain. In some embodiments, the costimulatory domain derived from CD28 comprises the sequence of SEQ ID NO: 32. In some embodiments, the costimulatory domain derived from CD28 comprises a sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 32.

[0311] In some embodiments, the CAR comprises two costimulatory domains, ie, a first costimulatory domain comprising a costimulatory domain derived from 4-1BB and a second costimulatory domain comprising a costimulatory domain derived from a CD28 costimulatory domain.

[0312] Chimeric Antigen Receptors (CARs) In some embodiments, the polynucleotide segment encoding a CAR encodes a CAR that includes an antigen-binding domain that targets an antigen associated with an autoimmune disease, an inflammatory disorder, or a cancer.

[0313] In some embodiments, the polynucleotide segment encoding the CAR encodes a CAR comprising an antigen binding domain that targets an antigen associated with an autoimmune disease or inflammatory disorder. In some embodiments, the antigen associated with an autoimmune disease or inflammatory disorder is, in the context of an associated MHC molecule, such as, but not limited to, an anti-HLA-A * 02. Anti-HLA-A * 24, or citrullinated peptides, insulin, MOG, GAD65, IA2, gliadin, and desmoglein.

[0314] In some embodiments, the polynucleotide segment encoding the CAR encodes a CAR comprising an antigen-binding domain that targets a cancer-associated antigen. In some embodiments, the cancer-associated antigen is selected from the group consisting of CD19, CD20, CD22, B7-H3, CEA, BCMA, CD23, Lym1, Lym2, CLEC5A, CDH179b, FLT3, GCC, Muc, CSF2RA, GFRa4, CD32, CD33, IL11Ra, IL13Ra, NYBRI, SLea, CD200R, TGFbetaR2, CD276, TROP2, LAMP1, PTK7, DLL3, CDH1, CDH6, CDH17, CDH19, TSHR, and tyrosinase.

[0315] In some embodiments, the polynucleotide segment encoding the CAR encodes a CAR comprising an antigen binding domain that targets a B cell antigen. In some embodiments, the B cell antigen is selected based in part on its expression during B cell differentiation, for example, as shown in FIG. 4. Non-limiting examples of antigen binding domains that target B cell antigens are described in WO 2020 / 010235, which is incorporated herein by reference in its entirety. In some embodiments, the antigen binding domain targets CD19. In some embodiments, the antigen binding domain targets CD20. In some embodiments, the antigen binding domain targets CD22. In some embodiments, the antigen binding domain targets B7-H3. In some embodiments, the antigen binding domain targets CD27. In some embodiments, the antigen binding domain targets CD38. In some embodiments, the antigen binding domain targets B cell maturation antigen (BCMA). In some embodiments, the antigen binding domain targets carcinoembryonic antigen (CEA).

[0316] In some embodiments, the polynucleotide segment encoding the CAR encodes a CAR comprising an anti-CD19 antigen binding domain. In some embodiments, the polynucleotide segment encoding the CAR encodes a second-generation CAR comprising an anti-CD19 antigen binding domain. In some embodiments, the polynucleotide segment encoding the CAR encodes a third-generation CAR comprising an anti-CD19 antigen binding domain.

[0317] In some embodiments, the polynucleotide segment encoding the CAR encodes an anti-CD19 CAR comprising an anti-CD19 antigen-binding domain, a hinge region, a transmembrane domain, a costimulatory domain, and an intracellular signaling domain. In some embodiments, the polynucleotide segment encoding the CAR encodes an anti-CD19 antigen-binding domain; a human CD8 hinge region; a TNFRSF19 transmembrane region; a 4-1BB costimulatory domain; and a CD3 zeta chain intracellular signaling domain. In some embodiments, the anti-CD19 CAR comprises the sequence of SEQ ID NO: 9. In some embodiments, the anti-CD19 CAR comprises a sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 9. In some embodiments, the first exogenous polynucleotide segment comprises the sequence of SEQ ID NO: 10. In some embodiments, the first exogenous polynucleotide segment comprises a sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 10.

[0318] Non-limiting examples of CARs comprising an anti-CD19 antigen binding domain include those described in U.S. Patent Application Publication No. 2020 / 0392200, WO 2020 / 010235, or WO 2012 / 079000, which are incorporated by reference in their entireties.

[0319] In some embodiments, the polynucleotide segment encoding the CAR encodes a CAR comprising an anti-CD20 antigen binding domain. In some embodiments, the polynucleotide segment encoding the CAR encodes a second-generation CAR comprising an anti-CD20 antigen binding domain. In some embodiments, the polynucleotide segment encoding the CAR encodes a third-generation CAR comprising an anti-CD20 antigen binding domain.

[0320] In some embodiments, the polynucleotide segment encoding the CAR encodes an anti-CD20 CAR comprising an anti-CD20 antigen-binding domain, a transmembrane domain, a costimulatory domain, and an intracellular signaling domain. In some embodiments, the polynucleotide segment encoding the CAR encodes an anti-CD20 antigen-binding domain; a human CD8 transmembrane region; a 4-1BB costimulatory domain; and a CD3 zeta chain intracellular signaling domain. In some embodiments, the anti-CD20 CAR comprises the sequence of SEQ ID NO: 16. In some embodiments, the anti-CD20 CAR comprises a sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 16. In some embodiments, the first exogenous polynucleotide segment comprises the sequence of SEQ ID NO: 17. In some embodiments, the first exogenous polynucleotide segment comprises a sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 17.

[0321] Non-limiting examples of CARs comprising an anti-CD20 antigen binding domain include those described in U.S. Patent Application Publication No. 2020 / 0392200, or WO 2020 / 010235, both of which are incorporated by reference herein in their entirety.

[0322] In some embodiments, the polynucleotide segment encoding the CAR encodes a CAR comprising an anti-CD22 antigen binding domain. In some embodiments, the polynucleotide segment encoding the CAR encodes a second-generation CAR comprising an anti-CD22 antigen binding domain. In some embodiments, the polynucleotide segment encoding the CAR encodes a third-generation CAR comprising an anti-CD22 antigen binding domain.

[0323] In some embodiments, the anti-CD22 CAR comprises the sequence of SEQ ID NO: 22. In some embodiments, the anti-CD22 CAR comprises a sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 22. In some embodiments, the first exogenous polynucleotide segment comprises the sequence of SEQ ID NO: 23. In some embodiments, the first exogenous polynucleotide segment comprises a sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 23.

[0324] Non-limiting examples of CARs, including anti-CD22 CARs, include those described in International Publication No. WO 2020 / 010235 and U.S. Patent Application Publication No. 2020 / 0392200, which are incorporated by reference in their entireties.

[0325] Non-limiting examples of CARs, including anti-B7-H3 CARs, include those described in U.S. Patent Application Publication Nos. 2016 / 0053017, 2017 / 0369585, and 2018 / 0346544, which are incorporated by reference in their entireties.

[0326] In some embodiments, the polynucleotide segment encoding the CAR encodes a CAR comprising an anti-B7-H3 antigen binding domain. In some embodiments, the polynucleotide segment encoding the CAR encodes a second-generation CAR comprising an anti-B7-H3 antigen binding domain. In some embodiments, the polynucleotide segment encoding the CAR encodes a third-generation CAR comprising an anti-B7-H3 antigen binding domain.

[0327] In some embodiments, the anti-B7-H3 CAR comprises the sequence of SEQ ID NO: 34. In some embodiments, the anti-B7-H3 CAR comprises a sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 34. In some embodiments, the first exogenous polynucleotide segment comprises the sequence of SEQ ID NO: 35. In some embodiments, the first exogenous polynucleotide segment comprises a sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 35.

[0328] In some embodiments, the polynucleotide segment encoding the CAR encodes a CAR comprising an anti-CD27 antigen binding domain. In some embodiments, the polynucleotide segment encoding the CAR encodes a second-generation CAR comprising an anti-CD27 antigen binding domain. In some embodiments, the polynucleotide segment encoding the CAR encodes a third-generation CAR comprising an anti-CD27 antigen binding domain.

[0329] In some embodiments, the polynucleotide segment encoding the CAR encodes a CAR comprising an anti-CD38 antigen binding domain. In some embodiments, the polynucleotide segment encoding the CAR encodes a second-generation CAR comprising an anti-CD38 antigen binding domain. In some embodiments, the polynucleotide segment encoding the CAR encodes a third-generation CAR comprising an anti-CD38 antigen binding domain.

[0330] In some embodiments, the polynucleotide segment encoding the CAR encodes a CAR comprising an anti-CEA antigen binding domain. In some embodiments, the polynucleotide segment encoding the CAR encodes a second-generation CAR comprising an anti-CEA antigen binding domain. In some embodiments, the polynucleotide segment encoding the CAR encodes a third-generation CAR comprising an anti-CEA antigen binding domain.

[0331] In some embodiments, the polynucleotide segment encoding the CAR encodes a CAR that comprises an anti-BCMA antigen binding domain. In some embodiments, the polynucleotide segment encoding the CAR encodes a second generation CAR that comprises an anti-BCMA antigen binding domain. In some embodiments, the polynucleotide segment encoding the CAR encodes a third generation CAR that comprises an anti-BCMA antigen binding domain. In some embodiments, the anti-BCMA CAR comprises the sequence of SEQ ID NOs: 41-49 and 54. In some embodiments, the anti-BCMA CAR comprises a sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NOs: 41-49 and 54.

[0332] The first exogenous polynucleotide further comprises one or more regulatory elements (e.g., any regulatory element described herein or known in the art) operably linked to the coding sequence of the CAR. In some embodiments, the regulatory element is a promoter.

[0333] In some embodiments, the regulatory element is CD4 + In some embodiments, the promoter comprises a promoter capable of directing expression of the CAR in T cells. + In some embodiments, the promoter drives constitutive expression of the CAR in T cells. + Drives expression of CAR in T cells.

[0334] In some embodiments, the first exogenous polynucleotide segment is a CD4 + It further comprises a segment encoding a selectable marker (eg, any selectable marker described herein or known in the art) that allows for selection of T cells.

[0335] In some embodiments, the first exogenous polynucleotide segment is expressed using a vector to express CD4 +In some embodiments, the vector is a plasmid vector. In some embodiments, the vector is a viral vector. In some embodiments, the vector is a retroviral vector (e.g., a gamma retroviral vector). In some embodiments, the vector is a lentiviral vector.

[0336] In some embodiments, the first exogenous polynucleotide segment is transduced into CD4 + The first exogenous polynucleotide segment is delivered to T cells, and the first exogenous polynucleotide segment comprises a lentiviral vector sequence. In certain embodiments, the lentiviral vector disclosed in Matrai et al., Molecular Therapy 18(3):477-490(2010) ("Matrai"), incorporated herein by reference, is used. In some embodiments, the first exogenous polynucleotide segment encoding a chimeric antigen receptor (CAR) and the second exogenous polynucleotide segment encoding interleukin-10 (IL-10) are present in a single lentiviral vector. In such cases, the first exogenous polynucleotide and the second exogenous polynucleotide segment are operably linked to a first promoter. For example, the lentiviral vector comprises a human PGK promoter, a first exogenous polynucleotide operably linked to a second exogenous polynucleotide segment via an internal ribosome entry site (IRES), or a self-cleaving peptide (e.g., a 2A self-cleaving peptide).

[0337] In some embodiments, the first exogenous polynucleotide segment is integrated into the T cell nuclear genome. In some embodiments, the first exogenous polynucleotide segment is not integrated into the nuclear genome. In some embodiments, the first exogenous polynucleotide segment is present in the cytoplasm of the T cell.

[0338] 6.3.3. Exogenous Polynucleotides Encoding IL-10 The single donor CD4IL-10 / CAR Cellular or polydonor CD4 IL-10 / CAR The cells are further genetically modified to include a second exogenous polynucleotide segment encoding IL-10. The second exogenous polynucleotide segment comprises the IL-10-encoding polynucleotide segment operably linked to one or more regulatory elements (e.g., any one or more regulatory elements described herein or known in the art).

[0339] The IL-10-encoding polynucleotide segment may encode human, bonobo, or rhesus macaque IL-10. In some embodiments, the IL-10-encoding polynucleotide segment encodes human IL-10 having the sequence of SEQ ID NO: 1. In some embodiments, the IL-10-encoding polynucleotide segment encodes a variant of human IL-10 having at least 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 1. In some embodiments, the IL-10-encoding polynucleotide segment has the nucleotide sequence of SEQ ID NO: 2. In some embodiments, the IL-10-encoding polynucleotide segment has at least 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 2.

[0340] In some embodiments, the exogenous polynucleotide encodes viral IL-10. In various embodiments, the exogenous polypeptide encodes IL-10 from HCMV, GMCMV, RhCMV, BaCMV, MOCMV, SMCMV, EBV, bonobo-HV, BaLCV, OvHV-2, EHV-2, CyHV-3, AngHV-1, ORFV, BPSV, PCPV, LSDV, SPV, GPV, or CNPV. In some embodiments, the exogenous polypeptide encodes viral IL-10 from EBV or ORFV.

[0341] In some embodiments, the viral IL-10 comprises the sequence of SEQ ID NO: 6. In some embodiments, the viral IL-10 comprises a sequence having at least 80%, 85%, 90%, 95%, 97%, 98%, 98%, or 99% sequence identity to SEQ ID NO: 6 or 18. In some embodiments, the exogenous polypeptide encoding the viral IL-10 comprises the sequence of SEQ ID NO: 7. In some embodiments, the exogenous polypeptide encoding the viral IL-10 comprises a sequence having at least 80%, 85%, 90%, 95%, 97%, 98%, 98%, or 99% sequence identity to SEQ ID NO: 7.

[0342] In some embodiments, the IL-10 is a protein comprising human IL-10 with 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid modifications. In some embodiments, the 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid modifications are substituted with amino acids of viral IL-10 at the corresponding amino acid positions.

[0343] In some embodiments, the polynucleotide segment encoding IL-10 encodes a variant of human IL-10 having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acid modifications compared to human IL-10 (e.g., SEQ ID NO: 1). In some embodiments, the 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions are substitution(s) with amino acid(s) of viral IL-10 at the corresponding amino acid position(s). In some embodiments, the polynucleotide segment encoding IL-10 encodes a variant of human IL-10 having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acid insertions, deletions, or modifications compared to human IL-10 (e.g., SEQ ID NO: 1). In some embodiments, the variant of human IL-10 has the sequence of SEQ ID NO: 56 or 57.

[0344] In some embodiments, the polynucleotide segment encoding IL-10 encodes IL-10 from house mouse (Mus musculus), "MOUSE" (SEQ ID NO: 58); brown rat (Rattus norvegicus), "RAT" (SEQ ID NO: 59); rhesus monkey (Macaca mulatta), "MACMU" (SEQ ID NO: 60); western gorilla (Gorilla gorilla), "GORILLA" (SEQ ID NO: 61); cynomolgus monkey (Macaca fascicularis), "CYNO" (SEQ ID NO: 62); olive baboon (Papio anubis), "OLIVE BABOON" (SEQ ID NO: 63); bonobo (Pan paniscus), "BONOBO" (SEQ ID NO: 64); chimpanzee (Pan troglodytes), "CHIMP" (SEQ ID NO: 65); and EBVB9 (SEQ ID NO: 66). In some embodiments, the polynucleotide segment encoding IL-10 encodes a protein having at least 90%, 95%, 98%, or 99% sequence identity to IL-10 of: house mouse (Mus musculus), "MOUSE" (SEQ ID NO: 58); brown rat (Rattus norvegicus), "RAT" (SEQ ID NO: 59); rhesus monkey (Macaca mulatta), "MACMU" (SEQ ID NO: 60); western gorilla (Gorilla gorilla), "GORILLA" (SEQ ID NO: 61); cynomolgus monkey (Macaca fascicularis), "CYNO" (SEQ ID NO: 62); olive baboon (Papio anubis), "OLIVE BABOON" (SEQ ID NO: 63); bonobo (Pan paniscus), "BONOBO" (SEQ ID NO: 64); chimpanzee (Pan troglodytes), "CHIMP" (SEQ ID NO: 65); and EBVB9 (SEQ ID NO: 66).

[0345] In some embodiments, the polynucleotide segment encoding IL-10 encodes a variant of human IL-10 having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acid substitutions, insertions, and / or deletions compared to human IL-10 (e.g., SEQ ID NO: 1). In some embodiments, the modification is a substitution, insertion, and / or deletion at corresponding positions with amino acids from Mus musculus, "MOUSE" (SEQ ID NO: 58); Rattus norvegicus, "RAT" (SEQ ID NO: 59); Rhesus macaque, "MACMU" (SEQ ID NO: 60); Gorilla gorilla, "GORILLA" (SEQ ID NO: 61); Macaca fascicularis, "CYNO" (SEQ ID NO: 62); Papio anubis, "OLIVE BABOON" (SEQ ID NO: 63); Bonobo, "BONOBO" (SEQ ID NO: 64); Chimpanzee, "CHIMP" (SEQ ID NO: 65); and EBVB9 (SEQ ID NO: 66). In some embodiments, the variant of human IL-10 has the sequence of SEQ ID NO: 67 or SEQ ID NO: 68.

[0346] In some embodiments, the polynucleotide segment encoding IL-10 encodes a variant of human IL-10 that has reduced immunostimulatory activity compared to human IL-10. In some embodiments, the variant of human IL-10 comprises an I105A substitution. In some embodiments, the variant of human IL-10 is produced using the method described in "A Single Amino Acid Determines the Immunostimulatory Activity of Interleukin 10," J Exp Med, 191, 2 (2000), pp. 213-223.

[0347] The second exogenous polynucleotide segment further comprises one or more regulatory elements operably linked to the coding sequence for IL-10, the one or more regulatory elements being operably linked to the coding sequence for transduced CD4 + Directs expression of encoded IL-10 in T cells.

[0348] In some embodiments, the regulatory element(s) is CD4 + In some embodiments, the promoter comprises a promoter capable of directing expression of IL-10 in T cells. + In some embodiments, the promoter drives constitutive expression of IL-10 in T cells. + Drives IL-10 expression in T cells.

[0349] In some embodiments, the second exogenous polynucleotide segment is a segment encoding a CD4 + It further comprises a segment encoding a selectable marker (eg, any selectable marker described herein or known in the art) that allows for selection of T cells.

[0350] In typical embodiments, the exogenous polynucleotide is delivered to the CD4+ T cell using a vector. In some embodiments, the vector is a plasmid vector. In some embodiments, the vector is a viral vector.

[0351] In some embodiments, the first exogenous polynucleotide segment encoding a CAR and the second exogenous polynucleotide segment encoding IL-10 are present in the same vector. In some embodiments, the first exogenous polynucleotide segment encoding a CAR and the second exogenous polynucleotide segment encoding IL-10 are present in the same viral vector. In some embodiments, the first exogenous polynucleotide segment encoding a CAR and the second exogenous polynucleotide segment encoding IL-10 are present in the same lentiviral vector.

[0352] In certain embodiments, the exogenous polynucleotide is delivered to the CD4+ T cell using a lentiviral vector, and the exogenous polynucleotide comprises a lentiviral vector sequence. In certain embodiments, the lentiviral vector disclosed in Matrai et al., Molecular Therapy 18(3):477-490(2010) ("Matrai"), which is incorporated herein by reference, is used.

[0353] In some embodiments, the second exogenous polynucleotide segment is integrated into the T cell nuclear genome. In some embodiments, the second exogenous polynucleotide segment is not integrated into the nuclear genome. In some embodiments, the second exogenous polynucleotide segment is present in the cytoplasm of the T cell.

[0354] In certain embodiments, the second exogenous polynucleotide segment has the sequence of SEQ ID NO: 5. In some embodiments, the exogenous polynucleotide has at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 5.

[0355] Regulatory Elements In some embodiments, the first and / or second exogenous polynucleotide segment each further comprises a regulatory element operably linked to the coding sequence of the CAR or IL- 10. In some embodiments, the regulatory element comprises a promoter sequence, an enhancer sequence, a non-coding sequence, or any combination thereof.

[0356] In some embodiments, the first exogenous polynucleotide segment further comprises a regulatory element operably linked to the coding sequence of the CAR, wherein the regulatory element encodes a transduced CD4 + Direct expression of the encoded CAR in T cells. In some embodiments, an inducible promoter is used to induce expression of the CAR when therapeutically appropriate.

[0357] In some embodiments, the second exogenous polynucleotide segment further comprises a regulatory element operably linked to the coding sequence of IL-10, wherein the regulatory element is a coding sequence of transduced CD4 + In some embodiments, an inducible promoter is used to induce expression of IL-10, where therapeutically appropriate. In some embodiments, the IL-10 promoter is used.

[0358] In some embodiments, the promoter is a tissue-specific promoter. In some embodiments, the promoter is a lineage-specific promoter. In some embodiments, the promoter is a promoter that can be used to drive ubiquitous expression of CAR or IL-10.

[0359] In some embodiments, the promoter is a native human promoter. In some embodiments, the promoter is a human elongation factor (EF) 1α promoter. In some embodiments, the promoter is a human phosphoglycerate kinase promoter (PGK). In some embodiments, the promoter is a human ubiquitin C promoter (UBI-C).

[0360] In some embodiments, the promoter is a synthetic promoter. In certain embodiments, the promoter is a minimal CMV core promoter. In certain embodiments, the promoter is an inducible or constitutive bidirectional promoter. In specific embodiments, the synthetic bidirectional promoter disclosed in Amendola et al., Nature Biotechnology, 23(1):108-116(2005) is used. This promoter can mediate the coordinated transcription of two mRNAs ubiquitously or in a tissue-specific manner. In certain embodiments, the bidirectional promoter induces the expression of CAR and a selection marker, or IL-10 and a selection marker.

[0361] In some embodiments, the promoter is a native human promoter. In some embodiments, the promoter is a human elongation factor (EF) 1α promoter. In some embodiments, the promoter is a human phosphoglycerate kinase promoter (PGK). In some embodiments, the promoter is a human ubiquitin C promoter (UBI-C).

[0362] In some embodiments, the first and / or second exogenous polynucleotide segment further comprises one or more non-coding sequences 3' to the coding sequence. Non-limiting examples of non-coding sequences 3' to the coding sequence include a 3' untranslated region (UTR), a poly(A) signal, and a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE).

[0363] In some embodiments, the exogenous polynucleotide comprises more than one coding sequence. For example, the exogenous polynucleotide comprises a first exogenous polynucleotide segment and a second exogenous polynucleotide segment on a single contiguous polynucleotide sequence (e.g., a single polynucleotide construct). In some embodiments, the one or more coding sequences comprise a sequence encoding a CAR and a sequence encoding a selectable marker. In some embodiments, the one or more coding sequences comprise a sequence encoding IL-10 and a sequence encoding a selectable marker. In some embodiments, the one or more coding sequences comprise a sequence encoding a CAR, a sequence encoding IL-10, and one or more sequences encoding a selectable marker. In some embodiments, the exogenous polynucleotide does not comprise a selectable marker.

[0364] In some embodiments, the multiple coding sequences are separated by one or more internal ribosome entry sites (IRES).

[0365] In some embodiments, the multiple coding sequences are separated by one or more self-cleaving peptides. In some embodiments, the self-cleaving peptide can be a 2A self-cleaving peptide. Non-limiting examples of self-cleaving peptides include 2A peptides (18-22 amino acids), including peptides from hand, foot, and mouth disease virus (F2A), porcine teschovirus 1 (P2A), Thosea asigna virus (T2A), or equine rhinitis A virus (E2A). In some embodiments, the first exogenous polynucleotide, the second exogenous polynucleotide, or both, comprise a sequence encoding a furin P2A peptide. In some embodiments, the first exogenous polynucleotide, the second exogenous polynucleotide, or both, comprise a sequence encoding a T2A peptide. In some embodiments, the first exogenous polynucleotide, the second exogenous polynucleotide, or both, comprise a sequence encoding an E2A peptide.

[0366] In some embodiments, the coding sequence further comprises a self-cleaving peptide between the heavy chain coding sequence and the light chain coding sequence, hi some embodiments, the self-cleaving peptide is selected from the group consisting of F2A, P2A, T2A and E2A.

[0367] Selection Markers In some embodiments, the first exogenous polynucleotide segment, the second exogenous nucleotide segment, or both, are capable of expressing a CD4 + and further comprising a polynucleotide segment encoding a selectable marker that allows for selection of T cells. In some embodiments, the first exogenous polynucleotide segment, the second exogenous nucleotide segment, or both do not comprise a selectable marker.

[0368] In some embodiments, the exogenous polynucleotide comprises a first exogenous polynucleotide segment and a second exogenous polynucleotide segment present in a single contiguous polynucleotide sequence (e.g., a single polynucleotide construct). In such cases, the exogenous polynucleotide is a contiguous polynucleotide sequence that is present in a single polynucleotide construct.+ It contains one or more selectable markers that allow for the selection of T cells.

[0369] In some embodiments, the selectable marker is ΔNGFR. In certain embodiments, the selectable marker is a polypeptide having the sequence of SEQ ID NO: 3. In certain embodiments, the selectable marker is a polypeptide having at least 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 3. In certain embodiments, the nucleotide sequence encoding the ΔNGFR selectable marker has the sequence of SEQ ID NO: 4. In some embodiments, the nucleotide sequence encoding the ΔNGFR selectable marker has at least 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 4.

[0370] In some embodiments, expression of the selectable marker correlates with expression of IL-10 from the exogenous polynucleotide. In some embodiments, expression of the selectable marker correlates linearly with expression of IL-10 from the exogenous polynucleotide. Thus, in some embodiments, expression of the selectable marker is measured to estimate expression of IL-10 from the exogenous polynucleotide.

[0371] In some embodiments, the selectable marker is a truncated form of an EGFR polypeptide. In some embodiments, the selectable marker is a truncated form of a human EGFR polypeptide, optionally a huEGFR polypeptide, as disclosed in Wang et al. "A transgene-encoded cell surface polypeptide for selection, in vivo tracking, and ablation of engineered cells," Blood, v. 118, no. 5 (2011), which is hereby incorporated by reference in its entirety.

[0372] In some embodiments, the exogenous polynucleotide further comprises a sequence encoding an antibiotic resistance gene. In some embodiments, the exogenous polynucleotide comprises a sequence encoding an ampicillin resistance gene.

