CD4 + T cell expressing IL-10 and chimeric antigen receptor and application thereof

Abstract

The present disclosure provides populations of CD4IL-10 / CAR cells (autologous or allogeneic single donors and allogeneic multiple donors) that are generated by genetically modifying CD4 + T cells to express IL-10 and chimeric antigen receptors. Also provided are methods of generating the CD4IL-10 / CAR cells and methods of using the CD4IL-10 / CAR cells for immune tolerance, treatment of GvHD, cell and organ transplantation, cancer, and other autoimmune and inflammatory diseases.

Description

[0001] 1. Cross-reference to related applications

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 295,491, filed on December 30, 2021, which is hereby incorporated by reference in its entirety.

[0003] 2. Sequence Listing

[0004] This application contains a sequence listing, which has been submitted in XML format and is hereby incorporated by reference in its entirety. The XML copy was created on December 24, 2022, named 37104-50735-Sequence-Listing.xml, and has a size of 95.2 kilobytes (KB).

[0005] 3. Background

[0006] Regulatory T cells belong to a small but important subset of T cells that maintain immune tolerance to self and non-pathogenic antigens and maintain immune homeostasis. There are two major populations of regulatory T cells—CD4 + FOXP3 + CD25 + T cells (FOXP3 + ) cells and type 1 regulatory T (Tr1) cells. FOXP3 + Both Tr1 and Tr1 cells downregulate pathogenic T cell responses in various preclinical models of organ and islet transplantation, graft-versus-host disease (GvHD), and various autoimmune and inflammatory diseases.

[0007] Tr1 cells have been shown to be effective in clinical studies. Administration of cloned antigen-specific autologous Tr1 cells to patients with persistent moderate to severe Crohn's disease resulted in objective, temporary remission (Desreumaux et al., Gastroenterology. 2012; 143(5): 1207-1217.e2.). In addition, after allogeneic hematopoietic stem cell transplantation (allo-HSCT), donor-derived CD4+ cells enriched in allogeneic specific Tr1 cells were used to treat Crohn's disease. + Adoptive transfer of T cell populations into leukemia patients results in rapid reconstitution of the immune system and protection against microbial and viral infections without severe GvHD. In responding patients, long-term remission and tolerance (>7 years) are achieved, leading to cure (Bacchetta et al., Front Immunol. 2014; 5:16).

[0008] Despite these encouraging results, the production of donor-derived or autologous Tr1 cells for large-scale treatment of patients with high unmet medical needs is not always feasible and is cumbersome, and the generation of large quantities of pure Tr1 cells cannot be achieved.

[0009] Recently, Locafaro and colleagues used a bidirectional lentiviral vector containing the human IL-10 gene to transduce purified CD4 + T cells circumvent some of these problems. The resulting single-donor CD4 IL-10 The cell population has the main functions of naturally occurring Tr1 cells. Like Tr1 cells, single donor CD4 IL-10 Cells produce high levels of IL-10 and downregulate allogeneic CD4 + T cells and allogeneic CD8 + In addition, they have direct cytotoxicity against both normal myeloid cells (including antigen presenting cells, APCs) and myeloid leukemia cells. In a humanized xenogeneic graft-versus-host disease (GvHD) model, these single donor CD4 IL-10 The cells were shown to be effective in reducing GvHD while maintaining graft-versus-leukemia (GvL) activity. See Locafaro et al. Mol Ther. 2017; 25(10): 2254-2269 and WO 2016 / 146,542.

[0010] CD4 IL-10 Methods for redirecting the cytotoxic capacity of cells to non-myeloid cells while retaining the immunosuppressive and immune homeostasis-maintaining activities of these cells.

[0011] 4. Overview

[0012] The present disclosure provides a novel immune cell CD4 IL-10 / CAR Cells engineered to express exogenous IL-10 and a chimeric antigen receptor (CAR). We have found that expression of CAR, in addition to IL-10, redirects the cytotoxic properties of these Tr1-like cells to therapeutically useful targets other than myeloid targets, while unexpectedly leaving the immunoregulatory properties of the cells intact. Also described is the use of CD4 IL-10 / CAR Cell therapy, CD4 IL-10 / CAR The use of cells for therapeutic purposes and CD4 IL-10 / CAR The invention also provides for the use of cells in the preparation of medicaments for the treatment of various diseases, wherein CAR directs the cytotoxic properties of these Tr1-like cells to therapeutically useful targets other than myeloid cells while leaving the immunoregulatory properties of the cells intact.

[0013] The present disclosure provides genetically modified CD4 + T cells (CD4 IL10 / CAR ), which comprises: (a) a first exogenous polynucleotide segment encoding a chimeric antigen receptor (CAR); and (b) a second exogenous polynucleotide segment encoding interleukin-10 (IL-10).

[0014] 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 the constitutive expression of the CAR.

[0015] In some embodiments, the CAR comprises an antigen binding domain, a hinge region, a transmembrane domain, and an intracellular signaling domain.

[0016] 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).

[0017] In some embodiments, the antigen binding domain targets an antigen associated with an autoimmune disease, an inflammatory disease, or cancer.

[0018] In some embodiments, the antigen is selected from the group consisting of: CD19, CD20, CD22, BCMA, CD27, CD38, B7-H3, CD23, Lyml, Lym2, CLEC5A, CDH179b, FLT3, GCC, Muc, CSF2RA, GFRa4, CD32, CD33, CEA, IL11Ra, IL13Ra, NYBRI, SLea, CD200R, TGFβR2, CD276, TROP2, LAMP1, PTK7, DLL3, CDH1, CDH6, CDH17, CDH19, TSHR, tyrosinase, HLA-A*02, HLA-A*24 or citrullinated peptides, insulin, MOG, GAD65, IA2, prolamins, and desmosomal mucins (in the context of relevant MHC molecules).

[0019] 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.

[0020] 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.

[0021] 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 NO: 50-53.

[0022] 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.

[0023] In some embodiments, the transmembrane domain is selected from a TNFRSF 19 transmembrane domain, a CD3 ζ transmembrane domain, a CD8 α transmembrane domain, a CD4 transmembrane domain, a CD28 transmembrane domain, or a B7 family inducible co-stimulatory (ICOS) transmembrane domain. In some embodiments, the transmembrane domain is derived from a CD8 α transmembrane domain.

[0024] In some embodiments, CAR further comprises one or more costimulatory domains. In some embodiments, CAR comprises two costimulatory domains.

[0025] 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.

[0026] In some embodiments, one of the one or more costimulatory domains is derived from CD28.

[0027] In some embodiments, the second costimulatory domain is derived from 4-1BB.

[0028] 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.

[0029] In some embodiments, CAR comprises: anti-CD19 antigen binding domain, anti-BCMA antigen binding domain or anti-CD20 antigen binding domain; human CD8 hinge region; TNFRSF 19 transmembrane region; 4-1BB costimulatory domain; and CD3 ζ chain intracellular signaling domain. In some embodiments, CAR comprises: anti-CD19 antigen binding domain, anti-BCMA antigen binding domain or anti-CD20 antigen binding domain; human CD8 hinge region; CD8α transmembrane region; 4-1BB costimulatory domain; and CD3 ζ chain intracellular signaling domain.

[0030] In some embodiments, the CAR comprises the sequence of SEQ ID NO:9, 16, 22, 34, 41-49 or 54.

[0031] In some embodiments, the first exogenous polynucleotide segment comprises the sequence of SEQ ID NO: 10, 17, 23, 35, or 55.

[0032] In some embodiments, the first exogenous polynucleotide segment is integrated into the T cell nuclear genome.

[0033] In some embodiments, the first exogenous polynucleotide segment is not integrated into the T cell nuclear genome.

[0034] In some embodiments, the first exogenous polynucleotide segment is in a vector.

[0035] 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.

[0036] In some embodiments, 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.

[0037] In some embodiments, the regulatory element drives constitutive expression of IL-10.

[0038] In some embodiments, the second exogenous polynucleotide segment is integrated into the T cell nuclear genome.

[0039] In some embodiments, the second exogenous polynucleotide segment is not integrated into the T cell nuclear genome.

[0040] In some embodiments, the second exogenous polynucleotide segment is in a vector.

[0041] In some embodiments, the first exogenous polynucleotide segment and the second exogenous polynucleotide segment are in the same vector. In some embodiments, the vector is a viral vector. In some embodiments, the vector is a lentiviral vector.

[0042] In some embodiments, CD4 + T cells constitutively express at least 100 pg IL-10 / 10 6 CD4 + T cells / mL culture medium.

[0043] In some embodiments, CD4 + T cells constitutively express at least 100 pg, 200 pg, 500 pg, 1 ng, 5 ng, 10 ng or 50 ng IL-10 / 106 CD4 + T cells / mL.

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

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

[0046] In some embodiments, CD4 + T cells were more expressed than unmodified CD4 + T cells express IL-10 at least 5-fold higher levels. + T cells were more expressed than unmodified CD4 + T cells expressed IL-10 at least 10-fold higher levels.

[0047] In some embodiments, CD4 + T cells expressing at least 100 pg IL-5 / 10 6 CD4 + T cells / mL. In some embodiments, CD4 + T cells expressing at least 100 pg, 200 pg, 500 pg, 1 ng, 5 ng, 10 ng or 50 ng IL-5 / 10 6 CD4 + T cells / mL.

[0048] In some embodiments, CD4 + T cells expressed at least 1 ng IL-5 / 10 after activation with anti-CD3 and anti-CD28 antibodies 6 CD4 + T cells / mL.

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

[0050] In some embodiments, CD4 + T cells expressing at least 100 pg IFN-γ / 10 6 CD4 + T cells / mL.

[0051] In some embodiments, CD4 + T cells expressing at least 100 pg, 200 pg, 500 pg, 1 ng, 5 ng, 10 ng or 50 ng IFN-γ / 10 6 CD4 + T cells / mL.

[0052] In some embodiments, CD4 + T cells expressed at least 1 ng IFN-γ / 10 after activation with anti-CD3 and anti-CD28 antibodies. 6 CD4 + T cells / mL.

[0053] In some embodiments, CD4 + T cells expressed at least 2 ng, 5 ng, 10 ng, 100 ng, 200 ng or 500 ng IFN-γ / 10 6 CD4 + T cells / mL.

[0054] In some embodiments, CD4 + T cells expressing at least 25 pg IL-4 / 10 6 CD4 + T cells / mL.

[0055] In some embodiments, CD4 + T cells expressing at least 25 pg, 50 pg, 75 pg, 100 pg, 200 pg, 500 pg, 1 ng, 5 ng, 10 ng or 50 ng IL-4 / 10 6 CD4 + T cells / mL.

[0056] In some embodiments, CD4 + T cells expressed at least 100 pg IL-4 / 10 after activation with anti-CD3 and anti-CD28 antibodies 6 CD4 + T cells / mL.

[0057] In some embodiments, CD4 +T cells expressing at least 100 pg, 200 pg, 300 pg, 400 pg, 500 pg, 600 pg, 700 pg, 800 pg, 900 pg, 1000 pg, 2 ng, 5 ng, 10 ng, 100 ng, 200 ng or 500 ng IL-4 / 10 after activation with anti-CD3 antibody and anti-CD28 antibody 6 CD4 + T cells / mL.

[0058] 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.

[0059] In some embodiments, the first exogenous polynucleotide segment or the second exogenous polynucleotide segment further comprises a sequence encoding a selectable marker.

[0060] In some embodiments, the selection 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.

[0061] In some embodiments, the selectable marker is a truncated form of the EGFR polypeptide.

[0062] In some embodiments, CD4 + T cells can respond to CD19 + Target cells produce in vitro cytotoxicity.

[0063] In some embodiments, CD4 + T cells can respond to CD19 + Target cells produce in vivo cytotoxicity.

[0064] In some embodiments, CD19 + The target cells are B cells that produce autoantibodies.

[0065] In some embodiments, CD19 + Target cells are CD19 + cancer cell.

[0066] In some embodiments, CD4 + T cells are capable of in vitro cytotoxicity against myeloid target cells.

[0067] In some embodiments, CD4 + T cells are capable of in vivo cytotoxicity against myeloid target cells.

[0068] In some embodiments, CD4 +T cells can respond to CD19 + Target cells and myeloid target cells produce cytotoxicity.

[0069] In some embodiments, the myeloid target cells express one or more of MHC class I, CD13, CD54, and CD112.

[0070] In some embodiments, CD19 + The cytotoxicity of the target cells is maintained after one or more in vitro restimulations. + The cytotoxicity of target cells is maintained after one or more in vitro expansions.

[0071] In some embodiments, the cytotoxicity to myeloid target cells is maintained after one or more in vitro restimulations. In some embodiments, the cytotoxicity to myeloid target cells is maintained after one or more in vitro expansions.

[0072] In some embodiments, CD4 + T cells can suppress allogeneic CD4 + T cell proliferation.

[0073] In some embodiments, CD4 + T cells can suppress allogeneic CD8 + T cell proliferation.

[0074] In some embodiments, CD4 + T cells can suppress allogeneic CD4 + T cell proliferation, allogeneic CD8 + T cell proliferation and PBMC proliferation.

[0075] In some embodiments, the inhibitory properties are maintained after one or more restimulations.

[0076] In another aspect, the disclosure features CD4 + A population of T cells comprising any of the genetically modified CD4 + cell.

[0077] In some embodiments, the CD4 + T cells are obtained from two, three, four, five, six, seven, eight, nine or ten different T cell donors and pooled.

[0078] In some embodiments, the CD4 + The T cells have a total of six, seven, eight, nine, ten, eleven, twelve or more different HLA haplotypes.

[0079] In some embodiments, all CD4 + The T cells are at least 1 / 10, 2 / 10, 3 / 10, 4 / 10, 5 / 10, 6 / 10, 7 / 10, 8 / 10 or 9 / 10 matched to each other at the HLA-A, HLA-B, HLA-C, HLA-DRBl and HLA-DQBl loci.

[0080] In some embodiments, all CD4 + The T cells are at least 1 / 8, 2 / 8, 3 / 8, 4 / 8, 5 / 8, 6 / 8, 7 / 8 or 8 / 8 matched to each other at the HLA-A, HLA-B, HLA-C and HLA-DRBl loci.

[0081] In some embodiments, all CD4 + The T cells have a 2 / 2 match with each other at the HLA-A locus.

[0082] In some embodiments, all CD4 + The T cells have a 2 / 2 match with each other at the HLA-B locus.

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

[0084] In some embodiments, all CD4 + The T cells have at least a 3 / 4 or 4 / 4 match with each other at the HLA-DRB1 and HLA-DQB1 loci.

[0085] In some embodiments, all CD4 + The T cells have less than 5 / 10, 6 / 10, 7 / 10, 8 / 10 or 9 / 10 matches with each other at the HLA-A, HLA-B, HLA C, HLA-DRBl and HLA-DQBl loci.

[0086] In some embodiments, all CD4 + The T cells have less than 4 / 8, 5 / 8, 6 / 8, 7 / 8 or 8 / 8 matches with each other at the HLA-A, HLA-B, HLA C and HLA DRB1 loci.

[0087] In some embodiments, all CD4 + T cells have less than a 2 / 2 match with each other at the HLA-A locus.

[0088] In some embodiments, all CD4+ The T cells have less than a 2 / 2 match with each other at the HLA-B locus.

[0089] In some embodiments, all CD4 + T cells have less than a 2 / 2 match with each other at the HLA-C locus.

[0090] In some embodiments, all CD4 + The T cells have less than a 4 / 2, 3 / 4, or 4 / 4 match with each other at the HLA-DRB1 and HLA DQB1 loci.

[0091] In some embodiments, all CD4 + T cells have the A*02 allele or are A*02 negative.

[0092] In some embodiments, all CD4 + T cells have the A*24 allele or are A*24 negative.

[0093] In some embodiments, NGFR + At least 30% of the cells in the population are CD4 + T cells express CAR.

[0094] In some embodiments, NGFR + At least 60% of the CD4+ T cells in the cell population express the CAR.

[0095] In some embodiments, NGFR + At least 90% of the CD4+ T cells in the cell population express the CAR.

[0096] In some embodiments, CD4 + T cells are in frozen suspension.

[0097] In some embodiments, CD4 + The T cells are in liquid suspension.

[0098] In some embodiments, the liquid suspension has been previously frozen.

[0099] In another aspect, the disclosure features any of the pharmaceutical compositions provided herein, comprising any of the CD4 + T cells or any CD4 + T cell populations.

[0100] In another aspect, the disclosure features the preparation of CD4 IL10 / CARThe method comprises the following steps: (a) obtaining primary CD4 T cells from one or more T cell donors; + T cells; and (b) modifying 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 + T cells.

[0101] In some embodiments, in step (a), primary CD4 + The T cells are obtained from two, three, four, five, six, seven, eight, nine or ten different T cell donors.

[0102] In some embodiments, the method further comprises combining the genetically modified CD4 + T cells.

[0103] In some embodiments, wherein in step (a), primary CD4 + The T cells are autologous to the patient.

[0104] In some embodiments, the method further comprises the following steps after step (a) and before step (b), or after step (b): incubating the CD4 + T cells.

[0105] In some embodiments, the method further comprises incubating the CD4 + T cells.

[0106] In some embodiments, one or more viral vectors are used to introduce the first exogenous polynucleotide segment, the second exogenous polynucleotide segment, or both into primary CD4 + In some embodiments, the viral vector is a lentiviral vector.

[0107] In some embodiments, the first exogenous polynucleotide segment comprises a coding sequence for a CAR.

[0108] In some embodiments, the CAR is specific for a target antigen associated with an autoimmune disease, inflammatory disease, or cancer.

[0109] In some embodiments, the target antigen is associated with an autoimmune disease, an inflammatory disease, or cancer.

[0110] 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.

[0111] In some embodiments, the CAR comprises the sequence of SEQ ID NO:9, 16, 22, 34, 41-49 or 54.

[0112] In some embodiments, the first exogenous polynucleotide segment comprises the sequence of SEQ ID NO: 10, 17, 23, 35, or 55.

[0113] 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.

[0114] In some embodiments, the first exogenous polynucleotide segment or the second exogenous polynucleotide segment further comprises a segment encoding a selectable marker.

[0115] In some embodiments, the encoded selection marker is ΔNGFR.In some embodiments, the encoded selection marker has the sequence of SEQ ID NO:3.

[0116] In some embodiments, the selectable marker is a truncated form of the EGFR polypeptide.

[0117] In some embodiments, the method further comprises the following steps after step (b): isolating the genetically modified CD4 + T cells, thereby generating enriched genetically modified CD4 + T cell populations.

[0118] In some embodiments, at least 30% or at least 60% of the genetically modified CD4 + T cells express IL-10 and CAR.

[0119] In some embodiments, at least 90% of the genetically modified CD4 + T cells express IL-10 and CAR.

[0120] In some embodiments, at least 40% of the genetically modified CD4 + T cells express a selectable marker.

[0121] In some embodiments, at least 75% of the genetically modified CD4 + T cells express a selectable marker.

[0122] In some embodiments, at least 90% of the genetically modified CD4 + T cells express a selectable marker.

[0123] In some embodiments, the method further comprises incubating the enriched genetically modified CD4 + Steps for T cell populations.

[0124] In some embodiments, the enriched genetically modified CD4 + The step of T cell population is performed in the presence of IL-2 and in the presence of beads coated with anti-CD3 antibody and anti-CD28 antibody or CD3 antibody and CD28 antibody.

[0125] In some embodiments, the method further comprises freezing the genetically modified CD4 + Next steps for T cells.

[0126] In some embodiments, at least two T cell donors are at least 1 / 10, 2 / 10, 3 / 10, 4 / 10, 5 / 10, 6 / 10, 7 / 10, 8 / 10, or 9 / 10 matched to each other at the HLA-A, HLA-B, HLA-C, HLA-DRBl, and HLA-DQBl loci.

[0127] In some embodiments, at least two T cell donors are at least 1 / 8, 2 / 8, 3 / 8, 4 / 8, 5 / 8, 6 / 8, 7 / 8, or 8 / 8 matched to each other at the HLA-A, HLA-B, HLA-C, and HLA-DRBl loci.

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

[0129] In some embodiments, at least two T cell donors are at least 3 / 4 or 4 / 4 matched to each other at the HLA-DRB1 and HLA-DQB1 loci.

[0130] In some embodiments, at least two T cell donors are less than 5 / 10, 6 / 10, 7 / 10, 8 / 10, or 9 / 10 matched to each other at the HLA-A, HLA-B, HLA C, HLA-DRBl, and HLA-DQBl loci.

[0131] In some embodiments, at least two T cell donors are less than 4 / 8, 5 / 8, 6 / 8, 7 / 8, or 8 / 8 matched to each other at the HLA-A, HLA-B, HLA C, and HLA DRB1 loci.

[0132] 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.

[0133] In some embodiments, at least two T cell donors have less than a 4 / 2, 3 / 4, or 4 / 4 match to each other at the HLA-DRB1 and HLA DQB1 loci.

[0134] In some embodiments, each of the at least two T cell donors has an A*02 allele or is A*02 negative. In some embodiments, each of the at least two T cell donors has an A*02 allele or is A*02 negative.

[0135] In some embodiments, in step (a), primary CD4 + T cells are obtained from one or more frozen stocks.

[0136] In some embodiments, in step (a), primary CD4 + T cells were obtained from unfrozen peripheral blood mononuclear cells from at least two different T cell donors.

[0137] In some embodiments, the method further comprises isolating CD4 + T cell steps.

[0138] In another aspect, the disclosure features a method for treating a hematological cancer comprising: administering to a hematological cancer patient a therapeutically effective amount of any of the provided CD4 IL-10 / CAR cells, any CD4 IL-10 / CAR Cell populations or any pharmaceutical compositions provided herein.

[0139] In some embodiments, the method further comprises administering a CD4 IL-10 / CAR Before or after the step of administering the allo HSCT transplant to the patient.

[0140] In another aspect, the disclosure features a method of treating a patient with a malignant tumor, comprising: administering to the patient an allo-HSCT transplant and administering a therapeutically effective amount of any CD4 IL-10 / CAR cells, any CD4 IL-10 / CAR Cell populations or any pharmaceutical compositions provided herein.

[0141] In some embodiments, CD4 IL-10 / CAR The amount of cells is also sufficient to inhibit or prevent graft-versus-host disease (GvHD) without inhibiting the graft-versus-leukemia (GvL) or graft-versus-tumor (GvT) efficacy of allo HSCT.

[0142] In some embodiments, the malignancy or hematological cancer is a myeloid leukemia.

[0143] In some embodiments, CD4 IL-10 / CAR The cells target and kill cancer cells that express CD13.

[0144] In some embodiments, CD4 IL-10 / CAR The cells target and kill cancer cells that express HLA class I.

[0145] In some embodiments, the myeloid leukemia is acute myeloid leukemia (AML).

[0146] In some embodiments, the malignancy or hematological cancer is CD19 + 、CD20 + 、CD22 + 、CD27 + 、CD38 + , BCMA+ or B7-H3 + Blood system cancer.

[0147] In some embodiments, CD19 + 、CD20 + 、CD22 + 、CD27 + 、CD38 + , BCMA+ or B7-H3 + The hematological cancer is selected from the group consisting of chronic lymphocytic leukemia, acute lymphoblastic leukemia (ALL) and non-Hodgkin's lymphoma.

[0148] In some embodiments, the allo-HSCT graft is obtained from a donor who is related or unrelated to the patient.

[0149] In some embodiments, CD4 IL-10 / CAR The cells are non-autologous to the patient.

[0150] In some embodiments, CD4 IL-10 / CAR The cells are autologous to the patient.

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

[0152] In some embodiments, CD4 IL-10 / CAR The cells do not lose reactivity to host alloantigens prior to administration to a patient.

[0153] In some embodiments, CD4 IL-10 / CAR The cells are Tr1-like cells.

[0154] In some embodiments, CD4 IL-10 / CAR The cells are polyclonal.

[0155] In some embodiments, CD4 IL-10 / CAR The cells are polyclonal and non-autologous to the patient.

[0156] In some embodiments, CD4 IL-10 / CAR The cells are polyclonal and autologous to the patient.

[0157] In some embodiments, CD4 IL-10 / CAR The cells are isolated from at least two donors and then genetically modified.

[0158] In some embodiments, none of the at least two donors is the same donor as the allo-HSCT donor.

[0159] In some embodiments, the allo-HSCT graft is obtained from a donor that is matched or mismatched to the patient.

[0160] In some embodiments, CD4 IL-10 / CAR Cells are targeted and killed by cells expressing CD19, CD20 or BCMA. In some embodiments, CD4 IL-10 / CAR In some embodiments, CD4 IL -10 / CAR Cells target and kill cancer cells expressing HLA class I and CD54. In some embodiments, CD4 IL-10 / CAR Cells target and kill cancer cells expressing CD112 and CD155. In some embodiments, CD4 IL-10 / CAR The cells target and kill cancer cells that express CD58.

[0161] In some embodiments, CD4 IL-10 / CAR The cells target and kill cancer cells in the patient.

[0162] In some embodiments, CD4 IL-10 / CAR The cells target and kill solid tumor cells in patients.

[0163] In another aspect, the disclosure features a method of treating a hematological cancer by allogeneic hematopoietic stem cell transplantation (allo-HSCT), comprising:

[0164] administering an allo-HSCT transplant to a patient;

[0165] The patient is administered an amount of CD4+ T cells sufficient to inhibit or prevent graft-versus-host disease (GvHD) without inhibiting the graft-versus-leukemia (GvL) or graft-versus-tumor (GvT) efficacy of the allo-HSCT transplant. IL-10 / CAR cell;

[0166] CD4 IL-10 / CAR The cells contain CD4 T cells that have been genetically modified by vector-mediated gene transfer of one or more vectors. + T cells, the vector comprising a coding sequence of human IL-10 under the control of a constitutive promoter or an inducible promoter and a coding sequence of CAR under the control of a constitutive promoter or an inducible promoter;

[0167] CD4 IL-10 / CAR The cells target and kill cancer cells in the patient;

[0168] CD4 IL-10 / CAR The cells have not lost reactivity to host alloantigens prior to administration to the patient; and

[0169] CD4 IL-10 / CAR The cells are non-autologous to the patient and are Tr1-like.

[0170] In another aspect, the disclosure features a method of treating a hematological cancer by allogeneic hematopoietic stem cell transplantation (allo-HSCT), comprising:

[0171] administering an allo-HSCT transplant to a patient;

[0172] Administering a certain amount of CD4 IL-10 / CAR cells, optionally wherein the amount is sufficient to inhibit or prevent graft-versus-host disease (GvHD) without inhibiting graft-versus-leukemia (GvL) or graft-versus-tumor (GvT) efficacy of the allo-HSCT transplant,

[0173] CD4 IL-10 / CAR The cells contain CD4 T cells that have been genetically modified by vector-mediated gene transfer of one or more vectors. +T cells, the vector comprising a coding sequence of human IL-10 under the control of a constitutive promoter or an inducible promoter and a coding sequence of CAR under the control of a constitutive promoter or an inducible promoter;

[0174] CD4 IL-10 / CAR The cells target and kill cancer cells in the patient;

[0175] CD4 IL-10 / CAR The cells have not lost reactivity to host alloantigens prior to administration to the patient; and

[0176] CD4 IL-10 / CAR The cells are autologous to the patient and are Tr1-like.

[0177] In another aspect, the disclosure features a method for preventing CD19 in a patient. + 、CD20 + 、CD22 + 、CD27 + 、CD38 + , BCMA + or B7-H3 + A method for the treatment of relapsed hematologic cancer comprising:

[0178] To those who have been identified as having CD19 + 、CD20 + 、CD22 + , BCMA+ or B7-H3 + Hematologic cancers may have CD19 + 、CD20 + 、CD22 + , BCMA + or B7-H3 + Patients at risk of recurrence of hematological cancer are administered a therapeutically effective amount of any CD4 IL-10 / CAR cells, any CD4 IL-10 / CAR Cell populations or any pharmaceutical composition provided herein. In some embodiments, administration of CD4 IL-10 / CAR cell.

[0179] In another aspect, the disclosure features a method for preventing B7-H3 in a patient. + A method for cancer recurrence, comprising: providing a patient identified as having B7-H3 + Administration of any CD4+ / - / - combination provided herein to a patient with cancer is sufficient to induce an anti-cancer effect. IL-10 / CAR cells, any CD4 IL-10 / CAR Cell populations or any pharmaceutical compositions provided herein.

[0180] In some embodiments, B7-H3 + Cancer is a solid tumor.

[0181] 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.

[0182] In another aspect, the disclosure features a method for treating a patient with minimal residual disease, comprising: administering to a patient identified as having or at risk of having minimal residual disease a therapeutically effective amount of any of the provided CD4+ / CD275 proteins sufficient to induce an anti-cancer effect. IL-10 / CAR cells, any CD4 IL-10 / CAR Cell populations or any pharmaceutical compositions provided herein.

[0183] In another aspect, the disclosure features a method of treating a patient comprising: administering to a patient in need of immune tolerance a therapeutically effective amount of any CD4 IL-10 / CAR cells, any CD4 IL-10 / CAR Cell populations or any pharmaceutical compositions provided herein.

[0184] In some embodiments, the method further comprises thawing the CD4 IL-10 / CAR cells or CD4 IL-10 / CAR A previous step of freezing suspension of cell populations.

[0185] In some embodiments, the patient suffers from an inflammatory disease or an autoimmune disease.

[0186] In some embodiments, the inflammatory disease 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, pemphigoid disease (vulgaris and deciduous), epidermolysis bullosa acquired, thrombotic thrombocytopenic purpura, idiopathic Platelet-derived purpura, autoantibody-induced vascular inflammation, autoantibody-induced carditis, rheumatoid arthritis, autoantibody-induced rheumatoid arthritis, neuromyelitis optica spectrum disorder, systemic lupus erythematosus (SLE), multiple sclerosis (MS), Sjögren's syndrome, autoimmune myopathy, type I diabetes mellitus, Addison's disease, pernicious anemia, autoimmune hepatitis, primary biliary cholangitis (PBC), autoimmune pancreatitis, goodpasture's disease, primary membranous nephropathy, ovarian insufficiency, autoimmune orchitis, dry eye disease, aplastic anemia, autoimmune neutropenia, and idiopathic interstitial pneumonia.

[0187] In some embodiments, 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.

[0188] In some embodiments, the patient has a disease or disorder involving overactivity of the NLPR3 inflammasome.

[0189] In some embodiments, the patient has type 2 diabetes, a neurodegenerative disease, a cardiovascular disease, or an inflammatory bowel disease.

[0190] In some embodiments, the patient has a disease or disorder involving increased production of IL-1 β by activated monocytes, macrophages, or dendritic cells.

[0191] In some embodiments, the patient has a disease or disorder involving increased production of IL-18 by activated monocytes, macrophages, or dendritic cells.

[0192] In some embodiments, the patient has a disease or disorder involving increased production of mature caspase 1 by activated monocytes, macrophages, or dendritic cells.

[0193] In some embodiments, the patient suffers from an allergic or atopic disease.

[0194] In some embodiments, wherein the allergic disease or atopic disease is selected from the group consisting of asthma, atopic dermatitis and rhinitis.

[0195] In some embodiments, the patient suffers from a food allergy.

[0196] In some embodiments, the patient has a solid tumor.

[0197] 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.

[0198] In some embodiments, the method further comprises administering a CD4 IL-10 / CAR cells, CD4 IL-10 / CAR Before or after the cell population or pharmaceutical composition, a step of performing an organ transplant on the patient. In some embodiments, the CD4 IL-10 / CAR cells, CD4 IL-10 / CAR The cell population or pharmaceutical composition prevents host rejection of an organ transplant or reduces the severity of host rejection of an organ transplant.

[0199] In some embodiments, the method further comprises administering a CD4 IL-10 / CAR cells, CD4 IL-10 / CAR The step of transplanting iPS cell-derived cells or tissues into a patient before or after 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 a cell transplant.

[0200] In some embodiments, the method further comprises administering a CD4 IL-10 / CAR cells, CD4 IL-10 / CAR The step of administering the recombinant AAV to the patient before or after the cell population or pharmaceutical composition. In some embodiments, the CD4 IL-10 / CAR cells, CD4 IL-10 / CAR The cell population or pharmaceutical composition reduces the immune response to the recombinant AAV.

[0201] In some embodiments, the method further comprises administering a CD4 IL-10 / CAR cells, CD4 IL-10 / CAR The step of administering a recombinant viral vector other than AAV to the patient before or after 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 a recombinant viral vector other than AAV.

[0202] In some embodiments, the method further comprises administering a CD4IL-10 / CAR cells, CD4 IL-10 / CAR The step of administering an immunogenic therapeutic protein to a patient before or after administering the immunogenic therapeutic protein to the patient. IL -10 / CAR cells, CD4 IL-10 / CAR The cell population or pharmaceutical composition reduces the immune response to the immunogenic therapeutic protein.In some embodiments, the immunogenic therapeutic protein is selected from the group consisting of a therapeutic antibody, a Factor VIII replacement, a cytokine, and a cytokine mutein.

[0203] In some embodiments, the patient has an excessive immune response to a viral or bacterial infection. In some embodiments, the patient has a coronavirus infection. In some embodiments, the patient has organ and / or tissue damage.

[0204] In some embodiments, the method further comprises the step of detecting a selected marker in a biological sample obtained from the patient, thereby detecting CD4 IL-10 / CAR The presence or absence of cells.

[0205] In some embodiments, the biological sample is a biopsy or blood from a patient.

[0206] In another aspect, the disclosure features a method of treating or inhibiting an autoimmune disease, an allergic disease, or an inflammatory disease in a patient, comprising: administering to a patient identified as having the autoimmune disease, the allergic disease, or the inflammatory disease a therapeutically effective amount of any of the provided CD4+ T cells sufficient to treat or inhibit the autoimmune disease, the allergic disease, or the inflammatory disease. IL-10 / CAR cells, any CD4 IL-10 / CAR Cell populations or any pharmaceutical compositions provided herein.

[0207] In another aspect, the disclosure features a method for reducing transplant rejection in a patient transplanted with hematopoietic stem cells, bone marrow cells, cord blood cells, tissue stem cells, or solid organs, comprising: administering to a patient identified as having rejection of the transplanted hematopoietic stem cells, bone marrow cells, tissue stem cells, or solid organs a therapeutically effective amount of any CD4+ / CD ... IL-10 / CAR cells, any CD4 IL-10 / CAR Cell populations or any pharmaceutical compositions provided herein.

[0208] In another aspect, the disclosure features a method for treating graft-versus-host disease (GvHD) in a patient, comprising: administering to a patient identified as having or at risk of having graft-versus-host disease (GvHD) a therapeutically effective amount of any of the provided CD4+ T cells sufficient to inhibit or prevent GvHD.IL-10 / CAR cells, any CD4 IL-10 / CAR Cell populations or any pharmaceutical compositions provided herein.

[0209] In some embodiments, graft-versus-host disease (GvHD) comprises acute GvHD.

[0210] In some embodiments, graft-versus-host disease (GvHD) comprises chronic GvHD.

[0211] In another aspect, the disclosure features a method for 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 therapeutically effective amount of any of the CD4 T cells provided herein sufficient to induce repair of the tissue or organ damage. IL-10 / CAR cells, any CD4 IL-10 / CAR Cell populations or pharmaceutical compositions provided herein.

[0212] In another aspect, the disclosure features a polynucleotide construct comprising: (a) a first polynucleotide segment encoding a chimeric antigen receptor (CAR); and (b) a second polynucleotide segment encoding interleukin-10 (IL-10).

[0213] In some embodiments, the first polynucleotide segment comprises a regulatory element operably connected to the coding sequence of the CAR. In some embodiments, the regulatory element drives the constitutive expression of the CAR.

[0214] 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.

[0215] In some embodiments, the method further comprises an internal ribosome entry site (IRES) or a self-cleaving peptide located 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.

[0216] In some embodiments, the CAR comprises an antigen binding domain, a hinge region, a transmembrane domain, and an intracellular signaling domain.

[0217] 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).

[0218] In some embodiments, the antigen binding domain targets an antigen associated with an autoimmune disease, an inflammatory disease, or cancer.

[0219] 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, IL11Ra, IL13Ra, NYBRI, SLea, CD200R, TGFβR2, CD276, TROP2, LAMP1, PTK7, DLL3, CDH1, CDH6, CDH17, CDH19, TSHR, tyrosinase, HLA-A2, citrullinated peptides, insulin, GAD65, IA2, prolamins, and desmosomal mucins.

[0220] 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.

[0221] 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.

[0222] 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 NO: 50-53.

[0223] 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.

[0224] In some embodiments, the transmembrane domain is selected from a TNFRSF 19 transmembrane domain, a CD3ζ transmembrane domain, a CD8α transmembrane domain, a CD4 transmembrane domain, a CD28 transmembrane domain, or a B7 family inducible co-stimulatory (ICOS) transmembrane domain. In some embodiments, the transmembrane domain is derived from CD8α.

[0225] In some embodiments, the CAR further comprises one or more costimulatory domains.

[0226] In some embodiments, the CAR comprises two costimulatory domains.

[0227] 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.

[0228] In some embodiments, one of the one or more costimulatory domains is derived from CD28.

[0229] In some embodiments, the second costimulatory domain is derived from 4-1BB.

[0230] 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.

[0231] 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 4a CD28 co-stimulatory domain; and a CD3 zeta chain intracellular signaling domain.

[0232] In some embodiments, the CAR comprises the sequence of SEQ ID NO:9, 16, 22, or 34.

[0233] In some embodiments, the first polynucleotide segment comprises the sequence of SEQ ID NO: 10, 17, 23, 35, or 55.

[0234] In some embodiments, the IL-10 is human IL-10. In some embodiments, the IL-10 is viral IL-10.

[0235] In some embodiments, 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.

[0236] In some embodiments, the first polynucleotide segment or the second polynucleotide segment further comprises a sequence encoding a selectable marker.

[0237] In some embodiments, the selection 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.

[0238] In some embodiments, the selectable marker is a truncated form of the EGFR polypeptide.

[0239] 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.

[0240] In another aspect, the disclosure features a polynucleotide construct comprising: a first polynucleotide segment having a sequence of SEQ ID NO: 10, 17, 23, 35, or 55; and a second polynucleotide segment having a sequence of SEQ ID NO:2.

[0241] In another aspect, the disclosure features a polynucleotide construct comprising: a first polynucleotide segment having a sequence of SEQ ID NO: 10, 17, 23, 35 or 55; a second polynucleotide segment having a sequence of SEQ ID NO: 2; and a third polynucleotide segment having a sequence of SEQ ID NO: 33 located between the first polynucleotide segment and the second polynucleotide segment.

