Novel large-scale CAR-T immune cell production method using lentiviral vector transfection

Abstract

The present disclosure provides novel and efficient methods and lentiviral vectors for making immune cell populations engineered to express chimeric antigen receptors (CARs), engineered T cell receptors (TCRs), and / or nucleic acid sequences encoding a polypeptide or functional derivative thereof that enhances immune cell function within less than 72 hours; an engineered cell produced by the method; a composition comprising the cell; and methods of treating a disease or condition using the cells.

Description

Cross - Reference to Related Applications

[0001] This application claims the priority of U.S. Provisional Application No. 63 / 414,829, filed on October 10, 2022, the content of which is hereby incorporated by reference in its entirety for all purposes. Technical Field

[0002] The present disclosure generally relates to efficient methods for manufacturing immune effector cells expressing chimeric antigen receptors (CARs) and / or engineered T - cell receptors (TCRs) and / or nucleic acid sequences encoding polypeptides or functional derivatives thereof that enhance immune cell function. Background Art

[0003] Adoptive immunotherapy involves transferring ex vivo - generated autologous antigen - specific T cells back into a patient and has shown to be a promising strategy for treating cancer, infections, and autoimmune diseases. T cells for adoptive immunotherapy are primary cells engineered to express a chimeric antigen receptor (CAR) or a recombinant T - cell receptor (TCR) and are expanded ex vivo to redirect primary immune cells against pathologic cells such as cancer cells. A CAR is a synthetic antibody - like molecule composed of a targeting moiety associated with one or more signaling domains in a single fusion molecule and is designed to confer antigen - specificity to T cells. CARs have successfully allowed T cells to be redirected against antigens expressed on the surface of tumor cells from various malignancies, including lymphomas and solid tumors.

[0004] The manufacture of gene - modified T cells is currently a complex process. There is a need for methods and processes for improving the generation of cell - therapy products expressing CARs or TCRs, enhancing product quality, and maximizing the therapeutic efficacy of CAR T - cell immunotherapy. The present invention provides methods and compositions to address these needs. Summary of the Invention

[0005] One aspect of the present disclosure provides a method for manufacturing an engineered population of immune cells, the method comprising: (a) enriching a population of lymphocytes, a population of immune cells, or a population of CD4 + and CD8 + cells obtained from a subject; (b) mixing the population of lymphocytes, the population of immune cells, or the population of CD4 + and CD8 + cells with one or more buffer solutions; and (c) transfecting the population of lymphocytes, the population of immune cells, or the population of CD4 + and CD8 + cells with an effective dose of a modifier; thereby generating a modified population of lymphocytes, a modified population of immune cells, or a modified population of CD4 + and CD8+ Cell population. In some embodiments, steps 1(a)-(c) are performed within 24 hours. In some embodiments, prior to enriching the immune cell population or the CD4 + and CD8 + cell population, the blood is separated by apheresis into plasma components, a mononuclear cell-containing layer, a platelet layer, and red blood cells to produce an apheresis product selected from: red blood cell apheresis, thrombus apheresis, coagulation cell apheresis, white blood cell apheresis, stem cells, plasma apheresis, and platelet apheresis. In some embodiments, the immune cell population or the CD4 + and CD8 + cell population is enriched by apheresis, elutriation, or gradient centrifugation.

[0006] Another aspect of the present disclosure provides a method for manufacturing an engineered immune cell population, the method comprising: (a) enriching a lymphocyte population, an immune cell population, or a CD4 + and CD8 + cell population from a donor leukapheresis; (b) mixing the lymphocyte population, the immune cell population, or the CD4 + and CD8 + cell population with one or more buffer solutions; and (c) transfecting the lymphocyte population, the immune cell population, or the CD4 + and CD8 + cell population with an effective dose of a modifier, thereby producing a lymphocyte population, a modified immune cell population, or a modified CD4 + and CD8 + cell population. In some embodiments, steps 1(a)-(c) are performed within 24 hours.

[0007] Another aspect of the present disclosure provides a method for manufacturing an engineered eukaryotic cell population, the method comprising: (a) obtaining a eukaryotic donor cell population from a subject; (b) mixing the eukaryotic donor cell population with one or more buffer solutions; and (c) transfecting the eukaryotic donor cell population with an effective dose of a modifier, thereby producing a modified eukaryotic donor cell population. In some embodiments, steps 1(a)-(c) are performed on the same day.

[0008] In some embodiments of the methods described herein, prior to the transfection step (c), the immune cell population, the CD4 + and CD8 + cell population, or the eukaryotic donor cell population is stimulated and / or activated with one or more stimulants.

[0009] In some embodiments of the methods described herein, the modifier is selected from small molecule agents, biologic agents, therapeutic agents, proteins, peptides, protein therapeutic agents, peptide therapeutic agents, chimeric antigen receptors, heterologous T cell receptors, viral vectors, vectors, retroviral vectors, lentiviral vectors, adenoviral vectors, and adeno-associated viral vectors.

[0010] In some embodiments of the methods described herein, the modifier is: (a) selected from retroviral vectors, lentiviral vectors, adenoviral vectors, or adeno-associated viral vectors; (b) a lentiviral vector; or (c) a retroviral vector.

[0011] In some embodiments of the methods described herein, the immune cell population, the CD4 + and CD8 + cell population, or the eukaryotic donor cell population is transfected with an effective dose of a lentiviral vector or a retroviral vector.

[0012] In some embodiments of the methods described herein, the lentiviral vector or the retroviral vector comprises a nucleic acid sequence encoding a chimeric antigen receptor (CAR), an engineered T cell receptor (TCR), and / or a nucleic acid sequence encoding a polypeptide that enhances immune cell function or a functional derivative thereof or a polypeptide that produces a therapeutic protein.

[0013] In some embodiments of the methods described herein, the immune cell population or the eukaryotic donor cell population is selected from mononuclear cells, lymphocyte-rich cells, B lymphocytes, T lymphocytes, CD4 + T lymphocytes, CD8 + T lymphocytes, dendritic cells, monocytes, natural killer (NK) cells, natural killer T (NKT) cells, regulatory T cells, CD4 + helper T cells, CD8 + cytotoxic T lymphocytes (CTLs), CD62L + cells, CD27 + cells, CCR7 + cells, CD45RO - cells, CD45RA + cells, neutrophils, basophils, eosinophils, megakaryocytes, stem cells, hematopoietic stem cells (HSCs), hematopoietic progenitor cells (HPCs), CD34 + cells, CD34 + peripheral blood stem cells, lymphokine-activated killer cells (LAKs), tumor-infiltrating lymphocytes (TILs), mesenchymal stem cells, mast cells, monocytes, macrophages, neutrophils, basophils, eosinophils, dendritic cells, megakaryocytes, and combinations thereof.