[0373] CD4 IL-10 / CAR Cellular or polydonor CD4 IL-10 / CAR Gene expression CD4 IL-10 / CAR In some embodiments, the cells express IL-10. IL-10 / CAR The cells constitutively express IL-10. In some embodiments, the CD4 IL-10 / CAR Upon activation, the cells express IL-10.

[0374] In some embodiments, CD4 IL-10 / CAR Cells (e.g., autologous single donor or allogeneic single donor) or polydonor CD4 IL-10 / CAR The cells are CD4 + T cells 10 6 In some embodiments, the CD4 IL-10 / CAR Cellular or polydonor CD4 IL-10 / CAR The cells are CD4 + T cells 10 6 Constitutively express at least 200 pg, 500 pg, 1 ng, 5 ng, 10 ng, or 50 ng of IL-10 per cell / mL culture medium.

[0375] In some embodiments, CD4 IL-10 / CAR Cells (e.g., autologous single donor or allogeneic single donor) or polydonor CD4 IL-10 / CAR The cells were activated with a combination of anti-CD3 and anti-CD28 antibodies or a combination of anti-CD3 and anti-CD28 antibody-coated beads, followed by CD4 + T cells 10 6 In some embodiments, polydonor CD4 IL-10 T cells were activated with anti-CD3 and anti-CD28 antibodies, or CD3 and CD28 antibody-coated beads, followed by CD4 + T cells 10 6 Express at least 5 ng, 10 ng, 100 ng, 200 ng, or 500 ng of IL-10 per cell / mL culture medium.

[0376] In various embodiments, IL-10 production is determined 12 hours, 24 hours, or 48 hours after activation using various methods for protein detection and measurement, such as ELISA, real-time polymerase chain reaction (PCR), spectroscopic procedures, colorimetry, amino acid analysis, radiolabeling, Edman degradation, HPLC, Western blotting, etc. In a preferred embodiment, IL-10 production is determined by ELISA 48 hours after activation with anti-CD3 and anti-CD28 antibodies.

[0377] In some embodiments, CD4 IL-10 / CAR Cells (e.g., autologous single donor or allogeneic single donor) or polydonor CD4 IL-10 / CAR The cells were unmodified CD4 + In some embodiments, polydonor CD4 IL-10 T cells are unmodified CD4 + express IL-10 at levels at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, or 50 times higher than T cells.

[0378] In some embodiments, CD4 IL-10 / CAR Cells (e.g., autologous single donor or allogeneic single donor) or polydonor CD4 IL-10 / CAR The cells are CD4 + T cells 10 6 In some embodiments, the CD4 IL-10 / CAR Cells (e.g., autologous single donor, or allogeneic single donor) or polydonor CD4 IL-10 / CAR The cells are CD4 + T cells 10 6 Express at least 100 pg, 200 pg, 500 pg, 1 ng, 5 ng, 10 ng, or 50 ng of IL-5 per cell / mL.

[0379] In some embodiments, CD4 IL-10 / CAR Cells (e.g., autologous single donor or allogeneic single donor) or polydonor CD4 IL-10 / CARThe cells were CD4 + T cells 10 6 In some embodiments, the CD4 IL-10 / CAR Cells (e.g., autologous single donor or allogeneic single donor) or polydonor CD4 IL-10 / CAR The cells were CD4 + T cells 10 6 Express at least 2 ng, 5 ng, 10 ng, 100 ng, 200 ng, or 500 ng of IL-5 per cell / mL.

[0380] In various embodiments, IL-5 production is determined 12 hours, 24 hours, or 48 hours after activation using various methods for protein detection and measurement, such as ELISA, real-time polymerase chain reaction (PCR), spectroscopic procedures, colorimetry, amino acid analysis, radiolabeling, Edman degradation, HPLC, Western blotting, etc. In a preferred embodiment, IL-5 production is determined by ELISA 48 hours after activation with anti-CD3 and anti-CD28 antibodies.

[0381] In some embodiments, CD4 IL-10 / CAR Cells (e.g., autologous single donor or allogeneic single donor) or polydonor CD4 IL-10 / CAR The cells are CD4 + T cells 10 6 In some embodiments, the CD4 IL-10 / CAR Cells (e.g., autologous single donor or allogeneic single donor) or polydonor CD4 IL-10 / CAR The cells are CD4 + T cells 10 6 Express at least 100 pg, 200 pg, 500 pg, 1 ng, 5 ng, 10 ng, or 50 ng of IFN-γ per cell / mL.

[0382] In some embodiments, CD4 IL-10 / CAR Cells (e.g., autologous single donor or allogeneic single donor) or polydonor CD4 IL-10 / CARThe cells were CD4 + T cells 10 6 In some embodiments, the CD4 IL-10 / CAR Cells (e.g., autologous single donor or allogeneic single donor) or polydonor CD4 IL-10 / CAR The cells were CD4 + T cells 10 6 Express at least 2 ng, 5 ng, 10 ng, 100 ng, 200 ng, or 500 ng of IFN-γ per cell / mL.

[0383] In various embodiments, IFN-γ production is determined 12 hours, 24 hours, or 48 hours after activation using various methods for protein detection and measurement, such as ELISA, real-time polymerase chain reaction (PCR), spectroscopic procedures, colorimetry, amino acid analysis, radiolabeling, Edman degradation, HPLC, Western blotting, etc. In a preferred embodiment, IFN-γ production is determined by ELISA 48 hours after activation with anti-CD3 and anti-CD28 antibodies.

[0384] In some embodiments, CD4 IL-10 / CAR Cells (e.g., autologous single donor or allogeneic single donor) or polydonor CD4 IL-10 / CAR The cells are CD4 + T cells 10 6 In some embodiments, the CD4 IL-10 / CAR Cells (e.g., autologous single donor or allogeneic single donor) or polydonor CD4 IL-10 / CAR The cells are CD4 + T cells 10 6 Express at least 25 pg, 50 pg, 75 pg, 100 pg, 200 pg, 500 pg, 1 ng, 5 ng, 10 ng, or 50 ng of IL-4 per cell / mL.

[0385] In some embodiments, CD4 IL-10 / CARCells (e.g., autologous single donor or allogeneic single donor) or polydonor CD4 IL-10 / CAR The cells were CD4 + T cells 10 6 In some embodiments, the CD4 IL-10 / CAR Cells (e.g., autologous single donor or allogeneic single donor) or polydonor CD4 IL-10 / CAR The cells were CD4 + T cells 10 6 Express at least 100pg, 200pg, 300pg, 400pg, 500pg, 600pg, 700pg, 800pg, 900pg, 1000pg, 2ng, 5ng, 10ng, 100ng, 200ng, or 500ng of IL-4 per cell / mL.

[0386] In various embodiments, IL-4 production is determined 12 hours, 24 hours, or 48 hours after activation using various methods for protein detection and measurement, such as ELISA, real-time polymerase chain reaction (PCR), spectroscopic procedures, colorimetry, amino acid analysis, radiolabeling, Edman degradation, HPLC, Western blotting, etc. In a preferred embodiment, IL-4 production is determined by ELISA 48 hours after activation with anti-CD3 and anti-CD28 antibodies.

[0387] In some embodiments, expression of one or more of IL-10, IL-4, IFN-γ, IL-22, and IL-5 is stable after one or more restimulations.

[0388] In some embodiments, CD4 IL-10 / CAR Cells (e.g., autologous single donor or allogeneic single donor) or polydonor CD4 IL-10 / CAR The cells further express a selectable marker. In some embodiments, CD4 IL-10 / CAR Cellular or polydonor CD4 IL-10 / CAR The cells express proteins typically expressed in Tr1 cells. In some embodiments, CD4 IL-10 / CARCellular or polydonor CD4 IL-10 / CAR The cells express marker proteins characteristic of Tr1 cells.

[0389] In some embodiments, CD4 IL-10 / CAR Cells (e.g., autologous single donor or allogeneic single donor) or polydonor CD4 IL-10 / CAR The cells express CD49b. In some embodiments, CD4 IL-10 / CAR Cellular or polydonor CD4 IL-10 / CAR In some embodiments, the cells express LAG-3. IL-10 / CAR Cellular or polydonor CD4 IL-10 / CAR The cells express TGF-β. In some embodiments, the cells express CD4 IL-10 / CAR Cellular or polydonor CD4 IL-10 / CAR The cells express IFN-γ. In some embodiments, the cells express CD4 IL-10 / CAR Cellular or polydonor CD4 IL-10 / CAR In some embodiments, the cells express GzB. IL-10 The cells release GzB when activated by myeloid antigen-presenting cells. In some embodiments, CD4 IL-10 / CAR Cellular or polydonor CD4 IL-10 / CAR The cells express perforin. In some embodiments, CD4 IL-10 / CAR Cellular or polydonor CD4 IL-10 / CAR The cells release perforin when activated by myeloid antigen-presenting cells. In some embodiments, CD4 IL-10 / CAR Cellular or polydonor CD4 IL-10 / CAR The cells express CD18. In some embodiments, CD4 IL-10 / CAR Cellular or polydonor CD4 IL-10 / CAR The cells express CD2. In some embodiments, CD4 IL-10 / CAR Cellular or polydonor CD4 IL-10 / CAR The cells express CD226. In some embodiments, the polydonor CD4 IL-10 In some embodiments, the cells express IL-22. IL-10 / CAR Cellular or polydonor CD4 IL-10 / CAR The cells express IL-10.

[0390] In some embodiments, CD4 IL-10 / CAR Cells (e.g., autologous single donor or allogeneic single donor) or polydonor CD4 IL-10 / CAR The cells exhibit at least one function of Tr1 cells, in various embodiments, the function is the secretion of IL-10, the secretion of TGF-β, and the specific killing of myeloid antigen-presenting cells through the release of granzyme B (GzB) and perforin.

[0391] CD4 IL-10 / CAR Additional characteristics of cells In some embodiments, CD4 IL-10 / CAR The cells comprise an anti-CD19 CAR. In some embodiments, the cells comprise a CD4 IL-10 / CAR The cells are CD19 + In some embodiments, CD4 IL-10 / CAR The cells are CD19 + In some embodiments, the CD19 + The target cells are autoantibody-producing B cells. In some embodiments, CD19 + Target cells are CD19 + In some embodiments, the CD19 + Cytotoxicity against target cells is maintained after one or more restimulations (see, for example, the production method described in Figures 5A-5C).

[0392] In some embodiments, CD4 IL-10 / CAR The cells are capable of in vitro cytotoxicity against myeloid target cells. In some embodiments, the CD4 IL-10 / CAR The cells are capable of in vivo cytotoxicity against myeloid target cells. In some embodiments, the CD4 IL-10 / CAR The cells are CD19 +The antibodies are capable of cytotoxicity against target cells and myeloid target cells. In some embodiments, the myeloid target cells express one or more of class I MHC, CD13, CD54, and CD112. In some embodiments, the cytotoxicity against myeloid target cells is maintained after one or more restimulations (see, for example, the production method described in Figures 5A-5C).

[0393] In some embodiments, CD4 IL-10 / CAR The cells were allogeneic CD4 + In some embodiments, CD4 IL-10 / CAR The cells were allogeneic CD4 + It is possible to inhibit T cell proliferation by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%.

[0394] In some embodiments, CD4 IL-10 / CAR The cells were allogeneic CD8 + In some embodiments, CD4 IL-10 / CAR The cells were allogeneic CD8 + It is possible to inhibit T cell proliferation by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%.

[0395] In some embodiments, CD4 IL-10 / CAR The cells were allogeneic CD4 + T cell proliferation and allogeneic CD8 + In some embodiments, CD4 IL-10 / CAR The cells were allogeneic CD4 + capable of suppressing T cell proliferation by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%, and +It is possible to inhibit T cell proliferation by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%.

[0396] In some embodiments, CD4 IL-10 / CAR Cell-mediated suppression is calculated as follows: 100-([CD4 IL-10 T cells (control) and CD4 IL-10 / CAR Responder proliferation in the presence of T cells / responder proliferation] × 100).

[0397] In some embodiments, the suppressive properties are stable after one or more restimulations.

[0398] 6.3.8. Process Products In some embodiments, CD4 IL-10 / CAR T cells (autologous, or allogeneic single-donor, or allogeneic polydonor CD4 IL-10 / CAR cells) are CD4 + In some embodiments, the CD4 T cells are obtained by modifying the T cells with an exogenous polynucleotide segment encoding a CAR and an exogenous polynucleotide segment encoding IL-10. +Modifying T cells with a first exogenous polynucleotide segment encoding a CAR and a second exogenous polynucleotide segment encoding IL-10 includes (i) a first transduction of a polynucleotide segment encoding IL-10 followed by a second transduction of an exogenous polynucleotide segment encoding a CAR; (ii) a first transduction of a polynucleotide segment encoding a CAR followed by a second transduction of an exogenous polynucleotide segment encoding IL-10; (iii) simultaneous transduction (i.e., co-transduction) of a polynucleotide segment encoding IL-10 and an exogenous polynucleotide segment encoding a CAR; or (iv) transduction of a single polynucleotide construct comprising both a polynucleotide segment encoding IL-10 and a polynucleotide segment encoding a CAR. In some embodiments, the polynucleotide encoding IL-10 further comprises a coding sequence for a marker protein (e.g., deltaNGFR).

[0399] In some embodiments, CD4 IL-10 / CAR Cells (single-donor or poly-donor CD4 IL-10 / CAR cells) are CD4 + The polynucleotide constructs are obtained by modifying T cells with (i) a first polynucleotide construct comprising a polynucleotide segment encoding a CAR and a second polynucleotide construct comprising a polynucleotide segment encoding IL-10, or (ii) a single polynucleotide construct comprising a polynucleotide segment encoding a CAR and a polynucleotide segment encoding IL-10.

[0400] In some embodiments, a polynucleotide construct comprising a first exogenous polynucleotide segment encoding a CAR, a second exogenous polynucleotide segment encoding IL-10, or the first and second exogenous polynucleotide segments is delivered to a CD4 + In some embodiments, the CD4 +T cells are transduced with a first viral vector (e.g., any viral vector described herein) containing a polynucleotide segment encoding IL-10, followed by a second transduction with a second viral vector (e.g., any viral vector described herein) containing a polynucleotide segment encoding a CAR. In another embodiment, CD4 + T cells are co-transduced with a first viral vector (e.g., any viral vector described herein) containing a first exogenous polynucleotide segment and a second viral vector containing a second exogenous polynucleotide segment. In yet another embodiment, CD4 + The T cells are transduced with a viral vector (eg, any viral vector described herein) containing both the first and second exogenous polynucleotide segments.

[0401] In some embodiments, polydonor CD4 IL-10 / CAR (i) Primary CD4 T cells obtained from at least two different T cell donors + (ii) pooling T cells; and (ii) pooled CD4 + T cells are generated by modifying them according to the methods described herein. In some embodiments, polydonor CD4 IL-10 / CAR T cells were (i) primary CD4 T cells from at least two different T cell donors; + (ii) obtaining T cells; and (iii) CD4 + (iii) individually modifying T cells according to the methods described herein, and then (iv) genetically modifying CD4 + It is generated by pooling T cells.

[0402] In some embodiments, CD4 IL-10 / CAR T cells are CD4 + In some embodiments, the T cells are cultured in the presence of a protein capable of activating CD4 IL-10 / CARThe T cells are cultured in the presence of anti-CD3 and anti-CD28 antibodies, or anti-CD3 and anti-CD28 antibody coated beads. IL-10 / CAR The T cells are cultured in the presence of anti-CD3 antibody, anti-CD28 antibody, and IL-2, or anti-CD3 antibody and anti-CD28 antibody coated beads and IL-2. IL-10 / CAR The T cells are cultured in the presence of a polymeric nanomatrix reagent to activate and expand human T cells via CD3 and CD28. In some embodiments, CD4 IL-10 / CAR The T cells are cultured in the presence of other T cell specific immune cell culture media, activators, and supplements.

[0403] In some embodiments, CD4 IL-10 / CAR (Single or polydonor CD4 IL-10 / CAR In some embodiments, the CD4 T cells are present in a frozen stock (e.g., frozen suspension). IL-10 / CAR (Single or polydonor CD4 IL-10 / CAR T cells) are present in liquid suspension.

[0404] CD4 IL10 / CAR T cell population In another aspect, the present disclosure provides a method for detecting a CD4 IL10 / CAR In some embodiments, the population is characterized as a CD4 T cell population. + T cells (e.g., primary CD4 + T cells).

[0405] In some embodiments, CD4 + T cell populations were obtained from autologous or allogeneic single T cell donors (single donor CD4 IL-10 / CAR CD4 obtained from + In some embodiments, primary CD4 T cells are + The T cells are derived from a single T cell donor. In some embodiments, primary CD4+ The T cells are derived from the same individual (i.e., the same patient). + T cell populations were derived from at least two different T cell donors (polydonor CD4 IL-10 / CAR CD4 obtained from + In some embodiments, primary CD4 T cells are + The T cells are derived from the donor of the allo-HSCT.

[0406] In some embodiments, CD4 + T cells were obtained and pooled from 2, 3, 4, 5, 6, 7, 8, 9, or 10 different T cell donors. IL-10 T cells and methods for making and using them are described in PCT / US2021 / 039464, which is incorporated by reference in its entirety. The methods and / or compositions described therein can be used to treat polydonor CD4 T cells. IL-10 / CAR The method can be employed to generate cells.

[0407] In some embodiments, primary culture CD4 + The T cells are derived from a donor selected from the donor after analyzing the donor's genetic information. In some embodiments, the T cells are derived from a donor (i.e., primary CD4 + The genetic information of the donor of the T cells is not analyzed. In some embodiments, primary CD4 + The T cells are derived from a donor selected based on their HLA haplotype. In some embodiments, the CD4 + The T cells collectively have 6, 7, 8, 9, 10, 11, 12, or more different HLA haplotypes. In some embodiments, all CD4 T cells in the population + The T cells have at least a 1 / 10, 2 / 10, 3 / 10, 4 / 10, 5 / 10, 6 / 10, 7 / 10, 8 / 10, or 9 / 10 match to each other at the HLA-A, HLA-B, HLA-C, HLA-DRB1, and HLA-DQB1 loci. +The T cells have at least a 1 / 8, 2 / 8, 3 / 8, 4 / 8, 5 / 8, 6 / 8, 7 / 8, or 8 / 8 match to each other at the HLA-A, HLA-B, HLA-C, and HLA-DRB1 loci.

[0408] In some embodiments, all CD4 + The T cells have a 2 / 2 match to each other at the HLA-A locus. In some embodiments, all CD4 T cells in the population + The T cells have a 2 / 2 match to each other at the HLA-B locus. In some embodiments, all CD4 + The T cells have a 2 / 2 match to each other at the HLA-C locus. In some embodiments, all CD4 T cells in the population + The T cells have at least a 3 / 4 or 4 / 4 match to each other at the HLA-DRB1 and HLA-DQB1 loci. In some embodiments, all CD4 T cells in the population + T cells are A * 02 or A * In some embodiments, all CD4+ T cells have 24 alleles. * 02 or A * 24 is negative.

[0409] In some embodiments, all CD4 + The T cells have less than a 5 / 10, 6 / 10, 7 / 10, 8 / 10, or 9 / 10 match to each other at the HLA-A, HLA-B, HLA-C, HLA-DRB1, and HLA-DQB1 loci. + The T cells have a 4 / 8, 5 / 8, 6 / 8, 7 / 8, or less than 8 / 8 match to each other at the HLA-A, HLA-B, HLA-C, and HLA-DRB1 loci. + The T cells have less than a 2 / 2 match to each other at the HLA-A locus. In some embodiments, all CD4 T cells in the population + The T cells have less than a 2 / 2 match with each other at the HLA-B locus. In some embodiments, all CD4 T cells in the population +The T cells have less than a 2 / 2 match to each other at the HLA-C locus. In some embodiments, all CD4 T cells in the population + The T cells have a 3 / 4 or less than 4 / 4 match to each other at the HLA-DRB1 and HLA-DQB1 loci.

[0410] In some embodiments, the CD4 + At least 30% (e.g., at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 98%) of the T cells express CAR and IL-10 as inferred by expression of ΔNGFR.

[0411] In some embodiments, CD4 + The T cells are in a frozen suspension. In some embodiments, CD4 + The T cells are in a liquid suspension. In some embodiments, the liquid suspension has been previously frozen.

[0412] Pharmaceutical Compositions In another aspect, pharmaceutical compositions are provided. In some embodiments, the pharmaceutical compositions comprise a single-donor CD4 IL-10 / CAR Any of the T cells, polydonor CD4 IL-10 / CAR Any of the T cells or polydonor CD4 IL-10 / CAR and a pharmaceutically acceptable carrier or diluent.

[0413] The pharmaceutical composition can be formulated for administration by any route suitable for human or veterinary medicine. In a typical embodiment, the composition is formulated for intravenous (IV) administration. In some embodiments, the composition is formulated for intravenous (IV) infusion. In embodiments formulated for IV administration, the pharmaceutical composition is in the form of a pyrogen-free, parenterally acceptable aqueous solution with suitable pH, isotonicity, and stability.

[0414] In some embodiments, the pharmaceutically acceptable carrier or diluent is saline, lactated Ringer's solution, or other physiologically compatible solution. In various embodiments, the pharmaceutical composition solution contains 2-20%, preferably 5%, human serum albumin.

[0415] In some embodiments, a unit dosage form of the pharmaceutical composition is provided that is adapted for administering the pharmaceutical composition by systemic administration, particularly intravenous administration.

[0416] In some embodiments, the unit dosage form comprises 10 4 ~10 11 CD4 IL-10 / CAR Cellular or polydonor CD4 IL-10 / CAR cells, 10 4 ~10 10 CD4 IL-10 / CAR Cellular or polydonor CD4 IL-10 / CAR cells, 10 4 ~10 9 CD4 IL-10 / CAR T cells or polydonor CD4 IL-10 / CAR cells, 10 5 ~10 10 CD4 IL-10 / CAR Cellular or polydonor CD4 IL-10 / CAR cells, 10 5 ~10 9 CD4 IL-10 / CAR Cellular or polydonor CD4 IL-10 / CAR cells, 10 5 ~10 8 CD4 IL-10 / CAR Cellular or polydonor CD4 IL-10 / CAR cells, or 10 5 ~10 7 CD4 IL-10 / CAR Cellular or polydonor CD4 IL-10 / CAR Contains cells.

[0417] In an exemplary embodiment, the unit dosage pharmaceutical composition is in liquid form.

[0418] 6.6. Single donor CD4 IL10 / CARCellular or polydonor CD4 IL10 / CAR How to make cells In another aspect, the present disclosure provides a method for detecting CD4 IL10 / CAR Cellular or polydonor CD4 IL10 / CAR A method for producing a cell is provided.

[0419] In some embodiments, the methods provided herein involve transducing a CD4 + T cells or pooled CD4 + In some embodiments, the CD4 + T cells or pooled CD4 + Modifying T cells with an exogenous polynucleotide segment encoding a CAR and an exogenous polynucleotide segment encoding IL-10 includes (i) a first transduction of a polynucleotide segment encoding IL-10 followed by a second transduction of an exogenous polynucleotide segment encoding a CAR; (ii) a first transduction of a polynucleotide segment encoding a CAR followed by a second transduction of an exogenous polynucleotide segment encoding IL-10; (iii) simultaneous transduction (i.e., co-transduction) of a polynucleotide segment encoding IL-10 and an exogenous polynucleotide segment encoding a CAR; or (iv) transduction of a single polynucleotide construct comprising both a polynucleotide segment encoding IL-10 and a polynucleotide segment encoding a CAR.

[0420] In some embodiments, a single polynucleotide construct comprising a polynucleotide segment encoding IL-10 and a polynucleotide segment encoding a CAR is a bidirectional vector in which IL-10 is under the control of one promoter (e.g., PGK or EF1a) and CAR is under the control of a second promoter (e.g., CMV). In such cases, if the construct comprises a polynucleotide sequence encoding a selectable marker, the polynucleotide sequence can be downstream of either IL-10 or CAR.

[0421] In some embodiments, a single polynucleotide construct comprising a polynucleotide segment encoding IL-10 and a polynucleotide segment encoding a CAR comprises, from 5' to 3', a sequence encoding IL-10 and a sequence encoding a CAR. In such embodiments, the sequence encoding IL-10 and the sequence encoding a CAR are operably linked to a single promoter (e.g., constitutive or inducible). In such embodiments, a sequence encoding an internal ribosome entry site (IRES) or a sequence encoding a 2A-peptide is located between the sequence encoding IL-10 and the sequence encoding a CAR.

[0422] In some embodiments, a single polynucleotide construct comprising a polynucleotide segment encoding IL-10 and a polynucleotide segment encoding a CAR comprises, from 5' to 3', a sequence encoding the CAR and a sequence encoding IL-10. In such embodiments, the sequence encoding the CAR and the sequence encoding IL-10 are operably linked to a single promoter (e.g., constitutive or inducible). In such embodiments, a sequence encoding an internal ribosome entry site (IRES) or a sequence encoding a 2A-peptide is located between the sequence encoding the CAR and the sequence encoding IL-10.

[0423] In some embodiments, the method comprises: (a) primary cultured CD4 T cells obtained from one or more T cell donors; +and (b) pooling the pooled CD4 T cells by introducing a first exogenous polynucleotide segment encoding a CAR and a second exogenous polynucleotide segment encoding IL-10 according to the methods provided herein. + In another embodiment, the method includes modifying T cells. In another embodiment, the method includes (a) generating primary CD4 T cells from one or more T cell donors. + (b) obtaining CD4 T cells from each donor by introducing a first exogenous polynucleotide segment encoding a CAR and a second exogenous polynucleotide segment encoding IL-10 (e.g., according to the methods provided herein). + Individually modifying T cells; then genetically modifying CD4 + T cells are pooled, thereby IL-10 / CAR Various methods known in the art can be used to transfect a first exogenous polynucleotide segment encoding a CAR, a second exogenous polynucleotide segment encoding IL-10, or both, into primary cultured CD4 + It can be introduced into T cells.