[0242] In another aspect, the disclosure features a polynucleotide construct comprising: a first polynucleotide segment having a sequence of SEQ ID NO: 10, 17, 23, 35 or 55; a second polynucleotide segment having a sequence of SEQ ID NO: 2; a third polynucleotide segment having a sequence of SEQ ID NO: 27 located between the first polynucleotide segment and the second polynucleotide segment; and a fourth polynucleotide segment having a sequence of SEQ ID NO: 4.

[0243] In another aspect, the disclosure features CD4 IL-10 cells, wherein the IL-10 is a viral IL-10. The viral IL-10 has a sequence of SEQ ID NO: 6 or 18. In some embodiments, the viral IL-10 is encoded by a polynucleotide having a sequence of SEQ ID NO: 7. In some embodiments, the IL-10 is a human IL-10, wherein one, two, three, four, five, six, seven, eight, nine, or ten amino acids from human IL-10 are replaced by the corresponding amino acid sequence from the viral IL-10. In some embodiments, the CD4 + T cells are transduced with exogenous viral IL-10 under the control of a constitutive promoter. In some embodiments, the expression control element drives viral IL-10 in activated CD4 +In some embodiments, the exogenous polynucleotide encoding viral IL-10 is integrated into the T cell nuclear genome. In some embodiments, the exogenous polynucleotide encoding viral IL-10 is not integrated into the T cell nuclear genome. In some embodiments, the exogenous polynucleotide encoding viral IL-10 has a sequence of SEQ ID NO: 7.

[0244] In another aspect, the disclosure features CD4 IL-10 cells, wherein IL-10 is Mus musculus, "MOUSE" (SEQ ID NO: 58); Rattus norvegicus, "RAT" (SEQ ID NO: 59); Macaca mulatta, "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 (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 IL-10 is a protein having at least 90%, 95%, 98% or 99% sequence identity to the IL-10 of Mus musculus, "MOUSE" (SEQ ID NO: 58); Rattus norvegicus, "RAT" (SEQ ID NO: 59); Rhesus macaque, "MACMU" (SEQ ID NO: 60); Gorilla, "GORILLA" (SEQ ID NO: 61); Crab-eating macaque, "CYNO" (SEQ ID NO: 62); Baboon, "OLIVEBABOON" (SEQ ID NO: 63); Bonobos, "BONOBO" (SEQ ID NO: 64); Chimpanzee, "CHIMP" (SEQ ID NO: 65); and EBVB9 (SEQ ID NO: 66).

[0245] In another aspect, the disclosure features CD4 IL-10The cell, wherein IL-10 is a variant of human IL-10. The human IL-10 variant has the sequence of SEQ ID NO: 67 or SEQ ID NO: 68. In some embodiments, the IL-10 is human IL-10 in which one, two, three, four, five, six, seven, eight, nine, or ten amino acids from human IL-10 are replaced by the corresponding amino acid sequence of IL-10 from another species (e.g., Mus musculus, "MOUSE" (SEQ ID NO:58); Rattus norvegicus, "RAT" (SEQ ID NO:59); Rhesus macaque, "MACMU" (SEQ ID NO:60); Gorilla, "GORILLA" (SEQ ID NO:61); Cynomolgus macaque, "CYNO" (SEQ ID NO:62); Baboon, "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, CD4 + T cells are transduced with exogenous IL-10 variants under the control of a constitutive promoter. In some embodiments, the expression control element drives the IL-10 variant to express in activated CD4 + Expression in T cells. In some embodiments, the exogenous polynucleotide encoding the IL-10 variant is integrated into the T cell nuclear genome. In some embodiments, the exogenous polynucleotide encoding the IL-10 variant is not integrated into the T cell nuclear genome.

[0246] In yet another aspect, the present disclosure provides for preparing viral IL-10CD4 IL-10 The method comprises the following steps:

[0247] (i) Obtaining primary CD4 T cells from a single donor + T cells; and

[0248] (ii) Modification of donor CD4 by introduction of exogenous polynucleotide encoding viral IL-10 + T cells,

[0249] Genetically modified CD4 + T cells.

[0250] In some embodiments, the method further comprises the following steps after step (i) or after step (ii): incubating primary CD4+ T cells.

[0251] In some embodiments, the method comprises further incubating primary CD4 + In some embodiments, the exogenous polynucleotide encoding viral IL-10 uses a vector.

[0252] In some embodiments, the exogenous polynucleotide encoding viral IL-10 comprises a segment encoding a selection marker. In some embodiments, the encoded selection marker is ΔNGFR. In some embodiments, the encoded selection marker has a sequence of SEQ ID NO: 3. In some embodiments, the encoded selection marker is a truncated EGFR polypeptide. In some embodiments, the encoded selection marker is a truncated human EGFR polypeptide.

[0253] In some embodiments, the method further comprises the following steps after step (ii): isolating the genetically modified CD4 + T cells, thereby generating enriched genetically modified CD4 + T cell populations.

[0254] In some embodiments, the method further comprises incubating the enriched genetically modified CD4 + In some embodiments, the enriched genetically modified CD4 + The step of T cell population is performed in the presence of IL-2 and in the presence of beads coated with anti-CD3 antibody and anti-CD28 antibody or CD3 antibody and CD28 antibody.

[0255] In some embodiments, in step (i), primary CD4 + T cells are obtained from frozen stocks. In some embodiments, in step (i), primary CD4 + T cells were obtained from unfrozen peripheral blood mononuclear cells from a single T cell donor.

[0256] In some embodiments, the method further comprises isolating CD4 + In some embodiments, peripheral blood mononuclear cells are obtained from buffy coats or apheresis. 5. Brief description of the accompanying drawings

[0258] Figure 1 is a non-limiting illustration of various chimeric antigen receptor (CAR) structures provided herein.

[0259] Figure 2AThe partial structure of the anti-CD19 CAR coding sequence is shown, which includes the scFv from the fully human anti-CD19 monoclonal antibody FMC63, the CD8α hinge, the CD8α transmembrane domain, the CD28 co-stimulatory domain and the CD3ζ activation domain, which is used to deliver the anti-CD19 CAR to CD4 + CD4 IL-10抗CD19 CAR lentiviral vectors in cells.

[0260] Figure 2B The method for delivering anti-CD19 CAR to CD4 + CD4 IL-10抗CD19CAR The cells were transfected with a lentiviral vector (pLV anti-CD19 CAR).

[0261] Figure 3A-3B The method for generating a lentiviral vector (LVV; LVV IL-10-NGFR ) is a bidirectional plasmid (pLVIL-10) used to deliver human IL-10 and ΔNGFR coding sequences to CD4 + CD4 IL-10 cells or CD4 IL-10抗CD19CAR cell. Figure 3A Partial structure of a bidirectional lentiviral vector used to deliver human IL-10 and ΔNGFR coding sequences into CD4+ T cells from multiple donors to generate multi-donor CD4IL-10 cells is shown. Figure 3B A method for delivering human IL-10 and ΔNGFR coding sequences to CD4 T cells from multiple donors is shown. + Multidonor CD4 T cells IL-10 The complete circular structure of the bidirectional lentiviral vector (hPGK.IL10.WPRE.mhCMV.ΔNGFR.SV40PA) in cells.

[0262] Figure 4 Antigen expression during naive B cell differentiation is shown for illustrative purposes.

[0263] Figures 5A-5C Shown is the generation of CD4 IL-10抗CD19CAR Schematic diagram of non-limiting examples of methods for activating human CD4 T cells. In each method, human CD4 T cells were activated with anti-CD3 / anti-CD28 beads (Miltenyi) (3:1 cell:bead ratio) in complete medium (X-vivo supplemented with 5% human AB serum and rhIL-2 (50 U / mL)). + T cells. Figure 5A Shows activated CD4 + T cells were activated 48 hours after inoculation with a vector encoding an anti-CD19 CAR (LVV CD19-CAR) and 24 hours later (i.e. 72 hours after activation) they were transduced with a lentiviral vector encoding human IL-10 and a truncated form of human NGF receptor (LVV IL-10-NGFR ) were transduced with bidirectional lentiviral vectors. Both LVVs were used at a multiplicity of infection (MOI) of 20. Figure 5B Shows activated CD4+ T cells 48 hours after activation with LVV IL-10-NGFR transduction, and 24 hours later (i.e. 24 hours after activation) with LVV CD19-CAR Transduction. Figure 5C Shows activated CD4 + T cells were activated with LVV 48 hours after IL-10-NGFR and LVV CD19-CAR Simultaneous transduction. By transducing only LVV 48 hours after activation IL-10-NGFR Transduction of activated CD4 + T cell generation control (no CAR expression). Figures 5A-5C In each of the 247 cells, transduction was performed in the presence of polybrene (8 μg / mL). Figures 5A-5C Each of the transduced cells (i.e., ΔNGFR+ cells) were purified using anti-CD271 microbeads (Miltenyi) and anti-CD19 CAR microbeads (Miltenyi) 10-28 days after transduction. The 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CD19CAR Cells or control CD4 IL-10 Cells) were characterized in vitro and in vivo. The culture medium was changed every 2-3 days as needed throughout the culture period.

[0264] Fig. 6A Shows the Figure 5A Control (left panel) and CD4 IL-10抗CD19 CAR (Right panel) Flow cytometry graph (x-axis is anti-CD19 CAR expression and y-axis is NGFR expression (surrogate for IL-10)). Figure 6B Shows the Figure 5B Control (left panel) and CD4 IL-10抗CD19CAR (Right panel) Flow cytometry graph (x-axis is CD19 and y-axis is NGFR). Figure 6C Shows the Figure 5C Control (left panel) and CD4 IL-10抗CD19 CAR (Right panel) Flow cytometry graph (x-axis is CD19 and y-axis is NGFR).

[0265] Figure 7 CD4 IL-10 T cells (control) and CD4 IL-10抗CD19 CAR Flow cytometry plot of T cells (x-axis is anti-CD19 CAR expression and y-axis is NGFR expression). Figure 5C The method shown here is for generating CD4 IL-10 T cells (control) and CD4 IL-10抗CD19 CAR T cells. The figure below shows Figure 5C The method shown in the CD4 IL-10 T cells (control) and CD4 IL-10抗CD19CAR T cells.

[0266] Figures 8A-8D CD4 isolated from three different donors (26.1, 26.2 and 26.3) is shown. + CD4 T cells IL-10 T cells (control) and CD4 IL-10 / CAR T cell cytokine production characteristics. Cytokine production characteristics include IL-10 ( Fig. 8A ), IFN-γ( Figure 8B )、IL-4( Figure 8C ) and IL-5( Fig.8D ).

[0267] Fig. 9 The CD4 isolated from donor 24.2 is shown. + CD4 T cells IL-10 T cells (control) and CD4 IL -10 / CAR Cytokine production signature of T cells.

[0268] Fig.10 The CD4 isolated from donor 24.2 is shown. + CD4 T cells IL-10抗CD19CAR Cells kill NALM6 cells expressing CD19 in vitro. ALL-CM is a myeloid cell line, and K562, a cell line sensitive to NK killing, was included as a control.

[0269] Fig.11A CD4 isolated from three different donors (26.1, 26.2 and 26.3) is shown. + CD4 T cells IL-10抗CD19CAR Cell killing of CD19 in vitro + NALM6 cells.

[0270] Fig. 11B The NALM6 cells and CD4 IL-10 T cells (control) or NALM6 cells with CD4 IL-10抗CD19CAR IL-10 levels in the supernatant on day 3 of cell co-culture. BLQ: below the quantitative level (31 pg / mL).

[0271] Fig.12 CD4 isolated after the second restimulation (TF2) is shown. IL-10 T cells (control) and CD4 IL -10抗CD19CAR Cells (see Figure 5C ) Flow cytometry of allogeneic CD4+ T cell proliferation assay after co-culture (counts relative to ). The figure above shows the relationship between IL-10抗CD19CAR After co-culture of cells, CD4 + The following figure shows the proliferation of T cells. IL-10 CD4 T cells (control) after co-culture + Proliferation of T cells. Flow cytometry plot showing "non-dividing" cells and "dividing" cells, where the dividing cells consist of fewer and fewer cells due to dilution after each division. CD4 IL-10 T cells (control) and CD4 IL-10抗CD19CAR Cell-mediated inhibition was calculated as follows: 100-([in CD4 IL-10 T cells (control) or CD4 IL-10抗CD19CAR Proliferation of responders in the presence of cells / proliferation of responders] × 100).

[0272] Fig.13 CD4 isolated after the second restimulation (TF2) is shown. IL-10 T cells (control) and CD4 IL -10抗CD19CAR Cells (see Figure 5C ) After co-culture, allogeneic CD8 + Flow cytometry of T cell proliferation assay (count relative to ). The figure above shows the relationship between IL-10抗CD19CAR CD8 + The following figure shows the proliferation of T cells. IL-10 T cells (control) after co-culture CD8 + Proliferation of T cells. Flow cytometry plot showing "non-dividing" cells and "dividing" cells, where the dividing cells consist of fewer and fewer cells due to dilution after each division. CD4 IL-10T cells (control) and CD4 IL-10抗CD19CAR Cell-mediated inhibition was calculated as follows: 100-([in CD4 IL-10 T cells (control) or CD4 IL-10抗CD19CAR Proliferation of responders in the presence of cells / proliferation of responders] × 100).

[0273] Fig.14 CD4 isolated after the third restimulation (TF3) is shown. IL-10 T cells (control) and CD4 IL -10抗CD19CAR Cells (see Figure 5C ) Flow cytometry of allogeneic PBMC proliferation assay after co-culture (counts relative to ). Flow cytometry plots showing "proliferating" cells, where dividing cells consist of fewer and fewer cells due to dilution after each division. CD4 IL-10 T cells (control) and CD4 IL-10抗CD19CAR Cell-mediated inhibition was calculated as follows: 100-([in CD4 IL-10 T cells (control) or CD4 IL-10抗CD19CAR Proliferation of responders in the presence of cells / proliferation of responders] × 100).

[0274] Fig.15 A schematic diagram of the experimental overview used to evaluate CD19+ tumor cell growth in mice is shown. On day 0, NSG mice were injected with (i) NALM6-luciferase (1 × 10 per mouse) 5 (ii) NALM6-luciferase (1×10 per mouse) 5 ) + PBMC (2.5×10 per mouse 6 (iii) NALM6-luciferase (1×10 per mouse) 5 )+CD4 IL -10抗CD19CAR Cells (2.5×10 per mouse 6 ), or (iv) NALM6-luciferase (1 × 10 per mouse 5 ) + PBMC (2.5×10 per mouse 6 )+CD4 IL-10抗CD19CAR Cells (2.5×10 per mouse 6 indivual).

[0275] Fig.16A Shown in injection Fig.15 Bioluminescence of NALM6-luciferase cells in NSG mice on days 3, 6, 9, and 12 following conditions (i) to (iv) described in FIG.

[0276] Fig. 16B Shows the Fig.16A Line graphs of quantification of bioluminescence in images. Fig.15 The total flux [p / s] of each of the conditions (i) to (iv) described in the Figure 2 changes over time (days after NALM6-luciferase injection). The P value was measured at day 12 using the Mann-Whitney test to compare conditions (ii) to (iv) with condition (i) (NALM6-luciferase alone).

[0277] Fig.17 Shows Fig.15 Line graph of weight loss over time for mice under conditions (i), (ii) and (iii) as described in. Weight loss was used as an indicator of xenogeneic graft-versus-host disease (GvHD).

[0278] Figures 18A-18C Single donor CD4 in a humanized mouse model of xeno-GvHD IL-10抗CD19CAR Cells and multiple donor CD4 IL-10抗CD19CAR cell. Fig.18A Single donor CD4 T cells tested in a humanized mouse model of xeno-GvHD induced by allogeneic PBMCs IL-10抗CD19CAR cells, wherein on day 3, irradiated NSG mice were injected intravenously (iv) with (i) allogeneic PBMCs (2.5E6 cells / mouse), (ii) a single donor CD4 IL-10抗CD19CAR cells (2.5E6 cells / mouse), (iii) multiple donor CD4 IL-10抗CD19CAR cells (2.5E6 cells / mouse), (iv) PBMC + single donor CD4 IL-10抗CD19CAR cells, (v) or PBMC + multiple donor CD4 IL-10抗CD19CAR cells (2.5E6 cells / mouse). Fig.18B CD4 T cells tested in a humanized mouse model of xeno-GvHD induced by allogeneic PBMCs IL-10抗CD19CAR Cells, where on day 3, irradiated NSG mice were injected intravenously (iv) with allogeneic PBMCs (2.5E6 cells / mouse) and / or single donor CD4 IL -10抗CD19CAR cell. Fig.18C CD4 T cells tested in a humanized mouse model of xeno-GvHD induced by allogeneic PBMCs are shown. IL-10抗CD19CAR Cells, where on day 3, irradiated NSG mice were injected intravenously (iv) with allogeneic PBMCs (2.5E6 cells / mouse) and / or multiple donor CD4 IL-10抗CD19CARCells (2.5E6 cells / mouse). PBMC: peripheral mononuclear cells; GvHD: graft-versus-host disease.

[0279] Fig.19 shows the method for generating CD4 IL-10 Exemplary scheme of cells.

[0280] Fig. 20A Human CD4 T cells transduced with LV-IL-10 / ΔNGFR (a bidirectional lentiviral vector encoding human IL-10 and a truncated form of the human NGF receptor) are shown. + CD4 + ΔNGFR + Percentage of cells (mean ± SD, n = 10) and vector copy number (VCN, mean ± SD, n = 10). Fig. 20B Human CD4 T cells transduced with LV-IL-10 / ΔNGFR and purified using anti-CD271 microbeads from two representative donors (Donor B and Donor C) are shown. + FACS analysis of CD4 and ΔNGFR expression in T cells.

[0281] Fig.21 Single donor CD4 IL-10 Cytokine production profiles of cells after second (TF2) and third (TF3) restimulation. TF2 and TF3 CD4 IL-10 Cells were left unstimulated (arrows indicate spontaneous / unstimulated samples) or stimulated for 48 hours with fixed CD3 (10 μg / mL) and soluble CD28 mAb (1 μg / mL). Culture supernatants were collected and the levels of IL-10, IL-4, IL-5, IFN-γ, and IL-22 were determined by ELISA. All samples were tested in triplicate. Mean ± SD is shown, n = 8 tested donors.

[0282] Fig.22A CD4 expressing granzyme B (GzB) after the second round of stimulation (TF2) analyzed by FACS IL-10 Percentage of cells. Box and whiskers for n=7 donors and a single donor are shown. Fig. 22B Shows when CD4 IL-10 Cells (10 5 cells / well) and K562 and ALL-CM cells (10 5 The box-and-whisker plots represent data from n=4 donors, and the dots represent data from a single donor.

[0283] Fig.23A and 23BSingle donor CD4 IL-10 cells can suppress allogeneic CD4 + T cell proliferation. Allogeneic PBMC cells were used (10 5 cells / well) and in the absence or presence of CD4 IL-10 Cells (10 5 Allogeneic mature dendritic cells (5 × 10 4 cells / well) and soluble anti-CD3 mAb. After 4 days of culture, flow cytometry was used to analyze the CD4 + After gating on ΔNGFR-T cells, The percentage of proliferating responding cells was determined by dilution. Fig.23A Results for Donor-C, Donor-E, and Donor-F are shown, and Fig. 23B Results for Donor-H, Donor-I, and Donor-L are shown. Percentages of proliferation and inhibition are indicated. CD4 IL-10 Cell-mediated inhibition was calculated as follows: 100-([in CD4 IL-10 proliferation of responders in the presence of cells / proliferation of responders alone] × 100).

[0284] Fig.24A and 24B Single donor CD4 IL-10 Cells can suppress allogeneic CD8 + T cell proliferation. Allogeneic PBMC cells were used (10 5 cells / well) and in the absence or presence of CD4 IL-10 Cells (10 5 Allogeneic mature dendritic cells (5 × 10 4 cells / well) and soluble anti-CD3 mAb. After 4 days of culture, flow cytometry was used to analyze the CD4 + ΔNGFR - After T cells were gated, The percentage of proliferating responding cells was determined by dilution. Fig.24A Results for Donor-C, Donor-E, and Donor-F are shown, and Fig. 24B Results for Donor-H, Donor-I, and Donor-L are shown. Percentages of proliferation and inhibition are indicated. CD4 IL-10 Cell-mediated inhibition was calculated as follows: 100-([in CD4 IL-10proliferation of responders in the presence of cells / proliferation of responders alone] × 100).

[0285] Fig.25 CD4 IL-10 The average level (+ / -SD) of cells produced compared to multiple donor CD4 IL-10 Cytokine production profile of cells after the third (TF3) restimulation. TF3CD4 IL-10 Cells were pooled at a 1:1:1 ratio and stimulated with immobilized CD3 (10 μg / mL) and soluble CD28 mAb (1 μg / mL) for 48 h. Culture supernatants were collected and the levels of IL-10, IL-4, IL-5, IFN-γ, and IL-22 were determined by ELISA. IL-10 Results for cells; grey bars represent mean ± SD, n = 8 individual donors.

[0286] Fig.26A CD4 IL-10 The mean % (+ / -SD) of cells expressing granzyme B compared to CD4 expressing granzyme B (GzB) from multiple donors IL-10 The percentage of cells after the third round of stimulation (TF3) was analyzed by FACS. Fig.26B Shows that when multiple donor CD4 IL-10 Cells (10 5 cells / well) and K562 and ALL-CM cells (10 5 The percentage of dead cells after 3 days of co-culture with 1:1 ratio of leukemia cells / well was counted by FACS. + CD33 + ). The point is multi-donor CD4 IL-10 Results are shown in Figure 2 and grey bars represent mean ± SD of n = 3 individual donors used to generate the combined pool.

[0287] Fig.27A and 27B Multiple donor CD4 IL-10 cells can suppress allogeneic CD4 + T cells and CD8 + T cell proliferation. Allogeneic PBMC cells were used (10 5 cells / well) and in the absence or presence of multiple donor CD4 IL-10 Cells (10 5Allogeneic mature dendritic cells (5 × 10 4 cells / well) and soluble anti-CD3 mAb. After 4 days of culture, flow cytometry was used to analyze the CD4 + ΔNGFR – T cells and CD8 + ΔNGFR – After T cells were gated, The percentage of proliferating responding cells was determined by dilution. Fig.27A The CD4 + Multiple donor CD4 IL-10 Cell results. Fig.27B The CD4 + Multiple donor CD4 IL-10 Results of CD4 cells. IL-10 Cell-mediated inhibition was calculated as follows: 100-([in CD4 IL-10 proliferation of responders in the presence of cells / proliferation of responders alone] × 100).

[0288] Fig.28 Figure 2 shows the use of human PBMCs and / or multiple donor CD4 IL-10 Cell-induced GvHD regimen.

[0289] Fig.29 It shows that after injection of PBMC (5×10 6 cells / mouse), multiple donors (three donors) CD4 IL-10 Cells (5×10 6 cells / mouse), or PBMC (5×10 6 cells / mouse) and multiple donor CD4 IL-10 Cells (three donors) (5×10 6 % of NSG mice demonstrated GvHD every day after combination with 100 cells / mouse).

[0290] Fig.30 It shows that after injection of PBMC (5×10 6 cells / mouse), multiple donors (three donors) CD4 IL-10 Cells (5×10 6 cells / mouse), or PBMC (5×10 6 cells / mouse) and multiple donor CD4 IL-10 Cells (three donors) (5×10 6 cells / mouse) in NSG miceIL-10 Migration of cells to spleen (left panel) and bone marrow (right panel). Box and whisker plots of n=8 donors and a single donor are shown.

[0291] Fig.31 The results for testing CD4+ T cells injected on day 0 after irradiation and multi-donor or single-donor (BC-H) CD4 IL-10 Cell-induced GvHD regimen.

[0292] Fig.32 The % of NSG mice that demonstrated GvHD for each day following injection are shown.

[0293] Figures 33A-33C The graft-versus-leukemia (GvL) effect based on the reduction of circulating leukemic cells and long-term leukemia-free survival was shown. Leukemia was measured as previously described (Locafaro G. et al Molecular Therapy 2017). On day 0, NSG mice were sublethally irradiated and injected intravenously with myeloid leukemia cells (ALL-CM) (5×10 6 indivual). Fig.33A It is a diagram of the experiment. Fig.33B The results show that PBMC (5×10 6 or single donor (from donor BC-I and donor BC-H) CD4 IL-10 Cells (2.5×10 6 Leukemia-free survival of animals. Fig.33C The results show that PBMC (5×10 6 or multiple donor CD4 IL-10 Cells (from donor BC-I and donor BC-H) (2.5×10 6 Leukemia-free survival of animals.

[0294] Figures 34A-34C The results show that ALL-CM cells (5 × 10 6 Long-term leukemia-free survival was measured in NSG mice with 40% paraformaldehyde (P < 0.05). Fig.34A A graphical representation of the experiment is shown. Fig.34B The results show that PBMCs (5×10 6 ) or mononuclear cells (PBMC) (5×10 6 + single donor (from donor BC-H and donor BC-I) CD4 IL-10 Cells (2.5×10 6 Data of animals of each species. Fig.18C The results show that PBMCs (5×106 ) or mononuclear cells (PBMC) (5×10 6 + multiple donor CD4 IL-10 Cells (BC-I / H) (2.5×10 6 Data of animals of each species.

[0295] Figures 35A-35G CD4 IL-10 Cellular inhibition of NLPR3 inflammasome activation. Fig.35A CD4 from a single donor (#1) is shown IL-10 Effect of cell supernatants on IL-1β production by LPS-activated monocytes. Fig.35B CD4 from another single donor (#2) is shown IL-10 Effect of cell supernatants on IL-1β production by LPS-activated monocytes. Fig.35C CD4 from a single donor (#1) is shown IL-10 Effect of cell supernatants on the enhancement of LPS-induced IL-1β production by nigericine (NIG), an inhibitor of NLPR3 inflammasome activator. Fig.35D CD4 from a single donor (#2) is shown IL -10 Effects of cell supernatants on the enhancement of LPS-induced IL-1β production by nigericin (NIG), an inhibitor of the NLPR3 inflammasome activator. Fig.35E CD4 IL-10 Bar graph showing the effect of cell supernatants and pooled cells from two different donors on LPS-induced IL-1β production by monocytes. Fig.35F The results show that multiple donor CD4 IL-10 Effect of cell supernatants on IL-1β production by monocytes. Figure 35G Shown are the expression of multiple donor CD4 IL-10 Effect of cell supernatants on IL-18 production induced by the combination of LPS and nigericin.

[0296] Fig.36 The experimental scheme for testing graft-versus-myeloid leukemia and xeno-GvHD effects is shown. On day 0, NSG mice were injected intravenously with ALL-CM cells (2.5×10 6On day 3, mice were divided into five groups, and each group received the following treatments: (i) no treatment as control, (ii) allogeneic PBMCs; (iii) allogeneic PBMCs and multiple donor CD4 IL-10 cells (BC-E, BC-V, BC-T, 1:1:1 pool); (iv) allogeneic PBMC and single donor CD4 IL-10 cells (BC-E); or (v) multiple donor CD4 IL-10 Cells, concentrations as Fig.36 shown.

[0297] Fig.37 Single donor CD4 IL-10 cells (BC-T, BC-V and BC-E) and multi-donor CD4 IL-10 Histogram of cytokine secretion profiles of cells (pooled: BC-E, BC-V and BC-T at 1:1:1).

[0298] Fig.38 Single donor CD4 IL-10 cells (BC-V and BC-E) and multiple donor CD4 IL-10 Cells (BC-V / E / T pool) to allogeneic CD4 + and CD8 + Inhibitory effect on T cell proliferation in vitro. (5×10 4 cells / well) and in the absence or presence of CD4 IL-10 Cells (5×10 4 Allogeneic mature dendritic cells (1×10 4 cells / well) and soluble anti-CD3 mAb. After 3 days of culture, flow cytometry was used to analyze the CD4 + ΔNGFR - (above) or CD8 + ΔNGFR - (Bottom) After T cells were gated, The percentage of proliferating responding cells was determined by dilution. Fig.38 Results from single donors BC-V and BC-E and pooled cells from donors BC-V, BC-E, and BC-T are shown. Percentages of proliferation and inhibition are indicated. CD4 IL-10 Cell-mediated inhibition was calculated as follows: 100-([in CD4 IL-10 proliferation of responders in the presence of cells / proliferation of responders alone] × 100).

[0299] Fig.39 Single donor CD4 IL-10 cells (BC-E and BC-V) or multiple donor CD4 IL-10 The results showed that the live cells (% of BC-E+BC-V+BC-T) were co-cultured with ALL-CM myeloid tumor cells or K562 cells. IL-10 The cells were selectively cytotoxic to ALL-CM myeloid neoplasms but not to K562 cells that lack MHC class I expression.

[0300] Fig.40 The results show that ALL-CM cells (2.5×10 6 Leukemia-free survival was measured in NSG mice with 100% leukemia-free survival. On day 3, mice were divided into five groups, and each group received the following treatments: (i) no treatment as control; (ii) allogeneic mononuclear cells (PBMCs); (iii) allogeneic PBMCs and multiple donor CD4 IL-10 cells (BC-V / T / ET); (iv) allogeneic PBMC and single donor CD4 IL-10 Cells (BC-E) or (v) multiple donor CD4 IL-10 Cells. The graph shows the leukemia-free survival of the animals in each group.

[0301] Fig.41 It shows that after injection of ALL-CM cells (2.5×10 6 ) and then on day 3 the following treatments were administered: (i) no treatment as control; (ii) allogeneic PBMCs; (iii) allogeneic PBMCs and multiple donor CD4 IL-10 cells (BC-E, BC-V, BC-T); (iv) allogeneic PBMC and single donor CD4 IL-10 Cells (BC-E) or (v) multiple donor CD4 IL-10 cell.

[0302] Fig.42 NSG mice dosed with 2.5E+06 PBMCs (allogeneic to donors C, E, F, and H) all succumbed to acute and lethal xeno-GvHD on day 22. Single donor CD4 IL-10 cells (Batch C) or multiple donor CD4 IL -10 Administration of cells (batch CEFH) in combination with PBMCs prevented lethal xeno-GvHD in 75% (3 / 4 mice) and 80% (4 / 5 mice), respectively.IL-10 Transfer of cells did not induce any signs of GvHD. Taken together, these results suggest that multidonor CD4 IL-10 Cells suppress pathogenic human T cell responses, which are related to single donor CD4 IL-10 cells, or slightly more effectively. PBMC: peripheral mononuclear cells; GvHD: graft-versus-host disease.

[0303] Fig.43A An alignment of IL-10 protein sequences from different species is shown, including human (SEQ ID NO: 1); Mus musculus, “MOUSE” (SEQ ID NO: 58); Rattus norvegicus, “RAT” (SEQ ID NO: 59); Rhesus macaque, “MACMU” (SEQ ID NO: 60); gorilla, “GORILLA” (SEQ ID NO: 61); cynomolgus macaque, “CYNO” (SEQ ID NO: 62); Polonium, “OLIVE BABOON” (SEQ ID NO: 63); bonobo, “BONOBO” (SEQ ID NO: 64); chimpanzee, “CHIMP” (SEQ ID NO: 65); and EBVB9 (SEQ ID NO: 66).

[0304] Fig.43B Sequences of IL-10 variants generated by substituting one or more amino acids of human IL-10 with amino acids of viral IL-10 (EBVB9) at the corresponding positions are provided. Sequences of exemplary variants, possible huIL-10 hybrid #1 (SEQ ID NO:67) and possible huIL-10 hybrid #2 (SEQ ID NO:68) are also provided. "*" indicates one or more amino acid positions that are substituted. "#" indicates a preferred I105 to A105 amino acid substitution of IL-10 hybrid #2 (SEQ ID NO:68).

[0305] Fig.43C An alignment of human IL-10 (SEQ ID NO: 1) and IL10 EBVB9 (SEQ ID NO: 66) is shown. "*" indicates one or more amino acid positions that are substituted in IL-10 hybrid #1. "#" indicates a preferred I105 to A105 amino acid substitution for IL-10 hybrid #2.

[0306] The accompanying drawings depict various embodiments of the present invention for illustration purposes only. One skilled in the art will readily recognize from the following discussion that alternative embodiments of the structures and methods shown herein may be employed without departing from the principles of the invention described herein.

[0307] 6. Detailed description

[0308] 6.1. Definitions

[0309] Unless otherwise defined, all technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which the invention belongs. As used herein, the following terms have the meanings given below.

[0310] "Graft-versus-leukemia effect" or "GvL" refers to the effect that occurs after allogeneic hematopoietic stem cell transplantation (HSCT) or bone marrow transplantation (BMT). T lymphocytes in the allogeneic transplant eliminate malignant residual host leukemia cells.

[0311] "Graft versus tumor effect" or "GvT" refers to the effect that occurs after allogeneic hematopoietic stem cell transplantation (HSCT) or bone marrow transplantation (BMT). T lymphocytes in the allogeneic transplant eliminate malignant residual host cancer cells, such as cells of myeloma as well as lymphocytic and myeloid leukemias, lymphomas, multiple myeloma, and possibly breast cancer. The term GvT is a general term for GvL.

[0312] The terms "treatment", "treating", etc. are used herein in the broadest sense as understood in the medical field. Specifically, these terms generally mean obtaining a desired pharmacological and / or physiological effect. As used herein, "treatment" covers any treatment of a disease or condition in a mammal (particularly a human), and includes: (a) preventing a disease or condition from occurring in a subject who may be susceptible to the disease or condition but has not yet been diagnosed with the disease or condition; (b) inhibiting the disease or condition (e.g., preventing its development); or (c) alleviating the disease or condition (e.g., causing the disease or condition to subside, providing an improvement in one or more symptoms). The improvement of any condition can be easily assessed according to standard methods and techniques known in the art. The subject population treated by the disease method includes a subject with an undesirable condition or disease, and a subject at risk of developing the condition or disease.

[0313] As used herein, "HLA matching" refers to a pair of individuals having matching HLA alleles in HLA class I (HLA-A, HLA-B and HLA-C) and class II (HLA-DRB1 and HLA-DQB1) loci, thereby making the individuals immunologically compatible with each other. HLA compatibility can be determined using any method available in the art, for example, as described in Tiervy, Haematologica 2016 Volume 101 (6): 680-687, which is incorporated herein by reference.

[0314] For a given locus, when each of the two alleles of one individual matches the two alleles of another individual, the pair has a 2 / 2 match. When only one of the two alleles of one individual matches one of the two alleles of another individual, the pair has a 1 / 2 match. When all ten alleles of one individual (two each of the HLA-A, HLA-B, HLA-C, HLA-DRB1, and HLA-DQB1 loci) match all ten alleles of another individual, the pair has a 10 / 10 match at the HLA-A, HLA-B, HLA-C, HLA-DRB1, and HLA-DQB1 loci.

[0315] In a preferred embodiment, allele level typing is used to determine HLA compatibility. Allele level typing corresponds to the unique nucleotide sequence of HLA genes, defined by using all the numbers in the first, second, third and fourth fields, such as A*02:01:01:01. Functionally, the third and fourth fields characterize alleles that are different from each other by the silent substitution in the coding sequence and the substitution in the non-coding sequence, respectively, and these substitutions are irrelevant unless they prevent the expression of HLA alleles (e.g., invalid allele B*15:01:01:02N). Deletion of invalid alleles will lead to mismatching, which is likely to be recognized by alloreactive T cells and produce harmful clinical effects. Substitution in non-coding sequences may affect expression levels (e.g., A24low allele A*24:02:01:02L). This variability may also have an impact on anti-HLA allorecognition.

[0316] As used herein, the term "HLA-mismatched" refers to a pair of individuals having mismatched HLA alleles at the HLA class I (HLA-A, HLA-B, and HLA-C) and class II (HLA-DRB1 and HLA-DQB1) loci, rendering the individuals immunologically incompatible with each other.

[0317] As used herein, the term "HLA partially mismatched" refers to a pair of individuals having mismatched HLA alleles in the HLA class I (HLA-A, HLA-B, HLA-C) and HLA class II (HLA-DRB1, HLA-DQB1) loci, such that the individuals are immunologically incompatible with each other to an acceptable degree. Some studies have identified acceptable mismatches. Some HLA class I incompatibilities are considered more acceptable.

[0318] "HLA haplotype" refers to a series of HLA loci alleles of a chromosome, one from the mother and one from the father. The genotypes of the HLA class I (HLA-A, HLA-B, and HLA-C) and class II (HLA-DRB1 and HLA-DQB1) loci can be used to determine the HLA haplotype.

[0319] The term "therapeutically effective amount" refers to an amount effective to treat a disease and thereby ameliorate symptoms of the disease.

[0320] The term "prophylactically effective amount" refers to an amount that completely or partially prevents a disease, condition, or symptoms thereof.

[0321] The term "amelioration" refers to any therapeutically beneficial outcome of the disease state being treated (eg, a neurodegenerative disease state), including prevention, lessening of severity or progression, remission, or cure thereof.

[0322] The term "CD4 IL-10 / CAR ” refers to any CD4 that has been genetically modified to express a chimeric antigen receptor IL-10 When CD4 IL -10 / CAR When referring to a specific chimeric antigen receptor, the nomenclature may include a designation of the specificity of the CAR, such as “CD4 IL -10 / 抗[抗原]CAR " cells. For example, CD4 IL-10 / CAR and CD4 IL-10 / CD19 CAR Can be used interchangeably.

[0323] As used herein, the term "autologous" means that the cells are derived from the same individual (eg, patient) into which the cells are reintroduced.

[0324] As used herein, the term "allogeneic" refers to cells taken from two or more different individuals of the same species that are not genetically identical.

[0325] 6.2. Other interpretation conventions

[0326] 41, 42, 43, 44, 45, 46, 47, 48, 49, and 50.

[0327] 6.3.CD4 IL-10 / CAR cell

[0328] In a first aspect, a CD4 + T cells or CD4 + A population of T cells that have been genetically modified to express a chimeric antigen receptor (CAR) from a first exogenous polynucleotide encoding the CAR and to express IL-10 (CD4 IL-10 / CAR cell).

[0329] 6.3.1.CD4 + T cells and T cell donors

[0330] The CD4+ T cells used to generate CD4+ T cells can be isolated from peripheral blood, cord blood, or other blood samples from donors (preferably human donors) using methods available in the art. IL-10 / CAR (Autologous or allogeneic single donor CD4 IL-10 / CAR or allogeneic multi-donor CD4 IL-10 / CAR ) group of CD4 + In typical embodiments, CD4 + T cells are isolated from peripheral blood. In certain embodiments, CD4 + T cells are isolated using leukapheresis and buffy coat. In certain embodiments, CD4 + T cells were isolated from peripheral blood obtained from a third party.