[0014] In some embodiments of the methods described herein, the concentration of the population of immune cells, the population of CD4 + and CD8 + cells, or the population of eukaryotic donor cells is (a) at least about 0.7×10 7 , at least about 0.8×10 7 , at least about 0.9×10 7 , at least about 1×10 7 , at least about 2×10 7 , at least about 4×10 7 , at least about 6×10 7 , at least about 8×10 7 , at least about 1×10 8 or at least about 5×10 8 cells / mL; (b) from about 0.5×10 6 cells / mL to about 4×10 6 cells / mL; (c) from about 0.5×10 6 cells / mL to about 1×10 8 cells / mL; or (d) from about 4.0×10 6 cells / mL to about 1×10 8 cells / mL.

[0015] In some embodiments of the methods described herein, transfection is: (a) selected from viral transfection, transduction, non-viral transfection, and mixtures of viral and non-viral transfection; (b) selected from electroporation, laser beam, gene injection, sonoporation, magnetofection, metal-coated nanoparticles, magnetically conjugated adeno-associated virus, particle / nanoparticle-mediated transfection, liposome transfection, lipid-based transfection, anionic liposomes, cationic liposome-mediated transfection, cationic polymers, polymer encapsulation, peptide-mediated transfection, calcium phosphate, dendrimers, flowfection, photoporation, soluporation, transient cell membrane disruption, deformation, extrusion, stretching, constriction, weakening, elongation, thinning, biolistic particle delivery systems, and combinations thereof; (c) electroporation of viral particles; (d) electroporation and viral transfection (transduction); (e) viral transfection and lipid-based transfection; or (f) viral transfection and liposome-based transfection.

[0016] In some embodiments of the methods described herein, (a) after or before transfection, the modified population of immune cells, the modified CD4 + and CD8 +The cell population or the population of modified eukaryotic donor cells is activated with one or more stimulants; and (b) after transfection, the modified immune cell population, the modified CD4 + and CD8 + cell population or the population of modified eukaryotic donor cells is expanded ex vivo.

[0017] In some embodiments, the methods described herein further comprise stimulating and activating the modified immune cell population, the modified CD4 + and CD8 + cell population or the population of modified eukaryotic donor cells with one or more stimulants to produce an activated modified immune cell population, an activated modified CD4 + and CD8 + cell population or an activated modified eukaryotic cell population.

[0018] In some embodiments, the methods described herein further comprise expanding the activated lymphocyte population, the activated modified immune cell population, the activated modified mononuclear cell population, the activated modified CD4 + and CD8 + cell population or the activated modified eukaryotic donor cell population for a predetermined time to produce an engineered lymphocyte population, an engineered immune cell population, an engineered CD4 + and CD8 + cell population or an engineered eukaryotic donor cell population. In some embodiments, the expansion step is carried out under the following conditions: (a) under shaking conditions or rotational conditions; (b) in a closed system; (c) using a serum-free medium; and / or (d) in the presence of one or more stimulants.

[0019] In some embodiments, the activated modified immune cell population, the activated modified CD4 + and CD8 + cell population or the activated modified eukaryotic donor cell population is expanded by at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold or at least about 25-fold.

[0020] In some embodiments, the methods described herein further comprise harvesting the modified lymphocyte population, the modified immune cell population, the modified CD4 + and CD8 +The cell population or the population of modified eukaryotic donor cells is for cryopreservation or administration. In some embodiments, harvesting comprises selecting and enriching the engineered lymphocytes, the engineered immune cells, the engineered CD4 + and CD8 + cells or the engineered donor eukaryotic cells. In some embodiments, harvesting further comprises formulating the engineered lymphocytes, the engineered immune cells, the engineered CD4 + and CD8 + cells or the engineered donor eukaryotic cells for cryopreservation or administration to a subject in need.

[0021] In some embodiments, the predetermined time for expanding a population of the modified activated cells (lymphocytes, immune cells, mononuclear cells, CD4 + and CD8 + cells or eukaryotic donor cells) described herein is: (a) less than about 24 hours, less than about 30 hours, less than about 48 hours, less than about 72 hours, less than about 96 hours, or less than about 120 hours; (b) less than about 0.5 hours, less than about 1 hour, less than about 2 hours, less than about 3 hours, less than about 4 hours, less than about 5 hours, less than about 6 hours, less than about 7 hours, less than about 8 hours, less than about 9 hours, less than about 10 hours, less than about 11 hours, less than about 12 hours, less than about 13 hours, less than about 14 hours, less than about 15 hours, less than about 16 hours, less than about 17 hours, less than about 18 hours, less than about 19 hours, less than about 20 hours, less than about 21 hours, less than about 22 hours, or less than about 23 hours; or (c) about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, or more days.

[0022] In some embodiments of the methods described herein, from enriching and / or obtaining the lymphocyte population, the immune cell population, the CD4 + and CD8 + cell population or the eukaryotic donor cell population to harvesting the engineered immune cells, the engineered CD4 + and CD8 +The time of the cell or the engineered eukaryotic donor cell is: (a) about 72 hours or less; (b) from about 18 hours to about 72 hours, from about 18 hours to about 36 hours, from about 18 hours to about 24 hours, from about 24 hours to about 72 hours, from about 24 hours to about 36 hours, or from about 36 hours to about 72 hours; (c) less than about 2 hours, less than about 3 hours, less than about 4 hours, less than about 5 hours, less than about 6 hours, less than about 7 hours, less than about 8 hours, less than about 9 hours, less than about 10 hours, less than about 11 hours, less than about 12 hours, less than about 13 hours, less than about 14 hours, less than about 15 hours, less than about 16 hours, less than about 17 hours, less than about 18 hours, less than about 19 hours, less than about 20 hours, less than about 21 hours, less than about 22 hours, or less than about 23 hours; (d) about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, or more days; or (e) about 1 day, about 3 days, about 4 days, about 5 days, or about 6 days.