[0424] In some embodiments, the method comprises culturing primary CD4 + T cells or genetically modified CD4 + In some embodiments, the method further comprises incubating the T cells in the presence of anti-CD3 and anti-CD28 antibodies, or anti-CD3 and anti-CD28 antibody coated beads. + T cells or genetically modified CD4 + In some embodiments, the method further comprises incubating the T cells in the presence of an anti-CD3 antibody, an anti-CD28 antibody and IL-2, or an anti-CD3 antibody and an anti-CD28 antibody coated bead and IL-2. + T cells or genetically modified CD4 + In some embodiments, the method further comprises incubating the T cells in the presence of a feeder cell mixture. +T cells or genetically modified CD4 + The method further comprises incubating the T cells in the presence of a nanopreparation of anti-CD3 and anti-CD28 antibodies. In some embodiments, the incubation is performed in the presence of a polymer nanomatrix reagent to activate and expand human T cells via CD3 and CD28. In some embodiments, the method further comprises incubating the T cells in the presence of a nanopreparation of anti-CD3 and anti-CD28 antibodies. In some embodiments, the incubation is performed in the presence of a polymer nanomatrix reagent to activate and expand human T cells via CD3 and CD28. IL-10 / CAR The T cells are cultured in the presence of other T cell-specific immune cell culture media, activators, and adjuvants.

[0425] In some embodiments, an incubation step is performed prior to introducing a first exogenous polynucleotide segment encoding a CAR and a second exogenous polynucleotide segment encoding IL-10 according to the methods provided herein. In some embodiments, the ... + (b) after pooling T cells; (c) after pooling CD4 + In some embodiments, the incubation step is performed before modifying the T cells by introducing a first exogenous polynucleotide segment encoding a CAR and a second exogenous polynucleotide segment encoding IL-10 according to the methods provided herein. In some embodiments, the incubation step is performed after (a) incubating primary CD4 T cells from one or more different T cell donors. + (b) after obtaining T cells, transfecting each donor's CD4 T cells by introducing a first exogenous polynucleotide segment encoding a CAR and a second exogenous polynucleotide segment encoding IL-10 according to the methods provided herein. + This is done before the T cells are individually modified.

[0426] In some embodiments, the incubation step is performed after step (b). In other words, in some embodiments, the incubation step is performed after step (b). In other words, in some embodiments, the incubation step is performed after step (b) of incubating pooled CD4+ cells by introducing a first exogenous polynucleotide segment encoding a CAR and a second exogenous polynucleotide segment encoding IL-10 according to the methods provided herein. + In some embodiments, the incubation step is performed after the step of modifying T cells. In some embodiments, the incubation step is performed after (b) modifying the CD4 T cells of each donor by introducing a first exogenous polynucleotide segment encoding a CAR and a second exogenous polynucleotide segment encoding IL-10 according to the methods provided herein. + After individually modifying T cells, but not genetically modified CD4 + T cells are pooled, thereby genetically modifying CD4 + In some embodiments, the incubation step is performed before obtaining the T cells. + T cells are pooled, thereby IL-10 / CAR This is performed after obtaining the cells.

[0427] In some embodiments, the incubation step is performed more than once. + This is done both before and after genetic modification of the T cells.

[0428] In some embodiments, the first exogenous polynucleotide segment, the second exogenous polynucleotide segment, or both are transfected into primary cultured CD4 + In some embodiments, the first and second exogenous polynucleotide segments are introduced into T cells. In some embodiments, the first and second exogenous polynucleotide segments are located in the same viral vector. In some embodiments, the viral vector is a lentiviral vector.

[0429] In some embodiments, the first exogenous polynucleotide comprises a segment encoding a CAR having the sequence of SEQ ID NOs: 9, 16, 22, 34, 41-49, and 54, and the second exogenous polynucleotide comprises a segment encoding an IL-10 having the sequence of SEQ ID NO: 1. In some embodiments, the first exogenous polynucleotide comprises a segment encoding a CAR having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NOs: 9, 16, 22, 34, 41-49, and 54, and the second exogenous polynucleotide comprises a segment encoding an IL-10 having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 1.

[0430] In some embodiments, the polynucleotide segment encoding the CAR has the sequence of SEQ ID NO: 10, 17, 23, 35, or 55, and the polynucleotide segment encoding IL-10 has the sequence of SEQ ID NO: 2. In some embodiments, the polynucleotide segment encoding the CAR has at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 10, 17, 22, 35, or 55, and the polynucleotide segment encoding IL-10 has at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 2. In some embodiments, the first exogenous polynucleotide segment, the second exogenous polynucleotide segment, or both, encodes a polynucleotide segment encoding a CD4+ antibody that is successfully transduced. + The selected marker further comprises a segment encoding a marker that allows for selection of T cells. In some embodiments, the encoded selectable marker is ΔNGFR. In certain embodiments, the encoded selectable marker has the sequence of SEQ ID NO: 4. In some embodiments, the encoded selectable marker is a truncated form of human EGFR polypeptide.

[0431] In some embodiments, the method comprises genetically modifying CD4 + Isolating T cells and thereby genetically modifying CD4 IL-10 / CARFurther comprising generating an enriched population of cells.

[0432] In some embodiments, the genetically modified CD4 + At least 40% (e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 98%) of the T cells express a selectable marker, and the selectable marker is a surrogate for IL-10 expression. In some embodiments, the genetically modified CD4 T cells in the enriched population + At least 75% (e.g., at least 80%, at least 90%, at least 95%, or at least 98%) of the T cells express a selectable marker, where the selectable marker is a surrogate for IL-10 expression. In some embodiments, the genetically modified CD4 T cells in the enriched population + At least 75% of the T cells express the selection marker. In some embodiments, the genetically modified CD4 T cells in the enriched population + At least 95% of the T cells express the selection marker. In some embodiments, the genetically modified CD4 T cells in the enriched population + At least 96, 97, 98, or 99% of the T cells express the selectable marker.

[0433] In some embodiments, the genetically modified CD4 + At least 75% (e.g., at least 80%, at least 90%, at least 95%, or at least 98%) of the T cells express IL-10. In some embodiments, the genetically modified CD4 T cells in the enriched population + At least 75% of the T cells express IL-10. In some embodiments, the genetically modified CD4 T cells in the enriched population + At least 95% of the T cells express IL-10. In some embodiments, the genetically modified CD4 + At least 96, 97, 98, or 99% of the T cells express IL-10.

[0434] In some embodiments, the genetically modified CD4 +At least 30% (e.g., at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 98%) of the T cells express a CAR. In some embodiments, the genetically modified CD4 T cells in the enriched population express a CAR. + At least 50% of the T cells express a CAR. In some embodiments, the genetically modified CD4 + At least 75% of the T cells express a CAR. In some embodiments, the genetically modified CD4 + At least 95% of the T cells express a CAR. In some embodiments, the genetically modified CD4 + At least 96, 97, 98, or 99% of the T cells express the CAR.

[0435] In some embodiments, the genetically modified CD4 + At least 30% (e.g., at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 98%) of the T cells express IL-10 and a CAR. In some embodiments, the genetically modified CD4 T cells in the enriched population express IL-10 and a CAR. + At least 50% of the T cells express IL-10 and a CAR. In some embodiments, the genetically modified CD4 T cells in the enriched population + At least 75% of the T cells express IL-10 and a CAR. In some embodiments, the genetically modified CD4 T cells in the enriched population + At least 95% of the T cells express IL-10 and a CAR. In some embodiments, the genetically modified CD4 T cells in the enriched population + At least 96, 97, 98, or 99% of the T cells express IL-10 and CAR.

[0436] In some embodiments, the method comprises genetically modifying CD4 +The method further comprises incubating the enriched population of T cells. In some embodiments, the incubation is performed in the presence of anti-CD3 and anti-CD28 antibodies, or anti-CD3 and anti-CD28 antibody coated beads. In some embodiments, the incubation is further performed in the presence of IL-2. In some embodiments, the incubation is performed in the presence of feeder cells. In some embodiments, the incubation is performed in the presence of a nanopreparation of anti-CD3 and anti-CD28 antibodies. In some embodiments, the incubation is performed in the presence of a polymeric nanomatrix reagent to activate and expand human T cells via CD3 and CD28. In some embodiments, the incubation is performed in the presence of CD4 IL-10 / CAR The T cells are cultured in the presence of other T cell-specific immune cell culture media, activators, and supplements.

[0437] In some embodiments, the method comprises genetically modifying CD4 + The method further comprises the step of freezing the T cells.

[0438] In some embodiments, primary culture CD4 + The T cells are derived from a donor selected based on their HLA haplotype. In some embodiments, the method further comprises selecting a T cell donor by analyzing its genetic information. In some embodiments, the method comprises analyzing the genetic information or HLA haplotype of a potential T cell donor.

[0439] In some embodiments, primary culture CD4 + T cells were cultured in primary CD4 + In some embodiments, primary CD4 T cells are derived from a donor with at least a partial HLA match to the host to be treated with the T cells or their modification. + The T cells are derived from a donor with at least a partial HLA match to the stem cell (HSC), tissue, or organ donor. +The T cells are derived from a donor who is HLA-matched to the stem cell (HSC), tissue, or organ donor. In some embodiments, primary CD4 + The T cells are obtained from a third-party donor who is not biologically related to the host. In some embodiments, primary CD4 + The T cells are obtained from a third-party donor who is not biologically related to the stem cell, tissue, or organ donor.

[0440] In some embodiments, in step (a), primary cultured CD4 + The T cells are obtained from 2, 3, 4, 5, 6, 7, 8, 9, or 10 different T cell donors. In some embodiments, at least two T cell donors have at least a 1 / 10, 2 / 10, 3 / 10, 4 / 10, 5 / 10, 6 / 10, 7 / 10, 8 / 10, or 9 / 10 match to each other at the HLA-A, HLA-B, HLA-C, HLA-DRB1, and HLA-DQB1 loci. In some embodiments, at least two T cell donors have at least a 1 / 8, 2 / 8, 3 / 8, 4 / 8, 5 / 8, 6 / 8, 7 / 8, or 8 / 8 match to each other at the HLA-A, HLA-B, HLA-C, and HLA-DRB1 loci. In some embodiments, at least two T cell donors have a 2 / 2 match to each other at the HLA-A locus. In some embodiments, at least two T cell donors have a 2 / 2 match to each other at the HLA-B locus. In some embodiments, at least two T cell donors have a 2 / 2 match to each other at the HLA-C locus. In some embodiments, at least two T cell donors have at least a 3 / 4 or 4 / 4 match to each other at the HLA-DRB1 and HLA-DQB1 loci. In some embodiments, each of the at least two T cell donors has an A * 02 or A * In some embodiments, the T cell donor has 24 alleles of HLA-A * In some embodiments, each of the at least two T cell donors is HLA-A 02 negative. * 02 or HLA-A * 24 is negative.

[0441] In some embodiments, at least two T cell donors have less than a 5 / 10, 6 / 10, 7 / 10, 8 / 10, or 9 / 10 match with each other at the HLA-A, HLA-B, HLA-C, HLA-DRB1, and HLA-DQB1 loci. In some embodiments, at least two T cell donors have less than a 4 / 8, 5 / 8, 6 / 8, 7 / 8, or 8 / 8 match with each other at the HLA-A, HLA-B, HLA-C, and HLA-DRB1 loci. In some embodiments, at least two T cell donors have less than a 2 / 2 match with each other at the HLA-A locus. In some embodiments, at least two T cell donors have less than a 2 / 2 match with each other at the HLA-B locus. In some embodiments, at least two T cell donors have less than a 2 / 2 match with each other at the HLA-C locus. In some embodiments, at least two T cell donors have a 3 / 4 or less than 4 / 4 match to each other at the HLA-DRB1 and HLA-DQB1 loci.

[0442] In some embodiments, in step (a), primary cultured CD4 + In some embodiments, the T cells are obtained from one or more cryopreservations. In some embodiments, in step (a), primary cultured CD4 + The T cells are obtained from unfrozen peripheral blood mononuclear cells of at least two different T cell donors. In some embodiments, the method comprises: + In some embodiments, the method further comprises isolating T cells from peripheral blood mononuclear cells. + The T cells are obtained from a liquid suspension. In some embodiments, the liquid suspension is obtained from a previously frozen stockpile.

[0443] In some embodiments, the method comprises detecting CD4 + In some embodiments, the method does not include anergizing CD4 T cells in the presence of recombinant IL-10 protein. + The recombinant IL-10 protein does not involve an anergizing step of T cells, and it inhibits the CD4 +In some embodiments, the method comprises administering to a subject a CD4 T cell in the presence of DC10 cells from the host. + It does not include a step of anergizing T cells.

[0444] In some embodiments, the methods provided herein comprise the step of pooling, purifying, restimulating, and expanding CD4+ cells by introducing an exogenous polynucleotide segment encoding a CAR and an exogenous polynucleotide segment encoding IL-10 after one or more of the steps of pooling, purifying, restimulating, and expanding. + T cells or pooled CD4 + This includes modifying T cells.

[0445] CD4 IL-10 / CAR Cellular or polydonor CD4 IL-10 / CAR How to use In another aspect, the present disclosure provides a method of treating a patient, comprising administering to a subject a CD4 IL-10 / CAR Cells (e.g., autologous single-donor or allogeneic single-donor), allogeneic poly-donor CD4 IL-10 / CAR Any of the T cells, polydonor CD4 IL-10 / CAR Methods are provided that include administering any of the cell populations or any of the pharmaceutical compositions provided herein to a patient in need thereof.

[0446] In some embodiments, the method comprises administering to a subject a subject, the subject being a patient, a subject ... IL-10 / CAR A further step is included prior to thawing the frozen suspension of cells.

[0447] In some embodiments, autologous or allogeneic single-donor CD4 IL-10 / CAR Cells, or allogeneic polydonor CD4 IL-10 / CAR The cells, or pharmaceutical composition, prevent or reduce the severity of a pathogenic T cell response in a patient.

[0448] In some embodiments, polydonor CD4 IL-10The cells or pharmaceutical composition prevent or reduce the severity of an inflammatory or autoimmune response.

[0449] In some embodiments, polydonor CD4 IL-10 / CAR The cells or pharmaceutical compositions prevent or reduce the severity of a pathogenic T cell response in a patient. In some embodiments, the polydonor CD4 IL-10 / CAR The cells or pharmaceutical composition reduce inflammation. In some embodiments, the polydonor CD4 IL-10 / CAR The cells or pharmaceutical compositions enhance tissue repair. In some embodiments, the polydonor CD4 IL-10 / CAR The cells or pharmaceutical compositions enhance immune tolerance to self and non-pathogenic antigens and maintain immune system homeostasis. In some embodiments, polydonor CD4 IL-10 / CAR The cells or pharmaceutical compositions down-regulate pathogenic T cell responses associated with organ transplantation, GvHD, and various autoimmune and inflammatory diseases. In some embodiments, the polydonor CD4 IL-10 / CAR The cells or pharmaceutical compositions treat an autoimmune disease. In some embodiments, the polydonor CD4 IL-10 / CAR The cells or pharmaceutical compositions reduce NLPR3 inflammasome hyperactivity or reduce symptoms associated with NLPR3 inflammasome hyperactivity. IL-10 / CAR The cells or pharmaceutical compositions induce tumor cell death or reduce tumor growth. In some embodiments, the polydonor CD4 IL-10 / CAR The cells or pharmaceutical compositions increase disease-free survival (e.g., absence of minimal residual disease). In some embodiments, polydonor CD4 IL-10 / CAR The cells or pharmaceutical composition induce wound healing or tissue repair.

[0450] In some embodiments, polydonor CD4 IL-10 The cells or pharmaceutical compositions are administered in an amount effective to prevent or reduce the severity of a pathogenic T cell response in a patient. IL-10The cells or pharmaceutical composition are administered in an amount effective to reduce inflammation. In some embodiments, polydonor CD4 IL-10 The cells or pharmaceutical compositions are administered in an amount effective to enhance tissue repair. In some embodiments, polydonor CD4 IL-10 The cells or pharmaceutical compositions are administered in an amount effective to enhance immune tolerance to self and non-pathogenic antigens and to maintain homeostasis of the immune system. In some embodiments, polydonor CD4 IL-10 The cells or pharmaceutical compositions are administered in an amount effective to down-regulate pathogenic T cell responses associated with organ transplantation, GvHD, and various autoimmune or inflammatory diseases. In some embodiments, polydonor CD4 IL-10 The cells or pharmaceutical composition are administered in an amount effective to treat an autoimmune disease. In some embodiments, the polydonor CD4 IL-10 The cells or pharmaceutical compositions are administered in an amount effective to reduce NLPR3 inflammasome overactivity or to reduce symptoms associated with NLPR3 inflammasome overactivity. IL-10 The cells or pharmaceutical compositions are administered in an amount effective to induce tumor cell death or reduce tumor growth. IL-10 The cells or pharmaceutical composition are administered in an amount effective to increase disease-free survival (eg, absence of minimal residual disease).

[0451] In some embodiments, single-donor CD4 IL-10 / CAR The cells (e.g., autologous or allogeneic) or pharmaceutical composition prevent or reduce the severity of a pathogenic T cell response in a patient. In some embodiments, a single donor CD4 IL-10 / CAR The cells or pharmaceutical composition reduce inflammation. In some embodiments, the cells or pharmaceutical composition reduce inflammation. IL-10 / CAR The cells or pharmaceutical compositions enhance tissue repair. In some embodiments, the cells or pharmaceutical compositions are derived from single-donor CD4 IL-10 / CAR The cells or pharmaceutical compositions enhance immune tolerance to self and non-pathogenic antigens and maintain homeostasis of the immune system. In some embodiments, single-donor CD4 IL-10 / CARThe cells or pharmaceutical compositions downregulate pathogenic T cell responses associated with organ transplantation, GvHD, and various autoimmune and inflammatory diseases. In some embodiments, single-donor CD4 IL-10 / CAR The cells or pharmaceutical compositions treat an autoimmune disease. In some embodiments, the cells or pharmaceutical compositions are derived from a single donor CD4 IL-10 / CAR The cells or pharmaceutical compositions reduce NLPR3 inflammasome hyperactivity or reduce symptoms associated with NLPR3 inflammasome hyperactivity. IL-10 / CAR The cells or pharmaceutical compositions induce tumor cell death or reduce tumor growth. In some embodiments, single-donor CD4 IL-10 / CAR The cells or pharmaceutical compositions increase disease-free survival (e.g., absence of minimal residual disease). In some embodiments, single-donor CD4 IL-10 / CAR The cells or pharmaceutical composition induce wound healing or tissue repair.

[0452] In some embodiments, the method of treatment includes increasing CD4 in the patient after administration. IL-10 / CAR In some embodiments, the method further comprises detecting a selectable marker in a biological sample obtained from the patient, thereby detecting CD4 IL-10 / CAR Cellular or polydonor CD4 IL-10 / CAR In some embodiments, the method includes detecting a selectable marker in the patient, and detecting the presence or absence of CD4 cells after administration to the patient using the detection of the selectable marker. IL-10 / CAR In some embodiments, the selectable marker is CD4 in the patient. IL-10 / CAR The cells are detected at multiple time points to track changes in their presence. In some embodiments, the biological sample is a biopsy or blood sample from the patient.

[0453] CD4 IL-10 / CAR Cells (autologous single-donor, allogeneic single-donor, or allogeneic poly-donor CD4 IL-10 / CAR The cells) are administered in a therapeutically effective amount. The amount can be determined based on body weight and other clinical factors. In some embodiments, the cells 103 ~10 11 In some embodiments, 10 cells / kg are administered. 3 ~10 10 In some embodiments, 10 cells / kg are administered. 3 ~10 9 In some embodiments, 10 cells / kg are administered. 3 ~10 8 In some embodiments, 10 cells / kg are administered. 3 ~10 7 In some embodiments, 10 cells / kg are administered. 3 ~10 6 In some embodiments, 10 cells / kg are administered. 3 ~10 5 In some embodiments, 10 cells / kg are administered. 3 ~10 4 1 / kg is administered.

[0454] In various embodiments, CD4 IL-10 / CAR Cells (autologous single-donor, allogeneic single-donor, or allogeneic poly-donor CD4 IL-10 / CAR In some embodiments, the CD4 IL-10 / CAR Cells (autologous single-donor, allogeneic single-donor, or allogeneic poly-donor CD4 IL-10 / CAR In some embodiments, the CD4 IL-10 / CAR Cells (autologous single-donor, allogeneic single-donor, or allogeneic poly-donor CD4 IL-10 / CAR The cells) are administered daily, every 3 days, every 7 days, every 14 days, every 21 days, or monthly.

[0455] CD4 IL-10 / CAR Cells (autologous single-donor, allogeneic single-donor, or allogeneic poly-donor CD4 IL-10 / CAR The cells may be administered according to different routes of administration, for example, systemically, subcutaneously, or intraperitoneally. In some embodiments, the cells are administered in saline or physiological solution, which may contain 2-20%, preferably 5%, human serum albumin.

[0456] 6.7.1. Methods for reducing or preventing GvHD In some embodiments, CD4 IL-10 / CAR Cells (autologous single-donor, allogeneic single-donor, or allogeneic poly-donor CD4 IL-10 / CAR cells), single-donor or poly-donor CD4 IL-10 / CAR The population of cells, or any of the pharmaceutical compositions provided herein, is used to treat a patient prior to, concurrently with, or following hematopoietic stem cell (HSC) transplantation (HSCT).

[0457] In various embodiments, the HSCT is a matched related HSCT or a matched unrelated HSCT. In various embodiments, the HSCT is a haploidentical HSCT, a mismatched related HSCT, or a mismatched unrelated HSCT.

[0458] In some embodiments, the patient has a hematological malignancy requiring treatment with allo-HSCT. In some embodiments, the hematological malignancy is mediated by abnormal myeloid cells. In some embodiments, the malignancy or hematological cancer is myeloid leukemia. In some embodiments, the malignancy or hematological cancer is CD19 + , CD20 + , CD22 + , BCMA + , or B7-H3 + In some embodiments, the cancer is a blood cancer. + , CD20 + , CD22 + , or B7-H3 + The hematological cancer is selected from chronic lymphocytic leukemia, acute lymphoblastic leukemia (ALL), and non-Hodgkin's lymphoma.

[0459] In some embodiments, the T cell donor is CD4 IL-10 / CAR (autologous single donor or allogeneic single donor) or allogeneic polydonor CD4 IL-10 / CAR The cells and HSCs are selected based on the genetic information of the patient to be treated with the cells and HSCs and / or the genetic information of the HSC donor. In some embodiments, the T cell donor is a polydonor CD4 IL-10The cells and HSCs are selected based on the HLA haplotype of the patient to be treated with the cells and HSCs and / or the HLA haplotype of the HSC donor. IL-10 Prior to administering the cells, the method further comprises analyzing the genetic information or HLA haplotype of the T cell donor. In some embodiments, the method further comprises analyzing the genetic information or HLA haplotype of the host. In some embodiments, the method further comprises analyzing the genetic information or HLA haplotype of the HSC donor.

[0460] In some embodiments, the T cell donor, the host, and the HSC donor are biologically unrelated. In some embodiments, the T cell donor, the host, and the HSC donor have different HLA haplotypes. In some embodiments, the T cell donor, the host, and the HSC donor have at least partial HLA haplotype mismatches. In some embodiments, the T cell donor is selected if it has an HLA haplotype that has an HLA match above a threshold.

[0461] In some embodiments, the HSC donor is partially HLA-mismatched to the patient. In some embodiments, the HSC donor has less than a 5 / 10, 6 / 10, 7 / 10, 8 / 10, 9 / 10, or 10 / 10 match to the patient at the HLA-A, HLA-B, HLA-C, HLA-DRB1, and HLA-DQB1 loci. In some embodiments, the HSC donor has less than a 4 / 8, 5 / 8, 6 / 8, 7 / 8, or 8 / 8 match to the patient at the HLA-A, HLA-B, HLA-C, and HLA-DRB1 loci. In some embodiments, the HSC donor has less than a 2 / 2 match to the patient at the HLA-A, HLA-B, or HLA-C loci. In some embodiments, the HSC donor has less than a 3 / 4 or 4 / 4 match to the patient at the HLA-DRB1 and HLA-DQB1 loci.

[0462] In some embodiments, one or more of the T cell donors are HLA-mismatched or partially HLA-mismatched to the patient. In some embodiments, one or more of the T cell donors have less than a 5 / 10, 6 / 10, 7 / 10, 8 / 10, 9 / 10, or 10 / 10 match to the patient at the HLA-A, HLA-B, HLA-C, HLA-DRB1, and HLA-DQB1 loci. In some embodiments, one or more of the T cell donors have less than a 4 / 8, 5 / 8, 6 / 8, 7 / 8, or 8 / 8 match to the patient at the HLA-A, HLA-B, HLA-C, and HLA-DRB1 loci. In some embodiments, one or more of the T cell donors have less than a 2 / 2 match to the patient at the HLA-A, HLA-B, or HLA-C loci. In some embodiments, one or more of the T cell donors have a 2 / 4, 3 / 4, or less than 4 / 4 match to the patient at the HLA-DRB1 and HLA-DQB1 loci.

[0463] In some embodiments, one or more of the T cell donors are HLA-mismatched or partially HLA-mismatched to the HSC donor. In some embodiments, one or more of the T cell donors have less than a 5 / 10, 6 / 10, 7 / 10, 8 / 10, 9 / 10, or 10 / 10 match to the HSC donor at the HLA-A, HLA-B, HLA-C, HLA-DRB1, and HLA-DQB1 loci. In some embodiments, one or more of the T cell donors have less than a 4 / 8, 5 / 8, 6 / 8, 7 / 8, or 8 / 8 match to the HSC donor at the HLA-A, HLA-B, HLA-C, and HLA-DRB1 loci. In some embodiments, one or more of the T cell donors have less than a 2 / 2 match to the HSC donor at the HLA-A, HLA-B, or HLA-C loci. In some embodiments, one or more of the T cell donors have a 3 / 4 or less than 4 / 4 match to the HSC donor at the HLA-DRB1 and HLA-DQB1 loci.

[0464] In some embodiments, when administered to a patient, CD4 IL-10 / CAR Cells (single donor or polydonor CD4 IL-10 / CARcells), single-donor or poly-donor CD4 IL-10 / CAR The population of cells, or any of the pharmaceutical compositions provided herein, prevents or reduces the severity of GvHD from transplanted hematopoietic stem cells.