[0331] In some embodiments, CD4 + T cells were obtained from pre-frozen blood stocks or pre-frozen peripheral blood mononuclear cell (PBMC) stocks or pre-frozen CD4 + T cell stock separation. In some embodiments, CD4 + T cells are isolated from peripheral blood or PBMCs that have not been previously frozen. In some embodiments, CD4 + T cells are isolated individually from blood or PBMC obtained from individual donors and then pooled. In some embodiments, CD4 + T cells are isolated from blood or PBMCs first pooled from multiple donors.

[0332] In some embodiments, CD4 + T cells are obtained from a single T cell donor. In some embodiments, CD4 + The T cells are obtained from two, three, four, five, six, seven, eight, nine or ten different T cell donors.

[0333] In some embodiments, CD4 + CD4 T cells IL-10 / CAR Patient access to cell therapy.

[0334] In some embodiments, one or more T cell donors are selected without regard to genotype. In some embodiments, one or more T cell donors are selected based on genotype.

[0335] In certain embodiments, one or more T cell donors are selected based on their HLA haplotype.

[0336] 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.

[0337] In some embodiments, all CD4 + The T cells are at least 1 / 10, 2 / 10, 3 / 10, 4 / 10, 5 / 10, 6 / 10, 7 / 10, 8 / 10, or 9 / 10 matched to each other at the HLA-A, HLA-B, HLA-C, HLA-DRB1, and HLA-DQB1 loci. In some embodiments, all CD4 + The T cells have at least 1 / 8, 2 / 8, 3 / 8, 4 / 8, 5 / 8, 6 / 8, 7 / 8, or 8 / 8 matches with each other at the HLA-A, HLA-B, HLA-C, and HLA-DRB1 loci. In some embodiments, all CD4 + T cells have a 2 / 2 match with each other at the HLA-A locus. In some embodiments, all CD4 + The T cells have a 2 / 2 match with each other at the HLA-B locus. In some embodiments, all CD4 + T cells have a 2 / 2 match with each other at the HLA-C locus. In some embodiments, all CD4 + The T cells have at least a 3 / 4 or 4 / 4 match with each other at the HLA-DRB1 and HLA DQB1 loci. In some embodiments, all CD4 + The T cells have the A*02 allele or the A*24 allele.

[0338] In some embodiments, none of the one or more T cell donors is a potential CD4 IL-10 / CAR In some embodiments, none of the one or more T cell donors is a CD4 IL-10 / CAR The cells are used together with a donor of stem cells (e.g., HSC), tissue, or organ for the treatment methods described herein. In some embodiments, one or more T cell donors are CD4 IL-10 / CARHosts for cell therapy.

[0339] In some embodiments, one or more of the T cell donors are HLA mismatched or partially HLA mismatched with the patient (host) to be treated. In some embodiments, one or more of the T cell donors have less than 5 / 10, 6 / 10, 7 / 10, 8 / 10, 9 / 10 or 10 / 10 matches with the patient at HLA-A, HLA-B, HLA-C, HLA-DRB1 and HLA-DQB1 loci. In some embodiments, one or more of the T cell donors have less than 4 / 8, 5 / 8, 6 / 8, 7 / 8 or 8 / 8 matches with the patient at HLA-A, HLA-B, HLA-C and HLA-DRB1 loci. In some embodiments, one or more of the T cell donors have less than 2 / 2 matches with the patient at HLA-A, HLA-B or HLA-C loci. In some embodiments, one or more of the T cell donors has less than a 2 / 4, 3 / 4, or 4 / 4 match to the patient at the HLA-DRB1 and HLA-DQB1 loci.

[0340] In some embodiments, one or more of the T cell donors are HLA mismatched or partially HLA mismatched with the HSC donor. In some embodiments, one or more of the T cell donors have a match of less than 5 / 10, 6 / 10, 7 / 10, 8 / 10, 9 / 10 or 10 / 10 with the HSC donor at the HLA-A, HLA-B, HLA-C, HLA-DRB1 and HLA-DQB1 loci. In some embodiments, one or more of the T cell donors have a match of less than 4 / 8, 5 / 8, 6 / 8, 7 / 8 or 8 / 8 with the HSC donor at the HLA-A, HLA-B, HLA-C and HLA-DRB1 loci. In some embodiments, one or more of the T cell donors have a match of less than 2 / 2 with the HSC donor at the HLA-A, HLA-B or HLA-C loci. In some embodiments, one or more of the T cell donors has less than 3 / 4 or 4 / 4 match with the HSC donor at the HLA-DRB1 and HLA-DQB1 loci.

[0341] In a preferred embodiment, none of the CD4 + T cells are immortalized.

[0342] 6.3.2. Exogenous polynucleotide encoding CAR

[0343] CD4 of the present disclosure IL10 / CAR The cell is a CD4 T cells that has been genetically modified to express a first exogenous polynucleotide segment encoding a chimeric antigen receptor (CAR) from a first exogenous polynucleotide segment encoding a 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 unique function, and the optimal molecular design of CAR can be achieved by many changes in the protein domains that constitute it, as described by Rafiq et al. (Nat. Rev. Clin. Onco., 17: 147–167 (2020), which is incorporated herein by reference in its entirety.

[0344] In some embodiments, the polynucleotide segment encoding CAR encodes a first generation CAR, a second generation CAR or a third generation CAR. In some embodiments, the first generation CAR includes an antigen binding domain, a hinge region, a transmembrane domain and an intracellular signaling domain. In some embodiments, the second generation CAR includes an antigen binding domain, a hinge region, a transmembrane domain, a co-stimulatory domain and an intracellular signaling domain. Non-limiting examples of second generation CARs include U.S. Patent Publication Nos. 2004 / 0043401, 2013 / 0287748, 2014 / 0227237, 2014 / 0099309 and 2014 / 0050708; WO 2012 / 079000; and those described in WO 2015 / 157252, which are incorporated herein by reference in their entirety.

[0345] In some embodiments, the second generation CAR is pCAR. In some embodiments, pCAR comprises a second generation CAR, which comprises: (i) (a) signaling region; (b) costimulatory signaling region; (c) transmembrane domain; and (d) a first binding element that specifically interacts with a first epitope on a target antigen; and (ii) a chimeric costimulatory receptor (CCR) comprising (e) a costimulatory signaling region different from (b); (f) a transmembrane domain; and (g) a second binding element that specifically interacts with a second epitope on a second target antigen. Non-limiting examples of pCAR are described in U.S. Patent No. 10,703,794, which is incorporated herein by reference in its entirety.

[0346] 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 CARs include U.S. Patent Publication Nos. 2014 / 0322275A1; 2019 / 0345217; 2019 / 0112380A1; and those described in 2020 / 0031904A1; which are incorporated herein by reference in their entirety.

[0347] In some embodiments, the antigen binding domain comprises a single-chain antibody fragment (e.g., scFv), a nanobody (e.g., a camelid V H H domain), cytokines, ligands or peptides (adenectins and DARPins).

[0348] 6.3.2.1. Antigen binding domain

[0349] In some embodiments, the antigen binding domain of CAR comprises a single-chain antibody fragment. In some embodiments, the single-chain antibody fragment comprises a single-chain Fv (scFv).

[0350] In some embodiments, the antigen binding domain targets an antigen associated with an autoimmune disease, an inflammatory disease, or cancer. In some embodiments, the antigen binding domain targets a self-antigen.

[0351] In some embodiments, the antigen binding domain targets an antigen associated with an autoimmune disease or an inflammatory disease. In some embodiments, the antigen associated with an autoimmune disease or an inflammatory disease is selected from the group consisting of, but not limited to, anti-HLA-A*02, anti-HLA-A*24 or citrullinated peptides, insulin, MOG, GAD65, IA2, prolamins, and desmosome mucins (in the context of relevant MHC molecules). In some embodiments, the antigen associated with an autoimmune disease or an inflammatory disease is selected from the group consisting of: CD19, CD20, CD22, CD27, BCMA, and CD38.

[0352] In some embodiments, the antigen binding domain targets a cancer-associated antigen. In some embodiments, the cancer-associated antigen 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, IL11Ra, IL13Ra, NYBRI, SLea, CD200R, TGFβR2, CD276, TROP2, LAMP1, PTK7, DLL3, CDH1, CDH6, CDH17, CDH19, TSHR, and tyrosinase.

[0353] 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., Figure 4As shown. Non-limiting examples of antigen binding domains targeting 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).

[0354] 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)).

[0355] In some embodiments, the scFv targeting CD19 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 scFv targeting CD19 is described in U.S. Patent Publication Nos. 2018 / 0355052 or 2020 / 0392200, or WO 2020 / 010235, each of which is incorporated herein by reference in its entirety.

[0356] 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.

[0357] 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.

[0358] 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 targeting CD20 include those described in WO 2020 / 010235 and U.S. Patent Publication No. 2020 / 0392200, which are incorporated herein by reference in their entirety.

[0359] 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.

[0360] 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.

[0361] 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 targeting CD22 include those described in WO 2020 / 010235 and U.S. Patent Publication No. 2020 / 0392200, which are incorporated herein by reference in their entirety.

[0362] 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.

[0363] 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.

[0364] 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).

[0365] Non-limiting examples of scFvs targeting BCMA include those described in US9765342B2 or WO 2010 / 104949, which are incorporated by reference in their entirety. For example, as described in WO2010 / 104949, the anti-BCMA scFv may include the antigen binding domain of A7D12.2, C11 D5.3, C12A3.2 or C13F12.1 antibodies.

[0366] 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.

[0367] In some embodiments, the single-chain antibody fragment comprises a single-chain Fv (scFv) that binds to B7-H3 (also known as CD276 (eg, cluster of differentiation 276 protein (CD276) (eg, OMIM Accession No. 605715))).

[0368] Non-limiting examples of scFvs targeting B7-H3 include those described in U.S. Patent Publication Nos. 2016 / 0053017 and 2018 / 0346544, which are incorporated herein by reference in their entirety. For example, as described in U.S. Patent Publication No. 2018 / 0346544, an anti-CD276 scFv may include an antigen binding domain of MGA271 (CD276.MG), CD276.N1, CD276.N2, CD276.N3, CD276.N4, or CD276.N5 antibodies.

[0369] 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.

[0370] 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.

[0371] In some embodiments, the single-chain antibody fragment comprises a single-chain Fv (scFv) that binds to CD27 (eg, OMIM Accession No. 186711).

[0372] In some embodiments, the single-chain antibody fragment comprises a single-chain Fv (scFv) that binds to CD38 (eg, OMIM Accession No. 107270).

[0373] 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)).

[0374] In some embodiments, the single-chain antibody fragment comprises a single-chain Fv (scFv) that binds to BCMA (eg, B-cell maturation antigen (BCMA) (eg, OMIM Accession No. 109545)).

[0375] 6.3.2.2. Hinge region

[0376] The hinge region connects the extracellular antigen binding domain to the intracellular signaling domain (e.g., one or more co-stimulatory domains and intracellular signaling domains) through a transmembrane domain. The hinge provides enough flexibility to overcome steric hindrance and provides enough length to promote the antigen binding domain to approach and bind to the target antigen. In some embodiments, the difference in the length and composition of the hinge can affect antigen binding and signal transduction by CAR. For example, the spacer sequence in the hinge region or the spacer sequence added to the hinge region promotes the antigen binding domain to approach and bind to the target antigen. In some embodiments, the hinge region affects cytokine production.

[0377] 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 with SEQ ID NO: 28.

[0378] 6.3.2.3. Transmembrane domain

[0379] The transmembrane region anchors the CAR to CD4 IL10 / CAR In some embodiments, the transmembrane domain affects the stability and function of the CAR.

[0380] In some embodiments, the transmembrane domain is selected from the group consisting of: a TNFRSF 19 transmembrane domain, a CD3 ζ transmembrane domain, a CD8 α transmembrane domain, a CD4 transmembrane domain, a CD28 transmembrane domain, or a B7 family inducible co-stimulatory (ICOS) transmembrane domain. In some embodiments, the transmembrane domain is derived from TNFRSF 19. In some embodiments, the transmembrane domain derived from TNFRSF 19 comprises the sequence of SEQ ID NO: 29. In some embodiments, the transmembrane domain derived from TNFRSF 19 comprises a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity with SEQ ID NO: 29.

[0381] 6.3.2.4. Intracellular signaling domain and co-stimulatory domain

[0382] Intracellular signaling domain activates CD4 IL10 / CAR T cells. In some embodiments, the intracellular signaling domain is involved in T cell function, metabolism, and persistence.

[0383] 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 (ITAM). In some embodiments, the immunoreceptor tyrosine-based activation motif (ITAM) is derived from CD3 ζ. In some embodiments, the intracellular signaling domain derived from CD3 ζ comprises a sequence of SEQ ID NO: 30. In some embodiments, the intracellular signaling domain derived from CD3 ζ comprises a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 30.

[0384] In some embodiments, the intracellular signaling domain further comprises one or more costimulatory domains. In this case, compared to a CD4 T cell comprising a CAR that does not have one or more costimulatory domains. + Compared to T cells, one or more co-stimulatory domains enhance T cell activation, function, metabolism, and persistence. + (e.g., CD4 IL10 / CAR )T cells produce IL-2 and can proliferate upon repeated antigen exposure.

[0385] In some embodiments, co-stimulatory domains derived from different sources (e.g., 4-1BB and CD28) are present in CD4 + (e.g., CD4 IL10 / CAR) T cells. For example, a CD4 + (e.g., CD4 IL10 / CAR )T cells undergo enhanced differentiation into effector memory T cells. In another example, a CD4 + (e.g., CD4 IL10 / CAR )T cells undergo enhanced differentiation into central memory T cells.

[0386] In some embodiments, the one or more costimulatory domains are 4-1BB, CD28, OX40, ICOS, CD27, MYD88-CD40, and KIR2DS2.

[0387] In some embodiments, one or more co-stimulatory domains are derived from 4-1BB. In some embodiments, the co-stimulatory domain derived from 4-1BB comprises the sequence of SEQ ID NO: 31. In some embodiments, the co-stimulatory 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.

[0388] In some embodiments, one or more costimulatory domains are derived from CD28. In some embodiments, 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 with SEQ ID NO:32.

[0389] In some embodiments, CAR comprises two costimulatory domains. In some embodiments, CAR comprises a first costimulatory domain and a second costimulatory domain, the first costimulatory domain comprising a costimulatory domain derived from 4-1BB, and the second costimulatory domain comprising a costimulatory domain derived from a CD28 costimulatory domain.

[0390] 6.3.2.5. Chimeric Antigen Receptor (CAR)

[0391] In some embodiments, the CAR encoding polynucleotide segment encodes a CAR comprising an antigen binding domain that targets an antigen associated with an autoimmune disease, inflammatory disease, or cancer.

[0392] In some embodiments, the CAR encoding polynucleotide segment encodes a CAR comprising an antigen binding domain, which targets an antigen associated with an autoimmune disease or an inflammatory disease. In some embodiments, the antigen associated with an autoimmune disease or an inflammatory disease is selected from the group consisting of, but not limited to, anti-HLA-A*02, anti-HLA-A*24 or citrullinated peptides, insulin, MOG, GAD65, IA2, prolamin, and desmosome mucin (in the context of relevant MHC molecules).

[0393] In some embodiments, the CAR encoding polynucleotide segment encodes a CAR comprising an antigen binding domain targeting 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, Lyml, Lym2, CLEC5A, CDH179b, FLT3, GCC, Muc, CSF2RA, GFRa4, CD32, CD33, IL11Ra, IL13Ra, NYBRI, SLea, CD200R, TGFβR2, CD276, TROP2, LAMP1, PTK7, DLL3, CDH1, CDH6, CDH17, CDH19, TSHR and tyrosinase.

[0394] In some embodiments, the CAR encoding polynucleotide segment encodes a CAR comprising an antigen binding domain that targets a B cell antigen. In some embodiments, the selection of a B cell antigen is based in part on its expression during B cell differentiation, e.g., Figure 4 As shown. Non-limiting examples of antigen binding domains targeting 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).

[0395] In some embodiments, the CAR encoding polynucleotide segment encodes a CAR comprising an anti-CD19 antigen binding domain. In some embodiments, the CAR encoding polynucleotide segment encodes a second generation CAR comprising an anti-CD19 antigen binding domain. In some embodiments, the CAR encoding polynucleotide segment encodes a third generation CAR comprising an anti-CD19 antigen binding domain.

[0396] In some embodiments, the CAR encoding polynucleotide segment encodes an anti-CD19 CAR, which comprises: an anti-CD19 antigen binding domain, a hinge region, a transmembrane domain, a co-stimulatory domain, and an intracellular signaling domain. In some embodiments, the CAR encoding polynucleotide segment encodes an anti-CD19 antigen binding domain; a human CD8 hinge region; a TNFRSF 19 transmembrane region; a 4-1BB co-stimulatory domain; and a CD3 ζ chain intracellular signaling domain. In some embodiments, the anti-CD19 CAR comprises a 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 with SEQ ID NO: 9. In some embodiments, the first exogenous polynucleotide segment comprises a 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 with SEQ ID NO: 10.

[0397] Non-limiting examples of CARs comprising an anti-CD19 antigen binding domain include: U.S. Patent Publication Nos. 2020 / 0392200, WO 2020 / 010235, or WO 2012 / 079000, which are incorporated herein by reference in their entireties.

[0398] In some embodiments, the CAR encoding polynucleotide segment encodes a CAR comprising an anti-CD20 antigen binding domain. In some embodiments, the CAR encoding polynucleotide segment encodes a second generation CAR comprising an anti-CD20 antigen binding domain. In some embodiments, the CAR encoding polynucleotide segment encodes a third generation CAR comprising an anti-CD20 antigen binding domain.

[0399] In some embodiments, the CAR encoding polynucleotide segment encodes an anti-CD20 CAR comprising: an anti-CD20 antigen binding domain, a transmembrane domain, a co-stimulatory domain, and an intracellular signaling domain. In some embodiments, the CAR encoding polynucleotide segment encodes an anti-CD20 antigen binding domain; a human CD8 transmembrane region; a 4-1BB co-stimulatory domain; and a CD3 ζ chain intracellular signaling domain. In some embodiments, the anti-CD20 CAR comprises a 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 with SEQ ID NO: 16. In some embodiments, the first exogenous polynucleotide segment comprises a 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 with SEQ ID NO: 17.

[0400] Non-limiting examples of CARs comprising an anti-CD20 antigen binding domain include: U.S. Patent Publication No. 2020 / 0392200 or WO 2020 / 010235, both of which are incorporated herein by reference in their entirety.

[0401] In some embodiments, the CAR encoding polynucleotide segment encodes a CAR comprising an anti-CD22 antigen binding domain. In some embodiments, the CAR encoding polynucleotide segment encodes a second generation CAR comprising an anti-CD22 antigen binding domain. In some embodiments, the CAR encoding polynucleotide segment encodes a third generation CAR comprising an anti-CD22 antigen binding domain.

[0402] In some embodiments, the anti-CD22 CAR comprises a 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 a 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.

[0403] Non-limiting examples of CARs, including anti-CD22 CARs, include those described in WO 2020 / 010235 and U.S. Patent Publication No. 2020 / 0392200, which are incorporated herein by reference in their entireties.

[0404] Non-limiting examples of CARs, including anti-B7-H3 CARs, include those described in U.S. Patent Publication Nos. 2016 / 0053017, 2017 / 0369585, and 2018 / 0346544, which are incorporated herein by reference in their entireties.

[0405] In some embodiments, the CAR encoding polynucleotide segment encodes a CAR comprising an anti-B7-H3 antigen binding domain. In some embodiments, the CAR encoding polynucleotide segment encodes a second generation CAR comprising an anti-B7-H3 antigen binding domain. In some embodiments, the CAR encoding polynucleotide segment encodes a third generation CAR comprising an anti-B7-H3 antigen binding domain.

[0406] In some embodiments, the anti-B7-H3 CAR comprises a 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 a 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.

[0407] In some embodiments, the CAR encoding polynucleotide segment encodes a CAR comprising an anti-CD27 antigen binding domain. In some embodiments, the CAR encoding polynucleotide segment encodes a second generation CAR comprising an anti-CD27 antigen binding domain. In some embodiments, the CAR encoding polynucleotide segment encodes a third generation CAR comprising an anti-CD27 antigen binding domain.

[0408] In some embodiments, the CAR encoding polynucleotide segment encodes a CAR comprising an anti-CD38 antigen binding domain. In some embodiments, the CAR encoding polynucleotide segment encodes a second generation CAR comprising an anti-CD38 antigen binding domain. In some embodiments, the CAR encoding polynucleotide segment encodes a third generation CAR comprising an anti-CD38 antigen binding domain.

[0409] In some embodiments, the CAR encoding polynucleotide segment encodes a CAR comprising an anti-CEA antigen binding domain. In some embodiments, the CAR encoding polynucleotide segment encodes a second generation CAR comprising an anti-CEA antigen binding domain. In some embodiments, the CAR encoding polynucleotide segment encodes a third generation CAR comprising an anti-CEA antigen binding domain.

[0410] In some embodiments, the CAR encoding polynucleotide segment encodes a CAR comprising an anti-BCMA antigen binding domain. In some embodiments, the CAR encoding polynucleotide segment encodes a second-generation CAR comprising an anti-BCMA antigen binding domain. In some embodiments, the CAR encoding polynucleotide segment encodes a third-generation CAR comprising an anti-BCMA antigen binding domain. In some embodiments, the anti-BCMACAR comprises SEQ ID NOs: 41-49 and 54 sequences. 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.

[0411] The first exogenous polynucleotide further comprises one or more regulatory elements (e.g., any regulatory elements described herein or known in the art) operably connected to the coding sequence of the CAR. In some embodiments, the regulatory element is a promoter.

[0412] In some embodiments, the regulatory element comprises a regulatory element capable of directing CAR to express in CD4 + In some embodiments, the promoter drives the CAR to express in CD4 + In some embodiments, the promoter drives the CAR to be expressed in activated CD4 T cells. + Expression in T cells.

[0413] In some embodiments, the first exogenous polynucleotide segment further comprises a segment encoding a selectable marker (e.g., any selectable marker described herein or known in the art) that allows for the selection of successfully transduced CD4 + T cells.

[0414] In some embodiments, the first exogenous polynucleotide segment is delivered to the 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.

[0415] In some embodiments, a lentiviral vector is used to deliver the first exogenous polynucleotide segment to the CD4 +T cells, and the first exogenous polynucleotide segment comprises a lentiviral vector sequence. In certain embodiments, a lentiviral vector disclosed in Mátrai et al., Molecular Therapy 18(3): 477–490 (2010) (“Mátrai”) is used, which is incorporated herein by reference. In some embodiments, a first exogenous polynucleotide segment encoding a chimeric antigen receptor (CAR); and a second exogenous polynucleotide segment encoding interleukin-10 (IL-10) are present in a single lentiviral vector. In this case, the first exogenous polynucleotide and the second exogenous polynucleotide segment are operably connected to the first promoter. For example, the lentiviral vector includes a human PGK promoter, a first exogenous polynucleotide operably connected to the second exogenous polynucleotide segment by an internal ribosome entry site (IRES) or a self-cleaving peptide (e.g., a 2A self-cleaving peptide).

[0416] 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 segment polynucleotide is present in the T cell cytoplasm.

[0417] 6.3.3. Exogenous polynucleotide encoding IL-10

[0418] Single Donor CD4 of the Disclosure IL-10 / CAR Cells or multiple donor CD4 IL-10 / CAR The cell has been further genetically modified to comprise a second exogenous polynucleotide segment encoding IL-10. The second exogenous polynucleotide segment comprises an IL-10 encoding polynucleotide segment operably linked to one or more regulatory elements (e.g., any of the one or more regulatory elements described herein or known in the art).

[0419] The IL-10 encoding polynucleotide segment may encode IL-10 of a human, bonobo, or rhesus monkey. In some embodiments, the IL-10 encoding polynucleotide segment encodes a human IL-10 having a sequence of SEQ ID NO: 1. In some embodiments, the IL-10 encoding polynucleotide segment encodes a human IL-10 variant having at least 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 1. In some embodiments, the IL-10 encoding polynucleotide segment has a nucleotide sequence of SEQ ID NO: 2. In some embodiments, the IL-10 encoding polynucleotide segment has at least 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 2.

[0420] In some embodiments, the exogenous polynucleotide encodes a viral IL-10. In various embodiments, the exogenous polypeptide encodes an IL-10 from HCMV, GMCMV, RhCMV, BaCMV, MOCMV, SMCMV, EBV, Bonobo-HV, BaLCV, OvHV-2, EHV-2, CyHV-3, AngHV-1, ORFV, BPSV, PCPV, LSDV, SPV, GPV or CNPV. In some embodiments, the exogenous polypeptide encodes a viral IL-10 from EBV or ORFV.

[0421] 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.

[0422] In some embodiments, the IL-10 is a protein comprising human IL-10 with one, two, three, four, five, six, seven, eight, nine, or ten amino acid modifications. In some embodiments, the one, two, three, four, five, six, seven, eight, nine, or ten amino acid modifications are substitutions with viral IL-10 amino acids at the corresponding amino acid positions.

[0423] In some embodiments, the IL-10 encoding polynucleotide segment encodes a variant of human IL-10, which has one, two, three, four, five, six, seven, eight, nine, ten or more amino acid substitutions compared to human IL-10 (e.g., SEQ ID NO: 1). In some embodiments, one, two, three, four, five, six, seven, eight, nine or ten amino acid substitutions are substitutions with viral IL-10 amino acids at the corresponding amino acid positions. In some embodiments, the IL-10 encoding polynucleotide segment encodes a variant of human IL-10, which has one, two, three, four, five, six, seven, eight, nine, ten or more amino acid insertions, deletions or modifications compared to human IL-10 (e.g., SEQ ID NO: 1). In some embodiments, the variant of human IL-10 has a sequence of SEQ ID NO: 56 or 57.

[0424] In some embodiments, the IL-10 encoding polynucleotide segment encodes Mus musculus, "MOUSE" (SEQ ID NO:58); Rattus norvegicus, "RAT" (SEQ ID NO:59); Macaca mulatta, "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 Panpaniscus, "BONOBO" (SEQ ID NO:64); Chimpanzee Pan troglodytes, "CHIMP" (SEQ ID NO:65); and EBVB9 (SEQ ID NO:66). In some embodiments, the IL-10 encoding polynucleotide segment encodes a protein having at least 90%, 95%, 98% or 99% sequence identity to IL-10 of Mus musculus, "MOUSE" (SEQ ID NO:58); Rattus norvegicus, "RAT" (SEQ ID NO:59); Rhesus macaque, "MACMU" (SEQ ID NO:60); Gorilla, "GORILLA" (SEQ ID NO:61); Crab-eating macaque, "CYNO" (SEQ ID NO:62); Baboon, "OLIVE BABOON" (SEQ ID NO:63); Bonobo, "BONOBO" (SEQ ID NO:64); Chimpanzee, "CHIMP" (SEQ ID NO:65); and EBVB9 (SEQ ID NO:66).

[0425] In some embodiments, the IL-10 encoding polynucleotide segment encodes a variant of human IL-10 having one, two, three, four, five, six, seven, eight, nine, ten or more amino acid substitutions, insertions and / or deletions compared to human IL-10 (e.g., SEQ ID NO: 1). In some embodiments, the modification is a substitution, insertion and / or deletion of an amino acid with Mus musculus, "MOUSE" (SEQ ID NO: 58); Rattus norvegicus, "RAT" (SEQ ID NO: 59); Rhesus macaque, "MACMU" (SEQ ID NO: 60); Gorilla, "GORILLA" (SEQ ID NO: 61); Crab-eating macaque, "CYNO" (SEQ ID NO: 62); Baboon, "OLIVE BABOON" (SEQ ID NO: 63); Bonobos, "BONOBO" (SEQ ID NO: 64); Chimpanzee, "CHIMP" (SEQ ID NO: 65); and EBVB9 (SEQ ID NO: 66) at the corresponding position. In some embodiments, the variant of human IL-10 has the sequence of SEQ ID NO: 67 or SEQ ID NO: 68.

[0426] In some embodiments, the IL-10 encoding polynucleotide segment encodes a human IL-10 variant having reduced immunostimulatory activity compared to human IL-10. In some embodiments, the variant of human IL-10 includes an I105A substitution. In some embodiments, the variant of human IL-10 is prepared using the method described in A Single Amino Acid Determines the Immunostimulatory Activity of Interleukin 10, J Exp Med, 191, 2, 2000, p. 213-223.

[0427] The second exogenous polynucleotide segment further comprises one or more regulatory elements operably linked to the IL-10 coding sequence, wherein the one or more regulatory elements direct the encoded IL-10 to be expressed in the transduced CD4 + Expression in T cells.

[0428] In some embodiments, the regulatory element comprises a regulatory element capable of directing IL-10 in CD4 + In some embodiments, the promoter drives IL-10 expression in CD4 + In some embodiments, the promoter drives IL-10 in activated CD4 T cells. +Expression in T cells.

[0429] In some embodiments, the second exogenous polynucleotide segment further comprises a segment encoding a selectable marker (e.g., any selectable marker described herein or known in the art) that allows for the selection of successfully transduced CD4 + T cells.

[0430] In typical embodiments, a vector is used to deliver the exogenous polynucleotide into CD4+ T cells. In some embodiments, the vector is a plasmid vector. In some embodiments, the vector is a viral vector.

[0431] In some embodiments, the first exogenous polynucleotide segment encoding CAR and the second exogenous polynucleotide segment encoding IL-10 are in the same vector. In some embodiments, the first exogenous polynucleotide segment encoding CAR and the second exogenous polynucleotide segment encoding IL-10 are in the same viral vector. In some embodiments, the first exogenous polynucleotide segment encoding CAR and the second exogenous polynucleotide segment encoding IL-10 are in the same lentiviral vector.

[0432] In certain embodiments, a lentiviral vector is used to deliver the exogenous polynucleotide to CD4+ T cells, and the exogenous polynucleotide comprises a lentiviral vector sequence. In certain embodiments, a lentiviral vector disclosed in Mátrai et al., Molecular Therapy 18(3):477–490 (2010) (“Mátrai”) is used, which is incorporated herein by reference.

[0433] 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 T cell cytoplasm.

[0434] 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.

[0435] 6.3.4. Regulatory elements

[0436] In some embodiments, the first exogenous polynucleotide segment and / or the second exogenous polynucleotide segment further comprises a regulatory element operably connected to the coding sequence of CAR or IL-10, respectively. In some embodiments, the regulatory element comprises a promoter sequence, an enhancer sequence, a non-coding sequence, or any combination thereof.

[0437] 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 directs the encoded CAR to be expressed in the transduced CD4 + Expression in T cells. In some embodiments, an inducible promoter is used to induce expression of the CAR when it is therapeutically appropriate.

[0438] 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 directs the encoded IL-10 to be expressed in the transduced CD4 + In some embodiments, an inducible promoter is used to induce expression of IL-10, where therapeutically appropriate. In some embodiments, an IL-10 promoter is used.

[0439] 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 the ubiquitous expression of CAR or IL-10.

[0440] In some embodiments, the promoter is a native human promoter. In some embodiments, the promoter is the human elongation factor (EF) 1α promoter. In some embodiments, the promoter is the human phosphoglycerate kinase promoter (PGK). In some embodiments, the promoter is the human ubiquitin C promoter (UBI-C).

[0441] In some embodiments, the promoter is a synthetic promoter. In certain embodiments, the promoter is a minimal CMV core promoter. In a specific embodiment, the promoter is an inducible or constitutive bidirectional promoter. In a specific embodiment, a synthetic bidirectional promoter disclosed in Amendola et al., Nature Biotechnology, 23 (1): 108-116 (2005) is used. The promoter can mediate the coordinated transcription of two mRNAs in a ubiquitous or 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.

[0442] In some embodiments, the promoter is a native human promoter. In some embodiments, the promoter is the human elongation factor (EF) 1α promoter. In some embodiments, the promoter is the human phosphoglycerate kinase promoter (PGK). In some embodiments, the promoter is the human ubiquitin C promoter (UBI-C).

[0443] In some embodiments, the first exogenous polynucleotide segment and / or the 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 3' untranslated regions (UTRs), poly(A) signals, and woodchuck hepatitis virus post-transcriptional regulatory elements (WPREs).

[0444] In some embodiments, the exogenous polynucleotide comprises more than one coding sequence.For example, the exogenous polynucleotide includes a first exogenous polynucleotide segment and a second exogenous polynucleotide segment on a single continuous polynucleotide sequence (e.g., a single polynucleotide construct).In some embodiments, one or more coding sequences include a sequence encoding CAR and a sequence encoding a selection marker.In some embodiments, one or more coding sequences include a sequence encoding IL-10 and a sequence encoding a selection marker.In some embodiments, one or more coding sequences include a sequence encoding CAR, a sequence encoding IL-10, and one or more sequences encoding a selection marker.In some embodiments, the exogenous polynucleotide does not contain a selection marker.

[0445] In some embodiments, the multiple coding sequences are separated by one or more internal ribosome entry sites (IRES).

[0446] In some embodiments, a plurality of 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 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.

[0447] In some embodiments, the coding sequence further comprises a self-cleaving peptide located between the heavy chain coding sequence and the light chain coding sequence. In some embodiments, the self-cleaving peptide is selected from the group consisting of F2A, P2A, T2A and E2A.

[0448] 6.3.5. Selection of markers

[0449] In some embodiments, the first exogenous polynucleotide segment, the second exogenous polynucleotide segment, or both further comprise a polynucleotide segment encoding a selectable marker that allows for the selection of successfully transduced CD4 + In some embodiments, the first exogenous polynucleotide segment, the second exogenous polynucleotide segment, or both do not contain a selectable marker.

[0450] In some embodiments, 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 this case, the exogenous polynucleotide includes one or more selection markers that allow selection of successfully transduced CD4 + T cells.

[0451] In some embodiments, the selection marker is ΔNGFR. In certain embodiments, the selection marker is a polypeptide having the sequence of SEQ ID NO:3. In certain embodiments, the selection marker is a polypeptide having at least 90%, 95%, 98% or 99% sequence identity with SEQ ID NO:3. In a specific embodiment, the nucleotide sequence encoding the ΔNGFR selection marker has the sequence of SEQ ID NO:4. In some embodiments, the nucleotide sequence encoding the ΔNGFR selection marker has at least 90%, 95%, 98% or 99% sequence identity with SEQ ID NO:4.

[0452] In some embodiments, the expression of the selection marker is correlated with the expression of IL-10 from the exogenous polynucleotide. In some embodiments, the expression of the selection marker is linearly correlated with the expression of IL-10 from the exogenous polynucleotide. Thus, in some embodiments, the expression of the selection marker is measured to infer the expression of IL-10 from the exogenous polynucleotide.

[0453] In some embodiments, the selection marker is a truncated form of the EGFR polypeptide. In some embodiments, the selection marker is a truncated form of the human EGFR polypeptide, optionally huEGFR disclosed in Wang et al. "A transgene-encoded cellsurface polypeptide for selection, in vivo tracking, and ablation of engineered cells", Blood, v.118, n.5 (2011), which is incorporated herein by reference in its entirety.

[0454] 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.

[0455] 6.3.6.CD4 IL-10 / CAR Cells or multiple donor CD4 IL-10 / CAR Gene expression

[0456] CD4 IL-10 / CAR In some embodiments, the CD4 IL-10 / CAR The cells constitutively express IL-10. In some embodiments, the CD4 IL-10 / CAR Cells express IL-10 when activated.

[0457] In some embodiments, CD4 IL-10 / CAR cells (eg, autologous single donor or allogeneic single donor) or multiple donor CD4 IL-10 / CAR Cells constitutively express at least 100 pg IL-10 / 10 6 CD4 + T cells / mL culture. In some embodiments, CD4 IL-10 / CAR Cells or multiple donor CD4 IL-10 / CAR Cells constitutively express at least 200 pg, 500 pg, 1 ng, 5 ng, 10 ng or 50 ng IL-10 / 10 6 CD4 + T cells / mL culture.

[0458] In some embodiments, after activation with a combination of anti-CD3 and anti-CD28 antibodies, or beads coated with anti-CD3 and anti-CD28 antibodies, CD4 IL-10 / CAR cells (eg, autologous single donor or allogeneic single donor) or multiple donor CD4 IL-10 / CAR Cells expressing at least 1ng or 2ng IL-10 / 10 6 CD4 + In some embodiments, after activation with beads coated with anti-CD3 and anti-CD28 antibodies or CD3 and CD28 antibodies, multiple donor CD4 IL -10 T cells expressing at least 5ng, 10ng, 100ng, 200ng or 500ng IL-10 / 10 6 CD4 + T cells / mL culture.

[0459] In various embodiments, the amount of IL-10 produced is determined 12 hours, 24 hours, or 48 hours after activation using various protein detection and measurement methods (e.g., ELISA, real-time polymerase chain reaction (PCR), spectroscopic procedures, colorimetry, amino acid analysis, radiolabeling, Edman degradation, HPLC, Western blot, etc.). In a preferred embodiment, the amount of IL-10 produced is determined by ELISA 48 hours after activation with anti-CD3 antibodies and anti-CD28 antibodies.

[0460] In some embodiments, CD4 IL-10 / CAR cells (eg, autologous single donor or allogeneic single donor) or multiple donor CD4 IL-10 / CAR Unmodified CD4 + T cells express IL-10 at least 5-fold higher levels. IL-10 T cells were more expressed than unmodified CD4 + T cells express IL-10 at levels at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, or 50 fold higher.

[0461] In some embodiments, CD4 IL-10 / CAR cells (eg, autologous single donor or allogeneic single donor) or multiple donor CD4 IL-10 / CAR Cells expressing at least 100 pg IL-5 / 10 6 CD4 + T cells / mL. In some embodiments, CD4 IL -10 / CAR cells (eg, autologous single donor or allogeneic single donor) or multiple donor CD4 IL-10 / CAR Cells expressing at least 100 pg, 200 pg, 500 pg, 1 ng, 5 ng, 10 ng or 50 ng IL-5 / 10 6 CD4 + T cells / mL.

[0462] In some embodiments, after activation with anti-CD3 antibody and anti-CD28 antibody, CD4 IL-10 / CAR cells (eg, autologous single donor or allogeneic single donor) or multiple donor CD4 IL-10 / CAR Cells expressing at least 1ng IL-5 / 10 6 CD4 + In some embodiments, after activation with anti-CD3 antibody and anti-CD28 antibody, CD4 IL-10 / CAR cells (eg, autologous single donor or allogeneic single donor) or multiple donor CD4 IL-10 / CARCells expressing at least 2ng, 5ng, 10ng, 100ng, 200ng or 500ng IL-5 / 10 6 CD4 + T cells / mL.