[0023] In some embodiments of the methods described herein, the electroporation step, the activation step, and / or the amplification step are performed in a closed system, a semi-closed, and / or a functionally closed system. In some embodiments, the closed system is selected from a closed bag system, an automated closed cell sample handling system, and a bioreactor. In some embodiments, (a) the one or more stimulants are selected from agonistic antibodies, cytokines, recombinant costimulatory molecules, anti-CD3 antibodies or fragments thereof, anti-CD28 antibodies or fragments, small molecule inhibitors, and / or combinations thereof; (b) the one or more stimulants are cytokines selected from interleukin-2 (IL-2), interleukin-3 (IL-3), interleukin-6 (IL-6), interleukin-7 (IL-7), interleukin-7 receptor (IL-7R), interleukin-11 (IL-11), interleukin-12 (IL-12), interleukin-15 (IL-15), interleukin-15 receptor (IL-15R), interleukin-18 (IL-18), interleukin-18 receptor (IL-18R), interleukin-21 (IL-21), granulocyte macrophage colony-stimulating factor, interferon alpha, beta, or gamma, erythropoietin, and combinations thereof. In some embodiments, the one or more stimulants are conjugated to beads or nanostructures.

[0024] In some embodiments of the methods described herein, (a) the one or more stimulators are anti-CD3 and anti-CD28 antibodies or fragments thereof; (b) the one or more stimulators are anti-CD3 and anti-CD28 antibodies or fragments thereof and one or more cytokines; (b) the nanostructure is a nanomatrix; (c) the cytokine is selected from IL-2, IL-7, IL-6, IL-15, IL-15Ra or IL-21; (d) the cytokine is selected from IL-15 and IL-7, IL-7 and IL-21, IL-7 and IL-2, IL-15 and IL-2, IL-7, IL-15 and IL-21, IL-15 and IL-15Ra, or IL-7, IL-15 and IL-15Ra; and / or (e) the one or more stimulators are a nanomatrix and one or more cytokines. In some embodiments, the nanomatrix (a) comprises a matrix of mobile polymer chains and anti-CD3 and anti-CD28 antibodies or fragments thereof; or (c) has a size of 1 to 500 nm.

[0025] In some embodiments of the methods described herein, the effective dose of the retroviral vector or the lentiviral vector comprises a multiplicity of infection (MOI) of about 0.01, about 0.02, about 0.03, about 0.04, about 0.05, about 0.06, about 0.07, about 0.08, about 0.09, about 0.1, about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1.0, about 1.25, about 1.5, about 2.0, about 3.0, about 4.0 or about 5.0.

[0026] In some embodiments, the effective dose of the retroviral vector or the lentiviral vector comprises: (a) about 2 μl of the lentiviral vector with an MOI of about 0.08; (b) about 5 μl of the lentiviral vector with an MOI of about 0.2; or (c) about 10 μl of the lentiviral vector with an MOI of about 0.4.

[0027] In some embodiments of the methods described herein, the lentiviral vector is based on a virus selected from the group consisting of retroviruses, alpharetroviruses, betaretroviruses, gammaretroviruses, deltaretroviruses and epsilonretroviruses. In some embodiments, the lentiviral vector is based on human immunodeficiency virus (HIV), equine infectious anemia virus (EIAV), visna-maedi virus (VMV), caprine arthritis-encephalitis virus (CAEV), feline immunodeficiency virus (FIV), bovine immunodeficiency virus (BIV), visna virus and simian immunodeficiency virus (SIV).

[0028] In some embodiments, the lentiviral vector is pseudotyped with an envelope glycoprotein (Env) from a virus selected from the group consisting of murine leukemia virus (MLV), vesicular stomatitis virus (VSV) Indiana strain, VSV New Jersey strain, Cocal virus, Chandipura virus, Piry virus, spring viremia of carp virus (SVCV), sigma virus, infectious hematopoietic necrosis virus (IHNV), Mokola virus, rabies virus CVS virus, Isfahan virus, Araguari virus, Calchaqui virus, Jurona virus, La Joyavirus, Maraba virus, feline endogenous retrovirus (RD114) envelope protein, Perinet virus, Yug Bugdanovac virus, prototype foamy virus (PFV), and gibbon ape leukemia virus (GaLV).

[0029] In some embodiments, the lentiviral vector is pseudotyped with an envelope glycoprotein (Env) selected from the group consisting of vesicular stomatitis virus (VSV) Indiana strain, VSV New Jersey strain, and Cocal virus.

[0030] In some embodiments, the lentiviral vector comprises a heterologous viral envelope protein (Env) selected from the group consisting of VSV-G of the Indiana strain, VSV-G of the New Jersey strain, Cocal virus envelope protein, Isfahan virus envelope protein, Chandipura virus envelope protein, Piry virus envelope protein, murine leukemia virus (MLV) envelope glycoprotein, SVCV virus envelope protein, and variants thereof. In some embodiments, the lentiviral vector comprises a nucleotide sequence encoding a VSV-G envelope protein or a variant of the VSV G protein. In some embodiments of the methods described herein, the lentiviral vector is a lentiviral particle.

[0031] In some embodiments of the methods described herein, the CAR comprises an antigen-binding domain, a transmembrane domain, a co-stimulatory domain, and an intracellular domain, and wherein the antigen-binding domain is selected from (a) a full-length antibody or an antigen-binding fragment thereof, (b) a Fab, (c) a single-chain variable fragment (scFv), and (d) a single-domain antibody.

[0032] In some embodiments, the antigen-binding domain specifically binds to a target antigen selected from the group consisting of: CD4, CD5, CD19, CD20, CD22, CD79b, CD79a, CD33, CD30, CD70, BCMA, GPC2, CD123, CD133, EGFR, EGFRvIII, mesothelin, HER2, PSMA, PSCA, FAP, CEA, GD2, IL-13Ra2, glypican-3, CIAX, LI-CAM, CA 125, CTAG1B, TnMUC1, mucin 1, and folate receptor alpha (FRa), GFRα-4, NYESO, WT1, (AFP) / HLA-A2, AXL, B7-H3, CA-IX, CD3, CD7, CD8, CD38, CD44v6, CD80, CD86, CD117, CD147, CD276, CEA, claudin 18.2, c-Met, DLL3, DR5, EpCAM, EphA2, FAP, folate binding protein (FBP), glycolipid F77, glypican-3 (GPC3), glypican-2, HLA-A2, ICAMI, IL3Ra, LAGE-I, Lewis Y, LMPI (EBV), MAGE-Al, MAGE-A3, MAGE-A4, Melan A, MG7 (glycosylated CEA), MMP, MUCI, connexin 4 / FAP, NKG2D ligands, MIC-A, MIC-B, ULBP I to 6, NY-ESO-1, P16, PD-L1, ROR1, ROR2, TIM-3, TM4SF1, VEGFR2, and any combination thereof.