[0465] In some embodiments, when administered to a patient, CD4 IL-10 / CAR Cells (single-donor or poly-donor CD4 IL-10 / CAR cells), single-donor or poly-donor CD4 IL-10 / CAR The population of cells, or any of the pharmaceutical compositions provided herein, prevents or reduces the severity of pathogenic T cell responses by transplanted hematopoietic cells. In certain embodiments, CD4 IL-10 / CAR or polydonor CD4 IL-10 / CAR The cells prevent or reduce GvHD.

[0466] In some embodiments, polydonor CD4 IL-10 The cells or pharmaceutical compositions prevent or reduce the severity of tissue damage induced by pathogenic T cells or inflammation.

[0467] 6.7.2. Methods of Treating Cancer In some embodiments, CD4 IL-10 / CAR Cells (autologous single-donor, allogeneic single-donor, or allogeneic poly-donor CD4 IL-10 / CAR cells), single-donor or poly-donor CD4 IL-10 / CAR The population of cells, or any of the pharmaceutical compositions provided herein, are used to treat cancer. In some embodiments, the population of cells is CD4 IL-10 / CAR Cells (autologous single-donor, allogeneic single-donor, or allogeneic poly-donor CD4 IL-10 / CAR cells), single-donor or poly-donor CD4 IL-10 / CAR The population of cells, or any of the pharmaceutical compositions provided herein, are used to treat malignant diseases.

[0468] In a preferred embodiment, CD4 IL-10 / CAR Cells (autologous single-donor, allogeneic single-donor, or allogeneic poly-donor CD4 IL-10 / CARcells) directly mediate anti-tumor effects and, in certain embodiments, mediate anti-leukemia effects.

[0469] In some embodiments, CD4 IL-10 / CAR Cells (autologous single-donor, allogeneic single-donor, or allogeneic poly-donor CD4 IL-10 / CAR cells), single-donor or poly-donor CD4 IL-10 / CAR The population of cells, or any of the pharmaceutical compositions provided herein and the like, is administered before or after hematopoietic stem cell transplantation (HSCT), peripheral blood stem cell (PBSC), umbilical cord blood (CB), or bone marrow (BM) transplantation.

[0470] In some embodiments, the neoplastic cells express CD19. In some embodiments, the neoplastic cells express CD20. In some embodiments, the neoplastic cells express CD22. In some embodiments, the neoplastic cells express BCMA. In some embodiments, the neoplastic cells express B7-H3. In some embodiments, the neoplastic cells express CD13. In some embodiments, the neoplastic cells express HLA-class I. In some embodiments, the neoplastic cells express CD54. In some embodiments, the neoplastic cells express CD13, HLA-class I and CD54. In some embodiments, the neoplastic cells express CD112. In some embodiments, the neoplastic cells express CD58. In some embodiments, the neoplastic cells express CD155. In some embodiments, the tumor expresses CD112, CD58, or CD155. In various embodiments, the tumor is a solid tumor or a hematological tumor. In some embodiments, the tumor is a solid tumor. In some embodiments, the solid tumor expresses B7-H3. In some embodiments, the solid tumor is selected from the group consisting of breast cancer, brain cancer, lung cancer, liver cancer, stomach cancer, spleen cancer, colon cancer, kidney cancer, pancreatic cancer, prostate cancer, uterine cancer, skin cancer, head cancer, neck cancer, sarcoma, neuroblastoma, and ovarian cancer.

[0471] In some embodiments, the patient is diagnosed with adrenal gland cancer, anal cancer, bile duct cancer, bladder cancer, bone cancer, adult brain / CNS tumors, pediatric brain / CNS tumors, breast cancer, male breast cancer, cancer of unknown primary site, Castleman's disease, cervical cancer, colon / rectal cancer, endometrial cancer, esophageal cancer, Ewing's family of tumors, eye cancer, gallbladder cancer, gastrointestinal carcinoid tumors, gastrointestinal stromal tumors (GIST), gestational trophoblastic disease, Hodgkin's disease, Kaposi's sarcoma, kidney cancer, laryngopharyngeal cancer, acute lymphoblastic leukemia (ALL), acute myeloid leukemia (including AML, myeloid sarcoma, and leukemia cutis), chronic lymphocytic leukemia (CLL), chronic myeloid (CML) leukemia, chronic myelomonocytic leukemia (CMML), childhood leukemia, liver cancer, lung cancer, non-small cell lung cancer, thyroid ... cell lung cancer, small cell lung cancer, pulmonary carcinoid tumor, lymphoma, cutaneous lymphoma, malignant mesothelioma, multiple myeloma, myelodysplastic syndrome, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, non-Hodgkin's lymphoma, childhood non-Hodgkin's lymphoma, oral and oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, penile cancer, pituitary tumor, prostate cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma - adult soft tissue cancer, skin cancer, skin cancer - basal cell and squamous cell, skin cancer - melanoma, skin cancer - Merkel cell, small intestine cancer, stomach cancer, testicular cancer, thymic cancer, thyroid cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Waldenstrom's macroglobulinemia, and Wilms' tumor.

[0472] In some embodiments, the cancer is a myeloid tumor. In certain embodiments, the cancer is AML or CML. In some embodiments, the cancer is a myeloid tumor. In some embodiments, the cancer is ALL.

[0473] In some embodiments, the method is used to treat hematological cancers that affect the blood, bone marrow, and lymph nodes. In various embodiments, the hematological cancer is lymphoma (e.g., Hodgkin's lymphoma), lymphocytic leukemia, or myeloma. In various embodiments, the hematological cancer is acute or chronic myeloid (myeloid) leukemia (AML, CML), or myelodysplastic syndrome.

[0474] In some embodiments, the malignancy or hematological cancer is CD19+ , CD20 + , CD22 + , BCMA + , or B7-H3 + In some embodiments, the cancer is a blood cancer. + , CD20 + , CD22 + , BCMA + , or B7-H3 + The blood cancers are chronic lymphocytic leukemia, acute lymphoblastic leukemia (ALL), and non-Hodgkin's lymphoma.

[0475] In some embodiments, CD4 IL-10 / CAR Cells (autologous single-donor, allogeneic single-donor, or allogeneic poly-donor CD4 IL-10 / CAR cells) are used to treat patients with blood cancer, and the method includes: IL-10 / CAR Cells (autologous single-donor, allogeneic single-donor, or allogeneic poly-donor CD4 IL-10 / CAR cells), single-donor or poly-donor CD4 IL-10 / CAR The method includes administering to the patient a therapeutically effective amount of a population of cells, or any of the pharmaceutical compositions provided herein.

[0476] In some embodiments, CD4 IL-10 / CAR The cells are CD19 + The method is used to treat patients with hematological cancers, and the method comprises administering to the patient a CD4 IL-10 / 抗CD19 CAR Cells (autologous single-donor, allogeneic single-donor, or allogeneic poly-donor CD4 IL-10 / 抗CD19 CAR ), single-donor or polydonor CD4 IL-10 / 抗CD19 CAR The method includes administering to the patient a therapeutically effective amount of a population of cells, or any of the pharmaceutical compositions provided herein.

[0477] In some embodiments, CD4 IL-10 / CAR The cells were CD20 + The method is used to treat patients with hematological cancers, and the method comprises administering to the patient a CD4 IL-10 / 抗CD20 CAR Cells (autologous single-donor, allogeneic single-donor, or allogeneic poly-donor CD4 IL-10 / 抗CD20 CAR ), single-donor or polydonor CD4 IL-10 / 抗CD20 CARThe method includes administering to the patient a therapeutically effective amount of a population of cells, or any of the pharmaceutical compositions provided herein.

[0478] In some embodiments, CD4 IL-10 / CAR The cells were CD22 + The method is used to treat patients with hematological cancers, and the method comprises administering to the patient a CD4 IL-10 / 抗CD22 CAR Cells (autologous single-donor, allogeneic single-donor, or allogeneic poly-donor CD4 IL-10 / 抗CD22 CAR ), single-donor or polydonor CD4 IL-10 / 抗CD22 CAR The method includes administering to the patient a therapeutically effective amount of a population of cells, or any of the pharmaceutical compositions provided herein.

[0479] In some embodiments, CD4 IL-10 / CAR The cells are B7-H3 + The method is used to treat patients with cancer (e.g., solid tumors), and the method comprises: IL-10 / 抗B7-H3 CAR Cells (autologous single-donor, allogeneic single-donor, or allogeneic poly-donor CD4 IL-10 / 抗B7-H3 CAR ), single-donor or polydonor CD4 IL-10 / 抗B7-H3 CAR The method includes administering to the patient a therapeutically effective amount of a population of cells, or any of the pharmaceutical compositions provided herein.

[0480] In some embodiments, CD4 IL-10 / CAR The cells express BCMA + The method is used to treat patients with cancer (e.g., solid tumors), and the method comprises: IL-10 / 抗BCMA CAR Cells (autologous single-donor, allogeneic single-donor, or allogeneic poly-donor CD4 IL-10 / 抗BCMA CAR ), single-donor or polydonor CD4 IL-10 / 抗BCMA CAR The method includes administering to the patient a therapeutically effective amount of a population of cells, or any of the pharmaceutical compositions provided herein.

[0481] In some embodiments, the cancer is refractory or resistant to therapeutic intervention.

[0482] In some embodiments, the methods provided herein include detecting CD19 in a patient. + , CD20+ , CD22 + , BCMA + , or B7-H3 + Used to prevent recurrence of blood or solid cancers, CD19 + , CD20 + , CD22 + BCMA + , or B7-H3 + Patients identified as having hematologic or solid cancers, or CD19 + , CD20 + , CD22 + , BCMA + , or B7-H3 + Patients identified as at risk for recurrence of hematologic or solid tumors receive sufficient CD4 IL-10 / CAR Cells (autologous single-donor, allogeneic single-donor, or allogeneic poly-donor CD4 IL-10 / CAR cells), single-donor or poly-donor CD4 IL-10 / CAR The method comprises administering a therapeutically effective amount of the cell population, or any of the pharmaceutical compositions provided herein.

[0483] In some embodiments, CD4 IL-10 / CAR Cells (autologous single-donor, allogeneic single-donor, or allogeneic poly-donor CD4 IL-10 / CAR The therapeutic agent (cells) is used in combination with a therapeutic intervention. The combination may be simultaneous or at different times. Preferably, the therapeutic intervention is selected from the group consisting of chemotherapy, radiation therapy, allo-HSCT immunosuppression, blood transfusion, bone marrow transplant, growth factors, and biologics.

[0484] In some embodiments, CD4 IL-10 / CAR Cells (autologous single-donor, allogeneic single-donor, or allogeneic poly-donor CD4 IL-10 / CAR cells) are used to treat patients with malignant diseases, and the method comprises administering an allo-HSCT graft to the patient and IL-10 / CAR Cells (autologous single-donor, allogeneic single-donor, or allogeneic poly-donor CD4 IL-10 / CAR cells), single-donor or poly-donor CD4 IL-10 / CARThe method includes administering to the patient a therapeutically effective amount of a population of cells, or any of the pharmaceutical compositions provided herein.

[0485] In some embodiments, CD4 IL-10 / CAR Cells (autologous single-donor, allogeneic single-donor, or allogeneic poly-donor CD4 IL-10 / CAR CD4 cells) are used to treat cancer patients undergoing allogeneic HSCT (allo-HSCT) to prevent GvHD and to induce long-term tolerance (in addition to direct antitumor effects). IL-10 / CAR The amount of cells is sufficient to suppress or prevent graft-versus-host disease (GvHD) without suppressing the efficacy of graft-versus-leukemia (GvL) or graft-versus-tumor (GvT) in allo-HSCT (mediated by donor T cells present in the stem cell preparation).

[0486] Also provided herein are methods of treating patients with minimal residual disease, comprising administering to a patient identified as having, or at risk for having, minimal residual disease, a CD4+ antibody sufficient to induce an anti-cancer effect. IL-10 / CAR Cells (autologous single-donor, allogeneic single-donor, or allogeneic poly-donor CD4 IL-10 / CAR cells), single-donor or poly-donor CD4 IL-10 / CAR Also provided are methods that include administering a therapeutically effective amount of a population of cells, or any of the pharmaceutical compositions provided herein.

[0487] In some embodiments, CD4 IL-10 / CAR Cells (autologous single-donor, allogeneic single-donor, or allogeneic poly-donor CD4 IL-10 / CAR cells), single-donor or poly-donor CD4 IL-10 / CAR The population of cells, or any of the pharmaceutical compositions provided herein, induces cell death of tumor-infiltrating, tumor-growth promoting myeloid lineage cells (eg, monocytes, macrophages, neutrophils).

[0488] In some embodiments, CD4 IL-10 / CAR Cells (autologous single-donor, allogeneic single-donor, or allogeneic poly-donor CD4 IL-10 / CARcells), single-donor or poly-donor CD4 IL-10 / CAR The population of cells, or any of the pharmaceutical compositions provided herein, may be B cells (e.g., CD19 + B cells, CD20 + B cells, CD22 + B cells, BCMA + , B7-H3 + B cells, or B7-H3 + Induces cell death in solid tumor cells.

[0489] 6.7.3. Methods of Treating Inflammatory or Autoimmune Diseases In some embodiments, single-donor or poly-donor CD4 IL-10 / CAR The cells are administered to treat an inflammatory or autoimmune disease. In some embodiments, single-donor or poly-donor CD4 IL-10 / CAR The cells are administered to treat a disease or disorder associated with hyperactivity of the NLPR3 inflammasome.

[0490] The NOD-like receptor family (NLR) protein NLRP3 is an intracellular signaling molecule that senses danger signals from pathogens, the environment, or endogenous sources. After activation, NLPR3 interacts with caspase-1 to form a complex called the inflammasome. This leads to the activation of caspase-1, which cleaves the pro-inflammatory cytokines IL-1β and IL-18 into their active forms and mediates a type of inflammatory cell death known as pyroptosis.

[0491] In some embodiments, single-donor or poly-donor CD4 IL-10 / CAR The cells are administered to treat an inflammatory disease selected from Muckle-Wells syndrome (MWS), familial cold autoinflammatory syndrome (FCAS), and neonatal-onset multisystem inflammatory disease (NOMID). In some embodiments, single-donor or poly-donor CD4 IL-10 / CARThe cells are administered to treat a chronic disease selected from metabolic syndrome, type 2 diabetes, atherosclerosis, Alzheimer's disease, Parkinson's disease, ALS, nonalcoholic steatohepatitis, osteoarthritis, silicosis, asbestosis, gout, and pulmonary fibrosis. In some embodiments, single-donor or poly-donor CD4 IL-10 / CAR The cells are administered to treat Crohn's disease, ulcerative colitis, multiple sclerosis and systemic lupus erythematosus, or inflammatory eye diseases such as diabetic retinopathy, acute glaucoma, and age-related macular degeneration.

[0492] In some embodiments, single-donor or poly-donor CD4 IL-10 / CAR The cells are administered to treat a disease associated with NLRP3. The disease may be selected from the group consisting of CAPS, NASH, Alzheimer's disease, Parkinson's disease, cardiovascular disease, osteoarthritis, gout, pseudogout, nephrolithiasis, type II diabetes, Sjogren's syndrome, sickle cell disease (SCD), AMD, infectious diseases, cerebral malaria, asbestosis, contact hypersensitivity, sunburn, silicosis, pustular fibrosis, inflammatory bowel disease, ALS, myelodysplastic syndrome, and uveitis.

[0493] In some embodiments, the disease is a brain disorder selected from Parkinson's disease, Alzheimer's disease, age-related cognitive impairment, frontotemporal dementia, traumatic brain injury, intracerebral hemorrhage, sepsis-related encephalopathy, cerebral ischemia, subarachnoid hemorrhage, epilepsy, acrylamide poisoning, opioid-induced neuroinflammation, chronic migraine, perioperative neurocognitive impairment, post-stroke cognitive impairment, post-cardiac arrest cognitive impairment, social isolation-induced cognitive impairment, anxiety, and post-traumatic stress disorder.

[0494] In some embodiments, the disease is a pulmonary disorder selected from asthma, IR lung injury, ARDS / COPD, particulate matter-induced lung injury, radiation pneumonitis, pulmonary hypertension, sarcoidosis, cystic fibrosis, and allergic rhinitis.

[0495] In some embodiments, the disease is a cardiac disorder selected from atherosclerosis, heart failure, hypertension, myocardial infarction, atrial fibrillation, cardiac damage induced by metabolic dysfunction, heart failure, and endothelial dysfunction.

[0496] In some embodiments, the disease is a gastrointestinal disease, such as colitis. In some embodiments, the disease is a liver disorder selected from acute liver failure, immune circadian rhythm regulation, NASH, diabetic cognitive dysfunction, IR liver injury, idiosyncratic drug-induced liver injury, and liver fibrosis. In some embodiments, the disease is a pancreatic or renal disorder selected from diabetic encephalopathy, diabetic atherosclerosis, insulin resistance, islet transplant rejection, chronic crystal nephropathy, renal fibrosis, I / R kidney injury, obesity-related kidney disease, and renal hypertension. In some embodiments, the disease is a skin or eye disorder selected from psoriasis and retinal neovascularization. In some embodiments, the disease is a reproductive disorder, such as premature birth. In some embodiments, the disease is an immune disorder selected from primary dysmenorrhea, innate immunity, innate immunity to adaptive immunity, systemic lupus erythematosus-lupus nephritis, and multiple sclerosis. In some embodiments, the disease is a genetic disorder selected from Muckle-Wells syndrome, rheumatoid arthritis, sickle cell disease, and VCP-related disease. In some embodiments, the disease is a pain disorder selected from multiple sclerosis-related neuropathic pain, chronic prostatitis / chronic pelvic pain, cancer-induced bone pain, and hyperalgesia. In some embodiments, the disease is cancer, for example, human squamous cell carcinoma of the head and neck. In some embodiments, the disease is an infectious disorder, for example, a bacterial, viral, or parasitic infection.

[0497] In some embodiments, single-donor or poly-donor CD4 IL-10 / CAR The cells are used in combination with currently available treatments for NLRP3-associated diseases, such as biologic agents that target IL-1, including the recombinant IL-1 receptor antagonist anakinra, the neutralizing IL-1β antibody canakinumab, and the soluble decoy IL-1 receptor rilonacept.

[0498] In some embodiments, single-donor or poly-donor CD4IL-10 / CAR The cells are administered to treat a disease selected from type 2 diabetes, metabolic syndrome, cardiovascular disease, SLE, MS, CD, ulcerative colitis (UC), osteoarthritis, non-alcoholic steatohepatitis (NASH), Parkinson's disease, ALS, pulmonary fibrosis, silicosis, asbestosis, diabetic retinopathy, and age-related macular degeneration.

[0499] In some embodiments, single-donor or poly-donor CD4 IL-10 / CAR The cells are administered to treat inflammation, which may be associated with, but is not limited to, coronary artery disease (CAD), type 2 diabetes, neurodegenerative diseases, or inflammatory bowel disease.

[0500] In some embodiments, single-donor or poly-donor CD4 IL-10 / CAR The cells are administered to treat a disease or disorder associated with increased IL-1β production by activated monocytes, macrophages, or dendritic cells. In some embodiments, single-donor or poly-donor CD4 IL-10 / CAR The cells are administered to treat a disease or disorder associated with increased IL-18 production by activated monocytes, macrophages, or dendritic cells. In some embodiments, single-donor or poly-donor CD4 IL-10 / CAR The cells are administered to treat a disease or disorder associated with increased production of mature caspase-1 by activated monocytes, macrophages, or dendritic cells.

[0501] In some embodiments, single-donor or poly-donor CD4 IL-10 / CAR The cells are administered to reduce IL-1β production by activated monocytes, macrophages, or dendritic cells. In some embodiments, single-donor or poly-donor CD4 IL-10 / CAR The cells are administered to reduce IL-18 production by activated monocytes, macrophages, or dendritic cells. In some embodiments, single-donor or poly-donor CD4 IL-10 / CAR The cells are administered to reduce mature caspase-1 production by activated monocytes, macrophages, or dendritic cells.

[0502] 6.7.4. How to Treat Other Disorders In some embodiments, CD4 IL-10 / CAR Cells (autologous single-donor, allogeneic single-donor, or allogeneic poly-donor CD4 IL-10 / CAR In some embodiments, the methods provided herein involve administering CD4 IL-10 / CAR Cells (autologous single-donor, allogeneic single-donor, or allogeneic poly-donor CD4 IL-10 / CAR cells), single-donor or poly-donor CD4 IL-10 / CAR The method comprises administering a therapeutically effective amount of the population of cells, or any of the pharmaceutical compositions provided herein, to a patient in need of immune tolerance.

[0503] In some embodiments, CD4 IL-10 / CAR Cells (autologous single-donor, allogeneic single-donor, or allogeneic poly-donor CD4 IL-10 / CAR cells), single-donor or poly-donor CD4 IL-10 / CAR The population of cells, or any of the pharmaceutical compositions provided herein, is administered to a patient to treat an autoimmune disease.

[0504] In some embodiments, the autoimmune disease is autoimmune uveitis, psoriasis, vitiligo, alopecia areata, psoriatic arthritis, inflammatory bowel disease, Hashimoto's thyroiditis, autoimmune vasculitis, ulcerative colitis, bullous diseases, scleroderma, celiac disease, Graves' disease, systemic sclerosis, myasthenia gravis, anti-NMDA encephalitis, pemphigus diseases (both vulgaris and foliaceus), epidermolysis bullosa acquisita, thrombotic thrombocytopenic purpura, idiopathic thrombotic purpura, autoantibody-induced vascular inflammation, autoantibody-induced carditis, rheumatoid arthritis, The autoimmune disease is selected from the group consisting of rheumatoid arthritis, autoantibody-induced rheumatoid arthritis, neuromyelitis optica spectrum disorder, systemic lupus erythematosus (SLE), multiple sclerosis (MS), Sjögren's syndrome, autoimmune myopathy, type 1 diabetes, Addison's disease, pernicious anemia, autoimmune hepatitis, primary biliary cholangitis (PBC), autoimmune pancreatitis, Goodpasture's disease, primary membranous nephropathy, ovarian failure, autoimmune orchitis, dry eye disease, and idiopathic interstitial pneumonia. In some embodiments, the autoimmune disease is Crohn's disease, ulcerative colitis, celiac disease, type 1 diabetes, lupus, psoriasis, psoriatic arthritis, or rheumatoid arthritis. In some embodiments, the patient has an allergic or atopic disease. The allergic or atopic disease may be selected from the group consisting of asthma, atopic dermatitis, and rhinitis. In some embodiments, the patient has a food allergy.

[0505] In some embodiments, CD4 IL-10 / CAR Cells (autologous single-donor, allogeneic single-donor, or allogeneic poly-donor CD4 IL-10 / CAR cells), single-donor or poly-donor CD4 IL-10 / CAR The population of cells, or any of the pharmaceutical compositions provided herein, is administered to prevent or reduce the severity of pathogenic T cell responses to cell and organ transplants other than HSCT. In some embodiments, the method comprises administering CD4 IL-10 / CAR Cells (autologous single-donor, allogeneic single-donor, or allogeneic poly-donor CD4 IL-10 / CAR cells), single-donor or poly-donor CD4 IL-10 / CARThe method further comprises administering an organ transplant to the patient either before or after administering the population of cells or any of the pharmaceutical compositions provided herein. In certain embodiments, the organ is a kidney, heart, lung, liver, or pancreatic islet cells. In a preferred embodiment, CD4 IL-10 / CAR Cells (autologous single-donor, allogeneic single-donor, or allogeneic poly-donor CD4 IL-10 / CAR cells), single-donor or poly-donor CD4 IL-10 / CAR The population of cells, or any of the pharmaceutical compositions provided herein, prevents or reduces the severity of host rejection of an organ transplant.

[0506] In some embodiments, CD4 IL-10 / CAR Cells (autologous single-donor, allogeneic single-donor, or allogeneic poly-donor CD4 IL-10 / CAR cells), single-donor or poly-donor CD4 IL-10 / CAR The population of cells, or any of the pharmaceutical compositions provided herein, is administered to prevent or reduce an immune response associated with gene therapy, for example, the administration of a recombinant adenovirus, adeno-associated virus (AAV), herpes simplex virus (HSV), retrovirus, lentivirus, non-integrating lentivirus, alphavirus, flavivirus, rhabdovirus, measles virus, Newcastle disease virus, poxvirus, or picornavirus. In these embodiments, the method involves administering a recombinant adenovirus, adeno-associated virus (AAV), herpes simplex virus (HSV), retrovirus, lentivirus, non-integrating lentivirus, alphavirus, flavivirus, rhabdovirus, measles virus, Newcastle disease virus, poxvirus, or picornavirus to a CD4 IL-10 / CAR The method further comprises administering the cells or pharmaceutical composition to the patient either before or after administration.

[0507] In some embodiments, CD4 IL-10 / CAR Cells (autologous single-donor, allogeneic single-donor, or allogeneic poly-donor CD4 IL-10 / CAR cells), single-donor or poly-donor CD4 IL-10 / CARThe population of cells, or any of the pharmaceutical compositions provided herein, is administered to prevent or reduce an immune response to a recombinant viral vector other than AAV. In these embodiments, the method comprises administering a CD4 IL-10 / CAR cells, CD4 IL-10 / CAR The method further comprises administering to the patient a recombinant viral vector other than AAV either before or after administration of the cell population or pharmaceutical composition. Non-limiting examples of viral vectors other than AAV include herpes simplex virus (HSV), retrovirus, lentivirus, alphavirus, flavivirus, rhabdovirus, measles virus, Newcastle disease virus, poxvirus, or picornavirus.

[0508] In some embodiments, CD4 IL-10 / CAR Cells (autologous single-donor, allogeneic single-donor, or allogeneic poly-donor CD4 IL-10 / CAR cells), single-donor or poly-donor CD4 IL-10 / CAR The population of cells, or any of the pharmaceutical compositions provided herein, is administered to prevent or reduce immune responses associated with transplantation of iPS-derived tissues or cells, including, but not limited to, cardiomyocytes, hepatocytes, epithelial cells, cartilage, bone and muscle cells, and neurons.