[0463] In various embodiments, the amount of IL-5 produced is determined 12 hours, 24 hours, or 48 hours after activation using various protein detection and measurement methods (e.g., ELISA, real-time polymerase chain reaction (PCR), spectroscopic procedures, colorimetry, amino acid analysis, radiolabeling, Edman degradation, HPLC, Western blot, etc.). In a preferred embodiment, the amount of IL-5 produced is determined by ELISA 48 hours after activation with anti-CD3 antibodies and anti-CD28 antibodies.

[0464] In some embodiments, CD4 IL-10 / CAR cells (eg, autologous single donor or allogeneic single donor) or multiple donor CD4 IL-10 / CAR Cells expressing at least 100 pg IFN-γ / 10 6 CD4 + T cells / mL. In some embodiments, CD4 IL -10 / CAR cells (eg, autologous single donor or allogeneic single donor) or multiple donor CD4 IL-10 / CAR Cells expressing at least 100 pg, 200 pg, 500 pg, 1 ng, 5 ng, 10 ng or 50 ng IFN-γ / 10 6 CD4 + T cells / mL.

[0465] In some embodiments, after activation with anti-CD3 antibody and anti-CD28 antibody, CD4 IL-10 / CAR cells (eg, autologous single donor or allogeneic single donor) or multiple donor CD4 IL-10 / CAR Cells expressing at least 1 ng I IFN-γ / 10 6 CD4 + In some embodiments, after activation with anti-CD3 antibody and anti-CD28 antibody, CD4 IL-10 / CAR cells (eg, autologous single donor or allogeneic single donor) or multiple donor CD4 IL-10 / CAR Cells expressing at least 2 ng, 5 ng, 10 ng, 100 ng, 200 ng or 500 ng IFN-γ / 10 6 CD4 + T cells / mL.

[0466] In various embodiments, the amount of IFN-γ produced is determined 12 hours, 24 hours, or 48 hours after activation using various protein detection and measurement methods (e.g., ELISA, real-time polymerase chain reaction (PCR), spectroscopic procedures, colorimetry, amino acid analysis, radiolabeling, Edman degradation, HPLC, Western blot, etc.). In a preferred embodiment, the amount of IFN-γ produced is determined by ELISA 48 hours after activation with anti-CD3 antibodies and anti-CD28 antibodies.

[0467] In some embodiments, CD4 IL-10 / CAR cells (eg, autologous single donor or allogeneic single donor) or multiple donor CD4 IL-10 / CAR Cells expressing at least 25 pg IL-4 / 10 6 CD4 + T cells / mL. In some embodiments, CD4 IL-10 / CAR cells (eg, autologous single donor or allogeneic single donor) or multiple donor CD4 IL-10 / CAR The cells 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 / 10 6 CD4 + T cells / mL.

[0468] In some embodiments, after activation with anti-CD3 antibody and anti-CD28 antibody, CD4 IL-10 / CAR cells (eg, autologous single donor or allogeneic single donor) or multiple donor CD4 IL-10 / CAR Cells expressing at least 100 pg IL-4 / 10 6 CD4 + In some embodiments, after activation with anti-CD3 antibody and anti-CD28 antibody, CD4 IL-10 / CAR cells (eg, autologous single donor or allogeneic single donor) or multiple donor CD4 IL-10 / CAR The cells express at least 100 pg, 200 pg, 300 pg, 400 pg, 500 pg, 600 pg, 700 pg, 800 pg, 900 pg, 1000 pg, 2 ng, 5 ng, 10 ng, 100 ng, 200 ng or 500 ng IL-4 / 10 6 CD4 + T cells / mL.

[0469] In various embodiments, the amount of IL-4 produced is determined 12 hours, 24 hours, or 48 hours after activation using various protein detection and measurement methods (e.g., ELISA, real-time polymerase chain reaction (PCR), spectroscopic procedures, colorimetry, amino acid analysis, radiolabeling, Edman degradation, HPLC, Western blot, etc.). In a preferred embodiment, the amount of IL-4 produced is determined by ELISA 48 hours after activation with anti-CD3 antibodies and anti-CD28 antibodies.

[0470] 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.

[0471] In some embodiments, CD4 IL-10 / CAR cells (eg, autologous single donor or allogeneic single donor) or multiple donor CD4 IL-10 / CAR The cells further express a selectable marker. In some embodiments, CD4 IL-10 / CAR Cells or multiple donor CD4 IL-10 / CAR The cells express proteins that are normally expressed in Tr1 cells. In some embodiments, CD4 IL-10 / CAR Cells or multiple donor CD4 IL-10 / CAR The cells expressed Tr1 cell characteristic marker proteins.

[0472] In some embodiments, CD4 IL-10 / CAR cells (eg, autologous single donor or allogeneic single donor) or multiple donor CD4 IL-10 / CAR The cells express CD49b. In some embodiments, CD4 IL-10 / CAR Cells or multiple donor CD4 IL-10 / CAR In some embodiments, the CD4 IL-10 / CAR Cells or multiple donor CD4 IL-10 / CAR In some embodiments, the CD4 IL-10 / CAR Cells or multiple donor CD4 IL-10 / CAR In some embodiments, the CD4 IL-10 / CAR Cells or multiple donor CD4 IL-10 / CAR In some embodiments, multiple donor CD4 IL-10 Cells release GzB when activated with myeloid antigen-presenting cells. In some embodiments, CD4 IL-10 / CAR Cells or multiple donor CD4 IL -10 / CAR In some embodiments, the CD4 IL-10 / CAR Cells or multiple donor CD4 IL-10 / CARCells release perforin when activated with myeloid antigen-presenting cells. In some embodiments, CD4 IL-10 / CAR Cells or multiple donor CD4 IL -10 / CAR The cells express CD18. In some embodiments, CD4 IL-10 / CAR Cells or multiple donor CD4 IL-10 / CAR The cells express CD2. In some embodiments, CD4 IL-10 / CAR Cells or multiple donor CD4 IL-10 / CAR Cells express CD226. In some embodiments, multiple donor CD4 IL-10 In some embodiments, the CD4 IL-10 / CAR Cells or multiple donor CD4 IL-10 / CAR Cells express IL-10.

[0473] In some embodiments, CD4 IL-10 / CAR cells (eg, autologous single donor or allogeneic single donor) or multiple donor CD4 IL-10 / CAR The cell exhibits at least one function of a Tr1 cell. In various embodiments, the function is secretion of IL-10, secretion of TGF-β, and specific killing of myeloid antigen presenting cells by releasing granzyme B (GzB) and perforin.

[0474] 6.3.7.CD4 IL-10 / CAR Other properties of cells

[0475] In some embodiments, CD4 IL-10 / CAR The cell comprises an anti-CD19 CAR. In some embodiments, the CD4 IL -10 / CAR Cells can express CD19 + Target cells produce in vitro cytotoxicity. In some embodiments, CD4 IL-10 / CAR Cells can express CD19 + Target cells produce in vivo cytotoxicity. In some embodiments, CD19 + The target cell is a B cell that produces autoantibodies. In some embodiments, CD19 + Target cells are CD19 + Cancer cells. In some embodiments, + Target cell cytotoxicity is maintained after one or more restimulations (see, e.g., Figures 5A-5C The generation method described in ).

[0476] 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 / CARThe cells are capable of in vivo cytotoxicity against myeloid target cells. In some embodiments, CD4 IL-10 / CAR Cells can express CD19 + In some embodiments, the myeloid target cells express one or more of class I MHC, CD13, CD54, and CD112. In some embodiments, the cytotoxicity to the myeloid target cells is maintained after one or more restimulations (see, e.g., Figures 5A-5C The generation method described in ).

[0477] In some embodiments, CD4 IL-10 / CAR cells are able to suppress allogeneic CD4 + T cell proliferation. In some embodiments, CD4 IL-10 / CAR cells can transfer allogeneic CD4 + T cell proliferation is inhibited 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%.

[0478] In some embodiments, CD4 IL-10 / CAR cells are able to suppress allogeneic CD8 + T cell proliferation. In some embodiments, CD4 IL-10 / CAR cells are able to transfer allogeneic CD8 + T cell proliferation is inhibited 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%.

[0479] In some embodiments, CD4 IL-10 / CAR cells are able to suppress allogeneic CD4 + T cell proliferation and allogeneic CD8 + T cell proliferation. In some embodiments, CD4 IL-10 / CAR cells can transfer allogeneic CD4 + T cell proliferation is inhibited 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 allogeneic CD8 + T cell proliferation is inhibited 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%.

[0480] In some embodiments, CD4 IL-10 / CAR Cell-mediated inhibition was calculated as follows: 100-([in CD4 IL-10 T cells (control) and CD4 IL-10 / CARProliferation of responders in the presence of T cells / proliferation of responders] × 100).

[0481] In some embodiments, the inhibitory properties are stable after one or more restimulations.

[0482] 6.3.8. Products produced by the method

[0483] In some embodiments, the CD4 T cells are modified with an exogenous polynucleotide segment encoding a CAR and an exogenous polynucleotide segment encoding an IL-10. + T cells to obtain CD4 IL-10 / CAR Cells (autologous or allogeneic single donor or allogeneic multiple donor CD4 IL-10 / CAR In some embodiments, the CD4 T cells are modified with a first exogenous polynucleotide segment encoding a CAR and a second exogenous polynucleotide segment encoding an IL-10. + T cells include: (i) a first transduction with a polynucleotide segment encoding IL-10, followed by a second transduction with an exogenous polynucleotide segment encoding CAR; (ii) a first transduction with a polynucleotide segment encoding CAR, followed by a second transduction with an exogenous polynucleotide segment encoding IL-10; (iii) simultaneous transduction (i.e., co-transduction) with a polynucleotide segment encoding IL-10 and an exogenous polynucleotide segment encoding CAR; or (iv) transduction with a single polynucleotide construct comprising a polynucleotide segment encoding IL-10 and a polynucleotide segment encoding CAR. In some embodiments, the polynucleotide encoding IL-10 further comprises a coding sequence for a marker protein (e.g., ΔNGFR).

[0484] In some embodiments, the CD4 T cells are modified by (i) a first polynucleotide construct comprising a polynucleotide segment encoding a CAR and a second polynucleotide construct comprising a polynucleotide segment encoding an IL-10, or (ii) a single polynucleotide construct comprising a polynucleotide segment encoding a CAR and a polynucleotide segment encoding an IL-10. + T cells acquire CD4 IL -10 / CAR Cells (single or multiple donor CD4 IL-10 / CAR cell).

[0485] In some embodiments, the first exogenous polynucleotide segment encoding CAR, the second exogenous polynucleotide segment encoding IL-10, or a polynucleotide construct comprising the first and second exogenous polynucleotide segments is introduced into the CD4 T cells via a viral vector or a plasmid vector. + In some embodiments, a first viral vector (e.g., any viral vector described herein) containing a polynucleotide segment encoding IL-10 is used to transduce a CD4 T cell.+ T cells are then transduced a second time with a second viral vector (e.g., any viral vector described herein) containing a polynucleotide segment encoding a CAR. In another embodiment, 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 are used to transduce the CD4 + In yet another embodiment, a CD4 T cell is co-transduced with a viral vector (e.g., any viral vector described herein) containing the first and second exogenous polynucleotide segments. + T cells.

[0486] In some embodiments, multiple donor CD4 IL-10 / CAR Generated by: (i) combining primary CD4 T cell lines obtained from at least two different T cell donors + T cells; and (ii) modifying the combined CD4 + In some embodiments, multiple donor CD4 IL-10 / CAR T cells were generated by: (i) obtaining primary CD4 T cells from at least two different T cell donors; + (ii) modifying each donor's CD4 T cells according to the methods described herein; + T cells; and then (iii) combining genetically modified CD4 + T cells.

[0487] In some embodiments, CD4 IL-10 / CAR T cells are already able to activate CD4 + T cells are cultured in the presence of proteins. In some embodiments, CD4 IL-10 / CAR T cells are cultured in the presence of anti-CD3 antibody and anti-CD28 antibody, or beads coated with anti-CD3 antibody and anti-CD28 antibody. IL-10 / CAR T cells are cultured in the presence of anti-CD3 antibody, anti-CD28 antibody and IL-2, or in the presence of beads coated with anti-CD3 antibody and anti-CD28 antibody and IL-2. IL-10 / CAR T cells are cultured in the presence of polymer nanomatrix agents to activate and expand human T cells via CD3 and CD28. In some embodiments, CD4 IL-10 / CAR T cells are cultured in the presence of other T cell specific immune cell culture media, activators and supplements.

[0488] In some embodiments, CD4 IL-10 / CAR (single or multiple donor CD4 IL-10 / CART cells) are in a frozen stock solution (e.g., a frozen suspension). In some embodiments, the CD4 IL-10 / CAR (single or multiple donor CD4 IL-10 / CAR T cells) are in liquid suspension.

[0489] 6.4.CD4 IL-10 / CAR T cell population

[0490] In another aspect, the disclosure features a CD4 IL-10 / CAR In some embodiments, the population further comprises CD4 + T cells (e.g., primary CD4 + T cells).

[0491] In some embodiments, CD4 + The T cell population contains T cells from autologous or allogeneic single T cell donors (single donor CD4 IL-10 / CAR CD4 T cells + In some embodiments, primary CD4 + T cells are derived from a single T cell donor. In some embodiments, primary CD4 + T cells are from the same individual (i.e., the same patient). In some embodiments, the CD4 + The T cell population contains T cells from at least two different T cell donors (multiple donor CD4 IL-10 / CAR CD4 T cells + In some embodiments, primary CD4 + T cells were obtained from donors in allo-HSCT.

[0492] In some embodiments, CD4 + T cells are obtained from two, three, four, five, six, seven, eight, nine, or ten different T cell donors and pooled. IL-10 T cells and methods of making and using them are described in PCT / US2021 / 039464, which is incorporated herein by reference in its entirety. The methods and / or compositions described therein can be used to generate multiple donor CD4 IL-10 / CAR cell.

[0493] In some embodiments, primary CD4 + T cells are from donors that have been screened from donors after analyzing the donor's genetic information. In some embodiments, the donors (i.e., primary CD4 + In some embodiments, the primary CD4 +T cells are from donors selected based on their HLA haplotype. In some embodiments, the CD4 + T cells have a total of six, seven, eight, nine, ten, eleven, twelve or more different HLA haplotypes. In some embodiments, all CD4 + The T cells are at least 1 / 10, 2 / 10, 3 / 10, 4 / 10, 5 / 10, 6 / 10, 7 / 10, 8 / 10, or 9 / 10 matched to each other at the HLA-A, HLA-B, HLA-C, HLA-DRB1, and HLA-DQB1 loci. In some embodiments, all CD4 + The T cells are at least 1 / 8, 2 / 8, 3 / 8, 4 / 8, 5 / 8, 6 / 8, 7 / 8 or 8 / 8 matched to each other at the HLA-A, HLA-B, HLA-C and HLA-DRBl loci.

[0494] In some embodiments, all CD4 + T cells have a 2 / 2 match with each other at the HLA-A locus. In some embodiments, all CD4 + The T cells have a 2 / 2 match with each other at the HLA-B locus. In some embodiments, all CD4 + T cells have a 2 / 2 match with each other at the HLA-C locus. In some embodiments, all CD4 + The T cells have at least a 3 / 4 or 4 / 4 match with each other at the HLA-DRB1 and HLA-DQB1 loci. In some embodiments, all CD4 + T cells have the A*02 or A*24 allele. In some embodiments, all CD4 + T cells are A*02 or A*24 negative.

[0495] In some embodiments, all CD4 + T cells have less than 5 / 10, 6 / 10, 7 / 10, 8 / 10, or 9 / 10 matches with each other at the HLA-A, HLA-B, HLA-C, HLA-DRB1, and HLA-DQB1 loci. In some embodiments, all CD4 + T cells have less than 4 / 8, 5 / 8, 6 / 8, 7 / 8, or 8 / 8 matches with each other at the HLA-A, HLA-B, HLA-C, and HLA-DRB1 loci. In some embodiments, all CD4 + T cells have less than a 2 / 2 match with each other at the HLA-A locus. In some embodiments, all CD4 +T cells have less than a 2 / 2 match with each other at the HLA-B locus. In some embodiments, all CD4 + T cells have less than a 2 / 2 match with each other at the HLA-C locus. In some embodiments, all CD4 + The T cells have less than 3 / 4 or 4 / 4 matches with each other at the HLA-DRB1 and HLA DQB1 loci.

[0496] In some embodiments, 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 CD4 + T cells express CAR and IL-10.

[0497] In some embodiments, CD4 + T cells are in frozen suspension. In some embodiments, CD4 + The T cells are in a liquid suspension. In some embodiments, the liquid suspension has been previously frozen.

[0498] 6.5. Pharmaceutical Compositions

[0499] In another aspect, a pharmaceutical composition is provided. In some embodiments, the pharmaceutical composition comprises any of the single donor CD4 IL-10 / CAR T cells, any of the multiple donor CD4 IL-10 / CAR T cells or any of the multiple donor CD4 IL-10 / CAR T cell population, and a pharmaceutically acceptable carrier or diluent.

[0500] The pharmaceutical composition can be formulated for use by any suitable human or veterinary route of administration. In typical embodiments, the composition is formulated for intravenous (IV) administration. In some embodiments, the composition is formulated for intravenous (IV) infusion. In embodiments formulated for IV administration, the pharmaceutical composition is in the form of an acceptable aqueous solution for parenteral administration, which is pyrogen-free and has suitable pH value, isotonicity and stability.

[0501] In some embodiments, the pharmaceutically acceptable carrier or diluent is saline, lactated Ringer's solution or other physiologically compatible solutions. In various embodiments, the pharmaceutical composition solution comprises 2-20%, preferably 5% human serum albumin.

[0502] In some embodiments, unit dosage forms of pharmaceutical compositions are provided that are suitable for administering the pharmaceutical compositions by systemic administration, particularly intravenous administration.

[0503] In some embodiments, the unit dosage form contains 10 4 Up to 10 11 CD4 IL-10 / CAR Cells or multiple donor CD4 IL-10 / CAR Cells, 10 4 Up to 10 10 CD4 IL-10 / CAR Cells or multiple donor CD4 IL-10 / CAR Cells, 10 4 Up to 10 9 CD4 IL-10 / CAR T cells or multiple donor CD4 IL-10 / CAR Cells, 10 5 Up to 10 10 CD4 IL-10 / CAR Cells or multiple donor CD4 IL-10 / CAR Cells, 10 5 Up to 10 9 CD4 IL-10 / CAR Cells or multiple donor CD4 IL-10 / CAR Cells, 10 5 Up to 10 8 CD4 IL-10 / CAR Cells or multiple donor CD4 IL-10 / CAR cells, or 10 5 Up to 10 7 CD4 IL-10 / CAR Cells or multiple donor CD4 IL-10 / CAR cell.

[0504] In typical embodiments, the pharmaceutical composition in unit dosage form is in liquid form.

[0505] 6.6. Preparation of single donor CD4 IL10 / CAR Cells or multiple donor CD4 IL10 / CAR Cell Methods

[0506] In another aspect, the present disclosure provides methods for preparing CD4 IL10 / CAR Cells or multiple donor CD4 IL10 / CAR Cellular methods.

[0507] In some embodiments, the methods provided herein comprise modifying the CD4 by introducing an exogenous polynucleotide segment encoding a CAR and an exogenous polynucleotide segment encoding an IL-10. + T cells or combined CD4 + In some embodiments, the CD4 T cells are modified with an exogenous polynucleotide segment encoding a CAR and an exogenous polynucleotide segment encoding an IL-10.+ T cells or combined CD4 + T cells include: (i) a first transduction with a polynucleotide segment encoding IL-10, followed by a second transduction with an exogenous polynucleotide segment encoding CAR; (ii) a first transduction with a polynucleotide segment encoding CAR, followed by a second transduction with an exogenous polynucleotide segment encoding IL-10; (iii) simultaneous transduction (i.e., co-transduction) with a polynucleotide segment encoding IL-10 and an exogenous polynucleotide segment encoding CAR; or (iv) transduction with a single polynucleotide construct comprising a polynucleotide segment encoding IL-10 and a polynucleotide segment encoding CAR.

[0508] In some embodiments, a single polynucleotide construct comprising a polynucleotide segment encoding IL-10 and a polynucleotide segment encoding CAR is a bidirectional vector, wherein IL-10 is under the control of a promoter (e.g., PGK or EF1a), and CAR is under the control of a second promoter (e.g., CMV). In this case, if the construct includes a polynucleotide sequence encoding a selection marker, the polynucleotide sequence can be located downstream of IL-10 or CAR.

[0509] In some embodiments, a single polynucleotide construct comprising a polynucleotide segment encoding IL-10 and a polynucleotide segment encoding CAR comprises a sequence encoding IL-10 and a sequence encoding CAR from 5' to 3'. In such embodiments, the sequence encoding IL-10 and the sequence encoding CAR are operably connected to a single promoter (e.g., a constitutive or inducible promoter). 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 CAR.

[0510] In some embodiments, a single polynucleotide construct comprising a polynucleotide segment encoding IL-10 and a polynucleotide segment encoding CAR comprises a sequence encoding CAR and a sequence encoding IL-10 from 5' to 3'. In such embodiments, the sequence encoding CAR and the sequence encoding IL-10 are operably connected to a single promoter (e.g., a constitutive or inducible promoter). 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 CAR and the sequence encoding IL-10.

[0511] In some embodiments, the method comprises the steps of: (a) combining primary CD4 T cells obtained from one or more T cell donors; +(b) modifying the combined CD4 T cells by introducing a first exogenous polynucleotide segment encoding CAR and a second exogenous polynucleotide segment encoding IL-10 according to the methods provided herein + In other embodiments, the method comprises the following steps: (a) obtaining primary CD4 T cells from one or more T cell donors; + (b) modifying the CD4 T cells of each donor by introducing a first exogenous polynucleotide segment encoding CAR and a second exogenous polynucleotide segment encoding IL-10 (e.g., according to the methods provided herein); + T cells; and then combined with genetically modified CD4 + T cells, thereby acquiring CD4 IL -10 / CAR Cells. Various methods known in the art can be used to introduce the first exogenous polynucleotide segment encoding CAR, the second exogenous polynucleotide segment encoding IL-10, or both into primary CD4 + T cells.

[0512] In some embodiments, the method further comprises incubating primary CD4 + T cells or genetically modified CD4 + In some embodiments, the method further comprises incubating primary CD4 T cells in the presence of anti-CD3 antibody, anti-CD28 antibody and IL-2 or beads coated with anti-CD3 antibody and anti-CD28 antibody and IL-2. + T cells or genetically modified CD4 + In some embodiments, the method further comprises incubating primary CD4 T cells in the presence of a feeder cell mixture. + T cells or genetically modified CD4 + In some embodiments, the method further comprises incubating primary CD4 T cells in the presence of the nanoformulation of anti-CD3 antibody and anti-CD28 antibody. + T cells or genetically modified CD4 + In some embodiments, the incubation is performed in the presence of a polymer nanomatrix agent to activate and expand human T cells via CD3 and CD28. In some embodiments, CD4 IL-10 / CAR T cells are cultured in the presence of other T cell specific immune cell culture media, activators and supplements.

[0513] In some embodiments, the incubation step is performed before the first exogenous polynucleotide segment encoding CAR and the second exogenous polynucleotide segment encoding IL-10 are introduced according to the methods provided herein. In some embodiments, the incubation step is performed before (a) combining primary CD4 T cells obtained from one or more different T cell donors. + T cells; but in (b) modifying the combined CD4 T cells by introducing a first exogenous polynucleotide segment encoding 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) obtaining primary CD4 T cells from one or more different T cell donors. + T cells; but in (b) modifying the CD4 T cells of each donor by introducing a first exogenous polynucleotide segment encoding CAR and a second exogenous polynucleotide segment encoding IL-10 according to the method provided herein + T cells were performed before.

[0514] 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 which the combined CD4 T cells are modified by introducing a first exogenous polynucleotide segment encoding a CAR and a second exogenous polynucleotide segment encoding an IL-10 according to the methods provided herein. + 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 CAR and a second exogenous polynucleotide segment encoding IL-10 according to the methods provided herein. + T cells, but in combination with genetically modified CD4 + T cells were previously subjected to genetic modification to obtain CD4 + In some embodiments, the incubation step is performed after combining the genetically modified CD4 + T cells are then carried out to obtain CD4 IL-10 / CAR cell.

[0515] In some embodiments, the incubation step is performed more than once. In some embodiments, the incubation step is performed during CD4 + T cells before and after genetic modification.

[0516] In some embodiments, a viral vector is used to introduce the first exogenous polynucleotide segment, the second exogenous polynucleotide segment, or both into the primary CD4 + In some embodiments, the first exogenous polynucleotide segment and the second exogenous polynucleotide segment are located on the same viral vector. In some embodiments, the viral vector is a lentiviral vector.

[0517] In some embodiments, the first exogenous polynucleotide comprises a segment encoding a CAR having a sequence of SEQ ID NO: 9, 16, 22, 34, 41-49 and 54 and the second exogenous polynucleotide segment encodes an IL-10 having a 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 NO: 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.

[0518] In some embodiments, the CAR encoding polynucleotide segment has a sequence of SEQ ID NO: 10, 17, 23, 35 or 55, and the IL-10 encoding polynucleotide segment has a sequence of SEQ ID NO: 2. In some embodiments, the CAR encoding polynucleotide segment has at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity with SEQ ID NO: 10, 17, 22, 35 or 55, and the IL-10 encoding polynucleotide segment has at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity with SEQ ID NO: 2. In some embodiments, the first exogenous polynucleotide segment, the second exogenous polynucleotide segment, or both further comprises a segment encoding a marker that allows selection of successfully transduced CD4 + T cells. In some embodiments, the encoded selection marker is ΔNGFR. In certain embodiments, the encoded selection marker has the sequence of SEQ ID NO: 4. In some embodiments, the encoded selection marker is a truncated form of human EGFR polypeptide.

[0519] In some embodiments, the method further comprises isolating a genetically modified CD4 + Steps to generate enriched genetically modified CD4 IL-10 / CAR Cell population.

[0520] In some embodiments, 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 genetically modified CD4 +T cells express a selectable marker, wherein the selectable marker is a surrogate for IL-10 expression. In some embodiments, at least 75% (e.g., at least 80%, at least 90%, at least 95%, or at least 98%) of the genetically modified CD4 + The T cells express a selectable marker, wherein the selectable marker is a surrogate for IL-10 expression. In some embodiments, at least 75% of the genetically modified CD4 + T cells express a selectable marker. In some embodiments, at least 95% of the genetically modified CD4 + T cells express a selectable marker. In some embodiments, at least 96%, 97%, 98%, or 99% of the genetically modified CD4 + T cells express a selectable marker.

[0521] In some embodiments, at least 75% (e.g., at least 80%, at least 90%, at least 95%, or at least 98%) of the genetically modified CD4 + T cells express IL-10. In some embodiments, at least 75% of the genetically modified CD4 + T cells express IL-10. In some embodiments, at least 95% of the genetically modified CD4 + In some embodiments, at least 96%, 97%, 98%, or 99% of the genetically modified CD4 + T cells express IL-10.

[0522] In some embodiments, 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 genetically modified CD4 + In some embodiments, at least 50% of the genetically modified CD4 + In some embodiments, at least 75% of the genetically modified CD4 + In some embodiments, at least 95% of the genetically modified CD4 + In some embodiments, at least 96%, 97%, 98%, or 99% of the genetically modified CD4 + T cells express CAR.

[0523] In some embodiments, 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 genetically modified CD4 + T cells express IL-10 and CAR. In some embodiments, at least 50% of the genetically modified CD4 + T cells express IL-10 and CAR. In some embodiments, at least 75% of the genetically modified CD4 + T cells express IL-10 and CAR. In some embodiments, at least 95% of the genetically modified CD4 + T cells express IL-10 and CAR. In some embodiments, at least 96%, 97%, 98%, or 99% of the genetically modified CD4 + T cells express IL-10 and CAR.

[0524] In some embodiments, the method further comprises incubating the enriched genetically modified CD4 + In some embodiments, the incubation is performed in the presence of anti-CD3 antibody and anti-CD28 antibody, or beads coated with anti-CD3 antibody and anti-CD28 antibody. In some embodiments, the incubation is 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 nanoformulations of anti-CD3 antibody and anti-CD28 antibody. In some embodiments, the incubation is performed in the presence of polymer nanomatrix reagents to activate and expand human T cells through CD3 and CD28. In some embodiments, CD4 IL-10 / CAR T cells are cultured in the presence of other T cell specific immune cell culture media, activators and supplements.

[0525] In some embodiments, the method further comprises freezing the genetically modified CD4 + T cell steps.

[0526] In some embodiments, primary CD4 + The T cells are from a donor selected based on its HLA haplotype. In some embodiments, the method further comprises the step of selecting a T cell donor by analyzing its genetic information. In some embodiments, the method comprises the step of analyzing the genetic information or HLA haplotype of a potential T cell donor.

[0527] In some embodiments, primary CD4 + T cells from primary CD4+ The host treated with T cells or their modifications has a donor that is at least partially HLA-matched. In some embodiments, the primary CD4 + The T cells are from a donor that is at least partially HLA matched to the stem cell (HSC), tissue, or organ donor. In some embodiments, the CD4 + T cells are from a donor that is HLA matched to a stem cell (HSC), tissue, or organ donor. In some embodiments, primary CD4 + T cells are obtained from a third party donor who is not biologically related to the host. In some embodiments, primary CD4 + The T cells are obtained from a third party donor who is not biologically related to the stem cell, tissue or organ donor.

[0528] In some embodiments, in step (a), primary CD4 + T cells are obtained from two, three, four, five, six, seven, eight, nine or ten different T cell donors. In some embodiments, at least two T cell donors have at least 1 / 10, 2 / 10, 3 / 10, 4 / 10, 5 / 10, 6 / 10, 7 / 10, 8 / 10 or 9 / 10 matches with each other at HLA-A, HLA-B, HLA-C, HLA-DRB1 and HLA-DQB1 loci. In some embodiments, at least two T cell donors have at least 1 / 8, 2 / 8, 3 / 8, 4 / 8, 5 / 8, 6 / 8, 7 / 8 or 8 / 8 matches with each other at HLA-A, HLA-B, HLA-C and HLA-DRB1 loci. In some embodiments, at least two T cell donors have 2 / 2 matches with each other at HLA-A loci. In some embodiments, at least two T cell donors have 2 / 2 matches with each other at HLA-B loci. In some embodiments, at least two T cell donors have a 2 / 2 match to each other at the HLA-C locus. In some embodiments, at least two T cell donors have at least a 3 / 4 or 4 / 4 match to each other at the HLA-DRB1 and HLA-DQB1 loci. In some embodiments, each of at least two T cell donors has an A*02 or A*24 allele. In some embodiments, the T cell donor is HLA-A*02 negative. In some embodiments, each of at least two T cell donors is HLA-A*02 or HLA-A*24 negative.

[0529] In some embodiments, at least two T cell donors have less than 5 / 10, 6 / 10, 7 / 10, 8 / 10 or 9 / 10 matches with each other at HLA-A, HLA-B, HLA-C, HLA-DRB1 and HLA-DQB1 loci. In some embodiments, at least two T cell donors have less than 4 / 8, 5 / 8, 6 / 8, 7 / 8 or 8 / 8 matches with each other at HLA-A, HLA-B, HLA-C and HLA-DRB1 loci. In some embodiments, at least two T cell donors have less than 2 / 2 matches with each other at the HLA-A locus. In some embodiments, at least two T cell donors have less than 2 / 2 matches with each other at the HLA-B locus. In some embodiments, at least two T cell donors have less than 2 / 2 matches with each other at the HLA-C locus. In some embodiments, at least two T cell donors have less than 3 / 4 or 4 / 4 matches with each other at the HLA-DRB1 and HLA-DQB1 loci.

[0530] In some embodiments, in step (a), primary CD4 + T cells are obtained from one or more frozen stocks. In some embodiments, in step (a), primary CD4 + The T cells are obtained from unfrozen peripheral blood mononuclear cells of at least two different T cell donors. In some embodiments, the method further comprises isolating CD4 + In some embodiments, in step (a), primary CD4 + The T cells are obtained from a liquid suspension. In some embodiments, the liquid suspension is obtained from a previously frozen stock solution.

[0531] In some embodiments, the methods do not include CD4 + The step of de-reactive T cells in the presence of peripheral blood mononuclear cells (PBMC) from the host. In some embodiments, the method does not include allowing CD4 + The steps of T cell loss of responsiveness in the presence of recombinant IL-10 protein, wherein the recombinant IL-10 protein is not removed from CD4 + In some embodiments, the method does not include CD4 + The step whereby T cells lose their responsiveness in the presence of DC10 cells from the host.

[0532] In some embodiments, the methods provided herein include modifying the CD4 by introducing an exogenous polynucleotide segment encoding a CAR and an exogenous polynucleotide segment encoding an IL-10 after one or more of the steps of combining, purifying, restimulating, and expanding. +T cells or combined CD4 + T cells.

[0533] 6.7. Using CD4 IL-10 / CAR Cells or multiple donor CD4 IL-10 / CAR Methods

[0534] In another aspect, the present disclosure provides a method of treating a patient, comprising administering to a patient in need thereof any of the CD4 IL-10 / CAR cells (e.g., autologous single donor or allogeneic single donor), any allogeneic multiple donor CD4 IL-10 / CAR T cells, any of the multiple donor CD4 IL-10 / CAR cell population or any pharmaceutical composition provided herein.

[0535] In some embodiments, the method further comprises thawing the CD4 IL-10 / CAR A previous step of freezing the suspension of cells.

[0536] In some embodiments, the autologous single donor or allogeneic single donor CD4 IL-10 / CAR Allogeneic multi-donor CD4 IL-10 / CAR The cell or pharmaceutical composition prevents or reduces the severity of a pathogenic T cell response in a patient.

[0537] In some embodiments, multiple donor CD4 IL-10 The cell or pharmaceutical composition prevents or reduces the severity of an inflammatory or autoimmune response.

[0538] In some embodiments, multiple donor CD4 IL-10 / CAR The cell or pharmaceutical composition prevents or reduces the severity of a pathogenic T cell response in a patient. In some embodiments, multiple donor CD4 IL-10 / CAR Cells or pharmaceutical compositions reduce inflammation. In some embodiments, multiple donor CD4 IL-10 / CAR Cells or pharmaceutical compositions enhance tissue repair. In some embodiments, multiple donor CD4 IL-10 / CAR The cells or pharmaceutical compositions enhance immune tolerance to self and non-pathogenic antigens and maintain immune homeostasis. IL-10 / CAR The cells or pharmaceutical compositions downregulate pathogenic T cell responses associated with organ transplantation, GvHD, and various autoimmune and inflammatory diseases. IL-10 / CAR Cells or pharmaceutical compositions for treating autoimmune diseases. In some embodiments, multiple donor CD4 IL-10 / CARThe cell or pharmaceutical composition reduces the overactivity of the NLPR3 inflammasome or reduces symptoms associated with overactivity of the NLPR3 inflammasome. IL-10 / CAR The cell or pharmaceutical composition induces tumor cell death or reduces tumor growth. In some embodiments, multiple donor CD4 IL-10 / CAR The cell or pharmaceutical composition increases disease-free survival (e.g., absence of minimal residual disease). In some embodiments, multiple donor CD4 IL-10 / CAR The cells or pharmaceutical compositions induce wound healing or tissue repair.

[0539] In some embodiments, multiple donor CD4 IL-10 The cells or pharmaceutical compositions are administered in an amount effective to prevent or reduce the severity of a pathogenic T cell response in a patient. IL-10 The cells or pharmaceutical compositions are administered in an amount effective to reduce inflammation. IL-10 The cells or pharmaceutical compositions are administered in an amount effective to enhance tissue repair. IL-10 The cells or pharmaceutical compositions are administered in an amount effective to enhance immune tolerance to self and non-pathogenic antigens and maintain immune homeostasis. IL-10 The cells or pharmaceutical compositions are administered in an amount effective to downregulate pathogenic T cell responses associated with organ transplantation, GvHD, and various autoimmune or inflammatory diseases. IL-10 The cells or pharmaceutical compositions are administered in an amount effective to treat the autoimmune disease. IL -10 The cells or pharmaceutical compositions are administered in an amount effective to reduce overactivity of the NLPR3 inflammasome or to reduce symptoms associated with overactivity of the NLPR3 inflammasome. IL-10 The cells or pharmaceutical compositions are administered in an amount effective to induce tumor cell death or reduce tumor growth. IL-10 The cells or pharmaceutical compositions are administered in an amount effective to increase disease-free survival (eg, the absence of minimal residual disease).

[0540] In some embodiments, a single donor CD4 IL-10 / CAR Cells (e.g., autologous or allogeneic) or pharmaceutical compositions prevent or reduce the severity of a pathogenic T cell response in a patient. In some embodiments, a single donor CD4 IL-10 / CAR Cells or pharmaceutical compositions reduce inflammation. In some embodiments, a single donor CD4 IL-10 / CARCells or pharmaceutical compositions enhance tissue repair. In some embodiments, a single donor CD4 IL-10 / CAR The cell or pharmaceutical composition enhances immune tolerance to self and non-pathogenic antigens and maintains immune homeostasis. In some embodiments, a single donor CD4 IL -10 / CAR The cells or pharmaceutical compositions downregulate pathogenic T cell responses associated with organ transplantation, GvHD, and various autoimmune and inflammatory diseases. IL-10 / CAR Cells or pharmaceutical compositions for treating autoimmune diseases. In some embodiments, a single donor CD4 IL-10 / CAR The cell or pharmaceutical composition reduces the overactivity of the NLPR3 inflammasome or reduces symptoms associated with overactivity of the NLPR3 inflammasome. IL-10 / CAR The cell or pharmaceutical composition induces tumor cell death or reduces tumor growth. In some embodiments, a single donor CD4 IL -10 / CAR The cell or pharmaceutical composition increases disease-free survival (e.g., absence of minimal residual disease). In some embodiments, a single donor CD4 IL-10 / CAR The cells or pharmaceutical compositions induce wound healing or tissue repair.

[0541] In some embodiments, the method of treatment further comprises monitoring the CD4 IL-10 / CAR In some embodiments, the method comprises the steps of detecting a selected marker in a biological sample obtained from a patient, thereby detecting CD4 IL-10 / CAR Cells or multiple donor CD4 IL-10 / CAR In some embodiments, the method includes detecting a selectable marker in a patient and using the detection of the selectable marker to track and / or monitor the CD4 IL-10 / CAR In some embodiments, the markers are detected at multiple time points to track the CD4 IL -10 / CAR In some embodiments, the biological sample is a biopsy or a blood sample from a patient.