[0033] In some embodiments, the CAR transmembrane domain is selected from artificial hydrophobic sequences; transmembrane domains of type I transmembrane proteins, the α, β, or ζ chains of the T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, OX40 (CD134), 4-1BB (CD137), ICOS (CD278), or CD154; and transmembrane domains of killer cell immunoglobulin-like receptors (KIR).

[0034] In some embodiments, the co-stimulatory domain is the intracellular domain of a protein selected from the group consisting of: TNFR superfamily proteins, CD27, CD28, 4-1BB (CD137), OX40 (CD134), PD-1, CD7, LIGHT, CD83L, DAP10, DAP12, CD27, CD2, CD5, ICAM-1, LFA-1, Lck, TNFR-I, TNFR-II, Fas, CD30, CD40, ICOS (CD278), NKG2C, B7-H3 (CD276), and killer cell immunoglobulin-like receptor (KIR).

[0035] In some embodiments, the intracellular signaling domain comprises an intracellular domain selected from the group consisting of: the human CD3ζ chain (CD3ζ), FcγRIII, FcsRI, the cytoplasmic tail of an Fc receptor, a cytoplasmic receptor carrying an immunoreceptor tyrosine-based activation motif (ITAM), TCRζ, FcRγ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, and the cytoplasmic signaling domain of CD66d. In some embodiments of the methods described herein, the CAR further comprises a hinge region.

[0036] Another aspect of the present disclosure provides a method for delivering a nucleic acid encoding a chimeric antigen receptor (CAR), an engineered T cell receptor, or a therapeutic protein to a cell, the method comprising introducing into the cell a transfer plasmid comprising: (a) a polynucleotide sequence encoding at least one heterologous viral envelope protein engineered by the methods described herein; (b) a polynucleotide sequence encoding at least one retroviral rev protein; (c) a polynucleotide sequence encoding at least one retroviral gag protein and a retroviral pol protein; and / or (d) a polynucleotide sequence encoding the chimeric antigen receptor, the engineered T cell receptor (TCR), or the therapeutic protein. In some embodiments, at least a portion of one or more regions necessary for replication in the retroviral genome is mutated.

[0037] Another aspect of the present disclosure provides a lentiviral vector particle produced by the methods described herein.

[0038] Another aspect of the present disclosure provides a method of introducing a modification into a cell, the method comprising electroporating the cell with an effective dose of lentiviral vector particles described herein or prepared by the methods described herein, thereby producing a modified cell. In some embodiments, prior to electroporation, the cell is contacted with the effective dose of lentiviral vector. In some embodiments, after electroporation, the cell is contacted with the effective dose of lentiviral vector for up to about 4 hours. In some embodiments, the cell is contacted with the effective dose of lentiviral vector: (a) at least about 5 - 30 minutes, at least about 25 - 50 minutes, at least about 5 - 60 minutes, at least about 5 - 12 minutes, at least about 60 - 120 minutes, at least about 120 - 240 minutes after electroporation; (b) at least about 1 minute, at least about 2 minutes, at least about 5 minutes, at least about 10 minutes, at least about 20 minutes, at least about 30 minutes, at least about 45 minutes, at least about 60 minutes, at least about 75 minutes, at least about 90 minutes, at least about 100 minutes, at least about 110 minutes, at least about 120 minutes, at least about 150 minutes, at least about 160 minutes, at least about 170 minutes, at least about 180 minutes, at least about 190 minutes, at least about 200 minutes, at least about 220 minutes or at least about 240 minutes after electroporation.

[0039] In some embodiments, the cell is selected from immune cells, eukaryotic donor cells, mononuclear cells, enriched lymphocytes, B lymphocytes, T lymphocytes, CD4 + T lymphocytes, CD8 + T lymphocytes, dendritic cells, monocytes, natural killer (NK) cells, natural killer T (NKT) cells, regulatory T cells, CD4 + helper T cells, CD8 + cytotoxic T lymphocytes (CTL), CD62L + cells, CD27 + cells, CCR7 + cells, CD45RO - cells, CD45RA + cells, neutrophils, basophils, eosinophils, megakaryocytes, stem cells, hematopoietic stem cells (HSC), hematopoietic progenitor cells (HPC), CD34 + cells, CD34 + peripheral blood stem cells, lymphokine-activated killer cells (LAK), tumor-infiltrating lymphocytes (TIL), circulating tumor-specific T cells, mesenchymal stem cells, mast cells, monocytes, macrophages, neutrophils, basophils, eosinophils, dendritic cells, megakaryocytes, and combinations thereof.

[0040] In some embodiments, the cells are: (a) lymphoid cells selected from: T cells, B cells, natural killer (NK) cells, CD8 + T cells, CD4 + T cells, cytotoxic T lymphocytes, regulatory T cells, and any combination thereof; (b) myeloid cells selected from: monocytes, macrophages, neutrophils, basophils, eosinophils, dendritic cells, megakaryocytes, and any combination thereof; (c) stem cells, hematopoietic stem cells, hematopoietic progenitor cells, CD34 + cells, or CD34 + peripheral blood stem cells.

[0041] In some embodiments, the effective dose of the lentiviral vector particles comprises about 0.5 ul, about 1 ul, about 1.5 ul, about 2 ul, about 2.5 ul, about 3 ul, about 3.5 ul, about 4 ul, about 5 ul, about 6 ul, about 7 ul, about 8 ul, about 9 ul, about 10 ul, about 15 ul, or about 20 ul of the lentiviral vector. In some embodiments, the effective dose of the lentiviral vector particles comprises an multiplicity of infection (MOI) of about 0.01, about 0.02, about 0.03, about 0.04, about 0.05, about 0.06, about 0.07, about 0.08, about 0.09, about 0.1, about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1.0, about 1.25, about 1.5, about 2.0, about 3.0, about 4.0, or about 5.0.

[0042] In some embodiments, the effective dose of the lentiviral vector particles comprises: (a) about 2 ul of lentiviral vector particles with an MOI of about 0.08; (b) about 5 ul of lentiviral vector particles with an MOI of about 0.2; or (c) about 10 ul of lentiviral vector particles with an MOI of about 0.4.

[0043] One aspect of the present disclosure provides a modified cell, modified immune cell, modified CD4 + and CD8 + cells, or modified eukaryotic donor cells engineered by the methods described herein.