[0509] In some embodiments, the method comprises administering to a subject a subject, the subject being a patient, a subject ... IL-10 / CAR cells, CD4 IL-10 / CAR In some embodiments, the method further comprises administering an immunotherapeutic protein to the patient either before or after administration of the cell population or pharmaceutical composition. IL-10 / CAR cells, CD4 IL-10 / CAR The cell population, or pharmaceutical composition, reduces an immune response to an immunogenic therapeutic protein. In some embodiments, the immunotherapeutic protein is selected from a therapeutic antibody, a Factor VIII replacement, a cytokine, and a cytokine mutein.

[0510] In some embodiments, CD4 IL-10 / CARCells (autologous single-donor, allogeneic single-donor, or allogeneic poly-donor CD4 IL-10 / CAR cells), single-donor or poly-donor CD4 IL-10 / CAR The population of cells or any of the pharmaceutical compositions provided herein are administered to treat inflammation, which may be associated with, but is not limited to, coronary artery disease (CAD), type 2 diabetes, neurodegenerative disease, nonalcoholic steatohepatitis (NASH), or inflammatory bowel disease.

[0511] In some embodiments, CD4 IL-10 / CAR Cells (autologous single-donor, allogeneic single-donor, or allogeneic poly-donor CD4 IL-10 / CAR cells), single-donor or poly-donor CD4 IL-10 / CAR The population of cells, or any of the pharmaceutical compositions provided herein, is administered to treat a disease or disorder associated with hyperactivity of the NLPR3 inflammasome. IL-10 / CAR Cells (autologous single-donor, allogeneic single-donor, or allogeneic poly-donor CD4 IL-10 / CAR cells), single-donor or poly-donor CD4 IL-10 / CAR The population of cells, or any of the pharmaceutical compositions provided herein, is administered to treat a disease or disorder associated with increased IL-1β production by activated monocytes, macrophages, or dendritic cells. In some embodiments, the population of cells is administered to treat a disease or disorder associated with increased IL-1β production by activated monocytes, macrophages, or dendritic cells. IL-10 / CAR Cells (autologous single-donor, allogeneic single-donor, or allogeneic poly-donor CD4 IL-10 / CAR cells), single-donor or poly-donor CD4 IL-10 / CAR The population of cells, or any of the pharmaceutical compositions provided herein, is administered to treat a disease or disorder associated with increased IL-18 production by activated monocytes, macrophages, or dendritic cells. IL-10 / CAR Cells (autologous single-donor, allogeneic single-donor, or allogeneic poly-donor CD4 IL-10 / CAR cells), single-donor or poly-donor CD4 IL-10 / CARThe population of cells, or any of the pharmaceutical compositions provided herein, is administered to treat a disease or disorder associated with increased production of mature caspase-1 by activated monocytes, macrophages, or dendritic cells.

[0512] In some embodiments, CD4 IL-10 / CAR Cells (autologous single-donor, allogeneic single-donor, or allogeneic poly-donor CD4 IL-10 / CAR cells), single-donor or poly-donor CD4 IL-10 / CAR The population of cells, or any of the pharmaceutical compositions provided herein, is administered to reduce IL-1β production by activated monocytes, macrophages, or dendritic cells. In some embodiments, the population of cells is administered to reduce IL-1β production by activated monocytes, macrophages, or dendritic cells. IL-10 / CAR Cells (autologous single-donor, allogeneic single-donor, or allogeneic poly-donor CD4 IL-10 / CAR cells), single-donor or poly-donor CD4 IL-10 / CAR The population of cells, or any of the pharmaceutical compositions provided herein, is administered to reduce IL-18 production by activated monocytes, macrophages, or dendritic cells. IL-10 / CAR Cells (autologous single-donor, allogeneic single-donor, or allogeneic poly-donor CD4 IL-10 / CAR cells), single-donor or poly-donor CD4 IL-10 / CAR The population of cells, or any of the pharmaceutical compositions provided herein, is administered to reduce mature caspase-1 production by activated monocytes, macrophages, or dendritic cells.

[0513] In some embodiments, CD4 IL-10 / CAR Cells (autologous single-donor, allogeneic single-donor, or allogeneic poly-donor CD4 IL-10 / CAR cells), single-donor or poly-donor CD4 IL-10 / CAR The population of cells, or any of the pharmaceutical compositions provided herein, is administered to reduce an overactive immune response in a patient to a viral infection. In some embodiments, the virus is SARS-coV-2. In some embodiments, the population of cells is administered to a patient to reduce an overactive immune response in a patient to a viral infection ... IL-10 / CAR Cells (autologous single-donor, allogeneic single-donor, or allogeneic poly-donor CD4IL-10 / CAR cells), single-donor or poly-donor CD4 IL-10 / CAR The population of cells, or any of the pharmaceutical compositions provided herein, are administered to reduce an overactive immune response to bacterial infections, such as toxic shock, cytokine storm, therapeutic antibodies, Factor VIII replacement, cytokines, and cytokine muteins.

[0514] In another aspect, the disclosure provides a method of treating or inhibiting an autoimmune, allergic, or inflammatory disease in a patient, comprising administering to a patient identified as having an autoimmune, allergic, or inflammatory disease, a CD4+ antibody sufficient to treat or inhibit the autoimmune, allergic, or inflammatory disease. IL-10 / CAR Cells (autologous single-donor, allogeneic single-donor, or allogeneic poly-donor CD4 IL-10 / CAR cells), single-donor or poly-donor CD4 IL-10 / CAR The present invention provides methods comprising administering a therapeutically effective amount of a population of cells or any of the pharmaceutical compositions provided herein.

[0515] In another aspect, the present disclosure provides a method for reducing transplant rejection in a patient who has received a hematopoietic stem cell, bone marrow cell, or solid organ transplant, comprising administering to a patient identified as having rejection of the transplanted hematopoietic stem cell, bone marrow cell, or solid organ a CD4+ antibody sufficient to reduce rejection of the transplant. IL-10 / CAR Cells (autologous single-donor, allogeneic single-donor, or allogeneic poly-donor CD4 IL-10 / CAR cells), single-donor or poly-donor CD4 IL-10 / CAR The present invention provides methods comprising administering a therapeutically effective amount of a population of cells or any of the pharmaceutical compositions provided herein.

[0516] In another aspect, the disclosure provides a method of treating graft-versus-host disease (GvHD) in a patient, comprising administering to a patient identified as having or at risk for having graft-versus-host disease (GvHD) a CD4+ antibody sufficient to suppress or prevent GvHD. IL-10 / CARCells (autologous single-donor, allogeneic single-donor, or allogeneic poly-donor CD4 IL-10 / CAR cells), single-donor or poly-donor CD4 IL-10 / CAR The present invention provides methods for treating GvHD, comprising administering a therapeutically effective amount of a population of cells or any of the pharmaceutical compositions provided herein. In some embodiments, the GvHD is acute GvHD. In some embodiments, the GvHD is chronic GvHD.

[0517] In another aspect, the present disclosure provides a method of treating tissue or organ injury (e.g., wound healing) in a patient, comprising administering to a patient identified as having or at risk of having tissue or organ injury, a CD4 antibody as provided herein sufficient to induce repair of the tissue or organ injury. IL-10 / CAR Any of the cells provided herein, including CD4 IL-10 / CAR The present invention also features methods comprising administering a therapeutically effective amount of any of the cell populations or pharmaceutical compositions provided herein.

[0518] 6.8. Polynucleotides and Vectors The present disclosure also features one or more polynucleotide constructs comprising: (a) a first polynucleotide segment encoding a chimeric antigen receptor (CAR) (e.g., any CAR provided herein); and (b) a second polynucleotide segment encoding interleukin-10 (IL-10), and (c) a third polynucleotide segment encoding a truncated NGFR. In some embodiments, the first polynucleotide segment and the second and third polynucleotides are present in the same construct. In some embodiments, the first polynucleotide is present in a first polynucleotide construct, and the second and third polynucleotide segments are present in a second polynucleotide construct. In some embodiments, the third polynucleotide segment is optional.

[0519] In some embodiments, the first polynucleotide segment comprises a regulatory element (e.g., any exemplary regulatory element described herein (e.g., any promoter)) operably linked to the coding sequence of the CAR. In some embodiments, the regulatory element drives expression of the CAR.

[0520] In some embodiments, the second polynucleotide segment comprises a regulatory element (e.g., any of the exemplary regulatory elements described herein (e.g., any promoter)) operably linked to a coding sequence for IL-10. In some embodiments, the regulatory element drives constitutive expression of IL-10.

[0521] In some embodiments, the third polynucleotide segment comprises a regulatory element (e.g., any exemplary regulatory element (e.g., any promoter) described herein) operably linked to a coding sequence of a selectable marker (e.g., ΔNGFR). In some embodiments, the regulatory element drives constitutive expression of ΔNGFR.

[0522] In some embodiments, the polynucleotide construct comprises an internal ribosome entry site (IRES) or a self-cleaving peptide between the first polynucleotide segment and the second polynucleotide segment. In some embodiments, the polynucleotide construct comprises an internal ribosome entry site (IRES) or a self-cleaving peptide between the second polynucleotide segment and the first polynucleotide segment. In some embodiments, the self-cleaving peptide is selected from the group consisting of F2A, P2A, T2A, and E2A.

[0523] In some embodiments, the polynucleotide construct comprises a promoter, a self-cleaving peptide, or an IRES operably linked 5' to 3' to a first polynucleotide segment, and a second polynucleotide segment. In some embodiments, the polynucleotide construct comprises a promoter, a self-cleaving peptide, or an IRES operably linked 5' to 3' to a first polynucleotide segment, and a second polynucleotide segment, and a second promoter operably linked 5' to 3' to a third polynucleotide segment.

[0524] In some embodiments, the polynucleotide construct comprises a promoter, a self-cleaving peptide, or an IRES, and a first polynucleotide segment operably linked 5' to 3' to a second polynucleotide segment.

[0525] In some embodiments, the polynucleotide construct comprises a first promoter operably linked to a first polynucleotide segment and a second promoter operably linked to a second polynucleotide segment. In some embodiments, the polynucleotide construct comprises a first promoter operably linked to a first polynucleotide segment, a second promoter operably linked to a second polynucleotide segment, and a third promoter operably linked to a third polynucleotide segment.

[0526] In some embodiments, the polynucleotide construct encodes an antigen-binding domain that targets an antigen associated with an autoimmune disease, inflammatory disorder, or cancer. In some embodiments, the antigen is selected from the group consisting of CD19, CD20, CD22, CD27, BCMA, CD38, HLA, and the like, in the context of the associated MHC molecule. * A2, HLA * A24, or a citrullinated peptide, insulin, MOG, GAD65, IA2, gliadin, and desmoglein.

[0527] In some embodiments, the polynucleotide construct comprises a sequence encoding an antigen-binding domain that targets a cancer-associated antigen, wherein the cancer-associated antigen is selected from the group consisting of CD19, CD20, CD22, CD23, CD27, CD38, CEA, BCMA, Lym1, Lym2, CLEC5A, CDH179b, FLT3, GCC, Muc, CSF2RA, GFRa4, CD32, CD33, IL11Ra, IL13Ra, NYBRI, SLea, CD200R, TGFbetaR2, CEA, CD276, TROP2, LAMP1, PTK7, DLL3, CDH1, CDH6, CDH17, CDH19, TSHR, B7-H3, and tyrosinase.

[0528] In some embodiments, the first polynucleotide segment comprises the sequence of SEQ ID NO: 10, 17, 23, 35, or 55.

[0529] In some embodiments, the second polynucleotide segment comprises the sequence of SEQ ID NO:2.

[0530] In some embodiments, the polynucleotide construct further comprises one or more selectable markers (e.g., any of the selectable markers provided herein). In some embodiments, the first polynucleotide segment, the second polynucleotide segment, or both further comprise a sequence encoding a selectable marker.

[0531] In some embodiments, the polynucleotide construct comprises ΔNGFR as a selectable marker.

[0532] In some embodiments, the polynucleotide construct comprises a ΔNGFR comprising the sequence of SEQ ID NO:4.

[0533] In some embodiments, the polynucleotide construct comprises a truncated form of EGFR as a selectable marker.

[0534] In some embodiments, one or more polynucleotide constructs are one or more vectors.In some embodiments, the vector is a viral vector.Non-limiting examples of viral vectors include lentivirus, retrovirus, gamma retrovirus, adeno-associated virus, adenovirus, helper-dependent adenovirus, Sendai virus, or baculovirus.In some embodiments, the polynucleotide construct is a lentiviral vector.

[0535] In some embodiments, the first polynucleotide is present in a first lentiviral vector and the second polynucleotide segment is present in a second lentiviral vector.

[0536] In some embodiments, the lentiviral vector is capable of integrating into the T cell nuclear genome. In some embodiments, the lentiviral vector is not capable of integrating into the T cell nuclear genome. In some embodiments, an integration-deficient lentiviral vector is used. For example, some embodiments use an integration-deficient or other lentiviral vector disclosed in Matrai. In some embodiments, an integrase-deficient lentivirus is used. For example, an integrase-deficient lentivirus containing an inactivating mutation in integrase (D64V) described in Matrai et al., Hepatology 53:1696-1707 (2011), incorporated herein by reference, can be used. [Example]

[0537] 6.9.Example The following examples are offered by way of illustration and not by way of limitation.

[0538] 6.9.1. Summary of Experimental Findings Overall, the data from the experiments described below demonstrated that human CD4+ T cells can be transduced with two different lentiviral vectors: 1) an LVV encoding a synthetic CAR containing a human B cell surface antigen-binding domain, a CD8α hinge and CD8α transmembrane domain, a CD28 costimulatory domain, and a CD3 zeta activation domain, and 2) a bidirectional vector encoding the human IL-10 gene plus a truncated version of the NGFR gene. The resulting transduced CD4 + T cells expressed both the CD19 CAR (as measured by CD19 expression) and IL-10 / NGFR as measured by NGFR expression. After sequential or co-transduction of two different lentiviral vectors, dual-transduced CD4 + T cells (generally CD4 IL-10 / CAR cells, or CD4 if expressing an anti-CD19 CAR as described in Figures 2A-2B IL-10抗CD19CAR When chromatin-dependent transduction (referred to as chromatin-dependent transduction cells) was generated, no difference in transduction efficiency was found.

[0539] After activation through CD3 and CD28, CD4 IL-10 / CAR The cells are CD4 IL-10 The cells showed a cytokine production profile similar to that described for Tr1 cells. IL-10 / CAR The cells produced high levels of IL-10 and no or low levels of IL-4, IFN-γ, and IL-5. Notably, this cytokine production profile was stable even after long-term culture periods.

[0540] Interestingly, CD19 + Target cells and CD4 IL-10抗CD19CAR Co-culture of cells with CD4 IL-10抗CD19 CAR This resulted in specific activation of the cells and induction of IL-10 production.

[0541] CD4 IL-10抗CD19CAR The cells are CD4 IL-10 As observed for cells, in vitro PBMC expansion, allogeneic CD4+ T cell proliferation, and allogeneic CD8 + It suppressed T cell proliferation.

[0542] CD4 IL-10抗CD19CAR The cells are CD19 + Specific killing of target cells in vitro, but CD4 IL-10 Like cells, they maintain their ability to kill myeloid target cells, which is dependent on the expression of one or more of class I MHC, CD13, CD54, and CD112. IL-10抗CD19 CAR T cells express CD19 in vivo + Importantly, single-donor and poly-donor CD4 IL-10抗CD19 CAR Cells were single-donor and poly-donor CD4 IL-10 As described for cells, no xeno-GvHD was induced, whereas PBMCs induced severe xeno-GvHD. IL-10抗CD19 CAR The cells synergized with PBMCs in their antitumor effects, indicating that they were CD4 IL-10 In addition, single-donor and poly-donor CD4 IL-10抗CD19 CAR The cells protected mice from xeno-GvHD in a humanized mouse model of GvHD induced by allogeneic PBMCs (see Figures 18A-18C).

[0543] This data indicates that CD4 expressed on target cells IL-10抗CD19 CAR The specific interaction between CD19 and CD19 induces the activation of these cells, + It has been shown to result in specific killing of target cells and IL-10 production.

[0544] In summary, the data show that CD4 IL-10 / CART cells have been shown to have unique clinical utility for the treatment of all B-cell and T-cell mediated autoimmune diseases because they not only eliminate autoantibody-producing B cells but also suppress pathogenic autoimmune CD4 T cells through the production of IL-10. + T cells and CD8 + The data also suggest that CD4 IL-10抗CD19 CAR Single-donor or poly-donor CD4+ cells alone did not induce GvHD and did not interfere with the protective antitumor effect of PBMCs (GvL effect). IL-10抗CD19 CAR cells, especially in patients undergoing allogeneic HSCT. + It has also been shown to have unique clinical utility in treating / eliminating tumor cells and preventing tumor recurrence.

[0545] Importantly, even at high concentrations, CD4 IL-10抗CD19 CAR Cells (either autologous or allogeneic) alone did not induce GvHD. These results suggest that CD4 IL-10 / CAR We demonstrate that the cells can be used for the treatment of i) leukemias and other malignancies that express target antigens, ii) leukemias and other malignancies in patients undergoing allogeneic HSCT or BM transplantation to reduce GvHD while preserving the GvL or GvT therapeutic effects of HSCT, iii) autoimmune diseases through elimination of autoimmune B cells and down-regulation of pathogenic T cell responses and the NLPR3 inflammasome, iv) inflammatory diseases through down-regulation of pathogenic T cell responses and the NLPR3 inflammasome, v) cell and organ transplant rejection, vi) solid tumors that express tumor-associated antigens, including but not limited to B7-H3, and vii) immune-mediated diseases through tissue repair and wound healing.

[0546] [Example 1] CD4 IL-10 / 抗CD19 CAR Cell generation 6.9.2.1. Vector-Based Production of IL-10 / ΔNGFR and Anti-CD19 CAR Second-generation CD19 CARs were used in combination with CD4 IL-10抗CD19 CARThe CD19 CAR was used for the production of CD19 cells. The CD19 CAR contained an scFv (SEQ ID NO: 12) from a fully human anti-CD19 monoclonal antibody, a CD8α hinge and transmembrane domain, a CD28 costimulatory domain, and a CD3ζ (CD3 zeta) activation domain (Figure 2A). The CD19 CAR shown in Figure 2A was inserted into a lentiviral vector (LVV) (see Figure 2B). For an illustration of an exemplary CAR targeting CD19, CD20, CD22, BCMA, or B7H3, see Figure 1. The lentiviral vector was generated by ligating the coding sequence of an anti-CD19 scFv (FMC63) to a lentiviral backbone containing the human CD8α hinge and transmembrane domains, a CD28 costimulatory domain, and a CD3ζ intracellular signaling domain. Surface expression of the anti-CD19 CAR was monitored using an anti-CD19-CAR detection agent (Miltenyi).

[0547] The polynucleotide encoding IL-10 was administered to CD4 + T cells to induce CD4 IL-10抗CD19 CAR To produce the cells, a lentiviral vector (LVV) (FIG. 4) containing the coding sequence for both human IL-10 (SEQ ID NO: 2) and truncated NGFR (also known as CD271) (ΔNGFR) (SEQ ID NO: 4) was used. LVV containing the IL-10 and ΔNGFR coding sequences is further described in WO 2016 / 146542, which is incorporated herein by reference in its entirety.

[0548] A lentiviral vector was generated by ligating the coding sequence for human IL-10 from a 549-bp fragment of pH15C (ATCC 68192) into the plasmid pLVIL-10. The presence of a bidirectional promoter (the human PGK promoter plus the minimal core elements of the CMV promoter in opposite orientations) allowed for coexpression of two transgenes. The plasmid also contained the coding sequence for an antibiotic resistance gene (e.g., ampicillin or kanamycin). For an exemplary illustration of the IL-10 construct, see Figures 3A and 3B.

[0549] Lentiviral vectors were produced by Ca3PO4 transient four-plasmid cotransfection into 293T cells and concentrated by ultracentrifugation. 1 μM sodium butyrate was added to the culture for vector collection. Titers were estimated by limiting dilution in 293T cells, and vector particles were measured by HIV-1 Gag p24 antigen immunocapture (NEN Life Science Products; Waltham, MA). The infectivity of the vector was calculated as the ratio between titer and particles. For concentrated vectors, the titer was 1 × 10 8 ~6×10 9 transducing units / mL, and the infectivity is in the range of 5 x 10 4 ~5×10 5 The transducing units / ng range.

[0550] 6.9.2.2.CD4 IL-10 / CAR Cell production As shown in Figure 5A-5C, three different methods were used to identify CD4 IL-10抗CD19 CAR For each of the different methods, CD4 + T cells were purified from healthy human donors. Purified human CD4 + T cells were activated with anti-CD3 / anti-CD28 beads (Miltenyi) (3:1 cell:bead ratio) in complete culture medium (X-vivo supplemented with 5% human AB serum and rhIL-2 (50 U / mL)).

[0551] Figure 5A shows activated CD4 + T cells were injected with anti-CD19 CAR (LVV) 48 hours after activation. CD19-CAR ), and 24 hours later (i.e., 72 hours after activation) the cells were transduced with a lentiviral vector encoding human IL-10 and a truncated form of the human NGF receptor (LVV IL-10-NGFR ) were transduced with bidirectional lentiviral vectors encoding LVV. Both LVVs were used at a multiplicity of infection (MOI) of 20.

[0552] Figure 5B shows activated CD4 +T cells were treated with LVV 48 hours after activation. IL-10-NGFR and then 24 hours later (i.e., 27 hours after activation) LVV CD19-CAR indicates that the vector was transduced with

[0553] Figure 5C shows activated CD4 + T cells were treated with LVV 48 hours after activation. IL-10-NGFR and L.V.V. CD19-CAR The control CD4 IL10 Cells, LVV IL-10-NGFR 48 hours after activation with activated CD4 + T cells were generated by transduction. For each of Figures 5A-5C, transduction was performed in the presence of polybrene (8 μg / mL). In addition, for each of Figures 5A-5C, 10-28 days after transduction, the transduced cells (i.e., ΔNGFR + Cells) were purified using anti-CD271 microbeads (Miltenyi) and anti-CD19 CAR microbeads (Miltenyi). Purified cells were restimulated every 14 days as previously described (Andolfi et al. Mol. Ther. 20(9):1778-1790(2012) and Locafaro et al. Mol. Ther. 25(10):2254-2269(2017)). After the second (TF2) and third (TF3) restimulations, the resulting cells (CD4 IL-10抗CD19 CAR ) were characterized in vitro and in vivo. The culture medium was changed every 2–3 days throughout the entire culture period, as needed.

[0554] CD4 IL-10抗CD19 CAR Cells were analyzed by staining with anti-CD271 mAb (Biolegend) and anti-CD19 CAR detection reagent (Miltenyi) with TF3 (i.e., after the third restimulation) and analyzed by flow cytometry. Staining indicated CD4 expression, determined as co-expression of NGFR and anti-CD19 CAR for the three different transduction methods. IL-10抗CD19 CARThe cell identity and purity were 88.6% (Figure 6A from the method described in Figure 5A), 64.1% (Figure 6B from the method described in Figure 5B), and 52.6% (Figure 6C from the method described in Figure 5C), respectively. As expected, CD4 IL-10 The cells did not express the CD19 CAR (Figure 7).

[0555] As shown in Figure 7, CD4 IL-10抗CD19 CAR The cells expressed both IL-10 (as measured by NGFR expression) and anti-CD19 CAR. In this experiment, CD4+ cells from two different donors (26.1 and 26.2) were used. + T cells were then transfected with LVV at 48 hours after bead activation. IL-10-NGFR and L.V.V. 抗CD19 CAR were simultaneously transduced (Method 5C). IL-10 To generate T cells (control), CD4 + T cells were treated with LVV 48 hours after bead activation. IL-10-NGFR For this analysis, only CD4 IL-10 Cells (control) and CD4 IL-10抗CD19 CAR Cells were analyzed after a second round of restimulation with feeder cells (i.e., TF2). IL-10 Cells (control) and CD4 IL-10抗CD19 CAR Both LVV T cells were stained with anti-CD271 mAb (Biolegends) and CD19-CAR detection reagent (Miltenyi) and analyzed by flow cytometry. As shown in Figure 7, LVV T cells were stained with anti-CD271 mAb (Biolegends) and CD19-CAR detection reagent (Miltenyi) and analyzed by flow cytometry. IL-10-NGFR and L.V.V. CD19-CAR The majority of cells transduced with LVV expressed CD271 and CD19-CAR (Figure 7, right panel), whereas the majority of cells transduced with LVV expressed CD271 and CD19-CAR (Figure 7, right panel). IL-10-NGFR The majority of cells transduced with CD271 alone expressed CD271 but not CD19-CAR (Figure 7, left panel).

[0556] These results suggest that CD4 IL-10抗CD19 CARThis indicates that CD4 cells can be successfully generated regardless of the timing and order of transduction with two different LVVs. IL-10 / CAR These results suggest that IL-10 and CAR could be generated using a single LVV encoding both IL-10 and CAR.

[0557] 6.9.2.3.CD4 IL-10抗CD19 CAR The cells were naturally derived Tr1 cells and CD4 IL-10 have a cytokine production profile that is comparable to that of cells CD4 from three different healthy donors (26.1, 26.2, and 26.3) IL-10抗CD19 CAR Cells were generated by co-transduction of (i) LVV containing anti-CD19-CAR and (ii) LVV containing IL-10 and ΔNGFR according to the method described in Figure 5C. IL-10抗CD19 CAR To generate the cells, CD4 + T cells were treated with LVV 48 hours after bead activation. IL-10-NGFR and L.V.V. CD19-CAR were simultaneously transduced with CD4 IL-10 CD4 T cells from three different donors (26.1, 26.2, and 26.3) were used to generate T cells (control). + T cells were treated with LVV 48 hours after bead activation. IL-10-NGFR was transduced.

[0558] For this analysis, each CD4 IL-10 Cells (control) and CD4 IL-10 / CAR For T cells, 2 x 10 cells 5Cells (in 200 μL) were restimulated as previously described in Andolfi et al. Mol. Ther. 20(9): 1778-1790 (2012) and Locafaro et al. Mol. Ther. 25(10): 2254-2269 (2017). Fourteen days after the second round of restimulation (TF2), cells were stimulated with immobilized anti-CD3 and soluble anti-CD28 mAbs for 48 hours. Culture supernatants were collected, and the levels of IL-10, IL-4, IL-5, and IFN-γ were determined by ELISA.