[0542] CD4 IL-10 / CAR Cells (autologous single donor, allogeneic single donor, or allogeneic multiple donor CD4 IL-10 / CAR Cells) are administered in a therapeutically effective amount. This amount can be determined based on body weight and other clinical factors. In some embodiments, 10 3 Up to 10 11 In some embodiments, administration of 10 3 Up to 10 10In some embodiments, administration of 10 3 Up to 10 9 In some embodiments, administration of 10 3 Up to 10 8 In some embodiments, administration of 10 3 Up to 10 7 In some embodiments, administration of 10 3 Up to 10 6 In some embodiments, administration of 10 3 Up to 10 5 In some embodiments, administration of 10 3 Up to 10 4 cells / kg.

[0543] In various embodiments, CD4 IL-10 / CAR Cells (autologous single donor, allogeneic single donor, or allogeneic multiple donor CD4 IL-10 / CAR In some embodiments, the CD4 IL-10 / CAR Cells (autologous single donor, allogeneic single donor, or allogeneic multiple donor CD4 IL-10 / CAR In some embodiments, CD4 IL-10 / CAR Cells (autologous single donor, allogeneic single donor, or allogeneic multiple donor CD4 IL-10 / CAR cells) are administered daily, every 3 days, every 7 days, every 14 days, every 21 days or monthly.

[0544] CD4 IL-10 / CAR Cells (autologous single donor, allogeneic single donor, or allogeneic multiple donor CD4 IL-10 / CAR Cells) can be administered according to different administration routes (e.g., systemic administration, subcutaneous administration, or intraperitoneal administration). In some embodiments, cells are administered in saline or physiological solution that may contain 2-20%, preferably 5%, human serum albumin.

[0545] 6.7.1. Methods to reduce or prevent GvHD

[0546] In some embodiments, CD4 IL-10 / CAR Cells (autologous single donor, allogeneic single donor, or allogeneic multiple donor CD4 IL-10 / CAR cells), single donor or multiple donor CD4 IL-10 / CAR The cell populations or any pharmaceutical compositions provided herein are used to treat a patient prior to hematopoietic stem cell (HSC) transplantation (HSCT), concurrently with HSCT, or after HSCT.

[0547] In various embodiments, the HSCT is a matched related HSCT, or a matched unrelated HSCT. In various embodiments, the HSCT is a haploidentical HSCT, an unmatched related HSCT, or an unmatched unrelated HSCT.

[0548] In some embodiments, the patient has a hematologic malignancy that requires treatment with allo-HSCT. In some embodiments, the hematologic malignancy is mediated by abnormal myeloid cells. In some embodiments, the malignancy or hematologic cancer is a myeloid leukemia. In some embodiments, the malignancy or hematologic cancer is a CD19 + 、CD20 + 、CD22 + , BCMA + or B7-H3 + Hematologic cancers. In some embodiments, CD19 + 、CD20 + 、CD22 + or B7-H3 + The hematological cancer is selected from the group consisting of chronic lymphocytic leukemia, acute lymphoblastic leukemia (ALL) and non-Hodgkin's lymphoma.

[0549] In some embodiments, based on the CD4 IL-10 / CAR (autologous single donor or allogeneic single donor) or allogeneic multiple donor CD4 IL-10 / CAR T cell donors are selected based on the genetic information of the patient to be treated with cells and HSCs, and / or the genetic information of the HSC donor. In some embodiments, the T cell donors are selected based on the multiple donor CD4 IL-10 The T cell donor is selected by using the HLA haplotype of the patient to be treated with cells and HSC and / or the HLA haplotype of the HSC donor. In some embodiments, the method further comprises administering a CD4 IL-10 In some embodiments, the method further comprises the step of analyzing the genetic information or HLA haplotype of the T cell donor before the cells are grown. In some embodiments, the method further comprises the step of analyzing the genetic information or HLA haplotype of the host. In some embodiments, the method further comprises the step of analyzing the genetic information or HLA haplotype of the HSC donor.

[0550] In some embodiments, the T cell donor, the host, and the HSC donor are not biologically related. In some embodiments, the T cell donor, the host, and the HSC donor have different HLA haplotypes. In some embodiments, the T cell donor, the host, and the HSC donor are at least partially mismatched in HLA haplotype. In some embodiments, the T cell donor is selected when the T cell donor has an HLA haplotype with an HLA match exceeding a threshold.

[0551] In some embodiments, the HSC donor is partially HLA-unmatched with the patient. In some embodiments, the HSC donor has a match of less than 5 / 10, 6 / 10, 7 / 10, 8 / 10, 9 / 10 or 10 / 10 with the patient at the HLA-A, HLA-B, HLA-C, HLA-DRB1 and HLA-DQB1 loci. In some embodiments, the HSC donor has a match of less than 4 / 8, 5 / 8, 6 / 8, 7 / 8 or 8 / 8 with the patient at the HLA-A, HLA-B, HLA-C and HLA-DRB1 loci. In some embodiments, the HSC donor has a match of less than 2 / 2 with the patient at the HLA-A, HLA-B or HLA-C loci. In some embodiments, the HSC donor has a match of less than 3 / 4 or 4 / 4 with the patient at the HLA-DRB1 and HLA-DQB1 loci.

[0552] In some embodiments, one or more of the T cell donors are HLA-unmatched or partially HLA-unmatched with the patient. In some embodiments, one or more of the T cell donors have a match of less than 5 / 10, 6 / 10, 7 / 10, 8 / 10, 9 / 10, or 10 / 10 with the patient at the HLA-A, HLA-B, HLA-C, HLA-DRB1, and HLA-DQB1 loci. In some embodiments, one or more of the T cell donors have a match of less than 4 / 8, 5 / 8, 6 / 8, 7 / 8, or 8 / 8 with the patient at the HLA-A, HLA-B, HLA-C, and HLA-DRB1 loci. In some embodiments, one or more of the T cell donors have a match of less than 2 / 2 with the patient at the HLA-A, HLA-B, or HLA-C loci. In some embodiments, one or more of the T cell donors has less than a 2 / 4, 3 / 4, or 4 / 4 match to the patient at the HLA-DRB1 and HLA-DQB1 loci.

[0553] In some embodiments, one or more of the T cell donors are HLA-unmatched or partially HLA-unmatched with the HSC donor. In some embodiments, one or more of the T cell donors have a match of less than 5 / 10, 6 / 10, 7 / 10, 8 / 10, 9 / 10, or 10 / 10 with the HSC donor at the HLA-A, HLA-B, HLA-C, HLA-DRB1, and HLA-DQB1 loci. In some embodiments, one or more of the T cell donors have a match of less than 4 / 8, 5 / 8, 6 / 8, 7 / 8, or 8 / 8 with the HSC donor at the HLA-A, HLA-B, HLA-C, and HLA-DRB1 loci. In some embodiments, one or more of the T cell donors have a match of less than 2 / 2 with the HSC donor at the HLA-A, HLA-B, or HLA-C loci. In some embodiments, one or more of the T cell donors has less than 3 / 4 or 4 / 4 match with the HSC donor at the HLA-DRB1 and HLA-DQB1 loci.

[0554] In some embodiments, when administered to a patient, the CD4 IL-10 / CAR Cells (single or multiple donor CD4 IL-10 / CAR cells), single donor or multiple donor CD4 IL-10 / CAR The cell population or any pharmaceutical composition provided herein prevents GvHD of transplanted hematopoietic stem cells or reduces the severity of GvHD of transplanted hematopoietic stem cells.

[0555] In some embodiments, when administered to a patient, the CD4 IL-10 / CAR Cells (single or multiple donor CD4 IL-10 / CAR cells), single donor or multiple donor CD4 IL-10 / CAR The cell population or any pharmaceutical composition provided herein prevents or reduces the severity of a pathological T cell response of transplanted hematopoietic cells. In a specific embodiment, CD4 IL-10 / CAR or multiple donor CD4 IL-10 / CAR cells to prevent or reduce GvHD.

[0556] In some embodiments, multiple donor CD4 IL-10 The cell or pharmaceutical composition prevents or reduces the severity of tissue damage caused by pathogenic T cells or inflammation.

[0557] 6.7.2. Methods for treating cancer

[0558] In some embodiments, CD4 IL-10 / CAR Cells (autologous single donor, allogeneic single donor, or allogeneic multiple donor CD4 IL-10 / CARcells), single donor or multiple donor CD4 IL-10 / CAR The cell population or any pharmaceutical composition provided herein is used to treat cancer. In some embodiments, CD4 IL-10 / CAR Cells (autologous single donor, allogeneic single donor, or allogeneic multiple donor CD4 IL-10 / CAR cells), single donor or multiple donor CD4 IL-10 / CAR The cell population or any pharmaceutical composition provided herein is used to treat a malignant tumor.

[0559] In a preferred embodiment, CD4 IL-10 / CAR Cells (autologous single donor, allogeneic single donor, or allogeneic multiple donor CD4 IL-10 / CAR cells) directly mediate anti-tumor effects, and in certain embodiments, anti-leukemic effects.

[0560] In some embodiments, CD4 IL-10 / CAR Cells (autologous single donor, allogeneic single donor, or allogeneic multiple donor CD4 IL-10 / CAR cells), single donor or multiple donor CD4 IL-10 / CAR The cell population or any pharmaceutical composition provided herein and the allogeneic are administered before or after hematopoietic stem cell transplantation (HSCT), peripheral blood stem cell (PBSC) transplantation, cord blood (CB) transplantation, or bone marrow (BM) transplantation.

[0561] 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-I class. In some embodiments, the neoplastic cells express CD54. In some embodiments, the neoplastic cells express CD13, HLA-I class 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.

[0562] In some embodiments, the patient has a cancer selected from the group consisting of: adrenal cancer, anal cancer, bile duct cancer, bladder cancer, bone cancer, adult brain / CNS tumors, childhood brain / CNS tumors, breast cancer, male breast cancer, cancer of unknown primary site, Castleman's disease, cervical cancer, colon / rectum cancer, endometrial cancer, esophageal cancer, Ewing 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, laryngeal and hypopharyngeal cancer, leukemia, acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML, including myeloid sarcoma and cutaneous leukemia), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), chronic myeloid leukemia (CML), chronic myelomonocytic ... CMML, childhood leukemia, liver cancer, lung cancer, non-small cell lung cancer, small cell lung cancer, carcinoid tumors of the lung, lymphoma, lymphoma of the skin, malignant mesothelioma, multiple myeloma, myelodysplastic syndrome, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, non-Hodgkin lymphoma, childhood non-Hodgkin lymphoma, oral cavity and oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, penile cancer, pituitary tumors, prostate cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma - adult soft tissue cancer, skin cancer, skin cancer - basal cell carcinoma and squamous cell carcinoma, skin cancer - melanoma, skin cancer - Merkel cell carcinoma, small intestine cancer, stomach cancer, testicular cancer, thymic cancer, thyroid cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Waldenstrom's macroglobulinemia and Wilms' tumor.

[0563] In some embodiments, the cancer is a myeloid tumor. In specific embodiments, the cancer is AML or CML. In some embodiments, the cancer is a myeloid tumor. In some embodiments, the cancer is ALL.

[0564] In some embodiments, the method is used to treat a hematologic cancer that affects the blood, bone marrow, and lymph nodes. In various embodiments, the hematologic cancer is a lymphoma (e.g., Hodgkin's lymphoma), a lymphocytic leukemia, a myeloma. In various embodiments, the hematologic cancer is an acute or chronic myeloid (myeloid) leukemia (AML, CML) or a myelodysplastic syndrome.

[0565] In some embodiments, the malignancy or hematological cancer is CD19 + 、CD20 + 、CD22 + , BCMA + or B7-H3 + Hematologic cancers. In some embodiments, CD19 + 、CD20 + 、CD22 + , BCMA +or B7-H3 + Hematological cancers are chronic lymphocytic leukemia, acute lymphoblastic leukemia (ALL) and non-Hodgkin lymphoma.

[0566] In some embodiments, CD4 IL-10 / CAR Cells (autologous single donor, allogeneic single donor, or allogeneic multiple donor CD4 IL-10 / CAR cells) for treating a patient with a hematological cancer, wherein the method comprises administering to the patient a therapeutically effective amount of CD4 IL-10 / CAR Cells (autologous single donor, allogeneic single donor, or allogeneic multiple donor CD4 IL-10 / CAR cells), single donor or multiple donor CD4 IL-10 / CAR Cell populations or any pharmaceutical compositions provided herein.

[0567] In some embodiments, CD4 IL-10 / CAR Cells for the treatment of patients with CD19 + A patient with a hematological cancer, wherein the method comprises administering to the patient a therapeutically effective amount of CD4 IL-10 / 抗CD19 CAR Cells (autologous single donor, allogeneic single donor, or allogeneic multiple donor CD4 IL-10 / 抗CD19 CAR cells), single donor or multiple donor CD4 IL-10 / 抗CD19 CAR Cell populations or any pharmaceutical compositions provided herein.

[0568] In some embodiments, CD4 IL-10 / CAR Cells for the treatment of patients with CD20 + A patient with a hematological cancer, wherein the method comprises administering to the patient a therapeutically effective amount of CD4 IL-10 / 抗CD20 CAR Cells (autologous single donor, allogeneic single donor, or allogeneic multiple donor CD4 IL-10 / 抗CD20 CAR cells), single donor or multiple donor CD4 IL-10 / 抗CD20 CAR Cell populations or any pharmaceutical compositions provided herein.

[0569] In some embodiments, CD4 IL-10 / CAR Cells for the treatment of patients with CD22 + A patient with a hematological cancer, wherein the method comprises administering to the patient a therapeutically effective amount of CD4 IL-10 / 抗CD22 CAR Cells (autologous single donor, allogeneic single donor, or allogeneic multiple donor CD4 IL-10 / 抗CD22 CAR cells), single donor or multiple donor CD4 IL-10 / 抗CD22 CAR Cell populations or any pharmaceutical compositions provided herein.

[0570] In some embodiments, CD4 IL-10 / CAR Cells for the treatment of B7-H3 +A patient with cancer (e.g., a solid cancer), wherein the method comprises administering to the patient a therapeutically effective amount of CD4 IL-10 / 抗B7-H3CAR Cells (autologous single donor, allogeneic single donor, or allogeneic multiple donor CD4 IL-10 / 抗B7-H3 CAR cells), single donor or multiple donor CD4 IL-10 / 抗B7-H3 CAR Cell populations or any pharmaceutical compositions provided herein.

[0571] In some embodiments, CD4 IL-10 / CAR Cells for the treatment of BCMA + A patient with cancer (e.g., a solid cancer), wherein the method comprises administering to the patient a therapeutically effective amount of CD4 IL-10 / 抗BCMA CAR Cells (autologous single donor, allogeneic single donor, or allogeneic multiple donor CD4 IL-10 / 抗BCMA CAR cells), single donor or multiple donor CD4 IL-10 / 抗BCMACAR Cell populations or any pharmaceutical compositions provided herein.

[0572] In some embodiments, the cancer is refractory or resistant to therapeutic intervention.

[0573] In some embodiments, the methods provided herein are used to prevent CD19 + 、CD20 + 、CD22 + , BCMA + or B7-H3 + Relapse of hematologic and solid cancers, including: + 、CD20 + 、CD22 + , BCMA + or B7-H3 + Hematologic cancer or solid cancer or in patients with CD19 + 、CD20 + 、CD22 + , BCMA + or B7-H3 + Administration of a therapeutically effective amount of CD4+ sufficient to induce an anticancer effect to patients at risk of recurrence of hematologic cancer or solid cancer IL-10 / CAR Cells (autologous single donor, allogeneic single donor, or allogeneic multiple donor CD4 IL-10 / CAR cells), single donor or multiple donor CD4 IL-10 / CAR Cell populations or any pharmaceutical compositions provided herein.

[0574] In some embodiments, CD4 IL-10 / CAR Cells (autologous single donor, allogeneic single donor, or allogeneic multiple donor CD4 IL-10 / CARCells) are used in combination with therapeutic interventions. The combination can be performed simultaneously or at different times. Preferably, the therapeutic intervention is selected from the group consisting of chemotherapy, radiotherapy, allo-HSCT, immunosuppression, blood transfusion, bone marrow transplantation, growth factors, and biological agents.

[0575] In some embodiments, CD4 IL-10 / CAR Cells (autologous single donor, allogeneic single donor, or allogeneic multiple donor CD4 IL-10 / CAR cells) for treating a patient suffering from a malignant tumor, wherein the method comprises administering to the patient an allo-HSCT transplant, and administering to the patient a therapeutically effective amount of CD4 IL-10 / CAR Cells (autologous single donor, allogeneic single donor, or allogeneic multiple donor CD4 IL-10 / CAR cells), single donor or multiple donor CD4 IL-10 / CAR Cell populations or any pharmaceutical compositions provided herein.

[0576] In some embodiments, CD4 IL-10 / CAR Cells (autologous single donor, allogeneic single donor, or allogeneic multiple donor CD4 IL-10 / CAR CD4 cells) are used to treat cancer patients undergoing allogeneic HSCT (allo-HSCT) to prevent GvHD and induce long-term tolerance (in addition to direct antitumor effects). IL-10 / CAR The amount of cells is sufficient to inhibit or prevent graft-versus-host disease (GvHD) without inhibiting the graft-versus-leukemia (GvL) or graft-versus-tumor (GvT) efficacy of allo-HSCT (mediated by donor T cells present in the stem cell preparation).

[0577] Also provided herein is a method for treating a patient with minimal residual disease, comprising: administering to a patient identified as having minimal residual disease or at risk of having minimal residual disease a therapeutically effective amount of CD4 IL-10 / CAR Cells (autologous single donor, allogeneic single donor, or allogeneic multiple donor CD4 IL-10 / CAR cells), single donor or multiple donor CD4 IL-10 / CAR Cell populations or any pharmaceutical compositions provided herein.

[0578] In some embodiments, CD4 IL-10 / CAR Cells (autologous single donor, allogeneic single donor, or allogeneic multiple donor CD4 IL-10 / CAR cells), single donor or multiple donor CD4 IL-10 / CAR The cell populations or any pharmaceutical compositions provided herein induce cell death of myeloid cells (eg, monocytes, macrophages, neutrophils) that infiltrate tumors and promote tumor growth.

[0579] In some embodiments, CD4 IL-10 / CAR Cells (autologous single donor, allogeneic single donor, or allogeneic multiple donor CD4 IL-10 / CAR cells), single donor or multiple donor CD4 IL-10 / CAR Cell populations or any pharmaceutical compositions provided herein induce B cells (e.g., CD19 + B cells, CD20 + B cells, CD22 + B cells, BCMA + 、B7-H3 + B cells or B7-H3 + solid tumor cells) cell death.

[0580] 6.7.3. Methods for treating inflammatory diseases or autoimmune diseases

[0581] In some embodiments, administration of single or multiple donor CD4 IL-10 / CAR cells to treat inflammatory diseases or autoimmune diseases. In some embodiments, administration of single or multiple donor CD4 IL-10 / CAR The cells can be used to treat diseases or disorders involving overactivity of the NLPR3 inflammasome.

[0582] The NOD-like receptor family (NLR) protein NLRP3 is an intracellular signaling molecule that senses danger signals from pathogenic, environmental or endogenous sources. Upon activation, NLPR3 interacts with caspase 1 to form a complex called the inflammasome. This leads to the activation of caspase 1, which cleaves the proinflammatory cytokines IL-1β and IL-18 into their active forms and mediates a type of inflammatory cell death called pyroptosis.

[0583] In some embodiments, administration of single or multiple donor CD4 IL-10 / CAR Cells are used to treat an inflammatory disease selected from the group consisting of Muckle-Wells syndrome (MWS), familial cold autoinflammatory syndrome (FCAS), and neonatal-onset multisystem inflammatory disease (NOMID). In some embodiments, administration of single or multiple donor CD4 IL -10 / CAR Cells are used to treat a chronic disease selected from the group consisting of 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, administration of single or multiple donor CD4 IL-10 / CAR cells to treat Crohn's disease, ulcerative colitis, multiple sclerosis and systemic lupus erythematosus or inflammatory eye diseases such as diabetic retinopathy, acute glaucoma and age-related macular degeneration.

[0584] In some embodiments, administration of single or multiple donor CD4 IL-10 / CAR Cells are used to treat diseases associated with NLRP3. The disease can be selected from the group consisting of CAPS, NASH, Alzheimer's disease, Parkinson's disease, cardiovascular disease, osteoarthritis, gout, pseudogout, nephrocalcinosis, type II diabetes, Sjögren's syndrome, sickle cell disease (SCD), AMD, infection, cerebral malaria, asbestosis, contact hypersensitivity, sunburn, silicosis, cystic fibrosis, inflammatory bowel disease, ALS, myelodysplastic syndrome and uveitis.

[0585] In some embodiments, the disease is a brain disease selected from the group consisting of Parkinson's disease, Alzheimer's disease, age-related cognitive impairment, frontotemporal dementia, traumatic brain injury, cerebral hemorrhage, sepsis-related encephalopathy, cerebral ischemia, subarachnoid hemorrhage, epilepsy, acrylamide poisoning, opioid-induced neuroinflammation, chronic migraine, perioperative neurocognitive disorder, post-stroke cognitive impairment, post-cardiac arrest cognitive impairment, social isolation-induced cognitive impairment, anxiety, and post-traumatic stress disorder.

[0586] In some embodiments, the disease is a pulmonary disease selected from the group consisting of asthma, IR lung injury, ARDS / COPD, particulate matter-induced lung injury, radiation pneumonitis, pulmonary hypertension, sarcoidosis, cystic fibrosis, and allergic rhinitis.

[0587] In some embodiments, the disease is a cardiac disease selected from the group consisting of atherosclerosis, heart failure, hypertension, myocardial infarction, atrial fibrillation, cardiac damage caused by metabolic dysfunction, heart failure, and endothelial dysfunction.

[0588] In some embodiments, the disease is a gastrointestinal disease, such as colitis. In some embodiments, the disease is a liver disease selected from the following: acute liver failure, immune circadian rhythm regulation, NASH, diabetic cognitive dysfunction, IR liver injury, liver injury caused by idiopathic drugs, and liver fibrosis. In some embodiments, the disease is a pancreatic or renal disease selected from the following: diabetic encephalopathy, diabetic-related atherosclerosis, insulin resistance, islet transplant rejection, chronic crystalline nephropathy, renal fibrosis, I / R renal injury, obesity-related nephropathy, and renal hypertension. In some embodiments, the disease is a skin or eye disease selected from the following: psoriasis and retinal angiogenesis. In some embodiments, the disease is a reproductive disorder, such as premature birth. In some embodiments, the disease is an immune disorder selected from the following: primary dysmenorrhea, innate immunity, innate to adaptive immunity, systemic lupus erythematosus-lupus nephritis, and multiple sclerosis. In some embodiments, the disease is a hereditary disease selected from the following: Muckle-Wells syndrome, rheumatoid arthritis, sickle cell disease, and VCP-related diseases. In some embodiments, the disease is a painful disease selected from the group consisting of: multiple sclerosis-related neuropathic pain, chronic prostatitis / chronic pelvic pain, bone pain caused by cancer, and hyperalgesia. In some embodiments, the disease is cancer, such as human head and neck squamous cell carcinoma. In some embodiments, the disease is an infectious disease, such as a bacterial, viral, or parasitic infection.

[0589] In some embodiments, single or multiple donor CD4 IL-10 / CAR The cells were used in combination with currently available treatments for NLRP3-related diseases, such as biologics targeting IL-1, including the recombinant IL-1 receptor antagonist anakinra, the IL-1β neutralizing antibody canakinumab, and the soluble decoy IL-1 receptor rilonacept.

[0590] In some embodiments, administration of single or multiple donor CD4 IL-10 / CAR The cells are used to treat a disease selected from the group consisting of type 2 diabetes, metabolic syndrome, cardiovascular disease, SLE, MS, CD, ulcerative colitis (UC), osteoarthritis, nonalcoholic steatohepatitis (Nash), Parkinson's disease, ALS, pulmonary fibrosis, silicosis, asbestosis, diabetic retinopathy, and age-related macular degeneration.

[0591] In some embodiments, administration of single or multiple donor CD4 IL-10 / CAR Cells to treat inflammation. Inflammation may be associated with, but not limited to, coronary artery disease (CAD), type 2 diabetes, neurodegenerative diseases, or inflammatory bowel disease.

[0592] In some embodiments, administration of single or multiple donor CD4 IL-10 / CAR Cells are used to treat diseases or disorders involving increased IL-1β production by activated monocytes, macrophages, or dendritic cells. In some embodiments, administration of single or multiple donor CD4 IL-10 / CAR Cells are used to treat diseases or disorders involving increased IL-18 production by activated monocytes, macrophages, or dendritic cells. In some embodiments, administration of single or multiple donor CD4 IL-10 / CAR The cells are used to treat a disease or disorder involving increased production of mature caspase 1 by activated monocytes, macrophages or dendritic cells.

[0593] In some embodiments, administration of single or multiple donor CD4 IL-10 / CAR cells to reduce the production of IL-1β by activated monocytes, macrophages or dendritic cells. In some embodiments, administration of single or multiple donor CD4 IL-10 / CAR cells to reduce the production of IL-18 by activated monocytes, macrophages or dendritic cells. In some embodiments, administration of single or multiple donor CD4 IL-10 / CAR cells to reduce the production of mature caspase 1 by activated monocytes, macrophages or dendritic cells.

[0594] 6.7.4. Methods for treating other diseases

[0595] In some embodiments, CD4 IL-10 / CAR Cells (autologous single donor, allogeneic single donor, or allogeneic multiple donor CD4 IL-10 / CAR In some embodiments, the methods provided herein comprise administering to a patient in need of immune tolerance a therapeutically effective amount of CD4 IL-10 / CAR Cells (autologous single donor, allogeneic single donor, or allogeneic multiple donor CD4 IL-10 / CAR cells), single donor or multiple donor CD4 IL-10 / CAR Cell populations or any pharmaceutical compositions provided herein.

[0596] In some embodiments, CD4 IL-10 / CAR Cells (autologous single donor, allogeneic single donor, or allogeneic multiple donor CD4 IL-10 / CAR cells), single donor or multiple donor CD4 IL-10 / CAR Cell populations or any pharmaceutical composition provided herein to treat autoimmune diseases.

[0597] In some embodiments, the 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, pemphigoid disease (vulgaris and deciduous), epidermolysis bullosa acquisita, thrombotic thrombocytopenic purpura, idiopathic thrombocytopenic purpura, autoantibody-induced hemoglobinuria ...immune uveitis, psoriasis, vitiligo, vitiligo, vitiligo, vitiligo, vitiligo, vitiligo In some embodiments, the patient suffers from an allergic disease or atopic disease. ...

[0598] In some embodiments, administration of CD4 IL-10 / CAR Cells (autologous single donor, allogeneic single donor, or allogeneic multiple donor CD4 IL-10 / CAR cells), single donor or multiple donor CD4 IL-10 / CAR Cell populations or any pharmaceutical compositions provided herein to prevent or reduce the severity of pathogenic T cell responses to cell and organ transplantation other than HSCT. In some embodiments, the method comprises administering a CD4 IL -10 / CAR Cells (autologous single donor, allogeneic single donor, or allogeneic multiple donor CD4 IL-10 / CAR cells), single donor or multiple donor CD4 IL-10 / CAR In some embodiments, the organ is a kidney, heart, lung, liver or pancreatic islet cells. In a preferred embodiment, the CD4 IL -10 / CAR Cells (autologous single donor, allogeneic single donor, or allogeneic multiple donor CD4 IL-10 / CAR cells), single donor or multiple donor CD4 IL-10 / CARThe cell population or any pharmaceutical composition provided herein prevents or reduces the severity of host rejection of an organ transplant.

[0599] In some embodiments, administration of CD4 IL-10 / CAR Cells (autologous single donor, allogeneic single donor, or allogeneic multiple donor CD4 IL-10 / CAR cells), single donor or multiple donor CD4 IL-10 / CAR Cell populations or any pharmaceutical composition provided herein to prevent or reduce an immune response associated with gene therapy (e.g., 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 further comprises administering a CD4 IL-10 / CAR A step of administering to the patient 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 before or after the cell or pharmaceutical composition.

[0600] In some embodiments, administration of CD4 IL-10 / CAR Cells (autologous single donor, allogeneic single donor, or allogeneic multiple donor CD4 IL-10 / CAR cells), single donor or multiple donor CD4 IL-10 / CAR Cell populations or any pharmaceutical composition provided herein to prevent or reduce an immune response to a recombinant viral vector other than AAV. In these embodiments, the method further comprises administering a CD4 IL-10 / CAR cells, CD4 IL-10 / CAR Before or after the cell population or pharmaceutical composition, a step of administering a recombinant viral vector other than AAV to the patient. 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.

[0601] In some embodiments, administration of CD4 IL-10 / CAR Cells (autologous single donor, allogeneic single donor, or allogeneic multiple donor CD4 IL-10 / CAR cells), single donor or multiple donor CD4 IL-10 / CAR Cell populations or any pharmaceutical compositions provided herein to prevent or reduce immune responses associated with transplantation of iPS-derived tissues or cells. iPS-derived tissues and cells include, but are not limited to, cardiomyocytes, hepatocytes, epithelial cells, cartilage, bone and muscle cells, neurons.

[0602] In some embodiments, the method further comprises administering a CD4 IL-10 / CAR cells, CD4 IL-10 / CAR The step of administering an immunogenic therapeutic protein to a patient before or after administering the immunogenic therapeutic protein to the patient. IL -10 / CAR cells, CD4 IL-10 / CAR The cell population or pharmaceutical composition reduces the immune response to the immunogenic therapeutic protein.In some embodiments, the immunogenic therapeutic protein is selected from the group consisting of a therapeutic antibody, a Factor VIII replacement, a cytokine, and a cytokine mutein.

[0603] In some embodiments, administration of CD4 IL-10 / CAR Cells (autologous single donor, allogeneic single donor, or allogeneic multiple donor CD4 IL-10 / CAR cells), single donor or multiple donor CD4 IL-10 / CAR Cell populations or any pharmaceutical composition provided herein to treat inflammation. Inflammation may be associated with, but not limited to, coronary artery disease (CAD), type 2 diabetes, neurodegenerative diseases, nonalcoholic steatohepatitis (NASH), or inflammatory bowel disease.

[0604] In some embodiments, administration of CD4 IL-10 / CAR Cells (autologous single donor, allogeneic single donor, or allogeneic multiple donor CD4 IL-10 / CAR cells), single donor or multiple donor CD4 IL-10 / CAR Cell populations or any pharmaceutical compositions provided herein to treat diseases or disorders involving overactivity of the NLPR3 inflammasome. In some embodiments, administration of CD4 IL -10 / CAR Cells (autologous single donor, allogeneic single donor, or allogeneic multiple donor CD4 IL-10 / CAR cells), single donor or multiple donor CD4 IL-10 / CAR Cell populations or any pharmaceutical composition provided herein to treat a disease or disorder involving increased IL-1β production by activated monocytes, macrophages, or dendritic cells. In some embodiments, administration of CD4 IL-10 / CAR Cells (autologous single donor, allogeneic single donor, or allogeneic multiple donor CD4 IL-10 / CAR cells), single donor or multiple donor CD4 IL-10 / CAR Cell populations or any pharmaceutical composition provided herein to treat a disease or disorder involving increased IL-18 production by activated monocytes, macrophages, or dendritic cells. In some embodiments, administration of CD4 IL-10 / CAR Cells (autologous single donor, allogeneic single donor, or allogeneic multiple donor CD4 IL-10 / CAR cells), single donor or multiple donor CD4 IL-10 / CARThe cell population or any pharmaceutical composition provided herein is for treating a disease or disorder involving increased production of caspase 1 by activated monocytes, macrophages or dendritic cells.

[0605] In some embodiments, administration of CD4 IL-10 / CAR Cells (autologous single donor, allogeneic single donor, or allogeneic multiple donor CD4 IL-10 / CAR cells), single donor or multiple donor CD4 IL-10 / CAR Cell populations or any pharmaceutical composition provided herein to reduce the production of IL-1β by activated monocytes, macrophages or dendritic cells. In some embodiments, administration of CD4 IL-10 / CAR Cells (autologous single donor, allogeneic single donor, or allogeneic multiple donor CD4 IL-10 / CAR cells), single donor or multiple donor CD4 IL-10 / CAR Cell populations or any pharmaceutical composition provided herein to reduce the production of IL-18 by activated monocytes, macrophages or dendritic cells. In some embodiments, administration of CD4 IL-10 / CAR Cells (autologous single donor, allogeneic single donor, or allogeneic multiple donor CD4 IL-10 / CAR cells), single donor or multiple donor CD4 IL-10 / CAR The cell population or any pharmaceutical composition provided herein is used to reduce the production of mature caspase 1 by activated monocytes, macrophages or dendritic cells.

[0606] In some embodiments, administration of CD4 IL-10 / CAR Cells (autologous single donor, allogeneic single donor, or allogeneic multiple donor CD4 IL-10 / CAR cells), single donor or multiple donor CD4 IL-10 / CAR Cell populations or any pharmaceutical composition provided herein to reduce an overactive immune response to a viral infection in a patient. In some embodiments, the virus is SARS-coV-2. In some embodiments, administration of CD4 IL-10 / CAR Cells (autologous single donor, allogeneic single donor, or allogeneic multiple donor CD4 IL-10 / CAR cells), single donor or multiple donor CD4 IL-10 / CAR Cell populations or any pharmaceutical composition provided herein to reduce an overactive immune response to a bacterial infection (e.g., toxic shock, cytokine storm, therapeutic antibodies, Factor VIII replacement, cytokines and cytokine muteins).

[0607] In another aspect, the present disclosure provides a method for treating or inhibiting an autoimmune disease, an allergic disease, or an inflammatory disease in a patient, comprising administering to a patient identified as having an autoimmune disease, an allergic disease, or an inflammatory disease a therapeutically effective amount of a CD4+ T cell receptor sufficient to treat or inhibit the autoimmune disease, an allergic disease, or an inflammatory disease. IL-10 / CAR Cells (autologous single donor, allogeneic single donor, or allogeneic multiple donor CD4 IL-10 / CAR cells), single donor or multiple donor CD4 IL-10 / CAR Cell populations or any pharmaceutical compositions provided herein.

[0608] In another aspect, the present disclosure provides a method for reducing transplant rejection in a patient transplanted with hematopoietic stem cells, bone marrow cells or solid organs, comprising administering to a patient identified as having rejection of the transplanted hematopoietic stem cells, bone marrow cells or solid organs a therapeutically effective amount of CD4 IL-10 / CAR Cells (autologous single donor, allogeneic single donor, or allogeneic multiple donor CD4 IL-10 / CAR cells), single donor or multiple donor CD4 IL-10 / CAR Cell populations or any pharmaceutical compositions provided herein.

[0609] In another aspect, the present disclosure provides a method for treating graft-versus-host disease (GvHD) in a patient, comprising: administering to a patient identified as having or at risk of having graft-versus-host disease (GvHD) a therapeutically effective amount of CD4 T cells sufficient to inhibit or prevent GvHD. IL-10 / CAR Cells (autologous single donor, allogeneic single donor, or allogeneic multiple donor CD4 IL-10 / CAR cells), single donor or multiple donor CD4 IL-10 / CAR Cell populations or any pharmaceutical composition provided herein. In some embodiments, GvHD is acute GvHD. In some embodiments, GvHD is chronic GvHD.

[0610] In another aspect, the disclosure features a method for treating tissue or organ damage (e.g., wound healing) in a patient, comprising: administering to a patient identified as having or at risk of having tissue or organ damage a therapeutically effective amount of any of the CD4 T cells provided herein sufficient to induce repair of the tissue or organ damage. IL-10 / CAR cells, any CD4 IL-10 / CAR Cell populations or pharmaceutical compositions provided herein.

[0611] 6.8. Polynucleotides and vectors

[0612] The present disclosure is also characterized by 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 form of NGFR. In some embodiments, the first polynucleotide segment and the second and third polynucleotides are in the same construct. In some embodiments, the first polynucleotide is in the first polynucleotide construct, and the second and third polynucleotide segments are in the second polynucleotide construct. In some embodiments, the third polynucleotide segment is optional.

[0613] 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 the expression of the CAR.

[0614] In some embodiments, the second 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 IL-10. In some embodiments, the regulatory element drives constitutive expression of IL-10.

[0615] In some embodiments, the third polynucleotide segment comprises a regulatory element (e.g., any exemplary regulatory element described herein (e.g., any promoter)) operably linked to a coding sequence for a selectable marker (e.g., ΔNGFR). In some embodiments, the regulatory element drives constitutive expression of ΔNGFR.

[0616] 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.

[0617] In some embodiments, the polynucleotide construct comprises from 5' to 3': a promoter, a self-cleaving peptide or an IRES, and a second polynucleotide segment operably linked to the first polynucleotide segment. In some embodiments, the polynucleotide construct comprises from 5' to 3': a promoter, a self-cleaving peptide or an IRES, and a second polynucleotide segment operably linked to the first polynucleotide segment, and a second promoter operably linked to the third polynucleotide segment.

[0618] In some embodiments, the polynucleotide construct includes, from 5' to 3': a promoter, a self-cleaving peptide or an IRES, and a first polynucleotide segment operably linked to a second polynucleotide segment.

[0619] In some embodiments, the polynucleotide construct comprises a first promoter operably linked to the first polynucleotide segment and a second promoter operably linked to the second polynucleotide segment. In some embodiments, the polynucleotide construct comprises a first promoter operably linked to the first polynucleotide segment, a second promoter operably linked to the second polynucleotide segment, and a third promoter operably linked to the third polynucleotide segment.

[0620] In some embodiments, the polynucleotide construct encodes an antigen binding domain that targets an antigen associated with an autoimmune disease, an inflammatory disease, or a cancer. In some embodiments, the antigen is selected from the group consisting of CD19, CD20, CD22, CD27, BCMA, CD38, HLA*A2, HLA*A24 or citrullinated peptides, insulin, MOG, GAD65, IA2, prolamins, and desmosomal mucins (in the context of associated MHC molecules).

[0621] In some embodiments, the polynucleotide construct includes a sequence encoding an antigen binding domain targeting a cancer-associated antigen. In some embodiments, the cancer-associated antigen is selected from the group consisting of: CD19, CD20, CD22, CD23, CD27, CD38, CEA, BCMA, Lyml, Lym2, CLEC5A, CDH179b, FLT3, GCC, Muc, CSF2RA, GFRa4, CD32, CD33, IL11Ra, IL13Ra, NYBRI, SLea, CD200R, TGFβR2, CEA, CD276, TROP2, LAMP1, PTK7, DLL3, CDH1, CDH6, CDH17, CDH19, TSHR, B7-H3, and tyrosinase.