[0044] One aspect of the present disclosure provides a population of modified cells, population of modified immune cells, population of modified CD4 + and CD8 + cells, or population of modified eukaryotic donor cells engineered by the methods described herein.

[0045] One aspect of the present disclosure provides a modified cell, a modified immune cell, a modified CD4 + and CD8 + cell, or a modified eukaryotic donor cell, comprising the lentiviral vector described herein.

[0046] One aspect of the present disclosure provides a population of modified cells, a population of modified immune cells, a population of modified CD4 + and CD8 + cells, or a population of modified eukaryotic donor cells, comprising the lentiviral vector described herein.

[0047] In some embodiments, the engineered or modified CD4 + and CD8 + cells or modified eukaryotic donor cells described herein are used for the production of a protein of interest. In some embodiments, the protein of interest is selected from industrial proteins or therapeutic proteins. In some embodiments, the protein of interest is selected from enzymes, regulatory proteins, receptors, peptides, peptide hormones, cytokines, membrane or transport proteins, vaccine antigens, antigen-binding proteins, immunostimulatory proteins, allergens, full-length antibodies or antibody fragments or derivatives, single-chain antibodies (scFv), Fab fragments, Fv fragments, single-domain antibodies (VH or VL fragments), domain antibodies, camelid single-domain antibodies (VHH), nanobodies, and combinations thereof.

[0048] One aspect of the present disclosure provides a composition comprising: (a) a modified cell, a modified immune cell, a modified CD4 + and CD8 + cell, or a modified eukaryotic donor cell, described herein or engineered by the methods described herein; (b) a population of modified cells, a population of modified immune cells, a population of modified CD4 + and CD8 + cells, or a population of modified eukaryotic donor cells, described herein or engineered by the methods described herein; or (c) the lentiviral vector described herein. In some embodiments, the composition further comprises a pharmaceutically acceptable excipient.

[0049] One aspect of the present disclosure provides a method of treating a disease or disorder in a subject, the method comprising administering to the subject in need thereof a therapeutically effective amount of: (a) a modified cell, a modified immune cell, a modified CD4 + and CD8 +a cell or a modified eukaryotic donor cell; (b) a population of modified cells, a population of modified immune cells, modified CD4 + and CD8 + cell population or a population of modified eukaryotic donor cells; or (c) a composition described herein, thereby treating the disease or disorder in the subject.

[0050] In some embodiments, the disease or disorder is selected from viral infection, bacterial infection, parasitic infection, cancer, malignancy, non-cancerous disorder, autoimmune disease, fibrotic disease, Alzheimer's disease, protein deficiency disorder, and factor VIII deficiency.

[0051] In some embodiments, the cancer is selected from breast cancer, triple-negative breast cancer, prostate cancer, ovarian cancer, glioma, glioblastoma, renal cell carcinoma, kidney cancer, mesothelioma, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, kidney cancer, liver cancer, lung cancer, lung adenocarcinoma, gallbladder cancer, colon cancer, cervical squamous cell carcinoma, non-small cell lung cancer, small cell lung cancer, Merkel cell carcinoma, hepatocellular carcinoma, esophageal cancer, brain cancer, melanoma, Hodgkin lymphoma, non-Hodgkin lymphoma, urothelial cancer, gastric cancer, blood cancer, lymphoma, leukemia, multiple myeloma, diffuse large B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, acute myeloid leukemia, B-cell acute lymphoblastic leukemia (ALL), pre-B ALL, and any combination thereof. In some embodiments, the modified immune cell, the modified CD4 + and CD8 + cell or the modified eukaryotic donor cell: (a) is autologous to the subject; (b) is allogeneic to the subject; or (c) is xenogeneic to the subject. In some embodiments, the modified cell, the modified immune cell, the modified CD4 + and CD8 + cell or the modified eukaryotic donor cell is allogeneic to the subject. In some embodiments, the subject is a human.

[0052] One aspect of the present disclosure provides a method for producing a therapeutic protein, the method comprising: (a) manufacturing a population of engineered immune cells or a population of engineered eukaryotic cells comprising the therapeutic protein using the methods described herein; (b) harvesting the therapeutic protein; and (c) isolating and purifying the therapeutic protein.

[0053] In some embodiments, the therapeutic protein is selected from enzymes, regulatory proteins, receptors, peptides, peptide hormones, cytokines, membrane or transport proteins, vaccine antigens, antigen-binding proteins, immunostimulatory proteins, allergens, full-length antibodies or antibody fragments or derivatives, single-chain antibodies (scFv), Fab fragments, Fv fragments, single-domain antibodies (VH or VL fragments), domain antibodies, camelid single-domain antibodies (VHH), nanobodies, and combinations thereof.

[0054] One aspect of the present disclosure provides a kit comprising: (a) a population of modified immune cells engineered by the methods described herein or a population of modified CD4 + and CD8 + cells or populations; or (b) a lentiviral vector described herein.

[0055] The foregoing summary and the following description of the drawings and detailed description are exemplary and explanatory. They are intended to provide further details of the present disclosure but should not be construed as limiting. Other objects, advantages, and novel features will be readily apparent to those skilled in the art from the following detailed description of the present disclosure. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] Figure 1 Shows a schematic diagram of a rapid T cell engineering platform (1A) transfected with an integrative lentiviral vector (LVV) at D0. The platform 1A includes obtaining and processing donor leukapheresis at day 0, selecting CD4 + and CD8 + cells, performing lentiviral vector transfection in a closed system, followed by cell activation and in vitro culture and expansion for at least about 1 hour to about 72 hours (e.g., culturing and expanding from D0 - D3) prior to harvest. The harvested engineered T cells are cryopreserved or administered to a subject in need.

[0057] Figure 2 Shows Figure 1 a schematic diagram of a rapid T cell engineering platform (1B) transfected with an integrative lentiviral vector (LVV) at D0, where the process starts from donor whole blood rather than donor leukapheresis.

[0058] Figure 3Shows a schematic diagram of a rapid immune cell engineering platform (1C) demonstrating transfection with an integrative lentiviral vector (LVV) at D0. This platform 1C includes obtaining and processing a donor leukapheresis product on day 0, selecting immune cells (leukocytes and / or other immune cells), performing lentiviral vector transfection in a closed system, followed by cell activation and in vitro culture and expansion for at least about 1 hour to about 72 hours (e.g., culturing and expanding from D0 - D3). The harvested engineered immune cells are cryopreserved or administered to a subject in need.