[0559] As shown in Figures 8A-8D, some individual differences in cytokine production levels were observed, but CD4 IL-10 / CAR The cells are CD4 IL-10 Similar to Tr1 cells and Tr1 cells (not shown), they produced high levels of IL-10, low levels of IL-4, and variable levels of IFN-γ and IL-5.

[0560] Similar results were observed in CD4 from donor 24.2 collected at TF3. IL-10抗CD19 CAR For this experiment, CD4 IL-10抗CD19 CAR To generate cells, CD4 from donor 24.2 + T cells, LVV IL-10-NGFR and L.V.V. CD19-CAR were co-transduced 48 hours after bead activation (see Figure 5C). IL-10 To generate T cells (control), CD4 + T cells were treated with LVV 48 hours after bead activation. IL-10-NGFR For this analysis, only CD4 IL-10 T cells (control) and CD4 IL-10 / CAR For T cells, 2 x 10 cells 5Cells (in 200 μL) were restimulated as previously described in Andolfi et al. Mol. Ther. 20(9): 1778-1790 (2012) and Locafaro et al. Mol. Ther. 25(10): 2254-2269 (2017). Briefly, on day 14 after the third round of restimulation (TF3), cells were stimulated with immobilized anti-CD3 and soluble anti-CD28 mAbs for 48 hours. Culture supernatants were collected, and IL-10, IL-4, IL-5, and IFN-γ levels were determined by ELISA.

[0561] As shown in Figure 9, even after long-term culture, these cells still produce high levels of IL-10, low levels of IL-4, and variable levels of IL-5 and IFN-γ.

[0562] Collectively, the data in Figures 8A-8D and 9 demonstrate that purified CD4 IL-10 LVV in cells IL-10-NGFR and L.V.V. CD19-CAR It is feasible to simultaneously transduce CD4 IL-10 CD4 cells have the same unique and characteristic cytokine production profile as T cells and Tr1 cells IL-10-CAR In addition, it has been shown to induce the generation of CD4 IL-10-CAR No differences in cytokine production profiles were observed when T were harvested with TF2 or TF3, indicating that these cytokine production profiles are stable over time.

[0563] [Example 2] CD4 IL-10抗CD19 CAR Cell cytotoxicity and CD19 + Activation after engagement with target cells CD4 IL-10It was known that cells can kill myeloid tumor cells such as ALL-CM under defined conditions (Andolfi et al. Mol Ther. 2012;20(9):1778-1790). This experiment demonstrated that co-expression of anti-CD19 CAR inhibits CD19 expression when co-cultured with a cell line expressing CD19 (e.g., NALM6). + Cells that can kill cells (CD4 IL-10抗CD19 CAR ) was designed to evaluate whether or not the β-glucanase gene produces

[0564] In particular, CD4 IL-10 Cells (control) and CD4 IL-10抗CD19 CAR All cells were CD4 isolated from donor 24.2. + T cells were generated and harvested after the third restimulation (TF3) (see Figure 5C), and were cloned into K562, NALM6 (CD19 + ), or ALL-CM target cells (10 5 The cells were co-cultured with 1:1 target cells (cells / well) for 3 days. After 3 days of co-culture, the presence of residual target cells in each co-culture was determined using flow cytometry.

[0565] The data are CD4 IL-10抗CD19 CAR The cells were then transfected with CD19-CAR and CD19 + Through engagement with target cells, CD19 + NALM6 cells were shown to be killed by CD4 IL-10 The cells are ineffective and CD19 + NALM6 cells (Fig. 10). IL-10抗CD19 CAR The cells are CD4 IL-10 It also killed the myeloid tumor cell line ALL CM, as well as CD4 IL-10抗CD19 CAR cells and CD4 IL-10 All cells had no or very low cytotoxicity against K562 cells, which are highly susceptible to nonspecific cytotoxicity and killing by NK cells (FIG. 10).

[0566] These results suggest that CD4 IL-10抗CD19 CAREngagement of CD19-CAR expressed on NALM6 lymphoma cells with CD19 expressed on CD4 IL-10抗CD19 CAR Triggers the cytotoxic mechanism of cells, CD19 + It has been shown to result in selective and efficient killing of target cells. IL-10抗CD19 CAR The cells are CD4 IL-10 Like the cells, they retain the ability to kill myeloid target cells that express one or more of CD13, class I MHC antigens, CD54, and CD112, as previously described (Andolfi et al. Mol Ther. 2012;20(9):1778-1790).

[0567] In addition to inducing selective cytotoxic activity, CD4 IL-10抗CD19 CAR and CD19 + NALM6 engagement with target cells is mediated by CD4 IL-10抗CD19 CAR This resulted in selective activation and IL-10 production by the cells.

[0568] For these experiments, CD4 IL-10 Cells (control) and CD4 IL-10抗CD19 CAR Cells were isolated from three different donors (26.1, 26.2, and 26.3) and CD4 + T cells were generated from CD19 T cells before assessing IL-10 production. + Selective killing of cells was performed using CD4 IL-10抗CD19 CAR The cells were confirmed.

[0569] In particular, CD4 isolated from three different donors (26.1, 26.2, and 26.3) + CD4 generated from T cells IL-10 Cells (control) and CD4 IL-10抗CD19 CAR Cells were harvested after the second restimulation (TF2) (see Figure 5C) and cloned into K562 cells, NALM6 (CD19 + ) cells, or ALL-CM target cells (10 5The cells were co-cultured with leukemia target cells (1:1 ratio) at 1:1 ratio (cells / well) for 3 days. After 3 days of co-culture, the presence of residual leukemia target cells in each co-culture was determined using flow cytometry. The K562 cell line, which is highly susceptible to nonspecific cytotoxicity and NK cell killing, served as a control. Data are shown in Figure 11A.

[0570] As shown in Figure 11A, CD4 generated from three different donors (26.1, 26.2, and 26.3) IL-10抗CD19 CAR The cells are CD19 + NALM6 cells were efficiently killed. IL-10抗CD19 CAR Although the cells maintained their ability to kill the myeloid tumor cell line ALL-CM, they were not or only weakly cytotoxic to K562 cells, which do not express CD19 but are known to be susceptible to nonspecific cytotoxicity and killing by NK cells (Fig. 11A). IL-10抗CD19 CAR also killed ALL-CM, a myeloid leukemia cell line. Significant amounts of IL-10 were measured in the supernatant of the cytotoxicity assay after only 3 days of coculture with NALM6 cells (Fig. 11B). In contrast, NALM6 cells and CD4 IL-10 In co-culture with the cells, IL-10 (upper limit of quantification: 31 pg / mL) could not be detected (Fig. 11B).

[0571] In summary, the data show that CD4 IL-10抗CD19 CAR We demonstrated that CD19-CAR cells are selectively activated by engagement of CD19 expressed on relevant target cells. This activation is mediated by: (i) CD19 + (ii) the cytotoxic / lytic activity of the cells, which results in the killing of the target cells, and (iii) CD4 IL-10抗CD19 CAR This resulted in the induction of IL-10 production, which is necessary for the immune regulatory and suppressor functions of CD4 IL-10抗CD19 CAR The cells are CD4 IL-10 These cells maintain the same cytotoxic activity against myeloid tumor target cells previously observed with CD4 IL-10 Cells are CD19 +The inability to kill NALM6 cells (Fig. 11B) was due to the CD4 IL-10抗CD19 CAR The cytotoxic activity of the cells was determined by the combination of anti-CD19 CAR and CD19 + This demonstrated that this was due to engagement with target cells.

[0572] [Example 3] CD4 IL-10抗CD19 CAR The cells were allogeneic CD4 + and CD8 + Suppresses both T cell proliferative responses CD4 collected at TF2 or TF3 IL-10抗CD19 CAR cells and CD4 IL-10 Both cells produce high levels of IL-10, and both were compared with allogeneic CD4+ cells obtained from healthy donors. + and CD8 + The ability to suppress T cell proliferation was tested.

[0573] Allogeneic CD4 + To assess T cell proliferation, allogeneic PBMC cells were incubated with eFluor® 670 (1 × 10 5 cells / well) and CD4 IL-10 cells (control), or CD4 isolated after the second restimulation IL-10抗CD19 CAR cells (1×10 5 mature allogeneic dendritic cells (DCs) (5 × 10 cells / well) (see Figure 5C) in the presence or absence of DCs. 4 cells / well) and stimulated with soluble anti-CD3 mAb at a 1:1 responder:suppressor ratio. After 4 days of culture, CD4 + The percentage of proliferating responder T cells was determined by flow cytometry using dilutions of eFluor® 670. For flow cytometry, cells were divided into CD4 + ΔNGFR - Gating was done on T cells.

[0574] homogeneous CD8 + To assess T cell proliferation, allogeneic PBMC cells were incubated with eFluor® 670 (1 × 10 5 cells / well) and CD4IL-10 cells (control) or CD4 isolated after the second restimulation IL-10抗CD19 CAR cells (1×10 5 cells / well) (see Figure 5C) in the presence or absence of allogeneic mature dendritic cells (DCs) (5 × 10 4 cells / well) and stimulated with soluble anti-CD3 mAb at a 1:1 responder:suppressor ratio. After 4 days of culture, CD8 + The percentage of proliferating responder T cells was determined by flow cytometry using dilutions of eFluor® 670. For flow cytometry, cells were divided into CD8 + ΔNGFR - Gating was done on T cells.

[0575] To assess proliferation of allogeneic PBMCs, allogeneic PBMC cells were incubated with eFluor® 670 (1 × 10 5 cells / well) and CD4 IL-10 (control) or CD4 isolated after the second restimulation IL-10抗CD19CAR cells (1×10 5 cells / well) (see Figure 5C) in the presence or absence of allogeneic mature dendritic cells (DCs) (5 × 10 4 cells / well) and stimulated with soluble anti-CD3 mAb at a 1:1 responder:suppressor ratio. After 4 days of culture, CD8 + The percentage of proliferating responder T cells was determined by flow cytometry using dilutions of eFluor® 670. For flow cytometry, cells were divided into CD3 + ΔNGFR - Gating was done on T cells.

[0576] As shown in Figure 12, CD4 T cells from three different healthy donors 26.1, 26.2, and 26.3 collected at TF2 IL-10抗CD19 CAR Each cell was an allogeneic CD4 + They also inhibited T cell proliferation by 78%, 48%, and 74%. +They also suppressed T cell proliferation by 77%, 41%, and 42%, respectively (Figure 13). IL-10 Comparable results were obtained for allogeneic CD4 + inhibited T cell proliferation by 83%, 81%, and 61%, respectively, and CD8 + T cell proliferation was inhibited by 77%, 41%, and 68%, respectively (Figures 12 and 13). IL-10抗CD19 CAR cells and CD4 IL-10 The cells were also effective in suppressing the proliferation of allogeneic T cells when harvested with TF3. IL-10抗CD19 CAR The cells are CD4 IL-10 As with cells, they inhibited proliferation of PBMCs by 69% and 60%, respectively (FIG. 14).

[0577] Taken together, these results suggest that CD4 IL-10 / CAR The cells are allogeneic CD4 + T cells and allogeneic CD8 + It has an immunosuppressive effect on both CD4 T cells and CD4 IL-10 The immunosuppressive effect of CAR is comparable to that of CD4 IL-10 The results also showed that the immunosuppressive function of CD4 cells isolated after TF3 was maintained even after prolonged culture periods (i.e., after TF3). IL-10抗CD19 CAR It was shown to be stable in T cells.

[0578] [Example 4] CD4 IL-10抗CD19 CAR Treatment or prevention of GvHD and cancer using cells CD4 IL-10抗CD19 CAR In vivo effects on cells CD4 IL-10抗CD19 CAR The cell population was analyzed to identify CD19 cells in a humanized xeno-GvHD disease and tumor-engrafted NSG mouse model. + It was tested for its effect on tumor growth, GvL function, and induction of GvHD.

[0579] NSG mice were irradiated with a sublethal dose of 175–200 Gr according to their body weight. On day 0, NSG mice were injected with (i) NALM6-luciferase (1 × 10 5 cells / mouse), (ii) NALM6-luciferase (1 × 10 5 cells / mouse) + PBMCs (2.5 × 10 6 cells / mouse), (iii) NALM6-luciferase (1 × 10 5 (units / mouse) + CD4 IL-10抗CD19 CAR cells (2.5×10 6 cells / mouse), or (iv) NALM6-luciferase (1 × 10 5 cells / mouse) + PBMCs (2.5 × 10 6 (units / mouse) + CD4 IL-10抗CD19 CAR cells (2.5×10 6 cells / mouse) were injected (see Figure 15).

[0580] The effect on leukemia cell growth was determined by standard bioluminescence assays (see Figures 16A-16B). In addition, mice were treated with PBMC and / or CD4 IL-10抗CD19 CAR Mice were monitored for the development of GvHD by measuring body weight on days 0, 9, 12, and 15 after cell administration (see Figure 17).

[0581] As shown in Figures 16A-16B, PBMCs inhibited the growth of NALM6 cells, a model of the graft-versus-leukemia effect of allo-HSCT in human patients (Figures 16A-16B), but they induced xeno-GvHD in mice, similar to the GvHD observed in human allo-HSCT therapy (Figure 17). In contrast, CD4 IL-10抗CD19 CAR The cells inhibited the growth of NALM6 cells (Figures 16A-16B) and did not cause any signs of xeno-GvHD as determined by weight loss (Figure 17). IL-10 / CAR Treatment with T cells had the greatest anti-leukemia effect (FIGS. 16A-16B) and the greatest anti-tumor effect (FIG. 17).

[0582] These data are based on CD4 IL-10抗CD19 CART cells are strongly cytotoxic and express CD19 in vivo. + They have been shown to selectively kill target cells. In addition, they do not interfere with the protective GvL effect of PBMCs; in fact, they act synergistically. In contrast to PBMCs, CD4 IL-10抗CD19 CAR Treatment with T cells alone or in combination with PBMCs did not induce xeno-GvHD.

[0583] [Example 5] CD4 IL-10抗CD19 CAR The cells have suppressive activity in vivo and effectively prevent severe xeno-GvHD Using additional testing as described in Figure 18A, single-donor or polydonor CD4 IL-10抗CD19 CAR We evaluated whether the cells induce GvHD and whether they protect against GvHD induced by PBMCs. Specifically, humanized NSG mice were sublethally irradiated and GvHD was induced by allogeneic PBMCs. Mice were treated according to Figure 18A. On day 3, mice were injected with (i) PBMCs (2.5E6 cells / mouse), (ii) single-donor (BC26.3) CD4 IL-10抗CD19 CAR cells (2.5E6 cells / mouse), (iii) polydonor (BC-26.1, BC26.2, and BC26.3) CD4 IL-10抗CD19 CAR cells (2.5E6 cells / mouse), (iv) PBMCs (2.5E6 cells / mouse) and single donor (BC26.3) CD4 IL-10抗CD19 CAR cells (2.5E6 cells / mouse), or (v) PBMCs (2.5E6 cells / mouse) and polydonor (BC-26.1, BC26.2, and BC26.3) CD4 IL-10抗CD19 CAR Cells (2.5e6 cells / mouse) were administered intravenously (see Figure 18A). GvHD was determined using a composite score of weight loss, coat appearance, skin appearance, huddling, and activity (Bondanza et al. 2006). Mice with a composite score ≥6 and / or weight loss ≥20% were humanely euthanized. PBMC donors were single-donor CD4 IL-10抗CD19CAR and polydonor CD4 IL-10抗CD19 CARThe donor was unrelated to the donor used to generate the .

[0584] As shown in Figures 18B and 18C, injection of 2.5E6 PBMC cells induced fulminant, rapidly fatal xeno-GvHD by day 16, whereas injection of 2.5E6 single-donor CD4 IL-10抗CD19 CAR cells (Figure 18B) or 2.5E6 polydonor CD4 IL-10抗CD19 CAR Mice receiving 2.5E6 PBMCs and 2.5E6 single-donor CD4 IL-10抗CD19 CAR cells (Figure 18B) or 2.5E6 PBMCs and 2.5E6 polydonor CD4 IL-10抗CD19 CAR Administration of both PBMC and single-donor CD4 IL-10抗CD19 CAR Cellular or polydonor CD4 IL-10抗CD19 CAR Co-administration of protected mice against PBMC-induced xeno-GvHD.

[0585] These results are consistent with those of a single-donor CD4 IL-10抗CD19 CAR Cell and polydonor CD4 IL-10抗CD19 CAR However, we have shown that single-donor CD4 IL-10抗CD19 CAR Cell and polydonor CD4 IL-10抗CD19 CAR effectively prevent severe xeno-GvHD induced by PBMCs, demonstrating that they have suppressive activity in vivo.

[0586] Other experiments Mice in the treatment group were cultured using CD4 IL-10 / CAR Cells are monitored for an additional period to determine the effect on long-term survival.

[0587] CD4 IL-10 / CAR The amount and localization of cells are also monitored in peripheral blood and tissues after administration. IL-10 / CAR The presence of cells is monitored in peripheral blood and at sites of inflammation: lymph nodes, spleen, intestinal tract, and bone marrow. Mice in the treatment group(s) are monitored for an additional 3 weeks to determine long-term survival.

[0588] Results demonstrate sustained reduction and prevention of xeno-GvHD.

[0589] [Example 6] CD4 IL-10 / CAR Treatment of chronic inflammation and autoimmune diseases using cells Activation of the NLPR3 inflammasome has been implicated in many chronic inflammatory and autoimmune diseases. The NLPR3 inflammasome can be activated by "danger signals" that result in caspase-1-mediated production of the pro-inflammatory cytokines IL-1β and IL-18 by monocytes / macrophages. A series of in vitro experiments was performed to investigate the role of CD4+ on the NLPR3 inflammasome and IL-1β / IL-18 production by human monocytes. IL-10 / 抗CD19 CAR Examine the effects on cells.

[0590] First, human PBMCs are isolated from peripheral blood by standard density gradient centrifugation on Ficoll / Paque (Sigma-Aldrich). Monocytes are isolated from human PBMCs by negative selection using the Monocyte Isolation Kit II (Miltenyi) according to the manufacturer's instructions. Negative selection is preferred because positive selection or adhesion can lead to unwanted activation of the cells. Isolated monocytes are cultured in 96-well microtiter plates in culture medium containing 3% toxin-free human AB serum, with a CD4 IL-10 / 抗CD19 CAR 5 × 10 cells in the presence of various dilutions of cell supernatants / 200 μl / well 4 Seed at 200 μl / cell.

[0591] Table 1 summarizes the treatment conditions applied to different sets of monocytes, each set containing six wells of cells. Monocytes were activated with a combination of LPS and nigericin, an NLPR3 inflammasome activator. The caspase 1 inhibitor Z-YVADfmk and the specific NLPR3 inhibitor MC950 were added as controls, as indicated.

[0592] [Table 1]

[0593] After treatment as outlined in Table 1, supernatants are collected from six wells for each group and IL-1β / IL-18 production is measured by ELISA specific for mature IL-1β or IL-18 (Biolegend). Cells collected from selected groups are analyzed by Western blot to determine levels of activated caspase 1.

[0594] Experimental data are available for autologous single-donor, allogeneic single-donor, and allogeneic polydonor CD4 IL-10 / 抗CD19 CAR We show that autologous single-donor, allogeneic single-donor, and allogeneic polydonor CD4 cells down-regulate IL-1β and IL-18 production by activated monocytes. IL-10 / 抗CD19 CAR In addition, autologous single-donor, allogeneic single-donor, and allogeneic polydonor CD4 cells down-regulate mature caspase-1 production by activated monocytes. IL-10 / 抗CD19 CAR and polydonor CD4 IL-10 IL-10 produced by IL-10 downregulates the inflammasome.

[0595] Similar experiments are performed on human macrophages or dendritic cells rather than monocytes. IL-10 / 抗CD19 CAR We demonstrate that cells further downregulate IL-1β, IL-18, and mature caspase-1 production from activated macrophages and dendritic cells.

[0596] These data are consistent with autologous single-donor, allogeneic single-donor, and allogeneic polydonor CD4 IL-10 / 抗CD19 CAR These results demonstrate that autologous single-donor, allogeneic single-donor, and allogeneic poly-donor CD4+ cells can be used to treat diseases or disorders associated with hyperactivation of the NLPR3 inflammasome. IL-10 / 抗CD19 CARCells can be used to treat chronic inflammatory and autoimmune diseases.NLPR3 inflammasome can be activated by exogenous or endogenous "danger signals", such as pathogen-associated molecular patterns (PAMPs), silica, asbestos, damaged mitochondria, necrotic cells, and stressed cell-derived danger-associated molecular patterns (DAMPs)-like products, and uric acid crystals.

[0597] [Example 7] Polydonor CD4 IL-10 Cell generation Vector production Polydonor CD4 IL-10 The cells were generated by transduction with a lentiviral vector containing the coding sequences for both human IL-10 and a truncated form of NGFR (ΔNGFR), as described in International Publication No. WO 2016 / 146542, which is incorporated herein by reference in its entirety (Figures 3A-3B). The sequence of the vector is provided in SEQ ID NO: 5. Briefly, the lentiviral vector was generated by ligating the coding sequence for human IL-10 from a 549-bp fragment of pH15C (ATCC 68192) into plasmid #1074.1071.hPGK.GFP.WPRE.mhCMV.dNGFR.SV40PA. The presence of a bidirectional promoter (the human PGK promoter plus the minimal core elements of the CMV promoter in opposite orientations) allows for coexpression of the two transgenes. The plasmid further contains the coding sequence for an antibiotic resistance gene (e.g., ampicillin or kanamycin).

[0598] Lentiviral vectors were produced by Ca3PO4 transient four-plasmid cotransfection into 293T cells and concentrated by ultracentrifugation. 1 μM sodium butyrate was added to the culture for vector collection. Titers were estimated by limiting dilution in 293T cells, and vector particles were measured by HIV-1 Gag p24 antigen immunocapture (NEN Life Science Products; Waltham, MA). Vector infectivity was calculated as the ratio between titer and particles. For concentrated vectors, titers were 5 × 10 8 ~6×10 9 transducing units / mL, and infectivity is 5 × l0 4 ~5×10 5 The transducing units / ng range.

[0599] CD4 IL-10 Cell production Figure 19 shows the CD4 IL-10 Schematic of the cell production process. CD4 + Purified human CD4 T cells + T cells were activated with soluble anti-CD3, soluble anti-CD28 mAb, and rhIL-2 (50 U / mL) for 48 h and then transduced with a bidirectional lentiviral vector encoding human IL-10 and a truncated form of the human NGF receptor (LV-IL-10 / ΔNGFR) at a multiplicity of infection (MOI) of 20.

[0600] After 11 days, transduced cells were analyzed by FACS for expression of ΔNGFR, and vector copy number (VCN) was quantified by digital droplet PCR (ddPCR).

[0601] CD4 from 10 different donors + The mean transduction efficiency of T cells was 45±17%, with a VCN of 2.7±0.6%. Figure 20A shows human CD4 T cells transduced with LV-IL-10 / ΔNGFR (a bidirectional lentiviral vector encoding a truncated form of human IL-10 and the human NGF receptor). + CD4 on T cells + ΔNGFR+ The percentage of cells (mean ± SD, n = 10, left bar) and vector copy number (VCN, mean ± SD, n = 10, right bar) are shown. + ΔNGFR + Cell frequencies and vector copy numbers were measured using CD4 IL-10 Quantification was performed in cells by digital droplet PCR (ddPCR).

[0602] ΔNGFR + T cells were purified using anti-CD271 mAb-coated microbeads to obtain >95% pure CD4 IL-10 A cell population was obtained. After purification, the cells were stained with CD4 and ΔNGFR markers and analyzed by FACS. The data showed that the purity obtained from the purification step was greater than 98%. Figure 20B shows FACS data from two representative donors (donor B and donor C) out of the 10 donors tested. Purified CD4 IL-10 Cells were restimulated three times at 14-day intervals, and their in vitro and in vivo function was tested after the second (TF2) and / or third restimulation (TF3) function.

[0603] Dormant CD4 IL-10 The cells constitutively produced IL-10, and upon activation, the levels of IL-10 produced were strongly enhanced.

[0604] CD4 IL-10 The cells have a cytokine production profile comparable to that of naturally occurring Tr1 cells Single donor CD4 IL-10 The cytokine production profile of the cells was analyzed after the second (TF2) and third (TF3) restimulation, and the results are provided in Figure 21. Specifically, CD4 IL-10 Cells (2 x 10 cells in 200 μl) 5(individuals) were restimulated as previously described (Andolfi et al. Mol Ther. 2012;20(9):1778-1790 and Locafaro et al. Mol Ther. 2017;25(10):2254-2269). On day 14, after the second (TF2) and third (TF3) rounds of restimulation, CD4 IL-10 Cells were left unstimulated (orange bars) or stimulated with immobilized anti-CD3 and soluble anti-CD28 mAb (gray bars) for 48 hours. Culture supernatants were collected, and the levels of IL-10, IL-4, IL-5, IFN-γ, and IL-22 were determined by ELISA. All samples were tested in triplicate. The mean ± SD of n=8 donors tested is shown. The results presented in Figure 21 show the effect of immobilized anti-CD3 and soluble anti-CD28 mAb on CD4 expression levels in cells stimulated with immobilized anti-CD3 and soluble anti-CD28 mAb. IL-10 Figure 1 shows that the cells exhibit the Tr1 cell cytokine production profile.

[0605] Although considerable variation was observed between different donors, the overall cytokine production profiles after the second (TF2) or third (TF3) restimulation were comparable and mirrored those of Tr1 cells (Roncarolo et al., Immunity, 2018). Similar to Tr1 cells, CD4 IL-10 The cells produced high levels of IL-10, IL-5, IFN-γ, and IL-22, but low levels of IL-4 and undetectable levels of IL-2.

[0606] CD4 IL-10 The cells express high levels of granzymes and selectively kill myeloid leukemia cells. CD4 IL-10 Cells were further analyzed for granzyme B (GzB) expression after a second round of restimulation (TF2). Data in Figure 22A show that most CD4 IL-10 All CD4 cells from seven different donors expressed GzB. IL-10 More than 95% of the cells expressed high levels of granzyme B.