[0622] In some embodiments, the first polynucleotide segment comprises the sequence of SEQ ID NO: 10, 17, 23, 35, or 55.

[0623] In some embodiments, the second polynucleotide segment comprises the sequence of SEQ ID NO:2.

[0624] In some embodiments, the polynucleotide construct further comprises one or more selection markers (eg, any selection marker provided herein). In some embodiments, the first polynucleotide segment, the second polynucleotide segment, or both further comprises a sequence encoding a selection marker.

[0625] In some embodiments, the polynucleotide construct includes ΔNGFR as a selectable marker.

[0626] In some embodiments, the polynucleotide construct includes a ΔNGFR comprising the sequence of SEQ ID NO:4.

[0627] In some embodiments, the polynucleotide construct includes a truncated form of EGFR as a selectable marker.

[0628] 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.

[0629] In some embodiments, the first polynucleotide is in a first lentiviral vector and the second polynucleotide segment is in a second lentiviral vector.

[0630] In some embodiments, the lentiviral vector is capable of integrating into the T cell nuclear genome. In some embodiments, the lentiviral vector is not capable of integrating into the T cell nuclear genome. In some embodiments, an integration-deficient lentiviral vector is used. For example, in some embodiments, an integration-deficient or other lentiviral vector disclosed in Mátrai is used. In some embodiments, an integrase-deficient lentivirus is used. For example, an integrase-deficient lentivirus containing an inactivating mutation (D64V) in the integrase can be used, as described in Mátrai et al., Hepatology 53: 1696-1707 (2011), which is incorporated herein by reference. 6.9. Example

[0631] The following examples are offered for illustrative purposes only and are not intended to be limiting.

[0632] 6.9.1. Summary of experimental observations

[0633] Overall, the data from the experiments described below demonstrate that human CD4+ T cells can be transduced with two different lentiviral vectors: 1) an LVV encoding a synthetic CAR comprising a human B cell surface antigen binding domain, a CD8α hinge and CD8α transmembrane domain, a CD28 co-stimulatory domain, and a CD3ζ activation domain; and 2) a bidirectional vector encoding a human IL-10 gene plus a truncated form of the NGFR gene. +T cells expressed both CD19 CAR (measured by CD19 expression) and IL-10 / NGFR (measured by NGFR expression). Dual-transduced CD4 + T cells (usually designated as CD4 IL-10 / CAR Cells or when expressing Figure 2A-2B The anti-CD19 CAR described in IL-10抗CD19CAR No difference in transduction efficiency was found when

[0634] After activation by CD3 and CD28, CD4 IL-10 / CAR Cells showed CD4 IL-10 Cells have similar cytokine production characteristics to those described for Tr1 cells. For example, CD4 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 remained stable after prolonged culture.

[0635] Interestingly, CD19 + Target cells and CD4 IL-10抗CD19CAR Co-culture of cells resulted in CD4 IL-10抗CD19CAR Specific activation of cells and induction of IL-10 production.

[0636] CD4 IL-10抗CD19CAR The cells inhibited PBMC proliferation in vitro, allogeneic CD4 + T cell proliferation and allogeneic CD8 + T cell proliferation, CD4 IL-10 This result was also observed in cells.

[0637] CD4 IL-10抗CD19CAR Cell-specific killing of CD19 in vitro + target cells, but with CD4 IL-10 Like cells, they retain the 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CD19CAR T cells strongly suppress CD19 in vivo + Growth of target cells. Importantly, there is no significant difference between single-donor and multi-donor CD4 IL-10 Cells are similar to those described for single and multiple donor CD4 IL-10抗CD19 CAR cells did not induce xeno-GvHD, whereas PBMCs induced severe xeno-GvHD. IL-10抗CD19 CAR The cells exerted a synergistic antitumor effect with PBMCs, indicating that they did not interfere with protective GvL responses, as with CD4 IL-10cells as described (Locafaro et al. Mol Ther. 2017; 25(10): 2254-2269). In addition, in a humanized mouse model of GvHD induced by allogeneic PBMCs, single-donor and multi-donor CD4 IL-10抗CD19 CAR cells protect mice from xeno-GvHD (see Figures 18A-18C ).

[0638] These data suggest that CD4 IL-10抗CD19 CAR Specific interaction with CD19 expressed on target cells induces activation of these cells, leading to specific killing of CD19 + target cells and produce IL-10.

[0639] In summary, the data suggest that CD4 IL-10 / CAR T cells have unique clinical utility in treating all B-cell and T-cell mediated autoimmune diseases because they can not only eliminate B cells that produce autoantibodies but also simultaneously suppress pathogenic autoimmune CD4 + T cells and CD8 + T cell responses. The data also showed that single or multiple donor CD4 IL-10抗CD19 CAR Cells in treatment / elimination of CD19 + It has unique clinical utility in the prevention of tumor relapse (especially in patients undergoing allogeneic HSCT) because CD4 IL-10抗CD19 CAR The cells themselves did not induce GvHD and did not interfere with the protective anti-tumor effect (GvL effect) of PBMCs.

[0640] Importantly, even at high concentrations, CD4 IL-10抗CD19 CAR These results suggest that CD4 IL-10 / CAR The cells can be used to treat i) leukemias and other malignancies expressing target antigens, ii) leukemias and other malignancies in patients receiving allogeneic HSCT or BM transplants to reduce GvHD while retaining the GvL or GvT therapeutic effects of HSCT, iii) autoimmune diseases, which are treated by removing autoimmune B cells and downregulating pathogenic T cell responses and NLPR3 inflammasomes, iv) inflammatory diseases, which are treated by downregulating pathogenic T cell responses and NLPR3 inflammasomes, v) cell and organ transplant rejection, vi) solid tumors expressing tumor-associated antigens (including but not limited to B7-H3), and vii) immune-mediated diseases, which are treated through tissue repair and wound healing.

[0641] 6.9.2. Example 1: CD4IL-10 / 抗CD19 CAR Cell generation

[0642] 6.9.2.1. Vector Production of IL-10 / ΔNGFR and Anti-CD19 CAR

[0643] Second-generation CD19 CAR is used to generate CD4 IL-10抗CD19 CAR The CD19 CAR includes a scFv (SEQ ID NO: 12) from a fully human anti-CD19 monoclonal antibody, a CD8α hinge and CD8α transmembrane domain, a CD28 co-stimulatory domain, and a CD3ζ (CD3 zeta) activation domain ( Figure 2A ).like Figure 2A As shown, CD19 CAR was inserted into a lentiviral vector (LVV) (see Figure 2B ). For illustrations of exemplary CARs targeting CD19, CD20, CD22, BCMA, or B7H3, see Figure 1 The lentiviral vector was generated by linking the coding sequence of the anti-CD19 scFv (FMC63) to a lentiviral backbone including the human CD8α hinge and CD8α transmembrane regions, the CD28 co-stimulatory domain, and the CD3ζ intracellular signaling domain. The surface expression of the anti-CD19 CAR was monitored using an anti-CD19-CAR detector (Miltenyi).

[0644] In order to introduce the polynucleotide encoding IL-10 into CD4 + T cells to produce CD4 IL-10抗CD19CAR cells, using a lentiviral vector (LVV) containing the coding sequence of human IL-10 (SEQ ID NO: 2) and the coding sequence of a truncated form of NGFR (also known as CD271) (ΔNGFR) (SEQ ID NO: 4) ( Figure 4 ). LVV containing IL-10 and ΔNGFR coding sequences are further described in WO2016 / 146542, which is hereby incorporated by reference in its entirety.

[0645] The lentiviral vector was generated by ligating a 549 bp fragment of the human IL-10 coding sequence of pH15C (ATCC 68192) into the plasmid pLVIL-10. The presence of a bidirectional promoter (human PGK promoter plus minimal core elements of the CMV promoter in reverse) allows co-expression of the two transgenes. The plasmid further contains the coding sequence of an antibiotic resistance gene (e.g., ampicillin or kanamycin). For an exemplary description of the IL-10 construct, see Figure 3A and Figure 3B .

[0646] Lentiviral vectors were produced by transient co-transfection of four plasmids into 293T cells with Ca3PO4 and concentrated by ultracentrifugation: 1 μM sodium butyrate was added to the culture for vector collection. Titers of 293T cells were estimated by limiting dilution, and vector particles were measured by HIV-1 Gag p24 antigen immunocapture (NEN Life Science Products; Waltham, MA). Vector infectivity was calculated as the ratio between titer and particles. For concentrated vectors, the titer range was 1 × 10 8 Up to 6×10 9 transduction units / mL, and the infectivity range is 5×10 4 Up to 5×10 5 Transduction units / ng.

[0647] 6.9.2.2.CD4 IL-10 / CAR Cell production

[0648] like Figures 5A-5C As shown, three different methods were used to generate 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 medium (X-vivo, supplemented with 5% human AB serum and rhIL-2 (50 U / mL)). + T cells.

[0649] Figure 5A Shows activated CD4 + T cells were activated 48 hours after inoculation with a vector encoding an anti-CD19 CAR (LVV CD19-CAR ) and 24 hours later (i.e. 72 hours after activation) they were transduced with a lentiviral vector encoding human IL-10 and a truncated form of human NGF receptor (LVV IL-10-NGFR ) were transduced with a bidirectional lentiviral vector. Both LVVs were used at a multiplicity of infection (MOI) of 20.

[0650] Figure 5B Shows activated CD4+ T cells 48 hours after activation with LVV IL-10-NGFR transduction, and 24 hours later (i.e. 72 hours after activation) with LVV CD19-CAR Transduction.

[0651] Figure 5C Shows activated CD4 + T cells were activated with LVV 48 hours after IL-10-NGFR and LVV CD19-CAR Simultaneous transduction. By transducing only LVV 48 hours after activationIL-10-NGFR Transduction of activated CD4 + T cell generation control CD4 IL10 Cells. Figures 5A-5C In each of the 247 cells, transduction was performed in the presence of polybrene (8 μg / mL). Figures 5A-5C In each of the 10-28 days after transduction, the transduced cells (i.e., ΔNGFR + cells). The 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 as needed throughout the culture period.

[0652] CD4 IL-10抗CD19 CAR Cells were analyzed at TF3 (i.e., after the third restimulation) by staining with anti-CD271 mAb (Biolegend) and anti-CD19 CAR detection reagent (Miltenyi) and analyzed by flow cytometry. Staining showed that for the three different transduction methods, CD4 IL-10抗CD19 CAR The cell identity and purity were 88.6% ( Fig. 6A , from Figure 5A Method described in ), 64.1% ( Figure 6B , from Figure 5B ) and 52.6% ( Figure 6C , from Figure 5C As expected, using Figure 5C CD4 IL-10 Cells do not express CD19 CAR ( Figure 7 ).

[0653] like Figure 7 As shown, CD4 IL-10抗CD19 CAR The cells expressed IL-10 (measured by the expression of NGFR) and anti-CD19 CAR. In this experiment, CD4 + T cells were activated with LVV 48 hours after bead activation IL-10-NGFR and LVV 抗CD19 CARSimultaneous transduction (Method 5C). To generate CD4 IL-10 T cells (control), CD4 from donors 26.1 and 26.2 + T cells were activated with LVV only 48 hours after bead activation IL-10-NGFR For this analysis, CD4 IL-10 cells (control) and CD4 IL-10抗CD19 CAR Cells were analyzed. IL -10 cells (control) and CD4 IL-10抗CD19CAR T cells were stained with anti-CD271 mAb (Biolegends) and CD19-CAR detection reagent (Miltenyi) and analyzed by flow cytometry. Figure 7 As shown, most of them use LVV IL-10-NGFR and LVV CD19 -CAR Transduced cells expressed CD271 and CD19-CAR ( Figure 7 , right), while most only use LVV IL-10-NGFR Transduced cells expressed CD271 but not CD19-CAR ( Figure 7 , left picture).

[0654] These results indicate that CD4 IL-10抗CD19 CAR Cells can be successfully generated independently of the timing and order of transduction using two different LVVs. Furthermore, this suggests that a single LVV encoding both IL-10 and CAR can be used to generate CD4 IL -10 / CAR .

[0655] 6.9.2.3.CD4 IL-10抗CD19 CAR The cells have similarities to naturally derived Tr1 cells and CD4 IL-10 Cell-equivalent cytokine production profiles

[0656] according to Figure 5C CD4 T cells from three different healthy donors (26.1, 26.2, and 26.3) were generated by simultaneous transduction with (i) LVV containing anti-CD19-CAR and (ii) LVV containing IL-10 and ΔNGFR. IL-10抗CD19 CAR Specifically, to generate CD4 IL-10抗CD19 CAR Cells, 48 ​​hours after bead activation, were treated with LVV IL-10-NGFR and LVV CD19-CAR Simultaneous transduction of CD4 + T cells. To generate CD4IL-10 T cells (control), 48 hours after bead activation, were treated with LVV IL-10-NGFR Transduced CD4 + T cells.

[0657] For this analysis, each CD4 IL-10 cells (control) and CD4 IL-10 / CAR 2×10 T cells each 5 Cells (in 200 μL) were restimulated according to the methods described in Andolfi et al. Mol. Ther. 20 (9): 1778-1790 (2012) and Locafaro et al. Mol. Ther. 25 (10): 2254-2269 (2017). On day 14 after the second round (TF2) of restimulation, cells were stimulated with fixed anti-CD3 mAb and soluble anti-CD28 mAb for 48 hours. Culture supernatants were collected and the levels of IL-10, IL-4, IL-5 and IFN-γ were determined by ELISA.

[0658] like Figures 8A-8D Although some individual differences in cytokine production were observed, as shown in the CD4 IL-10 cells and Tr1 cells (not shown), CD4 IL-10 / CAR The cells produce high levels of IL-10, low levels of IL-4, and variable levels of IFN-γ and IL-5.

[0659] CD4 from donor 24.2 collected at TF 3 IL-10抗CD19 CAR Similar results were obtained for cells ( Fig. 9 ). In order to generate the CD4 IL-10抗CD19 CAR Cells, 48 ​​hours after bead activation, were treated with LVV IL-10-NGFR and LVV CD19-CAR Simultaneously transduced CD4 + T cells (see Figure 5C ). In order to generate CD4 IL-10 T cells (control), 48 hours after bead activation, treated with LVV alone IL-10-NGFR Transduced CD4 from donor 24.2 + T cells. For this analysis, each CD4 IL-10 cells (control) and CD4 IL-10 / CAR 2×10 T cells each 5Cells (in 200 μL) were restimulated according to the methods 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 (TF3) of restimulation, cells were stimulated with fixed anti-CD3 mAb and soluble anti-CD28 mAb for 48 hours. Culture supernatants were collected and the levels of IL-10, IL-4, IL-5 and IFN-γ were determined by ELISA.

[0660] like Fig. 9 As shown, 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.

[0661] In conclusion, Figures 8A-8D and Fig. 9 The data in the literature show that using LVV IL-10-NGFR and LVV CD19-CAR Simultaneous transduction of purified CD4 IL-10 The cells are viable and lead to the generation of CD4 IL-10 CD4 T cells and Tr1 cells share a unique and characteristic cytokine production signature IL-10-CAR In addition, CD4 T cells were harvested at TF2 or TF3 IL-10-CAR No differences in cytokine production profiles were observed at T, indicating that these cytokine production profiles are stable over long periods of time.

[0662] 6.9.3. Example 2: CD4 IL-10抗CD19 CAR Cell cytotoxicity and CD19 + Activation after target cell engagement

[0663] Known CD4 IL-10 Cells are able to kill myeloid tumor cells, such as ALL-CM, under certain conditions (Andolfi et al. Mol Ther. 2012; 20(9): 1778-1790). This experiment aims to evaluate whether co-expression of anti-CD19 CAR can produce cells that can kill CD19 when co-cultured with cell lines expressing CD19 (such as NALM6). + Cells (CD4 IL-10抗CD19 CAR ).

[0664] Specifically, CD4 IL-10 cells (control) and CD4 IL-10抗CD19 CAR cells (all of which were isolated from donor 24.2 CD4+ T cell production) (see Figure 5C ) and mixed with K562, NALM6 (CD19 + ) or ALL-CM target cells (10 5 After three days of co-culture, flow cytometry was used to determine the presence of residual target cells in each co-culture.

[0665] Data show that CD4 IL-10抗CD19 CAR Cells are activated through CD19-CAR and CD19 + Engagement and killing of target cells by CD19 + NALM6 cells. In contrast, CD4 IL-10 Cells are ineffective and do not kill CD19 + NALM6 cells ( Fig.10 ). In addition, CD4 IL-10抗CD19 CAR Cells and CD4 IL-10 Like cells, it kills myeloid tumor cell line ALL CM. CD4 IL-10抗CD19 CAR Cells and CD4 IL-10 The cells had no or very low cytotoxic effects on K562 cells, which are particularly sensitive to nonspecific cytotoxicity and NK cell killing ( Fig.10 ).

[0666] These results indicate that CD4 IL - 10抗CD19 CAR Engagement of CD19-CAR expressed on NALM6 lymphoma cells with CD19 expressed on NALM6 lymphoma cells triggers CD4 IL-10抗CD19 CAR The cytotoxic mechanism of cells, thereby selectively and effectively killing CD19 + target cells. In addition, CD4 IL-10抗CD19 CAR Cells were stained with CD4 IL-10 Like cells, they retain the ability to kill myeloid target cells expressing one or more of CD13, class I MHC antigens, CD54, and CD112.

[0667] In addition to inducing selective cytotoxic activity, CD4 IL-10抗CD19 CAR With CD19 + Engagement of NALM6 target cells also results in CD4 IL-10抗CD19 CAR Selective activation of cells and production of IL-10.

[0668] In these experiments, CD4 IL-10 cells (control) and CD4 IL-10抗CD19 CARThe cells were isolated from three different donors (26.1, 26.2 and 26.3) of CD4 + T cell generation. Before evaluating IL-10 production, the CD4 IL-10抗CD19 CAR Selective killing of CD19 cells + cell.

[0669] Specifically, CD4 IL-10 cells (control) and CD4 IL-10抗CD19 CAR cells (which were isolated from three different donors (26.1, 26.2 and 26.3) + T cell production) (see Figure 5C ) and were mixed with K562 cells, NALM6 (CD19 + ) cells or ALL-CM target cells (10 5 After three days of co-culture, flow cytometry was used to determine the presence of residual leukemic target cells in each co-culture. The K562 cell line is highly sensitive to nonspecific cytotoxicity and killing by NK cells and was used as a control. Fig.11A shown.

[0670] like Fig.11A As shown, CD4 IL-10抗CD19 CAR Cells effectively kill CD19 + NALM6 cells.CD4 IL-10抗CD19 CAR The cells retained the ability to kill the myeloid neoplasm cell line ALL-CM, while showing no or only weak cytotoxicity against K562 cells, which do not express CD19 but are known to be sensitive to nonspecific cytotoxicity and killing by NK cells ( Fig.11A ). CD4 IL-10抗CD19 CAR It also killed the myeloid leukemia cell line ALL-CM. After only 3 days of co-culture with NALM6 cells, significant amounts of IL-10 ( Fig. 11B ). In contrast, in NALM6 cells and CD4 IL-10 No IL-10 was detected in the co-culture of cells (limit of quantification: 31 pg / mL) ( Fig. 11B ).

[0671] In summary, the data suggest that CD4 IL-10抗CD19 CAR The cells are selectively activated by CD19-CAR engagement with CD19 expressed on relevant target cells. This activation results in (i) cytotoxic / cytolytic activity of the cells, thereby killing CD19 +target cells and (ii) induce IL-10 production, which is a CD4 IL-10抗CD19 CAR Required for cellular immune regulation and suppression. IL-10抗CD19 CAR Myeloid tumor target cells remain similar to those previously identified in CD4 IL-10 Finally, CD4 IL-10 Cells did not kill CD19 + NALM6 cells ( Fig. 11B ), prove CD4 IL-10抗CD19 CAR The cytotoxic activity of the cells is due to the interaction of anti-CD19 CAR with CD19 + Engagement of target cells.

[0672] 6.9.4. Example 3: CD4 IL-10抗CD19 CAR Allogeneic CD4 + and CD8 + T cell proliferative response

[0673] Since CD4 IL-10抗CD19 CAR Cells and CD4 IL-10 Both cells produced high levels of IL-10, so they were tested to inhibit allogeneic CD4 + and CD8 + The ability of T cells to proliferate.

[0674] To assess allogeneic CD4 + T cell proliferation, with (1×10 per well 5 Allogeneic PBMCs were labeled with 400 cells and in the presence or absence of a second restimulation (see Figure 5C ) after separation of CD4 IL-10 cells (control) or CD4 IL-10抗CD19 CAR Cells (1×10 per well 5 In the case of 5 × 10 cells per well (at a 1:1 responder:suppressor ratio), mature allogeneic dendritic cells (DCs) (5 × 10 4 After 4 days of culture, flow cytometry was used to analyze Dilution determination of CD4 + The percentage of proliferating responding T cells. For flow cytometry, CD4 + ΔNGFR - T cells were gated.

[0675] To assess allogeneic CD8 + T cell proliferation, with (1×10 per well5 Allogeneic PBMCs were labeled with 400 cells and in the presence or absence of a second restimulation (see Figure 5C ) after separation of CD4 IL-10 cells (control) or CD4 IL-10抗CD19 CAR Cells (1×10 per well 5 Allogeneic mature dendritic cells (DC) (5 × 10 cells per well) were used in the presence of 4 After 4 days of culture, flow cytometry was used to analyze Dilution determination of CD8 + The percentage of proliferating responding T cells. For flow cytometry, CD8 + ΔNGFR - T cells were gated.

[0676] To assess the proliferation of allogeneic PBMCs, (1×10 per well 5 Allogeneic PBMCs were labeled with 400 cells and in the presence or absence of a second restimulation (see Figure 5C ) after separation of CD4 IL-10 (control) or CD4 IL -10抗CD19CAR Cells (1×10 per well 5 Allogeneic mature dendritic cells (DC) (5 × 10 cells per well) were used in the presence of 4 After 4 days of culture, flow cytometry was used to analyze Dilution determination of CD8 + The percentage of proliferating responding T cells. For flow cytometry, CD3 + ΔNGFR - T cells were gated.

[0677] like Fig.12 As shown, CD4 T cells from three different healthy donors 26.1, 26.2 and 26.3 were harvested at TF2. IL -10抗CD19 CAR Allogeneic CD4 + T cell proliferation was inhibited by 78%, 48%, and 74%, respectively. + The proliferation of T cells was inhibited by 77%, 41% and 42% respectively ( Fig.13 ). CD4 IL-10 Comparable results were obtained with cells that converted allogeneic CD4+ The proliferation of T cells was inhibited by 83%, 81% and 61% respectively, and CD8 + The proliferation of T cells was inhibited by 77%, 41% and 68% respectively ( Fig.12 and Fig.13 ). Finally, at the time of TF3 harvest, CD4 IL-10抗CD19 CAR Cells and CD4 IL-10 Cells can also effectively inhibit the proliferation of allogeneic T cells. IL-10抗CD19 CAR Cells and CD4 IL-10 cells, inhibiting the proliferation of PBMC by 69% and 60% respectively ( Fig.14 ).

[0678] Taken together, these results suggest that CD4 IL-10 / CAR Allogeneic CD4 + T cells and allogeneic CD8 + T cells have immunosuppressive effects, and the immunosuppressive effects are related to CD4 IL-10 cells were equivalent; co-expression of CAR did not eliminate or alter CD4 IL-10 The results also showed that after long-term culture (i.e., CD4 IL -10抗CD19 CAR T cells), the immunosuppressive function is stable.

[0679] 6.9.5. Example 4: Using CD4 IL-10抗CD19 CAR Cell therapy or prevention of GvHD and cancer CD4 IL-10抗CD19 CAR The role of cells in the body.

[0680] Testing CD4 in humanized xeno-GvHD disease and tumor transplantation NSG mouse models IL-10抗 CD19 CAR Cell populations expressing CD19 + Effects on tumor growth, GvL effect, and GvHD induction.

[0681] NSG mice were sublethally irradiated with 175-200 Gr according to their body weight. On day 0, NSG mice were injected with (i) NALM6-luciferase (1×10 5 (ii) NALM6-luciferase (1×10 per mouse) 5 ) + PBMC (2.5×10 per mouse 6 (iii) NALM6-luciferase (1×10 per mouse) 5 )+CD4 IL-10抗CD19 CAR Cells (2.5×10 per mouse 6), or (iv) NALM6-luciferase (1 × 10 per mouse 5 ) + PBMC (2.5×10 per mouse 6 )+CD4 IL-10抗CD19 CAR (2.5×10 per mouse 6 )(See Fig.15 ).

[0682] Effects on leukemia cell growth were determined by standard bioluminescence measurements (see Figures 16A-16B ). In addition, when PBMC and / or CD4 IL-10抗CD19 CAR After the cells were grown, the mice were monitored for the development of GvHD by measuring body weight on days 0, 9, 12, and 15 (see Fig.17 ).

[0683] like Figures 16A-16B As shown, although PBMC inhibited the growth of NALM6 cells ( Figures 16A-16B ), mimicking the graft-versus-leukemia effect of allo-HSCT in human patients, but they induce xeno-GvHD in mice ( Fig.17 ), similar to the GvHD observed in human allo-HSCT. IL-10抗CD19 CAR Cells inhibit the growth of NALM6 cells ( Figures 16A-16B ) and did not induce any signs of xeno-GvHD (determined by weight loss) ( Fig.17 ). Using PBMC+CD4 IL-10 / CAR T cell therapy has the greatest anti-leukemia effect ( Figures 16A-16B ) and the greatest antitumor effect ( Fig.17 ).

[0684] These data suggest that CD4 IL-10抗CD19 CAR T cells are highly cytotoxic and selectively kill CD19 in vivo + target cells. Furthermore, they do not interfere with the protective GvL effect of PBMCs; in fact, the two act synergistically. Unlike PBMCs, CD4 IL-10抗CD19 CAR T cell therapy or CD4 IL-10抗CD19 CAR Combination therapy of T cells and PBMCs did not induce xeno-GvHD.

[0685] 6.9.6. Example 5: CD4 IL-10抗CD19 CAR Cells have suppressive activity in vivo and effectively prevent severe xeno-GvHD

[0686] Fig.18A Another study described in the literature evaluated single-donor or multi-donor CD4 IL-10抗CD19CARSpecifically, humanized NSG mice were sublethally irradiated and GvHD was induced by allogeneic PBMCs. Fig.18A On day 3, mice were intravenously administered with (i) PBMC (2.5E6 cells / mouse), (ii) single donor (BC26.3) CD4 IL-10抗CD19 CAR cells (2.5E6 cells / mouse), (iii) multiple donors (BC-26.1, BC26.2 and BC26.3) CD4 IL-10抗CD19 CAR cells (2.5E6 cells / mouse), (iv) PBMC (2.5E6 cells / mouse), and single donor (BC26.3) CD4 IL-10抗CD19 CAR cells (2.5E6 cells / mouse), or (v) PBMC (2.5E6 cells / mouse) and multiple donors (BC-26.1, BC26.2, and BC26.3) CD4 IL-10抗CD19 CAR Cells (2.5e6 cells / mouse) (see Fig.18A A composite score of weight loss, coat appearance, skin appearance, hunched back, and mobility was used to determine GvHD (Bondanza et al. 2006). Mice with a composite score ≥ 6 and / or weight loss ≥ 20% were euthanized for humane reasons. IL-10抗CD19CAR and multiple donor CD4 IL-10抗CD19 CAR The donor is unrelated.

[0687] like Fig.18B and Fig.18C As shown, injection of 2.5E6 PBMC cells induced fulminant and rapidly lethal xeno-GvHD on day 16, whereas injection of 2.5E6 single-donor CD4 IL-10抗CD19 CAR cell( Fig.18B ) or 2.5E6 multiple donor CD4 IL -10抗CD19 CAR cell( Fig.18C ) mice did not develop any signs of xeno-GvHD. In addition, when mice received 2.5E6 PBMCs and 2.5E6 single-donor CD4 IL-10抗CD19 CAR cell( Fig.18B ) or 2.5E6 PBMC and 2.5E6 multi-donor CD4 IL -10抗CD19 CAR cell( Fig.18C ) , PBMC and single donor CD4 IL-10抗CD19 CAR Cells or multiple donor CD4 IL-10抗CD19 CAR Co-administration of the cells protected mice from PBMC-induced xeno-GvHD.

[0688] These results suggest that single donor CD4 IL-10抗CD19 CAR Cells and multiple donor CD4 IL-10抗CD19 CAR The cells do not induce xeno-GvHD in vivo. In addition, single donor CD4 IL-10抗CD19 CAR Cells and multiple donor CD4 IL-10抗CD19 CAR The cells effectively prevented PBMC-induced severe xeno-GvHD, indicating that they have suppressive activity in vivo.

[0689] Other experiments

[0690] The treated mice were monitored for an additional period of time to determine the CD4 IL-10 / CAR Effects of cellular on long-term survival.

[0691] After administration, CD4 IL-10 / CAR Specifically, the amount and location of CD4 IL-10 / CAR The treated mice were monitored for an additional 3 weeks to determine their long-term survival.

[0692] Results showed that the reduction and prevention of xeno-GvHD was persistent.

[0693] 6.9.7. Example 6: Using CD4 IL-10 / CAR Cell therapy for chronic inflammatory and autoimmune diseases

[0694] Many chronic inflammatory and autoimmune diseases involve activation of the NLPR3 inflammasome. The NLPR3 inflammasome can be activated by “danger signals,” which leads to caspase 1-mediated production of proinflammatory cytokines IL-1β and IL-18 by monocytes / macrophages. A series of in vitro experiments were performed to investigate the role of CD4 IL-10 / 抗CD19 CAR Cellular effects on the NLPR3 inflammasome and IL-1β / IL-18 production by human monocytes.

[0695] First, human PBMCs were isolated from peripheral blood by standard density centrifugation on Ficoll / Paque (Sigma-Aldrich). Monocytes were isolated from human PBMCs by negative selection using the Monocyte Isolation Kit II (Miltenyi) according to the manufacturer's instructions. Negative selection is preferred because positive selection or adhesion can lead to undesirable cell activation. CD4 IL -10 / 抗CD19 CAR The isolated mononuclear cells were cultured at 5 × 10 cells / 200 μl in the presence of various dilutions of supernatant. 4 Cells / 200 μl were plated in 96-well microtiter plates in toxin-free medium containing 3% human AB serum.

[0696] Table 1 summarizes the treatment conditions applied to different groups of monocytes, each group including 6 wells of cells. Monocytes were activated by a combination of LPS and the NLPR3 inflammasome activator nigericin. Caspase 1 inhibitor Z-YVADfmk and specific NLPR3 inhibitor MC950 were added as controls.

[0697]

[0698]

[0699] *Z-YVADfmk is a specific inhibitor of caspase 1. 20 μM Z-YVADfmk (Biovision, Enzo Life Sciences, or Axxora Life Sciences) dissolved in DMSO was used.

[0700] **LPS (Sigma-Aldrich) 100 ng / mL plus nigericin (Nig, Invivogen) 10 μM, MCC950 (Invivogen) 10 μM added during the last 30 minutes of LPS incubation.

[0701] *** By placing CD4 IL-10 or CD4 GFP cells at 1×10 6 / mL was incubated for 3 days and the supernatant was collected to obtain CD4 IL-10 and CD4 GFP or NGFR culture supernatants. IL-10 production levels were measured by IL-10 specific ELISA. Anti-IL-10R antibody (Biolegend) 30 μg / mL.

[0702] After treatment as outlined in Table 1, supernatants were collected from 6 wells per group and IL-1β / IL-18 production was measured by ELISA specific for mature IL-1β or IL-18 (Biolegend). Cells collected from selected groups were analyzed by Western blot to determine the levels of activated caspase 1.

[0703] Experimental data show that autologous single donor, allogeneic single donor and allogeneic multiple donor CD4 IL-10 / 抗CD19 CAR cells downregulated IL-1β and IL-18 produced by activated monocytes. They further showed that autologous single donor, allogeneic single donor, and allogeneic multiple donor CD4 IL-10 / 抗CD19CAR cells downregulate mature caspase 1 produced in activated monocytes. In addition, autologous single-donor, allogeneic single-donor, and allogeneic multiple-donor CD4 IL-10 / 抗CD19 CAR and multiple donor CD4 IL-10 The produced IL-10 downregulates the inflammasome.

[0704] Similar experiments were performed using human macrophages or dendritic cells instead of monocytes. IL -10 / 抗CD19 CAR The cells also downregulated IL-1β, IL-18, and mature caspase 1 produced by activated macrophages and dendritic cells.

[0705] These data suggest that autologous single-donor, allogeneic single-donor, and allogeneic multiple-donor CD4 IL-10 / 抗CD19 CAR The cells can be used to treat diseases or disorders involving over-activation of the NLPR3 inflammasome. Specifically, autologous single donor, allogeneic single donor, and allogeneic multiple donor CD4 IL-10 / 抗CD19 CAR The cells can be used to treat chronic inflammatory and autoimmune diseases. The NLPR3 inflammasome can be activated by exogenous or endogenous "danger signals" such as pathogen-associated molecular patterns (PAMPs), silica, asbestos, danger-associated molecular patterns (DAMPs) such as products of damaged mitochondria, necrotic and stressed cells, and uremic acid crystals.

[0706] 6.9.8. Example 7: Multiple Donor CD4 IL-10 Cell generation

[0707] Vector production

[0708] Multiple donor CD4 IL-10 Cells were generated by transduction with a lentiviral vector containing the coding sequences for human IL-10 and a truncated form of NGFR (ΔNGFR) ( Figure 3A-3B ), as described in WO2016 / 146542, which is incorporated herein by reference in its entirety. The sequence of the vector is provided as SEQ ID NO:5. In brief, a lentiviral vector is generated by connecting a 549bp fragment of the coding sequence of human IL-10 of pH15C (ATCC 68192) to plasmid #1074.1071.hPGK.GFP.WPRE.mhCMV.dNGFR.SV40PA. The presence of a bidirectional promoter (human PGK promoter plus the minimum core element of the reverse CMV promoter) allows co-expression of two transgenes. The plasmid further contains the coding sequence of an antibiotic resistance gene (e.g., ampicillin or kanamycin).

[0709] Lentiviral vectors were produced by transient co-transfection of four plasmids into 293T cells with Ca3PO4 and concentrated by ultracentrifugation: 1 μM sodium butyrate was added to the culture for vector collection. Titers of 293T cells were estimated by limiting dilution, and vector particles were measured by HIV-1 Gag p24 antigen immunocapture (NEN Life Science Products; Waltham, MA). Vector infectivity was calculated as the ratio between titer and particles. For concentrated vectors, the titer ranged from 5 × 10 8 Up to 6×10 9 transduction units / mL, and the infectivity range is 5×10 4 Up to 5×10 5 Transduction units / ng.

[0710] CD4 IL-10 Cell production

[0711] Fig.19 It is CD4 IL-10 Schematic diagram of the cell generation process. Purification of CD4 from healthy donors + Human CD4 T cells were activated with soluble anti-CD3 mAb, soluble anti-CD28 mAb and rhIL-2 (50 U / mL). + T cells were incubated for 48 h and then transduced with a bidirectional lentiviral vector encoding human IL-10 and a truncated form of human NGF receptor (LV-IL-10 / ΔNGFR) at a multiplicity of infection (MOI) of 20.

[0712] After 11 days, transduced cells were analyzed for ΔNGFR expression by FACS, and vector copy number (VCN) was quantified by digital droplet PCR (ddPCR).

[0713] CD4 from 10 different donors + The average transduction efficiency was 45 ± 17% for T cells and 2.7 ± 0.6% for VCN. Fig. 20A Human CD4 T cells transduced with LV-IL-10 / ΔNGFR (a bidirectional lentiviral vector encoding human IL-10 and a truncated form of the human NGF receptor) are shown. + CD4 + ΔNGFR + Percentage of cells (mean ± SD, n = 10 left column) and vector copy number (VCN, mean ± SD, n = 10 right column). Quantification of CD4 by digital droplet PCR (ddPCR) IL-10 CD4 + ΔNGFR + Frequency of cells and vector copy number.

[0714] Purification of ΔNGFR using anti-CD271 mAb-coated beads + T cells with a purity of >95% CD4 IL-10 Cell population. After purification, cells were stained with markers for CD4 and ΔNGFR and analyzed by FACS. The data showed that the purification step resulted in a purity of over 98%. Fig. 20B FACS data from two representative donors (Donor B and Donor C) out of 10 tested donors are shown. IL-10 Cells were restimulated three times at 14-day intervals and their in vitro and in vivo functions were tested after the second (TF2) and third (TF3) functional restimulations.

[0715] Resting CD4 IL-10 Cells constitutively produce IL-10. Upon activation, the levels of IL-10 produced are strongly increased.

[0716] CD4 IL-10 The cells have cytokine production characteristics comparable to naturally derived Tr1 cells.

[0717] Analysis of single donor CD4 after the second (TF2) and third (TF3) restimulation IL-10 The cytokine production characteristics of the cells and the results in Fig.21 Specifically, CD4 IL-10 Cells (2×10 in 200 μl) 5 Cells) were restimulated as previously described (Andolfi et al. Mol Ther. 2012; 20(9): 1778-1790 and Locafaro et al. Mol Ther. 2017; 25(10): 2254-2269). On day 14 after the second (TF2) restimulation and on day 14 after the third (TF3) restimulation, CD4 IL-10 Cells were not stimulated again (orange bars) or stimulated for 48 hours with fixed anti-CD3 mAb and soluble anti-CD28 mAb (grey bars). Culture supernatants were collected and the levels of IL-10, IL-4, IL-5, IFN-γ and IL-22 were determined by ELISA. All samples were tested in triplicate. Mean values ​​± SD are shown, n = 8 tested donors. Fig.21 The results presented in the present study showed that CD4 IL-10 The cells displayed cytokine production characteristic of Tr1 cells.

[0718] Although considerable variation between donors was observed, the overall cytokine production profile following a second (TF2) or third (TF3) restimulation was comparable and mirrored that of Tr1 cells (Roncarolo et al., Immunity, 2018). 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.

[0719] CD4 IL-10 Cells express high levels of granzyme B and selectively kill myeloid leukemia cells After the second round of restimulation (TF2), CD4 IL-10 Granzyme B (GzB) expression in cells. Fig.22A The data show that most CD4 IL-10 All CD4 cells from 7 different donors expressed GzB. IL-10 More than 95% of the cells expressed high levels of granzyme B.