[0059] Figure 4 Shows a demonstration of Figure 3 schematic diagram of a rapid immune cell engineering platform (1D) transfected with an integrative lentiviral vector (LVV) at D0, where the process starts from donor whole blood instead of a donor leukapheresis product.

[0060] Figure 5 Shows a schematic diagram of a rapid ex vivo non - cultured T cell engineering platform (2A) demonstrating transfection with an integrative lentiviral vector (LVV) at D0. This platform 2A includes obtaining and processing a donor leukapheresis product on day 0, selecting CD4 + and CD8 + cells, performing lentiviral vector transfection in a closed system, followed by harvesting the cells without culturing or expanding after transfection. The harvested engineered T cells are cryopreserved or administered to a subject in need.

[0061] Figure 6 Shows a demonstration of Figure 5 schematic diagram of a rapid ex vivo non - cultured T cell engineering platform (2B) transfected with an integrative lentiviral vector (LVV) at D0, where the process starts from donor whole blood instead of a donor leukapheresis product.

[0062] Figure 7 Shows a schematic diagram of a rapid ex vivo non - cultured immune cell engineering platform (3A) demonstrating transfection with an integrative lentiviral vector (LVV) at D0. This platform 3A includes obtaining and processing a donor leukapheresis product on day 0, selecting immune cells (leukocytes and / or other immune cells), performing lentiviral vector transfection in a closed system, followed by harvesting the cells. The harvested engineered cells are cryopreserved or administered to a subject in need.

[0063] Figure 8 Shows a demonstration of Figure 5 schematic diagram of a rapid ex vivo non - cultured immune cell engineering platform (3B) transfected with an integrative lentiviral vector (LVV) at D0, where the process starts from donor whole blood instead of a donor leukapheresis product.

[0064] Figure 9Shows a schematic diagram demonstrating a rapid eukaryotic cell engineering platform (4A) transfected with an integrative lentiviral vector (LVV) at D0. The platform 4A includes obtaining and processing donor eukaryotic cells (e.g., mammalian cells, human cells) on day 0, selecting a specific cell type (epithelial cells, mesenchymal cells, fibroblasts, neuronal cells, or stem cells), and performing lentiviral vector transfection in a closed system, followed by cell activation and ex vivo culture and expansion for at least about 1 hour to about 72 hours (e.g., culturing and expanding from D0 - D3) prior to harvesting. The harvested engineered eukaryotic cells are cryopreserved or used immediately.

[0065] Figure 10 Shows a schematic diagram demonstrating a rapid non - ex vivo culture eukaryotic cell engineering platform (4B) transfected with an integrative lentiviral vector (LVV) at D0. The platform 4B includes obtaining and processing donor eukaryotic cells on day 0, selecting a specific cell type (epithelial cells, mesenchymal cells, fibroblasts, neuronal cells, or stem cells), and performing lentiviral vector transfection in a closed system, followed by harvesting the cells. The harvested eukaryotic cells are cryopreserved or used immediately.

[0066] Figure 11 Shows a bar graph summarizing the results of a CD19 CART cell - Nalm6 co - culture assay, demonstrating that CD19 CAR T cells (ElectricCAR T cells) generated by the novel manufacturing process using lentiviral transfection disclosed herein are highly cytotoxic to target tumor cells, as demonstrated by Nalm6 in vitro killing. Representative means of two - three independent experiments (n = 2 - 3) are shown. Mean ± SEM, ***p < 0.005. Detailed Description I. Overview A. Classical Manufacturing Process

[0067] Adoptive cell transfer therapy with T cells, especially T cells transduced with chimeric antigen receptors (CARs), has shown promise in several hematological cancer trials. Despite such success, the manufacturing of genetically modified T cells remains a complex, expensive, and time - consuming process. The classical CAR T cell manufacturing process begins with enriching T cells from fresh or cryopreserved leukapheresis samples. Enrichment typically involves using positive or negative selection. Then, beads coated with anti - CD3 / anti - CD28 antibodies (e.g., ) Enriched T cells are activated with anti-CD3 / anti-CD28 antibody-coated polymers, nanoparticles, nanocolloids, and / or coactivators selected from reagents that stimulate ICOS, CD27, HVEM, LIGHT, CD40, 4-1BB, OX40, DR3, GITR, CD30, TIM1, CD2, or CD226. Once activated, the T cells are transfected with a nucleic acid molecule encoding a CAR molecule or an exogenous TCR immediately or up to 18 hours after the start of the activation step. Typically, the T cells are transduced with a lentiviral vector containing a nucleic acid molecule encoding a CAR molecule or an exogenous TCR. In some cases, the cells are electroporated with in vitro transcribed RNA. The transfected cells are then cultured (i.e., expanded) in vitro for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 days or longer. Once the culture has proceeded for a predetermined number of days, the cells are harvested. Harvesting the cells includes mechanically resuspending the engineered cells (e.g., T cells) by spinning or pipetting or otherwise agitating; and removing the mimetic / activation reagent with an appropriate buffer. The cells are also washed to remove unnecessary reagents and are reconstituted in cryopreservation medium or used for immediate administration to a subject. The cells are cryopreserved until administration is needed.

[0068] The invention of the present disclosure relates to a significant improvement over this classical manufacturing process, in which one or more manufacturing steps are deleted or shortened. Surprisingly, it has been found that this shortened and more efficient immune effector cell manufacturing process produces highly desirable and effective compositions. The shortened process relies on lentiviral transfection rather than conventional lentiviral transduction. B. Improved 1-day manufacturing process using lentiviral transfection.

[0069] The present disclosure provides novel and efficient methods for manufacturing immune effector cells (e.g., T cells or NK cells) engineered to express a CAR or TCR, methods for treating a subject's disease (e.g., cancer) using the engineered cells, and lentiviral vectors for use in the methods described herein. In some embodiments, the manufacturing process disclosed herein can manufacture immune effector cells engineered to express a CAR or TCR in less than about 24 hours (e.g., less than about 20 hours, less than about 15 hours, less than about 10 hours, less than about 5 hours, or less than about 3 hours or any other time range less than about 24 hours described herein). In some embodiments, the manufacturing process disclosed herein can manufacture immune effector cells engineered to express a CAR or TCR in less than about 72 hours (e.g., less than about 24 hours, less than about 30 hours, less than about 40 hours, less than about 48 hours, less than about 60 hours, or less than about 72 hours or any other time range less than about 24 hours described herein).