[0607] CD4 from the second round (TF2) restimulation IL-10 The cells were further analyzed for their cytotoxic effect against myeloid leukemia cells (ALL-CM) and erythroleukemia cell lines (K562). IL-10 cells (10 5 cells / well) to K562 and ALL-CM cells (10 5 The cells were co-cultured with the residual leukemia cell line (CD45 low CD33 + ) were counted by FACS for each target cell.

[0608] CD4 IL-10 The cells selectively killed myeloid leukemia cells (ALL-CM) as shown in Figure 22B. The percentage of ALL-CM cells killed varied between 62% and 100%, whereas killing of the erythroleukemia cell line K562 (highly sensitive to nonspecific cytotoxic activity) varied between 0 and 27% (four different donors tested). Taken together, these data support the conclusion that CD4 IL-10 We confirmed that the cells express granzyme B and effectively kill myeloid leukemia cells. As expected, CD4 IL-10 Some variation in cell killing capacity was observed.

[0609] CD4 IL-10 The cells were allogeneic CD4 + and CD8 + suppresses both T cell proliferation responses CD4 IL-10 The cells were also transfected with allogeneic CD4 + T cells or CD8 + Its effect on T cells was also analyzed. Specifically, allogeneic PBMC cells were treated with eFluor® 670 (10 5 cells / well) and CD4 IL-10 cells (10 5 The cells were cultured in the presence or absence of allogeneic mature dendritic (DC) cells (5 × 10 cells / well). 4Cells were stimulated with soluble anti-CD3 mAb at a 1:1 responder:suppressor ratio (cells / well). After 4 days of culture, the percentage of proliferating responder cells was determined by flow cytometry. + ΔNGFR - T cells or CD8 + ΔNGFR - After gating on T cells, the dilution of eFluor® 670 was determined. + CD4 T cells from six different unpooled donors (donors C, E, and F in Figure 23A and donors H, I, and L in Figure 23B) IL-10 The effect of the cells is shown as percentage of proliferation and inhibition. + CD4 T cells from six different unpooled donors (donors C, E, and F in Figure 24A and donors H, I, and L in Figure 24B) IL-10 The effect of cells is shown.

[0610] Results are based on unpooled, individually tested CD4 IL-10 The cells are allogeneic CD4 + and CD8 + demonstrated that CD4 down-regulates both the proliferation and proliferation responses of T cells. + The inhibitory effect on T cells varied between 51% and 96%, but the CD8 + The inhibitory effect on T cells varied between 62% and 73%.

[0611] Polydonor CD4 IL-10 Cell production and characterization CD4 IL-10 Cells were isolated from multiple donors and analyzed using CD4 + Cells were generated as described above and in Figure 19. CD4 IL-10 The cells were stimulated by a second (TF2) and third (TF3) restimulation. After the third stimulation, CD4 IL-10Cells were pooled in a 1:1:1 ratio and stimulated with immobilized anti-CD3 and soluble anti-CD28 mAb for 48 hours.

[0612] Polydonor CD4 IL-10 The cells were isolated from individual donor CD4 Il-10 have a cytokine production profile comparable to that of Tr1 and Tr2 cells Culture supernatants were collected and levels of IL-10, IL-4, IL-5, IFN-γ, and IL-22 were determined by ELISA. The results presented in Figure 25 show the results of polydonor CD4+ cells pooled from three different allogeneic donors (pooled 1:1:1). IL-10 Cytokine production of cells (red dots) was measured in CD4 IL-10 CD4 from cells (gray bars) IL-10 The cytokine production of polydonor CD4 IL-10 The cells produced high levels of IL-10, IL-5, IFN-γ, and IL-22, as well as low levels of IL-4 and undetectable levels of IL-2 (not shown). IL-10 It is feasible to pool cells and these polydonor CD4 IL-10 The cells are CD4 IL-10 These results demonstrate that the pooled homogeneous cell populations maintain the cytokine production signature of Tr1 and Tr2 cells. Importantly, the pooled homogeneous cell populations contained >95% viable cells, indicating that they did not kill each other.

[0613] Polydonor CD4 IL-10 The cells express high levels of granzyme B and kill myeloid leukemia cell lines. Polydonor CD4 IL-10 Cells were further analyzed for granzyme B (GzB) expression after a third round of restimulation (TF3). Data in Figure 26A show that most polydonor CD4 IL-10 Polydonor CD4 cells express GzB. IL-10 More than 95% of cells were CD4 from a single donorIL-10 They expressed granzyme B, which was equivalent to the cells' GzB expression.

[0614] CD4 from the third round (TF3) restimulation IL-10 The cells were further analyzed for their cytotoxic effect on myeloid leukemia cells (ALL-CM cell line) or K562. IL-10 cells (10 5 cells / well) to K562 and ALL-CM cells (10 5 The cells were co-cultured with the residual leukemia cell line (CD45 low CD33 + ) were counted by FACS for each target cell. The results presented in Figure 26B show some level of cytotoxicity against K562 cells, which are highly sensitive to nonspecific cytotoxicity and NK cell cytotoxicity. Nevertheless, single-donor-derived CD4 IL-10 A level of selectivity against myeloid leukemia cells (ALL-CM) equivalent to that of the ALL-CM cells was obtained.

[0615] Polydonor CD4 IL-10 The cells suppress the proliferative responses of both allogeneic CD4+ and CD8+ T cells. Polydonor CD4 IL-10 The cells were also transfected with allogeneic CD4 + T cells or CD8 + The effect of allogeneic PBMC cells on T cells was analyzed. 5 Polydonor CD4 IL-10 cells (10 5 cells / well) in the presence or absence of allogeneic mature dendritic (DC) cells (5 × 10 4 Cells were stimulated with soluble anti-CD3 mAb at a 1:1 responder:suppressor ratio. After 4 days of culture, the percentage of proliferating responder cells was determined by flow cytometry. + ΔNGFR - T cells and CD8 + ΔNGFR -After gating on T cells, the CD4 T cells were determined by dilution of eFluor® 670. Figure 27A shows CD4 T cells from donors C, E, and F. IL-10 Polydonor CD4 containing cells IL-10 Figure 27B shows the results of CD4+ cells from donors H, I, and L that had been frozen, stored, and thawed prior to testing. IL-10 Polydonor CD4 containing cells IL-10 The results for cells are shown.

[0616] Figure 27A shows a polydonor CD4 IL-10 Cells (from three different donors) were CD4 + and CD8 + The polydonor CD4 T cells tested after the cells had been frozen, stored, and thawed prior to testing showed a 96% and 74% suppression of T cell responses, respectively. IL-10 Comparable results were obtained with a second, different batch of cells (Figure 27B). + and CD8 + The suppression of T cell proliferation was 68% and 75%, respectively. These data are consistent with the polydonor CD4 IL-10 It has been shown that cells can be frozen and stored without loss of function.

[0617] Collectively, polydonor CD4 IL10 The data obtained for the cells indicates that these cell preparations can be pooled without any problems. They contain >95% viable cells and are single-donor CD4 IL-10 Maintains all relevant functions of the cell (cytokine production, cytotoxicity, and suppression of allogeneic T cell responses). Polydonor CD4 IL-10 The use of larger pools of cells allows for the identification of CD4 IL-10 This should reduce the natural variability observed between cell lots and provide large amounts of off-the-shelf CD4 for human therapy. IL-10 The cells should be provided.

[0618] Polydonor CD4 IL-10Cellular products have significant advantages in terms of a more homogeneous product that allows for the determination of well-defined, low lot-to-lot variability, potency, and release criteria. In addition, this allows for the development of continuous, large-scale cell production processes.

[0619] Polydonor CD4 IL-10 Other methods of producing cells Buffy coats from at least 3–5 different donors were pooled before lentiviral transduction. + Cells were isolated from buffy coats by positive selection using anti-CD4 antibodies. + The purity of the cells was checked by FACS. Alternatively, frozen human CD4 + Cells were obtained from at least 3-5 healthy donors. Frozen human CD4 + Cells were thawed before use. CD4 from buffy coat or cryopreservation + Cells were activated with a combination of CD3 and CD28 antibodies or CD3 and CD28 antibody-coated beads in the presence of IL-2 for 24–48 h. In some cases, CD4 antibodies from buffy coats or cryopreserved samples were used. + Cells were activated with soluble anti-CD3, soluble anti-CD28 mAbs, and rhIL-2 (50 U / mL) for 48 hours, and CD4 IL-10 To produce cells, they were transduced with a bidirectional lentiviral vector encoding human IL-10 as described above.

[0620] In some cases, T cell donors (or CD4 + First, the HLA haplotype of the CD4 + Cells are selectively pooled for use.

[0621] Polydonor CD4 IL-10 The cells were then incubated with activated CD4 + The cells were generated by transduction with a lentiviral vector containing the human IL-10 and ΔNGFR coding sequences described above.

[0622] On days 7–11, 5–9 days after transduction, cells were harvested and successfully transduced T cells were purified using an anti-NGFR antibody. This process is typically performed using polydonor CD4 IL-10 This results in a 95% pure population of cells.

[0623] Purified polydonor CD4 IL-10 Cells were counted and restimulated with a mixture of CD3 and CD28 antibodies, CD3 and CD28 antibody-coated beads, optionally in the presence of feeder cells, and IL-2 for an additional 8–10 days. In some cases, purified polydonor CD4 IL-10 Cells were restimulated in the presence of feeder cells.

[0624] After a total culture period of 14–18 days, CD4 IL-10 Cells were harvested, counted, and tested for their ability to produce IL-10, either spontaneously or after activation with CD3 and CD28 antibodies or CD3 and CD28 antibody-coated beads. In addition, GrzB and perforin levels were measured. These were compared with human T cells (PBMCs) and purified CD4 + and CD8 + The ability to suppress T cell proliferation is also tested.

[0625] In addition, IL-22 production was constitutively and also in 200,000 CD4+ cells in a volume of 200 microliters using a combination of CD3 and CD28 antibodies previously described for the production of other cytokines such as IFN-γ, IL-10, IL-4, and IL-5. IL-10 Measured after cell activation. Pooled CD4 IL-10 The cells were frozen and then stored.

[0626] [Example 8] Polydonor CD4 IL-10 Treatment or prevention of GvHD using cells Polydonor CD4 IL-10 In vivo effects on cells Polydonor CD4IL-10 The cell population was tested in a humanized xeno-GvHD disease model, the NSG mouse model, for its effect on xeno-GvHD induced by human PBMCs as illustrated in Figure 28. NSG mice were sublethally irradiated and injected with human PBMCs (5x10 6 cells / mouse), polydonor (3 donors) CD4 IL-10 cells (5×10 6 cells / mouse), or human PBMCs (5 × 10 6 cells / mouse) and polydonor CD4 IL-10 Cells (BC-C / E / F) (5×10 6 The mice were intravenously injected with a combination of 1000x1000x1000 cells / mouse. Xeno-GvHD was assessed based on weight loss (>20% weight loss), skin lesions, coat condition, activity, and huddling, as previously described (Bondanza et al. Blood 2006).

[0627] Figure 29 shows the % of NSG mice demonstrating xeno-GvHD on each day after injection. 6 Administration of 5 × 10 human PBMCs to irradiated NSG mice unexpectedly resulted in an unusually fulminant GvHD. All mice died by day 10, reflecting highly lethal xeno-GvHD. 6 Polydonor CD4 IL-10 Co-administration of cells delayed this fulminant GvHD, but mice were sacrificed on day 14 after reaching the defined humane 20% weight loss criterion for sacrifice (Figure 29). Nevertheless, these results suggest that polydonor CD4 IL-10 Importantly, the same dose (5 × 10) of PBMCs was used to delay very severe xeno-GvHD. 6 Polydonor CD4 IL-10 The cells did not induce any signs of xeno-GvHD.

[0628] Human CD4 IL-10 The presence of cells was also confirmed by human PBMCs (5 × 10 6individuals / mouse), polydonor (3 donors; B-C / E / F) CD4 IL-10 cells (5×10 6 cells / mouse), or human PBMCs (5 × 10 6 cells / mouse) and polydonor CD4 IL-10 Cells (three donors; B-C / E / F) (5 × 10 6 The polydonor CD4 combinations (100 / mouse) were also tested in the spleen (Figure 30, left panel) and bone marrow (Figure 30, right panel) of injected NSG mice 14 days post-injection. The results presented in Figure 30 show that polydonor CD4 IL-10 The cells migrated to the spleen and bone marrow. A low percentage of these cells was found to be present 14 days after cell injection. These results support the conclusion that polydonor CD4 IL-10 These results indicate that the cells delayed the fulminant xeno-GvHD induced by human PBMCs, but did not themselves induce any xeno-GvHD.

[0629] Polydonor CD4 IL-10 Cells were purified CD4 + Inhibits cell-mediated severe xeno-GvHD Polydonor CD4 IL-10 The cells were cultured at 2.5×10 6 We tested this in a humanized xeno-GvHD model induced by the administration of purified human CD4+ T cells. NSG mice were sublethally irradiated on day 0 and injected with human CD4+ T cells on day 3. + T cells (2.5×10 6 (number / mouse) alone or in polydonor CD4 IL-10 Cells (three different donors; BC-H / I / L) (2.5 × 10 6 CD4 from a single donor (BC-H) or a combination of CD4 from a single donor (BC-H) IL-10 cells (2.5×10 6 The mice were intravenously injected with a combination of 1000x1000x1000 cells / mouse. Xeno-GvHD was assessed based on weight loss (>20% weight loss), skin lesions, coat condition, activity, and huddling, as previously described (Bondanza et al. Blood 2006).

[0630] Figure 32 shows the % of NSG mice demonstrating GvHD on each day after injection. IL-10 (BC-H / I / L) cells are human allogeneic CD4 + This indicates that T cell-mediated xeno-GvHD can be inhibited. In this experiment, xeno-GvHD was so severe that all mice in the control group receiving CD4+ T cells died by day 20. In contrast, 2.5 × 10 6 Polydonor CD4 IL-10 Co-administration of single donor CD4 inhibited GvHD by 75%. IL-10 Cells were also protective, but to a lesser extent.

[0631] Other experiments Polydonor CD4 IL-10 The therapeutic effect of the cells is tested in four different groups of mice: (i) CD4 IL-10 (ii) mice administered human PBMCs from unrelated donors (xeno-GvHD positive control); (iii) polydonor CD4 IL-10 (iii) PBMCs and polydonor CD4 IL-10 Mice administered a 1:1 ratio combination of PBMCs and polydonor CD4 IL-10 Mice received a 2:1 ratio of cells or a combination of different ratios. PBMC and polydonor CD4 IL-10 In animals receiving the cell combination, some animals received PBMC and polydonor CD4 IL-10 Cells were administered simultaneously, and some animals received polydonor CD4 IL-10 Cells were administered, and some animals were polydonor CD4 IL-10 Cells are administered.

[0632] Mice were cultured using PBMC and / or polydonor CD4 IL-10After cell administration, mice are monitored for the development of GvHD by measuring body weight at 1, 2, 3, 4, and, if necessary, 5 weeks. In addition to weight loss, mice are examined for skin lesions, coat condition, and activity. Mice in the treatment groups are monitored for an additional period to determine polydonor CD4 IL-10 The effect on long-term cell survival is determined.

[0633] Polydonor CD4 IL-10 The quantity and localization of cells will also be monitored in peripheral blood and tissues after administration. Specifically, polydonor CD4 IL-10 The presence of cells is monitored in the peripheral blood and at sites of inflammation: lymph nodes, spleen, intestinal tract, and bone marrow. Mice in the treatment group(s) are monitored for an additional 3 weeks to determine long-term survival.

[0634] The results are polydonor CD4 IL-10 The cells demonstrate efficacy in reducing and preventing xeno-GvHD.

[0635] [Example 9] Inhibition of GvHD and treatment of cancer Polydonor CD4 IL-10 The cell populations are tested for their effects on xeno-GvHD induced by human PBMCs and anti-tumor effects in NSG mouse models transplanted with human PBMCs and AML tumor cells. AML cells (ALL-CM) are administered intravenously as previously described in WO 2016 / 146542. PBMCs or polydonor CD4 IL-10 The cells, or a combination thereof, are administered three days later.

[0636] Polydonor CD4 IL-10 Cells are obtained as described in Example 1. Polydonor CD4 IL-10 The therapeutic effect of the cells was evaluated by irradiating each cell with 5 × 10 6 Four different groups of mice (AML mice) administered ALL-CM cells will be tested: (i) AML mice with no additional treatment; (ii) polydonor CD4 IL-105 x 10 cells from an unrelated donor 6 AML mice administered 2.5 × 10 human PBMCs - PBMCs induce severe xeno-GvHD; (iii) 2.5 × 10 6 Polydonor CD4 IL-10 AML mice administered cells; and (iv) PBMCs and polydonor CD4 IL-10 AML mice were administered 1:1 or 2:1 ratios of cells, or a combination of different ratios. One additional group of mice received no ALL-CML cells but received 5x10 6 Administer human PBMCs.

[0637] Polydonor CD4 IL-10 The effect of the cells on xeno-GvHD induced by human PBMCs will be examined based on weight loss, skin lesions, coat condition, activity, mortality, and long-term survival. IL-10 The anti-tumor or graft-versus-leukemia (GvL) effect of the cells is tested based on the reduction of circulating tumor cells and long-term tumor-free survival.

[0638] Some mice are monitored for up to 7 weeks to monitor for long-term survival and complete tumor regression.

[0639] The results are polydonor CD4 IL-10 The cells demonstrate efficacy in both inhibiting xeno-GvHD and treating cancer.

[0640] [Example 10] Polydonor CD4 IL-10 Cell-based cancer treatment Polydonor CD4 IL-10 The cell populations will be tested in the ALL-CM leukemia model of T cell therapy in NSG mice.

[0641] NSG mice were sublethally irradiated and injected with myeloid leukemia cells (ALL-CM) (5 × 10 6 In the first group of animals, PBMCs (5×106 (individuals) or single donor (from donors BC-I and BC-H) CD4 IL-10 cells (2.5×10 6 ) on day 3. In the second group of animals, PBMCs (5 × 10 6 ) or polydonor CD4 IL-10 cells (2.5×10 6 (1000 mg / kg / day) were injected on day 3. The graft-versus-leukemia (GvL) effect was tested in animals based on the reduction of circulating leukemia cells and long-term leukemia-free survival. Leukemia was measured as previously described (Locafaro G. et al. Molecular Therapy 2017).

[0642] As provided in Figures 33B and 33C, all mice injected with ALL-CM myeloid leukemia cells showed extensive leukemia progression by day 17. 6 Administration of PBMCs resulted in a strong inhibition of leukemia progression. Interestingly, a comparable level of leukemia progression inhibition was observed with a lower number (2.5 x 10 6 (units) single donor CD4 IL10 (Figure 33B) or polydonor CD4 IL10 (Figure 33C). These data were obtained using single-donor and polydonor CD4 IL10 have been shown to have a strong direct antimyeloid leukemia effect.

[0643] Single donor CD4 IL10 and polydonor CD4 IL10 The graft-versus-leukemia (GvL) effect of was further tested in combination with PBMCs in mice injected with ALL-CM myeloid leukemia cells (see Figure 34A). 6 Administration of PBMCs resulted in strong inhibition of leukemia progression and single-donor CD4 IL10 (2.5×10 6 5×10 6 Administration of 2.5 x 10 PBMCs had a synergistic effect (Figure 34B). 6 Polydonor...

Claims

(a) a first exogenous polynucleotide segment encoding a chimeric antigen receptor (CAR); and (b) a second exogenous polynucleotide segment encoding interleukin-10 (IL-10); A genetically modified CD4 + T cell (CD4 IL-10 / CAR) comprising: the modified CD4 + T cells have cytotoxicity against a target and immunomodulatory properties; A genetically modified CD4 + T cell (CD4 IL-10 / CAR), wherein the modified CD4 + T cell secretes IL-10 expressed from the second exogenous polynucleotide segment. (a) the first exogenous polynucleotide segment comprises a first regulatory element operably linked to a coding sequence of the CAR; optionally, the regulatory element drives constitutive expression of the CAR; and / or (b) the CAR comprises an antigen-binding domain, a hinge region, a transmembrane domain, and an intracellular signaling domain; optionally: (i) the antigen-binding domain is a single-chain antibody fragment; optionally, the single-chain antibody fragment comprises a single-chain Fv (scFv); (ii) the antigen binding domain targets an antigen associated with an autoimmune disease, inflammatory disorder, or cancer; optionally, the antigen is CD19, CD20, CD22, CD27, CD38, B7-H3, CD23, Lym1, Lym2, CLEC5A, CDH179b, FLT3, GCC, Muc, BCMA, CSF2RA, GFRα4, CD32, CD33, CEA, IL11Ra, IL13Ra, NYBRI, SLea, CD200R, TGFbetaR2, CD276, TROP2, LAMP1, PTK7, DLL3, CDH1, CDH6, CDH17, CDH19, TSHR, tyrosinase, HLA-A*02, HLA-A*03, HLA-A*04, HLA-A*05, HLA-A*06, HLA-A*07, HLA-A*08, HLA-A*09, HLA-A*10, HLA-A*11, HLA-A*12, HLA-A*13, HLA-A*14, HLA-A*15, HLA-A*16, HLA-A*17, HLA-A*18, HLA-A*19 ... 24, or citrullinated peptides, insulin, MOG, GAD65, IA2, gliadin, and desmoglein; and / or (iii) the antigen-binding domain is: (A) comprises an anti-CD19 antigen-binding domain; optionally, the anti-CD19 antigen-binding domain has the sequence of SEQ ID NO: 11; or (B) an anti-BCMA antigen-binding domain; optionally, the anti-BCMA antigen-binding domain comprises a sequence of SEQ ID NO: 50-53; The genetically modified CD4 + T cell of claim 1 . (a) the hinge region is selected from a human CD8α hinge region, a human CD28 hinge region, an IgG1 hinge region, or an IgG4 hinge region; optionally, the hinge region is derived from human CD8α; (b) the transmembrane domain is selected from a TNFRSF 19 transmembrane domain, a CD3 zeta transmembrane domain, a CD8α transmembrane domain, a CD4 transmembrane domain, a CD28 transmembrane domain, or a B7 family-induced costimulatory (ICOS) transmembrane domain; optionally, the transmembrane domain is derived from CD8α; and / or (c) the CAR further comprises one or more costimulatory domains; optionally, (i) the CAR comprises two costimulatory domains; and / or (ii) the one or more costimulatory domains are selected from the group consisting of 4-1BB, CD28, OX40, ICOS, CD27, MYD88-CD40, and KIR2DS2; optionally, one of the one or more costimulatory domains is derived from CD28 and / or 4-1BB. The genetically modified CD4 + T cell of claim 1 . (a) the intracellular signaling domain comprises an immunoreceptor tyrosine-based activation motif (ITAM); (b) the immunoreceptor tyrosine-based activation motif (ITAM) is derived from CD3 zeta; (c) the CAR is: an anti-CD19 antigen-binding domain, an anti-BCMA antigen-binding domain, or an anti-CD20 antigen-binding domain; human CD8α hinge region; human CD8α transmembrane domain; CD28 costimulatory domain; and CD3 zeta chain intracellular signaling domain Includes; (d) the CAR comprises the sequence of SEQ ID NO: 9, 16, 22, 34, 41-49, or 54; optionally, the first exogenous polynucleotide segment comprises the sequence of SEQ ID NO: 10, 17, 23, 35, or 55; (e) the first exogenous polynucleotide segment comprises: (i) integrated into the T cell nuclear genome; or (ii) not integrated into the T-cell nuclear genome; Optionally, the first exogenous polynucleotide segment is present in a vector; optionally, the vector is a viral vector; optionally, the vector is a lentiviral vector; (f) the second exogenous polynucleotide segment comprises a second regulatory element operably linked to a coding sequence for IL-10; optionally, the second regulatory element drives constitutive or inducible expression of the IL-10; (g) the IL-10 is human IL-10; (h) the IL-10 is viral IL-10; (i) the IL-10 is a protein having the sequence of SEQ ID NO:1; optionally, the second exogenous polynucleotide segment has the sequence of SEQ ID NO:2; (j) the second exogenous polynucleotide segment comprises: (i) integrated into the T cell nuclear genome; or (ii) not integrated into the T-cell nuclear genome; Optionally, the second exogenous polynucleotide segment is present in a vector; optionally, the vector is a viral vector; optionally, the vector is a lentiviral vector; (k) the first exogenous polynucleotide segment and the second exogenous polynucleotide segment are present in the same vector; optionally, the vector is a viral vector; optionally, the vector is a lentiviral vector; (l) the CD4 + T cells constitutively express at least 100 pg of IL-10 per 10 6 CD4 + T cells / mL of culture medium; optionally, the CD4 + T cells constitutively express at least 100 pg, 200 pg, 500 pg, 1 ng, 5 ng, 10 ng, or 50 ng of IL-10 per 10 6 CD4 + T cells / mL; (m) the CD4 + T cells express IL-10 at a level at least 5-fold higher than unmodified CD4 + T cells; optionally, the CD4 + T cells express IL-10 at a level at least 10-fold higher than unmodified CD4 + T cells; (n) the first exogenous polynucleotide segment, the second exogenous polynucleotide segment, or both, further comprise a sequence encoding a selectable marker; optionally: (i) the selectable marker is ΔNGFR; optionally, the ΔNGFR has the sequence of SEQ ID NO:3; optionally, the second exogenous polynucleotide segment comprises the sequence of SEQ ID NO:4; or (ii) the selectable marker is a truncated form of an EGFR polypeptide; (o) the CD4 + T cells: (i) capable of in vitro cytotoxicity against myeloid target cells; or (ii) capable of in vivo cytotoxicity against myeloid target cells; Optionally: (A) the CD4 + T cells are capable of cytotoxicity against CD19 + target cells and myeloid target cells; (B) the myeloid target cells express one or more of class I MHC, CD13, CD54, and CD112; and / or (C) said cytotoxicity against CD19 + target cells or myeloid target cells is maintained after one or more in vitro restimulations; and / or (p) The CD4 + T cells: (i) It is capable of suppressing allogeneic CD4 + T cell proliferation; (ii) capable of suppressing allogeneic CD8 + T cell proliferation; or (iii) capable of suppressing allogeneic CD4 + T cell proliferation, allogeneic CD8 + T cell proliferation, and allogeneic PBMCs; Optionally, the suppressive properties are maintained after one or more in vitro restimulations. The genetically modified CD4 + T cell of claim 1 .