[0720] Further analysis of CD4 IL-10 The cells showed cytotoxic effects on myeloid leukemia cell lines (ALL-CM) and erythroid leukemia cell lines (K562). IL-10 Cells (10 5 / well) and K562 and ALL-CM cells (10 5 / well) were co-cultured at a ratio of 1:1 for 3 days. The residual leukemia cell lines (CD45 低 CD33 + ).

[0721] CD4 IL-10 Cells selectively kill myeloid leukemia cells (ALL-CM), such as Fig. 22B The percentage of ALL-CM cells killed varied between 62% and 100%, whereas the percentage of erythroid leukemia cell line K562 (highly sensitive to nonspecific cytotoxic activity) killed varied between 0 and 27% (4 different tested donors). Taken together, these data confirm that CD4 IL-10 The cells expressed granzyme B and effectively killed myeloid leukemia cells. As expected, CD4 IL-10 There is some variation in the killing ability of the cells.

[0722] CD4 IL-10 Allogeneic CD4 + and CD8 + T cell proliferative response

[0723] CD4 IL-10 Allogeneic CD4 + T cells or CD8+ Specifically, allogeneic PBMC cells were used (10 5 cells / well) and in the absence or presence of CD4 IL-10 Cells (10 5 Allogeneic mature dendritic cells (5 × 10 4 cells / well) and soluble anti-CD3 mAb. After 4 days of culture, CD4 + ΔNGFR – T cells or CD8 + ΔNGFR – After T cells were gated, The percentage of proliferating responding cells was determined by dilution. Fig.23A and 23B Shown are the results from six different unpooled donors ( Fig.23A Donor-C, Donor-E and Donor-F, and Fig. 23B CD4 IL-10 CD4 + T cells affected, as well as the percentage of proliferation and suppression. Fig.24A and 24B Shown are the results from six different unpooled donors ( Fig.24A Donor-C, Donor-E and Donor-F, and Fig. 24B CD4 IL-10 CD8 + Effects of T cells.

[0724] The results showed that CD4 IL-10 Cells (unpooled and tested individually) down-regulate allogeneic CD4 + and CD8 + T cell proliferation response. + The inhibitory effect on T cells ranged from 51% to 96%, while the inhibitory effect on CD8 + The inhibitory effect of T cells ranged from 62% to 73%.

[0725] Multiple donor CD4 IL-10 Cell generation and characterization

[0726] Using CD4 from multiple donors + Cells were cultured as described above and Fig.19 The generation of CD4 IL-10 CD4 cells per donor IL-10The cells were stimulated by a second (TF2) and third (TF3) restimulation. After the third stimulation, CD4 IL-10 Cells were combined at a 1:1:1 ratio and stimulated with immobilized anti-CD3 mAb and soluble anti-CD28 mAb for 48 hours.

[0727] Multiple donor CD4 IL-10 The cells have the same CD4 IL-10 Cytokine production of cells is comparable to that of Tr1 cells Bio characteristics.

[0728] The culture supernatants were collected and the levels of IL-10, IL-4, IL-5, IFN-γ, and IL-22 were determined by ELISA. Fig.25 The results presented in the study showed that multiple donor CD4 IL-10 Cytokine production of cells (red dots) and CD4 IL-10 CD4 IL-10 Cells are equivalent. Multiple donor 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). These data suggest that combined CD4 IL-10 The cells are viable and these multiple donor CD4 IL-10 The cells maintained a single donor-derived CD4 IL-10 Importantly, the pooled allogeneic cell populations contained >95% viable cells, indicating that they do not kill each other.

[0729] Multiple donor CD4 IL-10 The cells expressed high levels of granzyme B and killed myeloid leukemia cell lines.

[0730] After restimulation in round 3 (TF3), multiple donor CD4 IL-10 Granzyme B (GzB) expression in cells. Fig.26A The data showed that most multi-donor CD4 IL-10 Cells express GzB. More than 95% of multi-donor CD4 IL-10 Cells expressing granzyme B, with CD4 IL-10 The GzB expression of the cells was comparable.

[0731] Further analysis of CD4 IL-10 The cytotoxicity of cells against myeloid leukemia cells (ALL-CM cell line) or K562. IL-10 Cells (10 5 cells / well) and K562 and ALL-CM cells (10 5The residual leukemia cell lines (CD45 低 CD33 + ). Fig.26B The results presented in showed some degree of cytotoxicity against K562 cells, which were highly sensitive to nonspecific cytotoxicity and cytotoxicity of NK cells. Nevertheless, CD4 IL-10 The cells showed a comparable level of selectivity for myeloid leukemia cells (ALL-CM).

[0732] Multiple donor CD4 IL-10 The cells inhibit the proliferative response of allogeneic CD4+ and CD8+ T cells.

[0733] We also analyzed multiple donor CD4 IL-10 Allogeneic CD4 + T cells or CD8 + Specifically, allogeneic PBMC cells were used (10 5 cells / well) and in the absence or presence of multiple donor CD4 IL-10 Cells (10 5 Allogeneic mature dendritic cells (5 × 10 4 cells / well) and soluble anti-CD3 mAb. After 4 days of culture, CD4 + ΔNGFR – T cells and CD8 + ΔNGFR – After T cells were gated, The percentage of proliferating responding cells was determined by dilution. Fig.27A CD4 IL-10 Multiple donor CD4 IL-10 Cell results. Fig.27B CD4 IL-10 Multiple donor CD4 IL-10 Results for cells that were frozen, stored, thawed, and then tested.

[0734] Fig.27A Shown, multiple donor CD4 IL-10 Cells (from 3 different donors) expressed CD4 + and CD8 + T cell responses were suppressed by 96% and 74%, respectively. IL-10Comparable results were obtained with cells that were frozen, stored, and thawed prior to testing ( Fig.27B ). CD4 + and CD8 + The inhibition of T cell proliferation was 68% and 75%, respectively. These data suggest that multiple donor CD4 IL-10 Cells can be frozen and stored without loss of function.

[0735] In summary, using multiple donor CD4 IL-10 The data obtained with the cells showed that these cell preparations can be pooled without any problems. They contain >95% viable cells and maintain single donor CD4 IL-10 All relevant functions of cells (cytokine production, cytotoxic capacity and suppression of allogeneic T cell responses). IL-10 Larger pools of cells should reduce CD4 IL-10 The natural variation observed between cell batches should provide a large supply of readily available CD4 IL-10 cell.

[0736] Multiple donor CD4 IL-10 Cell products will have the significant advantage of a more homogeneous product, which will allow for clear batch-to-batch variability, potency and release criteria to be defined. In addition, it will enable the development of continuous large-scale cell production methods.

[0737] For the generation of multiple donor CD4 IL-10 Other cell methods

[0738] Prior to lentiviral transduction, buffy coats from at least 3-5 different donors were pooled. CD4 was isolated from buffy coats by positive selection using an anti-CD4 antibody. + Cells. FACS was used to examine the presence of incorporated CD4 + Cell purity. Optionally, obtain frozen human CD4 + Cells. Frozen human CD4 + Cells were thawed before use. CD4 + Cells were activated for 24-48 hours using a combination of CD3 and CD28 antibodies or a combination of CD3 and CD28 antibody-coated beads. In some cases, CD4 + Cells were activated with soluble anti-CD3 mAb, soluble anti-CD28 mAb, and rhIL-2 (50 U / mL) for 48 h and transduced with a bidirectional lentiviral vector encoding human IL-10 as described above to generate CD4 IL-10 cell.

[0739] In some cases, the T cell donor (or CD4 + cells) and a CD4 + Cells were selectively pooled and used.

[0740] The activated CD4 + Cells to generate multiple donor CD4 IL-10 cell.

[0741] On days 7-11, 5-9 days after transduction, cells were harvested and transduced T cells were successfully purified using anti-NGFR antibodies. This process typically yielded 95% pure multi-donor CD4 IL-10 Cell population.

[0742] Purified multi-donor CD4 IL-10 The cells are counted and restimulated for 8-10 days in the presence of IL-2, optionally in the presence of feeder cells, by a mixture of CD3 and CD28 antibodies, beads coated with CD3 and CD28 antibodies. In some cases, purified multi-donor CD4 IL-10 cell.

[0743] After a total culture period of 14-18 days, CD4 IL-10 Cells were cultured and tested for their ability to produce IL-10 spontaneously or after activation with CD3 and CD28 antibodies or beads coated with CD3 and CD28 antibodies. In addition, levels of GrzB and perforin were measured. They were also tested for their ability to inhibit the proliferation of human T cells (PBMCs) and purified CD4 + and CD8 + The ability of T cells to proliferate.

[0744] In addition, 200,000 CD4 IL-10 Constitutive production of IL-22 by cells and as previously described for the production of other cytokines (e.g., IFN-γ, IL-10, IL-4, and IL-5), 200,000 CD4+ cells in a 200 μl volume were measured after activation using a combination of CD3 and CD28 antibodies. IL-10 IL-22 production by cells. Combined CD4 IL-10 Cells were frozen before storage.

[0745] 6.9.9. Example 8: Using Multiple Donor CD4 IL-10 Cell therapy or prevention of GvHD

[0746] Multiple donor CD4 IL-10 In vivo effects of cells

[0747] Testing multiple donor CD4 in a humanized xenogeneic GvHD disease model (NSG mouse model) IL-10 Effects of cell populations on human PBMC-induced xenogeneic GvHD, such as Fig.28 NSG mice were sublethally irradiated and intravenously injected with human PBMC (5×10 6 cells / mouse), multiple donors (three donors) CD4 IL-10 Cells (5×10 6 cells / mouse), or human PBMC (5×10 6 cells / mouse) and multiple donor CD4 IL-10 Cells (BC-C / E / F) (5×10 6 xeno-GvHD was assessed based on weight loss (>20% body weight loss), skin lesions, coat condition, mobility, and hunched back as previously described (Bondanza et al. Blood 2006).

[0748] Fig.29 The percentage of NSG mice that developed xeno-GvHD each day after injection is shown. 6 Administration of personal PBMC to irradiated NSG mice unexpectedly resulted in unusual fulminant GvHD. All mice died by day 10, reflecting very virulent xeno-GvHD. Co-administration of 5 × 10 6 Multiple donor CD4 IL-10 cells delayed this fulminant GvHD, but mice were sacrificed on day 14 because they reached the pre-specified humane 20% body weight loss criterion ( Fig.29 ). Nevertheless, these results suggest that multiple donor CD4 IL-10 It can delay the development of severe xeno-GvHD. Importantly, PBMC (5×10 6 Multiple donor CD4 IL-10 The cells did not induce any signs of xeno-GvHD.

[0749] The spleens of NSG mice were also tested 14 days after injection. Fig.30 , left) and bone marrow ( Fig.30 , right) Human CD4 IL -10 These NSG mice were injected with human PBMC (5×10 6 cells / mouse), multiple donors (three donors; BC-C / E / F) CD4 IL-10 Cells (5×10 6cells / mouse), or human PBMC (5×10 6 cells / mouse) and multiple donor CD4 IL-10 Cells (three donors; BC-C / E / F) (5×10 6 cells / mouse). Fig.30 The results presented in the study showed that multiple donor CD4 IL-10 The cells migrated to the spleen and bone marrow. These cells were found to be present at low percentages 14 days after cell infusion. These results suggest that multiple donor CD4 IL-10 The cells delayed fulminant xeno-GvHD induced by human PBMCs and did not induce any xeno-GvHD themselves.

[0750] Multiple donor CD4 IL-10 cytotoxicity of purified CD4 + Severe xeno GvHD of cells.

[0751] Testing multiple donor CD4 in a humanized xeno GvHD model IL-10 cells, where 2.5×10 6 Purified human CD4 + T cell-induced GvHD disease, such as Fig.31 NSG mice were sublethally irradiated on day 0 and injected intravenously with human CD4 + T cells (2.5×10 6 cells / mouse), or human CD4 + T cells (2.5×10 6 cells / mouse) and multiple donor CD4 IL-10 Cells (three different donors; BC-H / I / L) (2.5×10 6 cells / mouse), or human CD4 + T cells (2.5×10 6 cells / mouse) and CD4 IL-10 Cells (2.5×10 6 xeno-GvHD was assessed based on weight loss (>20% body weight loss), skin lesions, coat condition, mobility, and hunched back as previously described (Bondanza et al. Blood 2006).

[0752] Fig.32 The percentage of NSG mice that developed GvHD is shown each day after injection. IL-10 (BC-H / I / L) cells can suppress the expression of human allogeneic CD4 +T cell-mediated xeno-GvHD. In this experiment, xenoGvHD was very severe, as all mice in the control group that received CD4+ T cells died by day 20. In contrast, co-administration of 2.5 × 10 6 Multiple donor CD4 IL-10 Suppresses GvHD by 75%. Single donor CD4 IL-10 Cells also have a protective effect, but the effect is weaker.

[0753] Other experiments

[0754] Multiple donor CD4 IL-10 Therapeutic effect of cells: (i) Mice received IL-10 Human PBMC from unrelated donors (xeno-GvHD positive control); (ii) mice received multiple donor CD4 IL -10 cells (negative control); (iii) mice received PBMC and multiple donor CD4 IL-10 and (iv) mice received PBMC and multiple donor CD4 IL-10 Combination of cells. In the case of receiving PBMC and multiple donor CD4 IL-10 Some animals received both PBMC and multiple donor CD4 IL-10 Some animals received CD4 IL-10 cells, and some animals received CD4 IL-10 cell.

[0755] In the administration of PBMC and / or multiple donor CD4 IL-10 After the cells are added, the mice are monitored for the development of GvHD by measuring body weight at weeks 1, 2, 3, 4, and, if necessary, week 5. In addition to weight loss, the mice will be examined for skin lesions, hair condition, and mobility. The mice in the treatment groups will be monitored for an additional period of time to determine whether multiple donor CD4 IL-10 Effects of cellular on long-term survival.

[0756] After administration, the peripheral blood and tissue CD4 IL-10 Specifically, peripheral blood and inflammatory sites (lymph nodes, spleen, intestine, and bone marrow) were monitored for CD4 IL-10 The treated mice were monitored for an additional 3 weeks to determine their long-term survival.

[0757] The results showed that multiple donor CD4IL-10 The cells effectively reduced and prevented xeno-GvHD.

[0758] 6.9.10. Example 9: Inhibition of GvHD and Treatment of Cancer

[0759] Testing of multi-donor CD4 in the NSG mouse model transplanted with human PBMCs and AML tumor cells IL-10 Effect of cell populations on human PBMC-induced xenogeneic GvHD and antitumor effects. AML cells (ALL-CM) were administered intravenously (iv) as previously described in WO 2016 / 146542. PBMC or multi-donor CD4 IL-10 cells or a combination thereof.

[0760] Multiple donor CD4 IL-10 Cells were obtained as described in Example 1. Multiple donor CD4 IL -10 The therapeutic effect of cells, each group of mice received irradiation and 5×10 6 ALL-CM cells (AML mice): (i) AML mice that did not receive additional treatment; (ii) AML mice that received CD4 IL-10 5×10 cells from unrelated donors 6 AML mice with personal PBMCs – PBMCs induce severe xeno-GvHD; (iii) received 2.5×10 6 Multiple donor CD4 IL-10 and (iv) mice that received PBMC and multiple donor CD4 IL-10 Another group of mice did not receive ALL-CML cells but received 5×10 6 Individual PBMCs.

[0761] Testing of multiple donor CD4 based on weight loss, skin lesions, coat condition, motility, mortality, and long-term survival IL-10 The effect of cells on human PBMC-induced xeno-GvHD was tested based on the reduction of circulating tumor cells and long-term tumor-free survival. IL-10 Anti-tumor or graft-versus-leukemia (GvL) effects of cells.

[0762] Some mice were monitored for up to seven weeks to monitor long-term survival and complete tumor remission.

[0763] The results showed that multiple donor CD4 IL-10 The cells effectively suppressed xeno-GvHD and treated cancer.

[0764] 6.9.11. Example 10: Using multiple donor CD4 IL-10 Cell therapy for cancer

[0765] Testing multi-donor CD4 T cell therapy in the ALL-CM leukemia model in NSG mice IL-10 Cell population.

[0766] On day 0, NSG mice were sublethally irradiated and injected intravenously with myeloid leukemia cells (ALL-CM) (5 × 10 6 )(See Fig.33A In the first group of animals, PBMC (5×10 6 or single donor (from donor BC-I and donor BC-H) CD4 IL-10 Cells (2.5×10 6 In the second group of animals, PBMC (5×10 6 or multiple donor CD4 IL-10 Cells (2.5×10 6 The graft-versus-leukemia (GvL) effect was tested in animals based on the reduction of circulating leukemic cells and long-term leukemia-free survival. Leukemia was measured as previously described (Locafaro G. et al Molecular Therapy 2017).

[0767] like Fig.33B and Fig.33C As shown, all mice injected with ALL-CM myeloid leukemia cells developed extensive leukemia progression on day 17. 6 Interestingly, lower numbers (2.5 × 10 6 Single donor CD4 IL10 ( Fig.33B ) or multiple donor CD4 IL10 ( Fig.33C ) achieved comparable levels of leukemia progression inhibition. These data suggest that single-donor and multi-donor CCD4 IL10 It has a strong direct anti-myeloid leukemia effect.

[0768] Single donor CD4 IL10 and multiple donor CD4 IL10 Graft versus leukemia (GvL) effect in combination with PBMC (see Fig.34A ). Apply 5×10 6 PBMCs resulted in strong inhibition of leukemia progression, and 5×10 6PBMC and single donor CD4 IL10 (2.5×10 6 The combined use has a synergistic effect ( Fig.34B ). Interestingly, 5×10 6 PBMC and 2.5×10 6 Multiple donor CD4 IL10 The combined administration of Fig.34C ). These data suggest that multiple donor CD4 IL10 Synergizes with PBMCs to mediate a strong GvL effect.

[0769] 6.9.12. Example 11: Using multiple donor CD4 IL-10 Cell therapy for chronic inflammatory and autoimmune diseases

[0770] Many chronic inflammatory and autoimmune diseases involve activation of the NLPR3 inflammasome. The NLPR3 inflammasome can be activated by “danger signals,” which leads to caspase 1-mediated production of proinflammatory cytokines IL-1β and IL-18 by monocytes / macrophages. A series of in vitro experiments were performed to investigate the effects of multiple donor CD4 IL-10 Cellular effects on the NLPR3 inflammasome and IL-1β / IL-18 production by human monocytes.

[0771] First, human PBMCs were isolated from peripheral blood by standard density centrifugation on Ficoll / Paque (Sigma-Aldrich). Monocytes were isolated from human PBMCs by negative selection using the Monocyte Isolation Kit II (Miltenyi) according to the manufacturer's instructions. Negative selection is preferred because positive selection or adhesion can lead to undesired cell activation. 5 or 1×10 5 Multiple donor CD4 IL-10 The isolated mononuclear cells were cultured at 5 × 10 cells / 200 μl / well. 4 Cells / 200 μl were plated in 96-well microtiter plates in toxin-free medium containing 3% human AB serum.

[0772] The treatment conditions applied to 17 groups of monocytes, each group comprising 6 wells of cells, are summarized in Table 2. It is known that LPS alone can activate human monocytes without the need for a secondary signal provided by ATP.

[0773]

[0774]

[0775] *YVADfmk is a specific inhibitor of caspase 1. 20 mM Z-YVADfmk (Biovision, Enzo Life Sciences, or Axxora Life Sciences) dissolved in DMSO was used.

[0776] **LPS (Signa-Aldrich) 100 ng / mL

[0777] *** By placing CD4 IL-10 or CD4 GFP cells at 1×10 6 / mL was incubated for 3 days and the supernatant was collected to obtain CD4 IL-10 Supernatants of CD4 GFP or NGFR cultures were collected. IL-10 production levels were measured by IL-10-specific ELISA.

[0778] After treatment as outlined in Table 2, supernatants were collected from 6 wells of each group and IL-1β / IL-18 production was measured by ELISA specific for mature IL-1β or IL-18 (Biolegend). Cells collected from 6 wells of groups 3, 10, 13, 14, and 17 were analyzed by Western blot to determine the level of activated caspase 1.

[0779] Experimental data show that multiple donor CD4 IL-10 cells downregulated IL-1β and IL-18 produced by activated monocytes. They further showed that multiple donor CD4 IL-10 cells down-regulate mature caspase 1 production in activated monocytes. In addition, multiple donor CD4 IL-10 and CD4 IL-10 The produced IL-10 downregulates the inflammasome.

[0780] Similar experiments were performed using human macrophages or dendritic cells instead of monocytes. IL-10 The cells also downregulated IL-1β, IL-18, and mature caspase 1 produced by activated macrophages and dendritic cells.

[0781] These results suggest that multiple donor CD4 IL-10 The cells can be used to treat diseases or disorders involving overactivity of the NLRP3 inflammasome. IL-10 The cells can be used to treat chronic inflammatory and autoimmune diseases. The NLPR3 inflammasome can be activated by exogenous or endogenous "danger signals" such as pathogen-associated molecular patterns (PAMPs), silica, asbestos, danger-associated molecular patterns (DAMPs) such as products of damaged mitochondria, necrotic and stressed cells, and uremic acid crystals.

[0782] 6.9.13. Example 12: Multiple Donor CD4 IL-10 Cell supernatants inhibit NLPR3 inflammasome activation and IL-1β and IL-18 production in human monocytes

[0783] CD14 was isolated from PBMC using a pan-monocyte isolation kit (Miltenyi Biotec, Bergisch Gladbach, Germany). + Monocytes were plated at 2 × 10 per well. 5 100 μL / well were plated in 96 flat-bottom microtiter wells and cultured in the presence of LPS. Z-YVADfmk (20 μM), MMC950 (10 μM), IL-10 (10 ng / mL), or various concentrations of single or pooled donor CD4 IL-10 The cells were further cultured in the presence of the cell supernatant.

[0784] CD4 T cells from single or pooled donors activated for 72 hours with a combination of anti-CD3 and anti-CD28 antibodies IL-10 Cells were harvested from supernatants as previously described (Andolfi et al. 2012, Mol. Therapy Vol. 20, 1778-1790, Locafaro et al. Mol Ther 2017, 25, 2254). Fig.35C and 35D ), monocytes were incubated with a combination of LPS and the NLPR3 inflammasome activator nigericin ("NIG"), with NIG added during the last 30 min of LPS activation.

[0785] The NLPR3 inflammasome is activated by LPS, leading to the production of mature caspase 1 and biologically active forms of IL-1β and IL- 18. Monocytes plated in the absence of LPS activation did not produce detectable levels of IL-10 during incubation (not shown).

[0786] CD4 from a single donor was added at concentrations of 50%, 25%, and 12.5%. IL-10 The supernatant of the cells (containing 1769 pg IL-10 / mL) inhibited the production of IL-1β by LPS-activated monocytes from donors #1 and #2 in a dose-dependent manner ( Fig.35A and Fig.35B ). GFP-transduced CD4+ cell supernatant was used as a control. Complete inhibition of IL-1β production was observed at 50% and 25% concentrations, while 50% concentration of GFP-transduced control CD4+ cell supernatant had no effect.

[0787] Different concentrations of CD4 IL -10 T cell supernatants (50%, 25%, or 12.5%), Z-YVADfmk, or MCC950. CD4+ T cells from single donors (BC-E) or pooled donors IL-10 The supernatant of the cells contained 5295 or 3532 pg IL-10 / mL, respectively. IL-10 The supernatant of the cells also very effectively inhibited LPS-induced IL-1β production enhanced by the NLPR3 inflammasome activator nigericin ( Fig.35C and Fig.35D ).

[0788] The data showed that a concentration of 50% CD4 IL-10 The cell supernatant was as effective as the irreversible caspase 1 inhibitor Z-YVADfmk (Guo et al. 2015, Nature Med 21, 677), the selective NLPR3 inflammasome inhibitor MCC950 (Coll et al. 2019, Nature Chem. Biol 15, 556) and recombinant IL-10, indicating that the supernatant containing IL-10 inhibited NLPR3 inflammasome activation and mature caspase 1 production, thereby strongly inhibiting the production of the proinflammatory cytokine IL-1β ( Figures 35A-35D ).

[0789] In a second series of experiments, supernatants from a single donor (BC-E) and pooled CD4 IL-10 Comparable results were obtained with CD4 IL-10 Cells were activated with a combination of CD3 and CD28 antibodies as described (Andolfi et al. 2012). After 3 days, CD4 IL-10 These supernatants contained 5295 and 3532 pg IL-10 / mL, respectively, and inhibited LPS-induced IL-1β production by monocytes from donor #3 in a dose-dependent manner ( Fig.35E ). The supernatant at 50% concentration was as effective as Z-YVADfmk and MCC950. Pooled CD4 IL-10 The inhibitory effect of the cell supernatant was completely neutralized by anti-IL-10 receptor antibody. Similarly, the pooled supernatant from 3 different donors contained 2589 pg IL-10 / mL and dose-dependently inhibited the production of IL-1β by monocytes from donor #4 ( Fig.35FThe inhibitory effect of the supernatant was completely neutralized by IL-10 receptor antibody, indicating that NLPR3 activation is mediated by IL-10. IL-10 IL-10 produced by T cells strongly inhibits the production of the proinflammatory cytokine IL-1β. As expected, anti-IL-10 receptor antibodies had no effect on the inhibition of IL-1β production mediated by Z-VADfmk and MCC950 ( Fig.35E ).

[0790] CD4 was further tested on monocytes from donor #4 activated by LPS and nigericin IL-10 Different concentrations of single donor (BC-V) or multiple donor (three donors; BC-T / U / V) CD4 IL-10 Cell supernatant, ZYVADfmk or MCC950. Figure 35G The data presented in show that pooled supernatants from 3 different donors (BCT-UV) down-regulated IL-18 production induced by the combination of LPS and nigericin.

[0791] Together, these data suggest that single-donor and multi-donor CD4 IL-10 IL-10 produced by the cells strongly downregulates the NLPR3 inflammasome, leading to strong suppression of the proinflammatory cytokines IL-1β and IL-18.

[0792] 6.9.14. Example 13: Single and Multiple Donor CD4 IL-10 Cells inhibit xeno GvHD and myeloid tumor growth in vivo

[0793] Single-donor (BC-T, BC-V, and BC-E) or multi-donor (BC-V / T / E) CD4 IL-10 Functional properties and quality of cells. Single-donor and multi-donor CD4 IL-10 The cells all produced high levels of IL-10, variable levels of IFN-γ, very low levels of IL-4, and undetectable levels of IL-2 (the latter not shown), reflecting the characteristic cytokine production profile of Tr1 cells ( Fig.37 ).

[0794] In addition, single-donor (BC-T, BC-V, and BC-E) or multi-donor (BC-V / T / E) CD4 IL-10 CD4 + and CD8 +The inhibitory capacity of T cell proliferation. PBMCs were labeled with eFLuor670 (Invitrogen). Labeled PBMCs (1×10 5 Single and multiple donor CD4 IL-10 Cells were added to 96-well round-bottom plates at a ratio of 1:1 in a final volume of 0.2 mL. After 4 days of co-culture, their inhibitory effect on the proliferation of eFluor670-labeled responder cells was determined by eFluor670 dilution using flow cytometry as described in (Locafaro et al. Mol Ther 2017, 25, 2254). Fig.38 Flow cytometry results are provided. Single and multiple donor CD4 IL-10 The cells strongly inhibited the proliferation of allogeneic CD4+ and CD8+ T cells in vitro by more than 80% ( Fig.38 ).

[0795] Further analysis of CD4 IL-10 Cytotoxicity of cells against myeloid leukemia cells (ALL-CM) and erythroid leukemia cell lines (K562). Single donor (BC-E and BC-V) or multiple donor (BC-V / T / E) CD4 IL-10 The cells were co-cultured with ALL-CM or K562 cells at a 1:1 ratio.

[0796] After 3 days, cells were harvested and viable CD45 低 , CD3-target cells and analyzed by FACS as described (Locafaro et al. Mol Ther 2017, 25, 2254). Single and multiple donor CD4 IL-10 The cells also mediated potent direct cytotoxic effects against ALL-CM myeloid tumor cells, but they were unable to kill sensitive K562 cells, which lack MHC class I expression required for cytotoxic activity ( Fig.39 Single donor (BC-E and BC-V) or multiple donor (BC-V / T / E) CD4 IL-10 The cells had comparable cytotoxic activity against both target cell lines (ALL-CM and K562).

[0797] We also used a humanized xeno GvHD disease model (intravenous injection of ALL-CM cells (2.5 × 10 6 Single donor (BC-E) and multiple donor (BC-V / T / E) CD4 IL-10 Cytotoxic effects on cells. Fig.36As shown, their effects on GvHD induced by human PBMCs from allogeneic donors and their effects on acute myeloid leukemia growth of the cell line ALL CM in a therapeutic setting were tested.

[0798] Female NOD scid gamma (NSG) mice aged eight to ten weeks were obtained from Charles-River Italia (Calco, Italy). The experimental protocol was approved by the Internal Committee for Animal Research of Ospedale San Raffaele (Institutional Animal Care and Use Committee (IACUC)). On day 0, mice received whole-body irradiation from a linear accelerator. ALL-CM cells (2.5 × 10 6 On day 0, mice in different groups were injected with no injection, allogeneic PBMC (2.5×10 6 ), and allogeneic PBMC (2.5×10 6 ) combined with a single donor (BC-E, 2.5×10 6 or multiple donor CD4 IL-10 Cells (from 3 different donors (BC-V / T / E, 2.5×10 6 ) in a 1:1:1 ratio), or on day 3 multiple donor CD4 IL-10 Cells (2.5×10 6 All cells were administered intravenously in a volume of 250 μL of Iscove's modified Dulbecco's medium. Mice were monitored 3-4 times per week.

[0799] NSG mice were divided into five groups of 5 mice each, and each group received the following treatments on day 0: (i) no treatment as control; (ii) allogeneic PBMCs; (iii) allogeneic PBMCs and multiple donor CD4 IL-10 cells (BC-V / T / E); (iv) allogeneic PBMC and single donor CD4 IL-10 cells (BC-E); or (v) multi-donor (BC-V / T / E) CD4 IL-10 Myeloid leukemia progression was measured as previously described (Locafaro et al. Mol Ther 2017, 25, 2254).

[0800] Administration of ALL-CM cells to NSG mice resulted in rapid expansion of these cells, and all mice died or had to be sacrificed by day 20. As expected, injection of PBMCs prevented leukemia progression. Single and multiple donor CD4 IL-10 Administration of the cells in combination with allogeneic PBMCs did not interfere with the anti-myeloid leukemia effects of PBMCs.

[0801] Three days after administration of ALL-CM cells (when these cells have already expanded to a large extent), multiple donor CD4 IL-10 (BC-V / T / E) cells resulted in inhibition of tumor growth. These results suggest that multiple donor CD4 IL-10 The cells have direct anti-myeloid leukemia therapeutic effects in vivo ( Fig.40 ).

[0802] However, despite their beneficial anti-myeloid leukemia effects, PBMCs induced a very severe form of xeno-GvHD, and all mice died by day 24 ( Fig.41 In this study, single-donor (BC-E) and multi-donor (BC-V / T / E) CD4 IL-10 The ability of cells to inhibit PBMC-induced xeno-GvHD after administration to NSG mice. On day 0, NSG mice were injected with ALL-CM cells (2.5×10 6 The mice were divided into five groups, and each group received the following treatments: (i) no treatment as control; (ii) allogeneic PBMCs; (iii) allogeneic PBMCs and multi-donor (BC-V / T / E) CD4 IL-10 cells (BC-E, BC-V, BC-T); (iv) allogeneic PBMC and single donor CD4 IL-10 cells (BC-E) or injected on day 3 with multiple donors (BC-V / T / E) CD4 IL-10 Cells. In animals, xeno-GvHD was measured by survival and weight loss. In addition, hunchback, coat condition, and skin integrity were monitored as described (Bondanza et al, Blood, 2006, 107, 1828). Mice were sacrificed for ethical reasons if weight loss was greater than 20%. Fig.41 The results show that ALL-CM cells (2.5×10 6 ) and subsequently with PBMC (with or without single-donor or multiple-donor CD4 IL-10 % of NSG mice without GvHD every day after treatment with (cells).

[0803] The results showed that multiple donor CD4 IL-10 The cells did not induce xeno-GvHD and downregulated xeno-GvHD induced by allogeneic PBMCs. Taken together, these results suggest that multi-donor CD4 IL-10 The cells downregulated severe xeno-GvHD, had a direct anti-myeloid leukemia effect in a therapeutic setting, and did not interfere with the protective anti-myeloid leukemia effects of PBMCs.

[0804] 6.9.15. Example 14: Adoptive transfer of multi-donor CD4IL-10 cells from four different donors

[0805] Testing of multiple donor CD4 derived from four different donors IL-10 Adoptive transfer of cells inhibited the transfer capacity of PBMC-induced xeno-GvHD.

[0806] In these experiments, single donor CD4 IL-10 cells (donor C; batch C) and multi-donor CD4 IL-10 In this model, NSG mice were sublethally irradiated on day 0 and injected (slow intravenous bolus) on day 3 with (i) 2.5E+06 allogeneic PBMCs, (ii) 2.5E+06 allogeneic PBMCs combined with 2.5E+06 single donor CD4 IL-10 Cells (Batch C), (iii) 2.5E+06 allogeneic PBMCs and 2.5E+06 multi-donor CD4 IL-10 cells (batch CEFH), or (iv) single injection of 2.5E+06 multi-donor CD4 IL-10 Cells (batch CEFH). A composite score of weight loss, fur appearance, skin appearance, hunched back, and mobility was used to determine GvHD (see Bondanza A, et al. Blood 2006; 107: 1828–36). Fig.42 As shown, at the end of the study only multi-donor CD4 IL-...

Claims

1. A genetically modified CD4 + T cells (CD4 IL-10 / CAR ), which contains: (a) a first exogenous polynucleotide segment encoding a chimeric antigen receptor (CAR); and (b) a second exogenous polynucleotide segment encoding interleukin-10 (IL-10).

2. The genetically modified CD4 + T cell, wherein the first exogenous polynucleotide segment comprises a first regulatory element operably linked to the coding sequence of the CAR.

3. The genetically modified CD4 + T cell, wherein the regulatory element drives constitutive expression of the CAR.

4. The genetically modified CD4 + T cell, wherein the CAR comprises an antigen binding domain, a hinge region, a transmembrane domain, and an intracellular signaling domain.

5. The genetically modified CD4 + T cell, wherein the antigen binding domain is a single chain antibody fragment.

6. The genetically modified CD4 + T cell, wherein the single-chain antibody fragment comprises a single-chain Fv (scFv).

7. The genetically modified CD4 according to any one of claims 4-6. + T cell, wherein the antigen binding domain targets an antigen associated with an autoimmune disease, an inflammatory disease, or cancer.

8. The genetically modified CD4 + T cells, wherein the antigen is selected from the group consisting of: CD19, CD20, CD22, CD27, CD38, B7-H3, CD23, Lyml, Lym2, CLEC5A, CDH179b, FLT3, GCC, Muc, BCMA, CSF2RA, GFRa4, CD32, CD33, CEA, IL11Ra, IL13Ra, NYBRI, SLea, CD200R, TGFβR2, CD276, TROP2, LAMP1, PTK7, DLL3, CDH1, CDH6, CDH17, CDH19, TSHR, tyrosinase, HLA-A*02, HLA-A*24 or citrullinated peptide, insulin, MOG, GAD65, IA2, prolamin and desmosomal mucin.

9. The genetically modified CD4 + T cell, wherein the antigen binding domain comprises an anti-CD19 antigen binding domain.

10. The genetically modified CD4 + T cell, wherein the anti-CD19 antigen binding domain has the sequence of SEQ ID NO:

11.

11. The genetically modified CD4 + T cell, wherein the antigen binding domain is an anti-BCMA antigen binding domain.

12. The genetically modified CD4 + T cell, wherein the anti-BCMA antigen binding domain comprises the sequence of SEQ ID NO: 50-53.

13. The genetically modified CD4 according to any one of claims 1 to 12. + T cell, wherein the hinge region is selected from the group consisting of a human CD8α hinge region, a human CD28 hinge region, an IgG1 hinge region, and an IgG4 hinge region.

14. The genetically modified CD4 + T cell, wherein the hinge region is derived from human CD8α.

15. The genetically modified CD4 according to any one of claims 1-14. + T cells, wherein the transmembrane domain is selected from the group consisting of a TNFRSF 19 transmembrane domain, a CD3ζ transmembrane domain, a CD8α transmembrane domain, a CD4 transmembrane domain, a CD28 transmembrane domain, or a B7 family inducible co-stimulatory (ICOS) transmembrane domain.

16. The genetically modified CD4 + T cell, wherein the transmembrane domain is derived from CD8α.

17. The genetically modified CD4 according to any one of claims 1-16. + T cell, wherein the CAR further comprises one or more costimulatory domains.

18. The genetically modified CD4 + T cell, wherein the CAR comprises two costimulatory domains.

19. The genetically modified CD4 according to any one of claims 1-18. + T cell, wherein the one or more co-stimulatory domains are selected from the group consisting of 4-1BB, CD28, OX40, ICOS, CD27, MYD88-CD40 and KIR2DS2.

20. The genetically modified CD4 + T cell, wherein one of the one or more costimulatory domains is derived from CD28.

21. The genetically modified CD4 according to any one of claims 18-20. + T cell, wherein the second co-stimulatory domain is derived from 4-1BB.

22. The genetically modified CD4 according to any one of claims 1-21. + T cell, wherein the intracellular signaling domain comprises an immunoreceptor tyrosine-based activation motif (ITAM).

23. The genetically modified CD4 according to any one of claims 1-22. + T cells, the immunoreceptor tyrosine-based activation motif (ITAM) is derived from CD3ζ.

24. The genetically modified CD4 according to any one of claims 1-23. + T cell, wherein the CAR comprises: 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 region; CD28 co-stimulatory domain; and CD3ζ chain intracellular signaling domain.

25. The genetically modified CD4 according to any one of claims 1-24. + T cell, wherein the CAR comprises the sequence of SEQ ID NO: 9, 16, 22, 34, 41-49 or 54.

26. The genetically modified CD4 according to claim 25 + T cell, wherein the first exogenous polynucleotide segment comprises the sequence of SEQ ID NO: 10, 17, 23, 35 or 55.

27. The genetically modified CD4 according to any one of claims 1-26. + T cell, wherein the first exogenous polynucleotide segment is integrated into the T cell nuclear genome.

28. The genetically modified CD4 according to any one of claims 1-27. + T cell, wherein the first exogenous polynucleotide segment is not integrated into the T cell nuclear genome.