[0070] One aspect of the present disclosure provides a method for manufacturing an engineered population of immune cells, the method comprising: (1) enriching a population of lymphocytes, a population of immune cells, or a population of CD4 + and CD8 + cell populations from donor whole blood or donor leukapheresis (e.g., frozen or fresh); (2) mixing the population of lymphocytes, the population of immune cells, or the population of CD4 + and CD8 + cell populations with one or more buffer solutions; and (3) transfecting the population of lymphocytes, the population of immune cells, or the population of CD4 + and CD8 + cell populations with an effective dose of a modifier; thereby producing a modified population of lymphocytes, a modified population of immune cells, or a modified population of CD4 + and CD8 + cell populations. In some embodiments, steps 1(a)-(c) are performed in about 24 hours or less.

[0071] Another aspect of the present disclosure provides a method for manufacturing an engineered population of eukaryotic cells, the method comprising: (1) obtaining a population of eukaryotic donor cells; (2) mixing the population of eukaryotic donor cells with one or more buffer solutions; and (3) transfecting the population of eukaryotic donor cells with an effective dose of a modifier, thereby producing a modified population of eukaryotic donor cells.

[0072] In some embodiments, after transfection, the modified population of lymphocytes, the modified population of immune cells, the modified population of CD4 + and CD8 + cell populations or the modified population of eukaryotic donor cells are not activated with one or more stimulants or not expanded.

[0073] In some embodiments, the methods described herein further comprise stimulating and / or activating with one or more stimulants, and expanding the modified population of lymphocytes, the modified population of immune cells, the modified population of CD4 + and CD8 + cell populations or the modified population of eukaryotic donor cells to produce an activated modified population of immune cells, an activated modified population of CD4 + and CD8 + cell populations or an activated modified population of eukaryotic cells. In one aspect, the methods described herein further comprise stimulating and / or activating the population of lymphocytes, the population of immune cells, the CD4 + and CD8 + cell populations or the population of eukaryotic donor cells with one or more stimulants prior to the transfection step.

[0074] The methods of immunotherapy (e.g., adoptive cell transfer therapy) for treating a disease or disorder described herein employ a mature and robust system (apheresis and its products) to provide a rapid, reliable, and efficient generation of clinical-grade CAR or TCR-engineered cells that can be immediately administered to a subject on-site. Immunotherapy using electroporated CAR T cells generated by the methods disclosed herein can reduce the entire adoptive cell transfer process to about one day (e.g., about 24 hours) or about 3 days or less (e.g., about 72 hours or less). Thus, the manufacturing time is reduced from about 12 - 15 days to about 1 day, about 2 days or less, or about 3 days or less. The manufacturing process disclosed herein is efficient because it requires fewer cells to generate CAR T cells. For example, traditional manufacturing processes require up to about 300 million cells, while the manufacturing process described herein works efficiently with about 3 million cells - a 100x reduction in cell number. This is because the cells from the methods of the present invention are very fresh and undamaged and are therefore more potent (e.g., less ex vivo / in vitro processing).

[0075] The manufacturing process disclosed herein improves the generation of cell therapy products expressing CAR or TCR, enhances the quality of CAR T cell products, and maximizes the therapeutic efficacy of CAR T cell products in the following manner.

[0076] First, compared to classical manufacturing processes (e.g., about 12 days), the manufacturing process disclosed herein reduces the turnaround manufacturing time to about 1 day (e.g., 24 hours) or less (or about 2 days or less or about 3 days or less). This short turnaround time allows for the timely infusion of CAR T cells (e.g., CD19, mesothelin, PSMA, TnMUC, BCMA, or GPC2 CAR-T cells) to the patient. Additionally, the manufacturing process preserves putative stem memory T (Tstem) cells, a cell subset associated with increased anti-tumor efficacy. Most of the unstimulated CAR T cells generated by the manufacturing process disclosed herein maintain a less differentiated phenotype (e.g., more than 50% of the transfected CAR T cells are naive CAR T cells (CD45RO - CCR7 + )) compared to less than 10% of the CAR T cell population in stimulated CAR T cells. A population with a high proportion of naive electroporated CAR T cells is a desirable improvement because these CAR T cells retain an unactivated (i.e., less differentiated) phenotype, which is known to be beneficial for the persistence and potency of CAR T cells in cancer patients.

[0077] In some embodiments, CART cells made by the methods disclosed herein can be administered to a subject with minimal ex vivo expansion (e.g., less than about 1 day, less than about 12 hours, less than about 8 hours, less than about 6 hours, less than about 4 hours, less than about 3 hours, less than about 2 hours, less than about 1 hour of ex vivo expansion, or no ex vivo expansion). If desired, in other respects, the manufacturing can be less than about 5 days, less than about 4 days, less than about 3 days, or less than about 2 days. Thus, the methods described herein provide a rapid manufacturing process for preparing improved CAR-expressing cell products for use in treating a subject's disease. C. Summary of experimental results

[0078] The shortened manufacturing process disclosed herein is made possible by a new strategy of transducing immune cells with a lentiviral vector that contains nucleic acids encoding a CAR, a TCR, and / or a polypeptide or a functional derivative thereof that enhances immune cell function.

[0079] First, in one embodiment, the manufacturing process relies on a hybrid transfection method that combines biolistic transfection (virus-based transfection or viral transduction) and physical transfection (electroporation). In particular, lentiviral particles are electroporated into immune cells. Second, the lentiviral vector used for transducing cells is replication-incompetent. Electroporation of lentiviral particles into cells accelerates viral transfection / transduction, which is followed by a standard cell culture process or an ultra-rapid cell-free culture process. Electroporation of the lentiviral vector into cells also significantly reduces the amount of lentiviral vector required for optimal transfection using the process disclosed herein.

[0080] For example, the electroporation process can include lentiviral Use Figure 7 and Figure 8 The lentiviral nucleofection workflow shown in generates ultra-rapidly electroporated CAR T cells. Generating electroporated CAR T cells using this manufacturing process does not require cytokines and / or an ex vivo culture process. In addition, nucleofection of the CAR lentiviral vector results in efficient integration of the CAR transgene into the T cell genome. Further analysis also shows that the vector copy number / cell of electroporated CAR T cells is substantially similar to that of conventional CAR T cells (e.g., about 1 - 1.5 copies / cell). In addition, mRNA encoding the CAR transgene (qRTPCR of the WPRE sequence) is expressed within about 1 hour after nucleofection. Overall, electroporation of the lentiviral vector significantly improves the manufacturing efficiency of CAR T cells using the process disclosed herein.