5. The CAR comprises an antigen-binding domain, a hinge region, a transmembrane domain, and an intracellular signaling domain, wherein the antigen-binding domain comprises an anti-CD19 antigen-binding domain; optionally, the anti-CD19 antigen-binding domain has the sequence of SEQ ID NO: 11; and the CD4+ T cell: (a) capable of in vitro cytotoxicity against CD19 + target cells; or (b) capable of in vivo cytotoxicity against CD19 + target cells; Optionally, (i) the CD19 + target cells are autoantibody-producing B cells or CD19 + cancer cells; (ii) the CD4 + T cells are capable of cytotoxicity against CD19 + target cells and myeloid target cells; optionally, the myeloid target cells express one or more of class I MHC, CD13, CD54, and CD112; and / or (iii) the cytotoxicity against CD19 + target cells or myeloid target cells is maintained after one or more in vitro restimulations; The genetically modified CD4 + T cell of claim 1 .

6. A CD4+ T cell population comprising the genetically modified CD4+ cells of any one of claims 1 to 5; optionally comprising: (a) the CD4 + T cells prior to genetic modification are obtained and pooled from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 T cell donors; optionally, the CD4 + T cells in said population collectively have 6, 7, 8, 9, 10, 11, 12, or more different HLA haplotypes; （b） (A) All CD4 + T cells in the population: (i) have at least a 1 / 10, 2 / 10, 3 / 10, 4 / 10, 5 / 10, 6 / 10, 7 / 10, 8 / 10, or 9 / 10 match to each other at the HLA-A, HLA-B, HLA-C, HLA-DRB1, and HLA-DQB1 loci; (ii) have at least a 1 / 8, 2 / 8, 3 / 8, 4 / 8, 5 / 8, 6 / 8, 7 / 8, or 8 / 8 match to each other at the HLA-A, HLA-B, HLA-C, and HLA-DRB1 loci; (iii) have a 2 / 2 match to each other at the HLA-A locus; (iv) have a 2 / 2 match to each other at the HLA-B locus; (v) have a 2 / 2 match to each other at the HLA-C locus; and / or (vi) have at least a 3 / 4 or 4 / 4 match to each other at the HLA-DRB1 and HLA-DQB1 loci; or (B) all CD4 + T cells in said population have less than a 5 / 10, 6 / 10, 7 / 10, 8 / 10, or 9 / 10 match to each other at the HLA-A, HLA-B, HLA C, HLA-DRB1, and HLA-DQB1 loci; optionally, all CD4 + T cells in said population: (i) have less than a 4 / 8, 5 / 8, 6 / 8, 7 / 8, or 8 / 8 match to each other at the HLA-A, HLA-B, HLA-C, and HLA-DRB1 loci; (ii) have less than a 2 / 2 match to each other at the HLA-A locus; (iii) have less than a 2 / 2 match to each other at the HLA-B locus; (iv) have less than a 2 / 2 match to each other at the HLA-C locus; and / or (v) have less than a 2 / 4, 3 / 4, or 4 / 4 match* to each other at the HLA-DRB1 and HLA-DQB1 loci; (c) all CD4 + T cells in said population: (i) have the A*02 allele or are all A*02 negative; or (ii) have the A*24 allele or are all A*24 negative; (d) at least 30% of the CD4 + T cells in the NGFR + cell population express the CAR; optionally, at least 60% of the CD4 + T cells in the NGFR + cell population express the CAR; optionally, at least 90% of the CD4 + T cells in the NGFR + cell population express the CAR; and / or (e) the CD4 + T cells: (i) in a frozen suspension; or (i) present in a liquid suspension; CD4 + T cell population.

7. A pharmaceutical composition comprising the genetically modified CD4 + T cells described in any one of claims 1 to 5.

8. A method for producing genetically modified CD4 + T cells (CD4 IL-10 / CAR) cells, comprising: (i) a first exogenous polynucleotide segment encoding a chimeric antigen receptor (CAR), and (ii) a second exogenous polynucleotide segment encoding IL-10; modifying primary CD4 + T cells obtained from one or more T cell donors by introducing A method comprising:

9. The method of claim 8, wherein: (a) the primary CD4+ T cells are obtained from 2, 3, 4, 5, 6, 7, 8, 9, or 10 different T cell donors; and optionally, the method further comprises pooling the genetically modified CD4+ T cells; (b) before or after modifying the primary CD4 + T cells, the method further comprising: further comprising incubating the CD4 + T cells in the presence of anti-CD3 and anti-CD28 antibodies, or anti-CD3 and CD28 antibody coated beads, or polymer nanomatrix reagents for activating and expanding human T cells via CD3 and CD28, or other T cell specific immune cell culture media, activators, and supplements; optionally, the method further comprises incubating the CD4 + T cells in the presence of IL-2; (c) the first exogenous polynucleotide segment, the second exogenous polynucleotide segment, or both are introduced into the primary CD4 + T cells using one or more viral vectors; optionally, the viral vectors are lentiviral vectors; (d) the CAR is specific for a target antigen associated with an autoimmune disease, an inflammatory disorder, or a cancer; optionally, the target antigen is associated with an autoimmune disease, an inflammatory disorder, or a cancer; (e) the CAR is an anti-CD19 CAR, anti-CD20 CAR, anti-CD22 CAR, anti-BCMA CAR, anti-B7-H3 CAR, anti-CD27 CAR, or anti-CD38 CAR; (f) the CAR has a sequence of SEQ ID NO: 9, 16, 22, 34, 41 to 49, or 54; (g) the first exogenous polynucleotide segment has the sequence of SEQ ID NO: 10, 17, 23, 35, or 55; (h) the IL-10 has the sequence of SEQ ID NO: 1; (i) the second exogenous polynucleotide segment has the sequence of SEQ ID NO:2; (j) the first exogenous polynucleotide segment, the second exogenous polynucleotide segment, or both, further comprise a segment encoding a selectable marker; optionally: (i) the encoded selectable marker is ΔNGFR; optionally, the encoded selectable marker has the sequence of SEQ ID NO:3; or (ii) the selectable marker is a truncated form of an EGFR polypeptide; (k) the method further comprises, after the step of modifying the primary CD4 + T cells: and optionally further comprising the step of isolating said genetically modified CD4 + T cells that express said selection marker, thereby generating an enriched population of genetically modified CD4 + T cells; (i) at least 30% or at least 60% of the genetically modified CD4 + T cells in the enriched population express IL-10 and a CAR; optionally, at least 90% of the genetically modified CD4 + T cells in the enriched population express IL-10 and a CAR; (ii) at least 75% of the genetically modified CD4 + T cells in the enriched population express a selection marker; optionally, at least 90% of the genetically modified CD4 + T cells in the enriched population express a selection marker; and / or (iii) after isolating the genetically modified CD4 + T cells, the method further comprises incubating the enriched population of genetically modified CD4 + T cells; Optionally, the step of incubating the enriched population of genetically modified CD4 + T cells is performed in the presence of (i) anti-CD3 and anti-CD28 antibodies, or (ii) anti-CD3 and anti-CD28 antibody coated beads in the presence of IL-2, or polymer nanomatrix reagents for activating and expanding human T cells via CD3 and CD28, or (iii) other T cell specific immune cell culture media, activators, and supplements; and / or (l) the method further comprises a subsequent step of freezing the genetically modified CD4 + T cells; The method of claim 8.

10. The method of claim 1, wherein the primary CD4+ T cells are obtained from 2, 3, 4, 5, 6, 7, 8, 9, or 10 different T cell donors; optionally, the method further comprises pooling the genetically modified CD4+ T cells: （a） (A) the at least two T cell donors: (i) have at least a 1 / 10, 2 / 10, 3 / 10, 4 / 10, 5 / 10, 6 / 10, 7 / 10, 8 / 10, or 9 / 10 match to each other at the HLA-A, HLA-B, HLA-C, HLA-DRB1, and HLA-DQB1 loci; (ii) have at least a 1 / 8, 2 / 8, 3 / 8, 4 / 8, 5 / 8, 6 / 8, 7 / 8, or 8 / 8 match to each other at the HLA-A, HLA-B, HLA-C, and HLA-DRB1 loci; (iii) have a 2 / 2 match to each other at the HLA-A locus; (iv) have a 2 / 2 match to each other at the HLA-B locus; (v) have a 2 / 2 match to each other at the HLA-C locus; and / or (vi) have at least a 3 / 4 or 4 / 4 match to each other at the HLA-DRB1 and HLA-DQB1 loci; or (B) the at least two T cell donors have less than a 5 / 10, 6 / 10, 7 / 10, 8 / 10, or 9 / 10 match to each other at the HLA-A, HLA-B, HLA C, HLA-DRB1, and HLA-DQB1 loci; optionally, the at least two T cell donors: (i) have less than a 4 / 8, 5 / 8, 6 / 8, 7 / 8, or 8 / 8 match to each other at the HLA-A, HLA-B, HLA-C, and HLA-DRB1 loci; (ii) have less than a 2 / 2 match to each other at the HLA-A locus; (iii) have less than a 2 / 2 match to each other at the HLA-B locus; (iv) have less than a 2 / 2 match to each other at the HLA-C locus; and / or (v) have less than a 2 / 4, 3 / 4, or 4 / 4 match to each other at the HLA-DRB1 and HLA-DQB1 loci; (b) each of the at least two T cell donors: (i) having the A*02 allele or being A*02 negative; or (ii) have the A*24 allele or are A*24 negative; (c) the primary CD4 + T cells are obtained from one or more cryopreservations; and / or (d) the primary CD4 + T cells are obtained from unfrozen peripheral blood mononuclear cells of the at least two different T cell donors; optionally, the method further comprises isolating CD4 + T cells from the peripheral blood mononuclear cells.

10. The method according to claim 8 or 9.

11. The pharmaceutical composition of claim 7 for use in the following method: (a) A method of treating a hematological cancer, the method comprising: administering to a patient with hematological cancer a therapeutically effective amount of said CD4 IL-10 / CAR cells, CD4 IL-10 / CAR cell population, or said pharmaceutical composition sufficient to induce an anti-cancer effect. optionally, said method further comprising administering an allo-HSCT graft to said patient before or after administration of said CD4 IL-10 / CAR; or (b) A method of treating a patient with a malignant disease, the method comprising: administering an allo-HSCT graft to said patient; and administering to said patient a therapeutically effective amount of said CD4 IL-10 / CAR cells, CD4 IL-10 / CAR cell population, or said pharmaceutical composition. optionally, the amount of said CD4 IL-10 / CAR cells is further sufficient to suppress or prevent graft-versus-host disease (GvHD) without suppressing graft-versus-leukemia (GvL) or graft-versus-tumor (GvT) efficacy of said allo-HSCT. Pharmaceutical compositions.

12. (a) The malignant disease or hematological cancer is myeloid leukemia; (b) the CD4 IL-10 / CAR cells target and kill cancer cells that express CD13; (c) the CD4 IL-10 / CAR cells target and kill cancer cells that express HLA-class I; (d) the malignant disease, hematological cancer, or myeloid leukemia is acute myeloid leukemia (AML); (e) the malignancy or hematological cancer is a CD19 + , CD20 + , CD22 + , BCMA + , CD27 + , CD38 + , or B7-H3 + hematological cancer; optionally, the CD19 + , CD20 + , CD22 + , BCMA + , CD27 + , CD38 + , or B7-H3 + hematological cancer is selected from chronic lymphocytic leukemia, acute lymphoblastic leukemia (ALL), and non-Hodgkin's lymphoma; (f) the allo-HSCT graft is obtained from a related or unrelated donor with respect to the patient; (g) the CD4 IL-10 / CAR cells: (i) is non-autologous to the patient; (ii) is autologous to said patient; (iii) is allogeneic to said patient; (iv) is not anergized to host alloantigens prior to administration to said patient; (v) are Tr1-like cells; (vi) polyclonal; (vii) is polyclonal and non-autologous to the patient; (viii) is polyclonal and autologous to the patient; or (ix) isolated from at least two donors and subsequently genetically modified; optionally, none of the at least two donors are allo-HSCT donors; (h) the allo-HSCT graft is obtained from a matched or mismatched donor with respect to the patient; (i) the CD4 IL-10 / CAR cells target and kill: (i) cells expressing CD19, CD20, or BCMA; (ii) cells expressing CD54; (iii) cancer cells expressing HLA-class I and CD54; (iv) cancer cells expressing CD112 and CD155; (v) CD58-expressing cancer cells; (vi) cancer cells in the patient; and / or (vii) solid tumor cells in the patient; and / or (j) the CD4 IL-10 / CAR cells suppress: (i) allogeneic CD4 + T cell expansion; (ii) allogeneic CD8 + T cell expansion; and / or (iii) allogeneic CD4 + T cell proliferation, allogeneic CD8 + T cell proliferation, and PBMCs; The pharmaceutical composition of claim 11.

13. The pharmaceutical composition of claim 7 for use in the following method: (a) A method for preventing recurrence of a CD19 + , or CD20 + , CD22 + , BCMA + , CD27 + , CD38 + , or B7-H3 + hematologic cancer in a patient, said method comprising: administering a therapeutically effective amount of said CD4 IL-10 / CAR cells, CD4 IL-10 / CAR cell population, or said pharmaceutical composition to a patient identified as being at risk for recurrence of a CD19 + , CD20 + , CD22 + , BCMA + , CD27 + , CD38 + , or B7-H3 + hematologic cancer. wherein said therapeutically effective amount is sufficient to induce an anti-cancer effect; (b) a method for preventing recurrence of a B7-H3 + cancer in a patient, said method comprising: administering to a patient identified as having a B7-H3 + cancer a therapeutically effective amount of said CD4 IL-10 / CAR cells, CD4 IL-10 / CAR cell population, or said pharmaceutical composition sufficient to induce an anti-cancer effect. optionally, the B7-H3 + cancer is a solid tumor; optionally, the solid tumor is selected from the group consisting of breast cancer, brain cancer, lung cancer, liver cancer, stomach cancer, spleen cancer, colon cancer, kidney cancer, pancreatic cancer, prostate cancer, uterine cancer, skin cancer, head cancer, neck cancer, sarcoma, neuroblastoma, and ovarian cancer; or (c) A method of treating a cancer patient with minimal residual disease, said method comprising: administering a therapeutically effective amount of said CD4 IL-10 / CAR cells, CD4 IL-10 / CAR cell population, or said pharmaceutical composition to a patient identified as having or at risk of having minimal residual disease. wherein the therapeutically effective amount is sufficient to induce an anti-cancer effect. Pharmaceutical compositions.

14. The pharmaceutical composition of claim 7 for use in a method of treating a patient in need of immune tolerance, said method comprising: administering to said patient a therapeutically effective amount of said CD4 IL-10 / CAR cells, CD4 IL-10 / CAR cell population, or said pharmaceutical composition. Optionally, a method comprising: (a) the method further comprises the prior step of thawing a frozen suspension of CD4 IL-10 / CAR cells or CD4 IL-10 / CAR cell population; (b) the patient has an inflammatory disease or an autoimmune disease; optionally, the inflammatory disease or autoimmune disease is selected from the group consisting of type 1 diabetes, autoimmune uveitis, autoimmune hepatitis, rheumatoid arthritis, psoriasis, psoriatic arthritis, vitiligo, alopecia areata, multiple sclerosis, systemic lupus, inflammatory bowel disease, Addison's disease, Graves' disease, Sjogren's syndrome, Hashimoto's thyroiditis, myasthenia gravis, autoimmune vasculitis, pernicious anemia, ulcerative colitis, bullous disease, scleroderma, and celiac disease; optionally, the inflammatory disease or autoimmune disease is Crohn's disease, ulcerative colitis, celiac disease, type 1 diabetes, lupus, psoriasis, psoriatic arthritis, ankylosing spondylitis, or rheumatoid arthritis; (c) the patient has a disease or disorder associated with hyperactivity of the NLPR3 inflammasome; (d) the patient has type 2 diabetes, a neurodegenerative disease, a cardiovascular disease, or an inflammatory bowel disease; (e) the patient has a disease or disorder associated with increased IL-1β production by activated monocytes, macrophages, or dendritic cells; (f) the patient has a disease or disorder associated with increased IL-18 production by activated monocytes, macrophages, or dendritic cells; (g) the patient has a disease or disorder associated with increased production of mature caspase-1 by activated monocytes, macrophages, or dendritic cells; (h) the patient has an allergic or atopic disease; optionally, the allergic or atopic disease is selected from the group consisting of asthma, atopic dermatitis, and rhinitis; (i) the patient has a food allergy; (j) the patient has a solid tumor; optionally, the solid tumor is selected from the group consisting of breast cancer, brain cancer, lung cancer, liver cancer, stomach cancer, spleen cancer, colon cancer, kidney cancer, pancreatic cancer, prostate cancer, uterine cancer, skin cancer, head cancer, neck cancer, sarcoma, neuroblastoma, and ovarian cancer; (k) the method further comprises the step of administering an organ transplant to the patient either before or after administration of the CD4 IL-10 / CAR cells, the CD4 IL-10 / CAR cell population, or the pharmaceutical composition; optionally, the CD4 IL-10 / CAR cells, the CD4 IL-10 / CAR cell population, or the pharmaceutical composition prevents or reduces the severity of host rejection of the organ transplant; (l) the method further comprises transplanting iPS cell-derived cells or tissue into the patient either before or after administration of the CD4 IL-10 / CAR cells, the CD4 IL-10 / CAR cell population, or the pharmaceutical composition; optionally, the CD4 IL-10 / CAR cells, the CD4 IL-10 / CAR cell population, or the pharmaceutical composition prevents or reduces the severity of host rejection of the cell transplant; (m) the method further comprises administering a recombinant AAV to the patient either before or after administration of the CD4 IL-10 / CAR cells, the CD4 IL-10 / CAR cell population, or the pharmaceutical composition; optionally, the CD4 IL-10 / CAR cells, the CD4 IL-10 / CAR cell population, or the pharmaceutical composition reduces an immune response to the recombinant AAV; (n) the method further comprises administering to the patient a recombinant viral vector other than AAV either before or after administration of the CD4 IL-10 / CAR cells, the CD4 IL-10 / CAR cell population, or the pharmaceutical composition; optionally, the CD4 IL-10 / CAR cells, the CD4 IL-10 / CAR cell population, or the pharmaceutical composition reduces an immune response to the recombinant viral vector other than AAV; optionally, the method further comprises administering to the patient an immunogenic therapeutic protein either before or after administration of the CD4 IL-10 / CAR cells, the CD4 IL-10 / CAR cell population, or the pharmaceutical composition; and optionally: (i) the CD4 IL-10 / CAR cells, the CD4 IL-10 / CAR cell population, or the pharmaceutical composition reduces an immune response to the immunogenic therapeutic protein; and / or (ii) the immunogenic therapeutic protein is selected from a therapeutic antibody, a Factor VIII replacement, a cytokine, and a cytokine mutein; (o) the patient has an exaggerated immune response to a viral or bacterial infection; optionally, the patient has a coronavirus infection; or (p) the method further comprises detecting a selectable marker in a biological sample obtained from the patient, thereby detecting the presence or absence of CD4 IL-10 / CAR cells; optionally, the biological sample is a biopsy or blood from the patient. Pharmaceutical compositions.

15. The pharmaceutical composition of claim 7 for use in the following method: (a) A method of treating or inhibiting an autoimmune, allergic, or inflammatory disease in a patient, said method comprising: administering to a patient identified as having an autoimmune, allergic, or inflammatory disease a therapeutically effective amount of said CD4 IL-10 / CAR cells, CD4 IL-10 / CAR cell population, or said pharmaceutical composition sufficient to treat or inhibit the autoimmune, allergic, or inflammatory disease. a method comprising: (b) A method for reducing graft rejection in a patient who has received a hematopoietic stem cell, bone marrow cell, umbilical cord blood cell, tissue stem cell, or solid organ transplant, said method comprising: administering to a patient identified as having rejection of transplanted hematopoietic stem cells, bone marrow cells, or solid organs a therapeutically effective amount of said CD4 IL-10 / CAR cells, CD4 IL-10 / CAR cell population, or said pharmaceutical composition sufficient to reduce graft rejection. a method comprising: (c) A method of treating graft-versus-host disease (GvHD) in a patient, said method comprising: administering to a patient identified as having or at risk of having graft-versus-host disease (GvHD) a therapeutically effective amount of said CD4 IL-10 / CAR cells, CD4 IL-10 / CAR cell population, or said pharmaceutical composition sufficient to suppress or prevent GvHD. optionally, said graft-versus-host disease (GvHD) is: (i) including acute GvHD; or (ii) including chronic GvHD; or (d) A method of treating tissue or organ damage in a patient, said method comprising: administering to a patient identified as having or at risk of having tissue or organ damage a therapeutically effective amount of said CD4 IL-10 / CAR cells, CD4 IL-10 / CAR cell population, or said pharmaceutical composition sufficient to induce repair of the tissue or organ damage. a method comprising: Pharmaceutical compositions.

16. (a) a first polynucleotide segment encoding a chimeric antigen receptor (CAR); and (b) a second polynucleotide segment encoding interleukin-10 (IL-10); A polynucleotide construct comprising:

17. (a) the first polynucleotide segment comprises a first regulatory element operably linked to a coding sequence of the CAR; optionally, the first regulatory element drives constitutive expression of the CAR; (b) said second polynucleotide segment comprises a second regulatory element operably linked to a coding sequence of said IL-10; optionally, said second regulatory element drives constitutive expression of said IL-10; (c) the polynucleotide construct further comprises an internal ribosome entry site (IRES) or a self-cleaving peptide between the first polynucleotide segment and the second polynucleotide segment; optionally, the self-cleaving peptide is selected from the group consisting of F2A, P2A, T2A, and E2A; (d) the CAR comprises an antigen-binding domain, a hinge region, a transmembrane domain, and an intracellular signaling domain; optionally, the antigen-binding domain comprises a single-chain antibody fragment; optionally, the single-chain antibody fragment comprises a single-chain Fv (scFv); (e) the antigen binding domain targets an antigen associated with an autoimmune disease, inflammatory disorder, or cancer; optionally, the antigen is CD19, CD20, CD22, BCMA, CD27, CD38, CEA, B7-H3, CD23, Lym1, Lym2, CLEC5A, CDH179b, FLT3, GCC, Muc, CSF2RA, GFRα4, CD32, CD33, IL11Ra, IL13Ra, NYBRI, SLea, CD200R, TGFbetaR2, CD276, TROP2, LAMP1, PTK7, DLL3, CDH1, CDH6, CDH17, CDH19, TSHR, tyrosinase, HLA-A*02, HLA-A* 24 citrullinated peptides, insulin, MOG, GAD65, IA2, gliadin, and desmoglein; (f) the antigen-binding domain is; (i) an anti-CD19 antigen-binding domain; optionally, the anti-CD19 antigen-binding domain has the sequence of SEQ ID NO: 11; or (ii) an anti-BCMA antigen-binding domain; optionally, the anti-BCMA antigen-binding domain has the sequence of any one of SEQ ID NOs: 50-53; (g) the hinge region is selected from a human CD8α hinge region, a human CD28 hinge region, an IgG1 hinge region, or an IgG4 hinge region; optionally, the hinge region is derived from human CD8α; (h) the transmembrane domain is selected from a TNFRSF 19 transmembrane domain, a CD3 zeta transmembrane domain, a CD8α transmembrane domain, a CD4 transmembrane domain, a CD28 transmembrane domain, or a B7 family-induced costimulatory (ICOS) transmembrane domain; optionally, the transmembrane domain is derived from C CD8α; (i) the CAR further comprises one or more costimulatory domains; optionally: (i) the CAR comprises two costimulatory domains; and / or (ii) the one or more costimulatory domains are selected from the group consisting of 4-1BB, CD28, OX40, ICOS, CD27, MYD88-CD40, and KIR2DS2; optionally, one of the one or more costimulatory domains is derived from CD28 and / or 4-1BB; (j) the intracellular signaling domain comprises an immunoreceptor tyrosine-based activation motif (ITAM); (k) the immunoreceptor tyrosine-based activation motif (ITAM) is derived from CD3 zeta; (l) the CAR is: an anti-CD19 antigen-binding domain, an anti-BCMA antigen-binding domain, or an anti-CD20 antigen-binding domain; human CD8 hinge region; CD8 transmembrane region; CD28 costimulatory domain; and CD3 zeta chain intracellular signaling domain Includes; (m) the CAR comprises the sequence of SEQ ID NO: 9, 16, 22, 34, 41-49, or 54; optionally, the first polynucleotide segment comprises the sequence of SEQ ID NO: 10, 17, 23, 35, or 55; (n) the IL-10 is: (i) human IL-10; (ii) is a viral IL-10; or (iii) a protein having the sequence of SEQ ID NO:1; optionally, the second polynucleotide segment has the sequence of SEQ ID NO:2; (o) the first polynucleotide segment or the second polynucleotide segment further comprises a sequence encoding a selectable marker; optionally, the selectable marker is: (i) is ΔNGFR; optionally, said ΔNGFR has the sequence of SEQ ID NO:3 or SEQ ID NO:4; or (ii) is a truncated form of the EGFR polypeptide; and / or (p) the construct is a vector; optionally, the vector is a viral vector; optionally, the vector is a lentiviral vector; 17. The polynucleotide construct of claim 16. (a) a first polynucleotide segment having the sequence of SEQ ID NO: 10, 17, 23, 35, or 55; and (b) a second polynucleotide segment having the sequence of SEQ ID NO:2 17. The polynucleotide construct of claim 16, optionally comprising: (i) a polynucleotide construct comprising: (c) a third polynucleotide segment between the first polynucleotide segment and the second polynucleotide segment, the third polynucleotide segment having the sequence of SEQ ID NO: 33 or (ii) a polynucleotide construct comprising: (c) a third polynucleotide segment between the first polynucleotide segment and the second polynucleotide segment, the third polynucleotide segment having the sequence of SEQ ID NO:27; and (d) a fourth polynucleotide segment having the sequence of SEQ ID NO:4 further comprising: Polynucleotide constructs.

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