29. The genetically modified CD4 + T cell, wherein the first exogenous polynucleotide segment is in a vector.

30. The genetically modified CD4 according to any one of claims 1-29. + T cell, wherein the second exogenous polynucleotide segment comprises a second regulatory element operably linked to a coding sequence for IL-10.

31. The genetically modified CD4 according to any one of claims 1-30. + T cells, wherein the IL-10 is human IL-10.

32. The genetically modified CD4 according to any one of claims 1-30. + T cells, wherein the IL-10 is viral IL-10.

33. The genetically modified CD4 according to any one of claims 1-30. + T cell, wherein the IL-10 is a protein having the sequence of SEQ ID NO:

1.

34. The genetically modified CD4 + T cell, wherein the second exogenous polynucleotide segment has the sequence of SEQ ID NO:

2.

35. The genetically modified CD4 according to any one of claims 30-34. + T cell, wherein the second regulatory element drives constitutive or inducible expression of the IL-10.

36. The genetically modified CD4 according to any one of claims 1-35. + T cell, wherein the second exogenous polynucleotide segment is integrated into the T cell nuclear genome.

37. The genetically modified CD4 according to any one of claims 1-35. + T cell, wherein the second exogenous polynucleotide segment is not integrated into the T cell nuclear genome.

38. The genetically modified CD4 + T cell, wherein the second exogenous polynucleotide segment is in a vector.

39. The genetically modified CD4 according to any one of claims 1-38. + T cell, wherein the first exogenous polynucleotide segment and the second exogenous polynucleotide segment are in the same vector.

40. The genetically modified CD4 according to any of claims 29, 38 or 39. + T cell, wherein the vector is a viral vector.

41. The genetically modified CD4 + T cells, wherein the vector is a lentiviral vector.

42. The genetically modified CD4 according to any one of claims 1-41. + T cells, of which the CD4 + T cells constitutively express at least 100 pg IL-10 / 10 6 CD4 + T cells / mL culture medium.

43. The genetically modified CD4 according to claim 42 + T cells, of which the CD4 + T cells constitutively express at least 100 pg, 200 pg, 500 pg, 1 ng, 5 ng, 10 ng or 50 ng IL-10 / 10 6 CD4 + T cells / mL.

44. The genetically modified CD4 according to any one of claims 1-43. + T cells, of which the CD4 + T cells expressed at least 1 ng IL-10 / 10 after activation with anti-CD3 and anti-CD28 antibodies 6 CD4 + T cells / mL.

45. The genetically modified CD4 according to claim 44 + T cells, of which the CD4 + T cells expressing at least 2ng, 5ng, 10ng, 100ng, 200ng or 500ng IL-10 / 10 after activation with anti-CD3 and anti-CD28 antibodies 6 CD4 + T cells / mL.

46. ​​The genetically modified CD4 according to any one of claims 1-45. + T cells, of which the CD4 + T cells were more expressed than unmodified CD4 + T cells expressed IL-10 at least 5-fold higher levels.

47. The genetically modified CD4 according to claim 46 + T cells, of which the CD4 + T cells were more expressed than unmodified CD4 + T cells expressed IL-10 at least 10-fold higher levels.

48. The genetically modified CD4 according to any one of claims 1-47. + T cell, wherein the first exogenous polynucleotide segment, the second exogenous polynucleotide segment, or both further comprises a sequence encoding a selectable marker.

49. The genetically modified CD4 of claim 48 + T cells, wherein the selection marker is ΔNGFR.

50. The genetically modified CD4 of claim 49 + T cell, wherein the ΔNGFR has the sequence of SEQ ID NO:

3.

51. The genetically modified CD4 + T cell, wherein the second exogenous polynucleotide segment comprises the sequence of SEQ ID NO:

4.

52. The genetically modified CD4 of claim 48 + T cells, wherein the selection marker is a truncated form of the EGFR polypeptide.

53. A genetically modified CD4 according to any one of claims 9-10 or claims 13-52 when dependent on claim 9. + T cells, of which the CD4 + T cells can respond to CD19 + Target cells produce in vitro cytotoxicity.

54. A genetically modified CD4 according to any one of claims 9-10 or claims 13-52 when dependent on claim 9. + T cells, of which the CD4 + T cells can respond to CD19 + Target cells produce in vivo cytotoxicity.

55. The genetically modified CD4 + T cells, in which the CD19 + The target cells are B cells that produce autoantibodies.

56. The genetically modified CD4 + T cells, in which the CD19 + Target cells are CD19 + cancer cell.

57. The genetically modified CD4 according to any one of claims 1-56. + T cells, of which the CD4 + T cells are capable of in vitro cytotoxicity against myeloid target cells.

58. The genetically modified CD4 according to any one of claims 1-56. + T cells, of which the CD4 + T cells are capable of in vivo cytotoxicity against myeloid target cells.

59. The genetically modified CD4 according to any one of claims 53-58. + T cells, of which the CD4 + T cells can respond to CD19 + Target cells and myeloid target cells produce cytotoxicity.

60. The genetically modified CD4 according to any one of claims 57-59. + T cells, wherein the myeloid target cells express one or more of class I MHC, CD13, CD54 and CD112.

61. The genetically modified CD4 according to any one of claims 53-60. + T cells, which express CD19 + Target cell cytotoxicity is maintained after one or more in vitro restimulations.

62. The genetically modified CD4 according to any one of claims 53-60. + T cells, wherein cytotoxicity against myeloid target cells is maintained after one or more in vitro restimulations.

63. The genetically modified CD4 according to any one of claims 1-62. + T cells, of which the CD4 + T cells can suppress allogeneic CD4 + T cell proliferation.

64. The genetically modified CD4 according to any one of claims 1-63. + T cells, of which the CD4 + T cells can suppress allogeneic CD8 + T cell proliferation.

65. The genetically modified CD4 according to any one of claims 1-64. + T cells, of which the CD4 + T cells can suppress allogeneic CD4 + T cell proliferation, allogeneic CD8 + T cell proliferation and allogeneic PBMCs.

66. The genetically modified CD4 according to any one of claims 63-65. + T cells, wherein the suppressive properties are maintained after one or more in vitro restimulations.

67. A CD4 + A T cell population comprising the genetically modified CD4 + cell.

68. The CD4 according to claim 67 + T cell population, in which CD4 + T cells are obtained from one, two, three, four, five, six, seven, eight, nine or ten T cell donors and pooled.

69. The CD4 according to claim 68 + T cell population, wherein the CD4 + The T cells have a total of six, seven, eight, nine, ten, eleven, twelve or more different HLA haplotypes.

70. The CD4 of any one of claims 67-69. + T cell population, wherein all CD4 + The T cells have at least 1 / 10, 2 / 10, 3 / 10, 4 / 10, 5 / 10, 6 / 10, 7 / 10, 8 / 10 or 9 / 10 match with each other at the HLA-A, HLA-B, HLA-C, HLA-DRBl and HLA-DQBl loci.

71. The CD4 of any one of claims 67-70. + T cell population, wherein all CD4 + The T cells are at least 1 / 8, 2 / 8, 3 / 8, 4 / 8, 5 / 8, 6 / 8, 7 / 8 or 8 / 8 matched to each other at the HLA-A, HLA-B, HLA-C and HLA-DRBl loci.

72. The CD4 of any one of claims 67-71. + T cell population, wherein all CD4 + The T cells have a 2 / 2 match with each other at the HLA-A locus.

73. The CD4 of any one of claims 67-72. + T cell population, wherein all CD4 + The T cells have a 2 / 2 match with each other at the HLA-B locus.

74. The CD4 of any one of claims 67-73. + T cell population, wherein all CD4 + The T cells have a 2 / 2 match with each other at the HLA-C locus.

75. The CD4 of any one of claims 67-74. + T cell population, wherein all CD4 + The T cells have at least a 3 / 4 or 4 / 4 match with each other at the HLA-DRB1 and HLA-DQB1 loci.

76. The CD4 of any one of claims 67-69. + T cell population, wherein all CD4 + The T cells have less than 5 / 10, 6 / 10, 7 / 10, 8 / 10, or 9 / 10 matches with each other at the HLA-A, HLA-B, HLAC, HLA-DRBl, and HLA-DQBl loci.

77. The CD4 according to claim 76 + T cell population, wherein all CD4 + The T cells have less than 4 / 8, 5 / 8, 6 / 8, 7 / 8 or 8 / 8 matches with each other at the HLA-A, HLA-B, HLA C and HLA DRB1 loci.

78. The CD4 of any one of claims 76-77. + T cell population, wherein all CD4 + T cells have less than a 2 / 2 match with each other at the HLA-A locus.

79. The CD4 of any one of claims 76-78. + T cell population, wherein all CD4 + T cells have less than a 2 / 2 match with each other at the HLA-B locus.

80. The CD4 of any one of claims 76-79. + T cell population, wherein all CD4 + T cells have less than a 2 / 2 match with each other at the HLA-C locus.

81. The CD4 of any one of claims 76-80. + T cell population, wherein all CD4 + The T cells have less than 2 / 4, 3 / 4 or 4 / 4 matches with each other at the HLA-DRB1 and HLA DQB1 loci.

82. The CD4 according to any one of claims 67 to 81. + T cell population, wherein all CD4 + T cells either have the A*02 allele or are all A*02 negative.

83. The CD4 according to any one of claims 67-81. + T cell population, wherein all CD4 + T cells either have the A*24 allele or are all A*24 negative.

84. The CD4 of any one of claims 67-83. + T cell population, in which NGFR + At least 30% of the cells in the population are CD4 + T cells express CAR.

85. The CD4 according to claim 84 + T cell population in which the NGFR + At least 60% of the cells in the population are CD4 + T cells express CAR.

86. The CD4 according to claim 85 + T cell population in which the NGFR + At least 90% of the cells in the population are CD4 + T cells express CAR.

87. The CD4 of any one of claims 67-86. + T cell population, in which the CD4 + T cells are in frozen suspension.

88. The CD4 of any one of claims 67-86 + T cell population, in which the CD4 + The T cells are in liquid suspension.

89. The CD4 according to claim 88 + A population of T cells, wherein the liquid suspension has been previously frozen.

90. A pharmaceutical composition comprising the genetically modified CD4 + T cell or a CD4 + T cell populations.

91. A method for preparing genetically modified CD4 + T cells (CD4 IL-10 / CAR ) cells, comprising the following steps: (a) Obtain primary CD4 T cells from one or more T cell donors + T cells; as well as (b) modifying the CD4 by introducing the following polynucleotide segments + T cells: (i) a first exogenous polynucleotide segment encoding a chimeric antigen receptor (CAR), and (ii) a second exogenous polynucleotide segment encoding IL-10.

92. The method of claim 91, wherein in step (a), the primary CD4 + The T cells are obtained from two, three, four, five, six, seven, eight, nine or ten different T cell donors.

93. The method of claim 92, further comprising combining the genetically modified CD4 + T cells.

94. The method of claim 91, wherein in step (a), the primary CD4 + The T cells are autologous to the patient.

95. The method according to any one of claims 91-94, further comprising the following steps after step (a) and before step (b), or after step (b): The CD4 + T cells to activate and expand human T cells through CD3 and CD28, or incubate the CD4 + T cells.

96. The method of claim 95, further comprising incubating the CD4 + T cells.

97. The method of any one of claims 91-96, wherein the first exogenous polynucleotide segment, the second exogenous polynucleotide segment, or both are introduced into the primary CD4 + in T cells.

98. The method of claim 97, wherein the viral vector is a lentiviral vector.

99. The method of any one of claims 91-98, wherein the CAR is specific for a target antigen associated with an autoimmune disease, an inflammatory disease, or cancer.

100. The method of claim 99, wherein the target antigen is associated with an autoimmune disease, an inflammatory disease, or cancer.

101. The method of any one of claims 91-100, wherein the CAR is an anti-CD19 CAR, an anti-CD20 CAR, an anti-CD22 CAR, an anti-BCMACAR, an anti-B7-H3 CAR, an anti-CD27 CAR or an anti-CD38 CAR.

102. The method of any one of claims 91-101, wherein the CAR has a sequence of SEQ ID NO: 9, 16, 22, 34, 41-49 or 54.

103. The method of any one of claims 91-102, wherein the first exogenous polynucleotide segment has a sequence of SEQ ID NO: 10, 17, 23, 35 or 55.

104. The method of any one of claims 91-103, wherein the IL-10 has the sequence of SEQ ID NO:

1.

105. The method of any one of claims 91-104, wherein the second exogenous polynucleotide segment has a sequence of SEQ ID NO:

2.

106. The method of any one of claims 91-105, wherein the first exogenous polynucleotide segment, the second exogenous polynucleotide segment, or both further comprises a segment encoding a selectable marker.

107. The method of claim 106, wherein the encoded selectable marker is ΔNGFR.

108. The method of claim 107, wherein the encoded selection marker has a sequence of SEQ ID NO:

3.

109. The method of claim 106, wherein the selection marker is a truncated form of an EGFR polypeptide.

110. The method according to any one of claims 91-109, further comprising the following steps after step (b): (c) Isolation of genetically modified CD4 expressing a selectable marker + T cells, thereby generating enriched genetically modified CD4 + T cell populations.

111. The method of claim 110, wherein at least 30% or at least 60% of the genetically modified CD4 + T cells express IL-10 and the CAR.

112. The method of claim 111, wherein at least 90% of the genetically modified CD4 + T cells express IL-10 and the CAR.

113. The method of any one of claims 110-112, wherein at least 75% of the genetically modified CD4 + The T cells express the selection marker.

114. The method of claim 113, wherein at least 90% of the genetically modified CD4 + The T cells express the selection marker.

115. The method according to any one of claims 110-114, further comprising incubating the enriched genetically modified CD4 + Steps for T cell populations.

116. The method of claim 115, wherein the enriched genetically modified CD4 + The T cell population step is performed in the presence of IL-2 or a polymer nanomatrix reagent, in the presence of (i) anti-CD3 antibody and anti-CD28 antibody, or (ii) beads coated with anti-CD3 antibody and anti-CD28 antibody to activate and expand human T cells via CD3 and CD28, or (iii) in the presence of other T cell-specific immune cell culture media, activators and supplements.

117. The method of any one of claims 91-116, further comprising freezing the genetically modified CD4 + Next steps for T cells.

118. The method of any one of claims 92-117, wherein at least two T cell donors are at least 1 / 10, 2 / 10, 3 / 10, 4 / 10, 5 / 10, 6 / 10, 7 / 10, 8 / 10 or 9 / 10 matched to each other at the HLA-A, HLA-B, HLA-C, HLA-DRB1 and HLA-DQB1 loci.

119. The method of any one of claims 92-118, wherein at least two T cell donors are at least 1 / 8, 2 / 8, 3 / 8, 4 / 8, 5 / 8, 6 / 8, 7 / 8 or 8 / 8 matched to each other at the HLA-A, HLA-B, HLA-C and HLA-DRB1 loci.

120. The method of any one of claims 92-119, wherein at least two T cell donors have a 2 / 2 match with each other at the HLA-A locus.

121. The method of any one of claims 92-120, wherein at least two T cell donors have a 2 / 2 match with each other at the HLA-B locus.

122. The method of any one of claims 92-121, wherein at least two T cell donors have a 2 / 2 match with each other at the HLA-C locus.

123. The method of any one of claims 92-122, wherein at least two T cell donors are at least 3 / 4 or 4 / 4 matched to each other at the HLA-DRB1 and HLA-DQB1 loci.

124. The method of any one of claims 92-117, wherein at least two T cell donors have a match of less than 5 / 10, 6 / 10, 7 / 10, 8 / 10 or 9 / 10 with each other at the HLA-A, HLA-B, HLA C, HLA-DRB1 and HLA-DQB1 loci.

125. The method of claim 124, wherein at least two T cell donors have less than 4 / 8, 5 / 8, 6 / 8, 7 / 8 or 8 / 8 matching with each other at the HLA-A, HLA-B, HLAC and HLADRB1 loci.

126. The method of any one of claims 124-125, wherein at least two T cell donors have less than a 2 / 2 match with each other at the HLA-A locus.

127. The method of any one of claims 124-126, wherein at least two T cell donors have less than a 2 / 2 match with each other at the HLA-B locus.

128. The method of any one of claims 124-127, wherein at least two T cell donors have less than a 2 / 2 match with each other at the HLA-C locus.

129. The method of any one of claims 124-128, wherein at least two T cell donors have less than a 4 / 2, 3 / 4, or 4 / 4 match with each other at the HLA-DRB1 and HLADQB1 loci.

130. The method of any one of claims 92-129, wherein each of at least two T cell donors has an A*02 allele or is A*02 negative.

131. The method of any one of claims 92-129, wherein each of at least two T cell donors has an A*24 allele or is A*24 negative.

132. The method according to any one of claims 92-131, wherein in step (a), the primary CD4 + T cells are obtained from one or more frozen stocks.

133. The method according to any one of claims 92-132, wherein in step (a), the primary CD4 + T cells were obtained from unfrozen peripheral blood mononuclear cells from at least two different T cell donors.

134. The method of claim 133, further comprising isolating CD4 + T cell steps.

135. A method for treating a hematological cancer, comprising: Administering a therapeutically effective amount of a CD4+ antibody according to any one of claims 1 to 66 sufficient to induce an anti-cancer effect to a patient with a hematological cancer IL-10 / CAR Cells, CD4 IL-10 / CAR A cell population or a pharmaceutical composition according to claim 90.

136. The method of claim 135, further comprising administering the CD4 IL-10 / CAR Prior to or after the step of administering an allo-HSCT transplant to said patient.

137. A method for treating a patient with a malignant tumor, comprising: administering an allo-HSCT transplant to said patient, and Administering a therapeutically effective amount of a CD4 IL-10 / CAR Cells, CD4 IL-10 / CAR A cell population or a pharmaceutical composition according to claim 90.

138. The method of claim 137, wherein CD4 IL-10 / CAR The amount of cells is also sufficient to inhibit or prevent graft-versus-host disease (GvHD) without inhibiting the graft-versus-leukemia (GvL) or graft-versus-tumor (GvT) efficacy of allo-HSCT.

139. The method of any one of claims 135-138, wherein the malignancy or hematological cancer is a myeloid leukemia.

140. The method of any one of claims 135-139, wherein the CD4 IL-10 / CAR The cells target and kill cancer cells that express CD13.

141. The method of any one of claims 135-140, wherein the CD4 IL-10 / CAR The cells target and kill cancer cells that express HLA class I.

142. The method of any one of claims 135-141, wherein the myeloid leukemia is acute myeloid leukemia (AML).

143. The method of any one of claims 135-138, wherein the malignancy or hematological cancer is CD19 + 、CD20 + 、CD22 + , BCMA + 、CD27 + 、CD38 + or B7-H3 + Blood system cancer.

144. The method of claim 143, wherein the CD19 + 、CD20 + 、CD22 + , BCMA + 、CD27 + 、CD38 + or B7-H3 + The hematological cancer is selected from the group consisting of chronic lymphocytic leukemia, acute lymphoblastic leukemia (ALL) and non-Hodgkin's lymphoma.

145. The method of any one of claims 136-144, wherein the allo-HSCT graft is obtained from a donor who is related or unrelated to the patient.

146. The method of any one of claims 135-144, wherein the CD4 IL-10 / CAR The cells are non-autologous to the patient.

147. The method of any one of claims 135-144, wherein the CD4 IL-10 / CAR The cells are autologous to the patient.

148. The method of any one of claims 135-144, wherein the CD4 IL-10 / CAR The cells are allogeneic to the patient.

149. The method of any one of claims 135-144, wherein the CD4 IL-10 / CAR The cells have not lost reactivity to host alloantigens prior to administration to the patient.

150. The method of any one of claims 135-144, wherein the CD4 IL-10 / CAR The cells are Tr1-like cells.

151. The method of any one of claims 135-144, wherein the CD4 IL-10 / CAR The cells are polyclonal.

152. The method of any one of claims 135-144, wherein the CD4 IL-10 / CAR The cells are polyclonal and non-autologous to the patient.

153. The method of any one of claims 135-144, wherein the CD4 IL-10 / CAR The cells are polyclonal and autologous to the patient.

154. The method of any one of claims 135-144, wherein the CD4 IL-10 / CAR The cells are isolated from at least two donors and then genetically modified.

155. The method of claim 154, wherein none of the at least two donors is the same donor as the allo-HSCT donor.

156. The method of any one of claims 135-155, wherein the allo-HSCT graft is obtained from a donor that is matched or mismatched to the patient.

157. The method of any one of claims 135-156, wherein the CD4 IL-10 / CAR The cells target and kill cells expressing CD19, CD20, or BCMA.

158. The method of any one of claims 135-157, wherein the CD4 IL-10 / CAR Cells target and kill cells expressing CD54.

159. The method of any one of claims 135-158, wherein the CD4 IL-10 / CAR The cells target and kill cancer cells expressing HLA class I and CD54.

160. The method of any one of claims 135-159, wherein the CD4 IL-10 / CAR The cells target and kill cancer cells that express CD112 and CD155.

161. The method of any one of claims 135-160, wherein the CD4 IL-10 / CAR The cells target and kill cancer cells that express CD58.

162. The method of any one of claims 135-161, wherein the CD4 IL-10 / CAR The cells target and kill cancer cells in the patient.

163. The method of any one of claims 135-162, wherein the CD4 IL-10 / CAR The cells target and kill solid tumor cells in the patient.

164. The method of any one of claims 136-163, wherein the CD4 IL-10 / CAR Allogeneic CD4 + T cell proliferation.

165. The method of any one of claims 136-164, wherein the CD4 IL-10 / CAR Allogeneic CD8 + T cell proliferation.

166. The method of any one of claims 136-163, wherein the CD4 IL-10 / CAR Allogeneic CD4 + T cell proliferation, allogeneic CD8 + T cell proliferation and PBMCs.

167. A method for treating hematological cancer by allogeneic hematopoietic stem cell transplantation (allo-HSCT), comprising: administering an allo-HSCT transplant to a patient; Administering a certain amount of CD4 IL-10 / CAR cells, optionally wherein the amount is sufficient to inhibit or prevent graft-versus-host disease (GvHD) without inhibiting graft-versus-leukemia (GvL) or graft-versus-tumor (GvT) efficacy of the allo-HSCT transplant, The CD4 IL-10 / CAR Cells contain CD4 + T cells, the CD4 + The T cells are genetically modified by vector-mediated gene transfer of a coding sequence for human IL-10 under the control of a constitutive or inducible promoter and vector-mediated gene transfer of a coding sequence for CAR under the control of a constitutive or inducible promoter; The CD4 IL-10 / CAR cells targeting and killing cancer cells in said patient; The CD4 IL-10 / CAR The cells have not lost reactivity to host alloantigens prior to administration to the patient; and The CD4 IL-10 / CAR The cells are non-autologous to the patient and are Tr1-like.

168. A method for treating hematological cancer by allogeneic hematopoietic stem cell transplantation (allo-HSCT), comprising: administering an allo-HSCT transplant to a patient; Administering a certain amount of CD4 IL-10 / CAR cells, optionally wherein the amount is sufficient to inhibit or prevent graft-versus-host disease (GvHD) without inhibiting graft-versus-leukemia (GvL) or graft-versus-tumor (GvT) efficacy of the allo-HSCT transplant, The CD4 IL-10 / CAR Cells contain CD4 + T cells, the CD4 + The T cells are genetically modified by vector-mediated gene transfer of a coding sequence for human IL-10 under the control of a constitutive or inducible promoter and vector-mediated gene transfer of a coding sequence for CAR under the control of a constitutive or inducible promoter; The CD4 IL-10 / CAR cells targeting and killing cancer cells in said patient; The CD4 IL-10 / CAR The cells have not lost reactivity to host alloantigens prior to administration to the patient; and The CD4 IL-10 / CAR The cells are autologous to the patient and are Tr1-like.

169. A method for preventing CD19 in patients + or CD20 + 、CD22 + , BCMA + 、CD27 + 、CD38 + or B7-H3 + A method for the treatment of relapsed hematologic cancer comprising: To be identified as having CD19 + 、CD20 + 、CD22 + , BCMA + 、CD27 + 、CD38 + or B7-H3 + Patients at risk of recurrence of hematological cancer are administered a therapeutically effective amount of a CD4 IL-10 / CAR Cells, CD4 IL-10 / CAR The cell population or pharmaceutical composition of claim 90, wherein the therapeutically effective amount is sufficient to induce an anti-cancer effect.

170. A method for preventing B7-H3 in patients + Methods for treating cancer recurrence, which include: To those who are diagnosed with B7-H3 + A patient with cancer is administered a therapeutically effective amount of a CD4 IL-10 / CAR Cells, CD4 IL-10 / CAR A cell population or a pharmaceutical composition according to claim 90.

171. The method of claim 170, wherein the B7-H3 + Cancer is a solid tumor.

172. The method of claim 171, wherein 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.

173. A method for treating a cancer patient having minimal residual disease, comprising: Administering a therapeutically effective amount of a CD4+ antibody according to any one of claims 1 to 66 to a patient identified as having minimal residual disease or at risk of having minimal residual disease. IL-10 / CAR Cells, CD4 IL-10 / CAR The cell population or pharmaceutical composition of claim 90, wherein the therapeutically effective amount is sufficient to induce an anti-cancer effect.

174. A method of treating a patient, comprising: Administering a therapeutically effective amount of a CD4 IL-10 / CAR Cells, CD4 IL-10 / CAR The pharmaceutical composition of claim 90.

175. The method of claim 174, further comprising thawing CD4 IL-10 / CAR cells or CD4 IL -10 / CAR A previous step of freezing suspension of cell populations.

176. The method of claim 174 or 175, wherein the patient suffers from an inflammatory disease or an autoimmune disease.

177. The method of claim 176, wherein the inflammatory 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, Sjögren's syndrome, Hashimoto's thyroiditis, myasthenia gravis, autoimmune vasculitis, pernicious anemia, ulcerative colitis, bullous diseases, scleroderma, and celiac disease.

178. The method of claim 177, wherein 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.

179. The method of claim 174, wherein the patient suffers from a disease or disorder involving overactivity of the NLPR3 inflammasome.

180. The method of claim 174, wherein the patient has type 2 diabetes, a neurodegenerative disease, a cardiovascular disease, or an inflammatory bowel disease.

181. The method of claim 174, wherein the patient has a disease or disorder involving increased production of IL-1 β by activated monocytes, macrophages, or dendritic cells.

182. The method of claim 174, wherein the patient has a disease or disorder involving increased production of IL-18 by activated monocytes, macrophages, or dendritic cells.

183. The method of claim 174, wherein the patient has a disease or disorder involving increased production of mature caspase 1 by activated monocytes, macrophages, or dendritic cells.

184. The method of claim 174, wherein the patient suffers from an allergic or atopic disease.

185. The method of claim 184, wherein the allergic or atopic disease is selected from the group consisting of asthma, atopic dermatitis, and rhinitis.

186. The method of claim 174, wherein the patient suffers from food allergies.

187. The method of claim 174, wherein the patient has a solid tumor.

188. The method of claim 187, wherein 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.

189. The method of claim 174, further comprising administering the CD4 IL-10 / CAR cells, the CD4 IL-10 / CAR The step of performing an organ transplant on the patient before or after administering the cell population or the pharmaceutical composition.

190. The method of claim 189, wherein the CD4 IL-10 / CAR cells, the CD4 IL-10 / CAR The cell population or the pharmaceutical composition prevents host rejection of the organ transplant or reduces the severity of host rejection of the organ transplant.

191. The method of claim 174, further comprising administering the CD4 IL-10 / CAR cells, the CD4 IL-10 / CAR The step of transplanting iPS cell-derived cells or tissues into the patient before or after the cell population or the pharmaceutical composition.

192. The method of claim 191, wherein the CD4 IL-10 / CAR cells, the CD4 IL-10 / CAR The cell population or the pharmaceutical composition prevents or reduces the severity of host rejection of the cell transplant.

193. The method of claim 174, further comprising administering the CD4 IL-10 / CAR cells, the CD4 IL-10 / CAR The step of administering the recombinant AAV to the patient before or after the cell population or the pharmaceutical composition.

194. The method of claim 193, wherein the CD4 IL-10 / CAR cells, the CD4 IL-10 / CAR The cell population or the pharmaceutical composition reduces the immune response to the recombinant AAV.

195. The method of claim 174, further comprising administering the CD4 IL-10 / CAR cells, the CD4 IL-10 / CAR The step of administering a recombinant viral vector other than AAV to the patient before or after the cell population or the pharmaceutical composition.

196. The method of claim 195, wherein the CD4 IL-10 / CAR cells, the CD4 IL-10 / CAR The cell population or the pharmaceutical composition reduces the immune response to the recombinant viral vector other than AAV.

197. The method of claim 196, further comprising administering the CD4 IL-10 / CAR cells, the CD4 IL-10 / CAR The immunogenic therapeutic protein is administered to the patient before or after administration of the cell population or the pharmaceutical composition.

198. The method of claim 197, wherein the CD4 IL-10 / CAR cells, the CD4 IL-10 / CAR The cell population or the pharmaceutical composition reduces the immune response to the immunogenic therapeutic protein.

199. The method of claim 197 or 198, wherein the immunogenic therapeutic protein is selected from a therapeutic antibody, a Factor VIII substitute, a cytokine, and a cytokine mutein.

200. The method of claim 174, wherein the patient has an exaggerated immune response to a viral or bacterial infection.

201. The method of claim 200, wherein the patient has a coronavirus infection.

202. The method of claim 174, further comprising the steps of: Detection of a selected marker in a biological sample obtained from the patient to detect CD4 IL-10 / CAR The presence or absence of cells.

203. The method of claim 202, wherein the biological sample is a biopsy or blood from the patient.

204. A method for treating or inhibiting an autoimmune disease, an allergic disease, or an inflammatory disease in a patient, comprising: Administering a therapeutically effective amount of a CD4+ antibody according to any one of claims 1 to 66 sufficient to treat or inhibit the autoimmune disease, allergic disease or inflammatory disease to a patient identified as having the autoimmune disease, allergic disease or inflammatory disease IL-10 / CAR Cells, CD4 IL-10 / CAR A cell population or a pharmaceutical composition according to claim 90.

205. A method for reducing transplant rejection in a patient transplanted with hematopoietic stem cells, bone marrow cells, cord blood cells, tissue stem cells or solid organs, comprising: A therapeutically effective amount of a CD4+ antibody according to any one of claims 1 to 66 sufficient to reduce transplant rejection is administered to a patient identified as having rejection of a transplanted hematopoietic stem cell, bone marrow cell or solid organ. IL-10 / CAR Cells, CD4 IL-10 / CAR A cell population or a pharmaceutical composition according to claim 90.

206. A method for treating graft-versus-host disease (GvHD) in a patient, comprising: Administering a therapeutically effective amount of a CD4+ antibody according to any one of claims 1 to 66 sufficient to inhibit or prevent GvHD to a patient identified as having or at risk of having GvHD IL-10 / CAR Cells, CD4 IL-10 / CAR A cell population or a pharmaceutical composition according to claim 90.

207. The method of claim 206, wherein the graft-versus-host disease (GvHD) comprises acute GvHD.

208. The method of claim 206, wherein the graft-versus-host disease (GvHD) comprises chronic GvHD.

209. A method for treating tissue or organ damage in a patient, comprising: Administering a therapeutically effective amount of a CD4+ antibody according to any one of claims 1 to 66 sufficient to induce repair of tissue or organ damage to a patient identified as having tissue or organ damage or at risk of having tissue or organ damage IL-10 / CAR Cells, CD4 IL-10 / CAR A cell population or a pharmaceutical composition according to claim 90.

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

211. The polynucleotide construct of claim 210, wherein the first polynucleotide segment comprises a first regulatory element operably linked to the coding sequence of the CAR.

212. The polynucleotide construct of claim 211, wherein the first regulatory element drives constitutive expression of the CAR.

213. The polynucleotide construct of any one of claims 210-212, wherein the second polynucleotide segment comprises a second regulatory element operably linked to the coding sequence of IL-10.

214. The polynucleotide construct of claim 213, wherein the second regulatory element drives constitutive expression of the IL-10.

215. The polynucleotide construct of any one of claims 210-214, further comprising an internal ribosome entry site (IRES) or a self-cleaving peptide located between the first polynucleotide segment and the second polynucleotide segment.

216. The polynucleotide construct of claim 215, wherein the self-cleaving peptide is selected from the group consisting of F2A, P2A, T2A and E2A.

217. The polynucleotide construct of any one of claims 210-216, wherein the CAR comprises an antigen binding domain, a hinge region, a transmembrane domain, and an intracellular signaling domain.

218. The polynucleotide construct of claim 217, wherein the antigen binding domain comprises a single-chain antibody fragment.

219. The polynucleotide construct of claim 218, wherein the single-chain antibody fragment comprises a single-chain Fv (scFv).

220. The polynucleotide construct of any one of claims 210-219, wherein the antigen binding domain targets an antigen associated with an autoimmune disease, an inflammatory disease, or cancer.

221. The polynucleotide construct of claim 220, wherein the antigen is selected from the group consisting of CD19, CD20, CD22, BCMA, CD27, CD38, CEA, B7-H3, CD23, Lyml, Lym2, CLEC5A, CDH179b, FLT3, GCC, Muc, CSF2RA, GFRa4, CD32, CD33, IL11Ra, IL13Ra, NYBRI, SLea, CD200R, TGFβR2, CD276, TROP2, LAMP1, PTK7, DLL3, CDH1, CDH6, CDH17, CDH19, TSHR, tyrosinase, HLA-A*02, HLA-A*24 citrullinated peptide, insulin, MOG, GAD65, IA2, prolamin, and desmosomal mucin.

222. The polynucleotide construct of any one of claims 210-221, wherein the antigen binding domain is an anti-CD19 antigen binding domain.

223. The polynucleotide construct of claim 222, wherein the anti-CD19 antigen binding domain has the sequence of SEQ ID NO:

11.

224. The polynucleotide construct of any one of claims 210-221, wherein the antigen binding domain is an anti-BCMA antigen binding domain.

225. The polynucleotide construct of claim 224, wherein the anti-BCMA antigen binding domain has the sequence of any one of SEQ ID NOs: 50-53.

226. A polynucleotide construct according to any one of claims 210-225, wherein the hinge region is selected from the human CD8α hinge region, the human CD28 hinge region, the IgG1 hinge region or the IgG4 hinge region.

227. The polynucleotide construct of claim 226, wherein the hinge region is derived from human CD8α.

228. The polynucleotide construct of any one of claims 210-227, wherein the transmembrane domain is selected from a TNFRSF 19 transmembrane domain, a CD3ζ transmembrane domain, a CD8α transmembrane domain, a CD4 transmembrane domain, a CD28 transmembrane domain, or a B7 family inducible co-stimulatory (ICOS) transmembrane domain.

229. The polynucleotide construct of claim 228, wherein the transmembrane domain is derived from C CD8α.

230. The polynucleotide construct of any one of claims 210-229, wherein the CAR further comprises one or more co-stimulatory domains.

231. A polynucleotide construct according to any claim 230, wherein the CAR comprises two co-stimulatory domains.

232. The polynucleotide construct of any one of claims 210-231, wherein the one or more co-stimulatory domains are selected from the group consisting of 4-1BB, CD28, OX40, ICOS, CD27, MYD88-CD40, and KIR2DS2.

233. The polynucleotide construct of claim 232, wherein one of the one or more co-stimulatory domains is derived from CD28.

234. The polynucleotide construct of any one of claims 231-233, wherein the second co-stimulatory domain is derived from 4-1BB.

235. The polynucleotide construct of any one of claims 210-234, wherein the intracellular signaling domain comprises an immunoreceptor tyrosine-based activation motif (ITAM).

236. A polynucleotide construct according to any one of claims 210-235, wherein the immunoreceptor tyrosine-based activation motif (ITAM) is derived from CD3ζ.

237. The polynucleotide construct of any one of claims 210-236, wherein the CAR comprises: anti-CD19 antigen binding domain, anti-BCMA antigen binding domain or anti-CD20 antigen binding; human CD8 hinge region; CD8 transmembrane region; CD28 co-stimulatory domain; and CD3ζ chain intracellular signaling domain.

238. A polynucleotide construct according to any one of claims 210-237, wherein the CAR comprises a sequence of SEQ ID NO: 9, 16, 22, 34, 41-49 or 54.

239. The polynucleotide construct of claim 238, wherein the first polynucleotide segment comprises the sequence of SEQ ID NO: 10, 17, 23, 35 or 55.

240. The polynucleotide construct of any one of claims 210-239, wherein the IL-10 is human IL-10.

241. The polynucleotide construct of any one of claims 210-239, wherein the IL-10 is a viral IL-10.

242. The polynucleotide construct of any one of claims 210-239, wherein the IL-10 is a protein having the sequence of SEQ ID NO:

1.

243. The polynucleotide construct of claim 242, wherein the second polynucleotide segment has the sequence of SEQ ID NO:

2.

244. The polynucleotide construct of any one of claims 210-243, wherein the first polynucleotide segment or the second polynucleotide segment further comprises a sequence encoding a selection marker.

245. The polynucleotide construct of claim 244, wherein the selection marker is ΔNGFR.

246. The polynucleotide construct of claim 245, wherein the ΔNGFR has the sequence of SEQ ID NO:

3.

247. A polynucleotide construct according to claim 245, wherein the polynucleotide construct comprises the sequence of SEQ IDNO:

4.

248. The polynucleotide construct of any one of claims 244-247, wherein the selection marker is a truncated form of an EGFR polypeptide.

249. A polynucleotide construct according to any one of claims 210-248, wherein the construct is a vector.

250. The polynucleotide construct of claim 249, wherein the vector is a viral vector.

251. The polynucleotide construct of claim 250, wherein the vector is a lentiviral vector.

252. A polynucleotide construct comprising: (a) a first polynucleotide segment having a 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.

253. A polynucleotide construct comprising: (a) a first polynucleotide segment having a sequence of SEQ ID NO: 10, 17, 23, 35 or 55; (b) a second polynucleotide segment having a sequence of SEQ ID NO: 2; and (c) a third polynucleotide segment having a sequence of SEQ ID NO: 33 located between the first polynucleotide segment and the second polynucleotide segment.

254. A polynucleotide construct comprising: (a) a first polynucleotide segment having a sequence of SEQ ID NO: 10, 17, 23, 35 or 55; (b) a second polynucleotide segment having a sequence of SEQ ID NO: 2; (c) a third polynucleotide segment having a sequence of SEQ ID NO: 27 located between the first polynucleotide segment and the second polynucleotide segment; and (d) a fourth polynucleotide segment having the sequence of SEQ ID NO:4.

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