[0081] Third, the present disclosure shows for the first time that when compared to traditional electroporation procedures (e.g., adding an expression vector to cells prior to electroporation), applying an electric current to cells for up to 4 hours (e.g., up to 2 hours) prior to adding a lentiviral vector to the cells increases CAR expression by about 10%-15%. Thus, a lentiviral vector is used as a cargo carrier in the form of particles; and physical transfection (such as electroporation) is used to deliver the lentiviral particles into the cells.

[0082] Tables 2 and 3 summarize the raw data from flow cytometry analysis of electroporated CD19 CAR T cells generated by the disclosed methods, and show that lentiviral vectors electroporated at very low multiplicities of infection (MOI) in unstimulated and stimulated primary human T cells produced large numbers of CAR-T cells. When compared to conventional methods, the transfection rates disclosed herein are very high, with traditional transduction methods (viral transduction only) resulting in about 1% to about 3% CAR expression in T cells.

[0083] Figure 11 Show that stimulated or unstimulated lentiviral vector-transfected CD19 CAR T cells (CART19 cells) efficiently killed target cells. In addition, when compared to lentiviral transduction over the same time period, the lentiviral vector increased the percentage of CAR + T cells (e.g., CD19 CAR T cells) by at least 10-fold (39.8% CAR + T cells compared to 3.5% CAR + T cells).

[0084] Since electroporated CAR T cells generated by the processes disclosed herein are different from conventional CAR T cells, at least in that electroporated CAR T cells are not activated ex vivo, while conventional CART cells are generated over a 7-12 day process. In addition, when compared to electroporated CAR T cells stimulated with, for example, CD3 / CD28 most unstimulated electroporated CART cells (more than 50%) maintain an unactivated or less differentiated phenotype (Tables 4 and 5). A high percentage of less differentiated CAR T cells (e.g., naive T cell populations) is a highly desirable and unexpected improvement because the less differentiated phenotype enhances the persistence and efficacy of CART cells in cancer patients. In addition, within 48 hours after co-culture, electroporated CAR T cells were shown to be as effective as conventional CAR T cells in killing CD19 + Nalm6 cells (Table 6). Electroporated CAR T cells are effective in killing target cells at effector:target ratios as low as 0.62:1 or lower.

[0085] For several reasons, the novel manufacturing method described herein provides the most efficient CAR T cell immunotherapy known to date. The electroporated CAR T cell manufacturing process reduces the entire CAR T manufacturing process to one day or up to 3 days if expansion (e.g., culturing) is required. Thus, the manufacturing time for electroporated CAR T cells is reduced from 12 - 15 days to approximately 1 day or up to 3 days. Additionally, fewer cells are required. For example, the traditional manufacturing process requires up to approximately 300 million cells, while the manufacturing process disclosed herein works efficiently with approximately 3 million cells because the cells are overall very fresh.

[0086] Finally, the manufacturing process is cost - effective because a batch of cells using conventional methods can cost $1 million per batch, which is sufficient to treat approximately 8 patients. However, the method disclosed herein will produce a batch of CAR T cells sufficient to infuse approximately 20 patients. Thus, the method disclosed herein doubles or triples the number of patients treated at the same cost and significantly reduces the cost of CAR T cells per patient.

[0087] Accordingly, the manufacturing process described herein provides the generation of effective clinical - grade CAR or TCR - engineered cells for immediate administration in about 1 day or less (or otherwise about 3 days or less or other time periods described herein), which is an improvement over manufacturing processes known in the art. II. Method for Generating Modified T Cells

[0088] The present disclosure provides a rapid and efficient manufacturing process for engineering modified cells (e.g., immune effector cells, electroporated CAR T cells), the modified cells comprising a CAR, an exogenous TCR, and / or an immune - enhancing factor that improves the fitness of the engineered immune cells; a composition comprising the engineered modified cells; and a method of using the engineered modified cells to treat a subject's disease (such as cancer). The rapid and efficient manufacturing method of the engineered immune cells disclosed herein provides engineered CAR T cells (i.e., electroporated) within less than 24 hours after transfection. The rapid turnaround is made possible by a combination of at least three factors: (1) using fresh apheresis products; (2) electroporating lentiviral vectors into purified apheresis products (e.g., purified T cells or purified immune cells) at a very low MOI; and / or (3) newly engineered lentiviral vectors. The CAR T cells engineered by the method disclosed herein are referred to as electroporated CAR T cells. The CAR engineered by the process disclosed herein is referred to as an electroporated CAR because electricity (e.g., electroporation) is used to drive the CAR - encoding vector (e.g., a lentiviral particle comprising a nucleic acid sequence encoding the CAR) into the cells. In particular, rather than passively transducing the cells with a lentiviral vector, the lentiviral vector is actively introduced into the cells by electroporation. A. Novel Electroporated CAR Manufacturing Platform

[0089] One aspect of the present disclosure provides a method for manufacturing engineered immune cell populations, the method comprising: (1) enriching a lymphocyte population, an immune cell population, or a CD4 + and CD8 + cell population obtained from a subject's blood; (2) mixing the lymphocyte population, the immune cell population, or the CD4 + and CD8 + cell population with one or more buffer solutions; and (3) transfecting the lymphocyte population, the immune cell population, or the CD4 + and CD8 + cell population with an effective dose of a modifier; thereby producing a modified lymphocyte population, a modified immune cell population, or a modified CD4 + and CD8 + cell population.

[0090] In some embodiments, prior to the enrichment step (e.g., enriching or selecting a lymphocyte population, an immune cell population, or a CD4 + and CD8 + cell population), the blood is separated by apheresis into a plasma component, a mononuclear cell-containing layer, a platelet layer, and red blood cells to produce an apheresis product selected from: red blood cell apheresis, thrombus apheresis, coagulation cell apheresis, white blood cell apheresis, stem cells, plasma apheresis, and platelet apheresis. In some embodiments, the apheresis product (e.g., a lymphocyte population, an immune cell population, or a CD4 + and CD8 + cell population) is enriched by apheresis, elutriation, or gradient centrifugation.

[0091] In some embodiments, in the method for generating CAR T cells (i.e., electroporated CAR) disclosed herein, apheresis samples are used at the point of care. Fresh apheresis samples are preferred because less immune cells are needed / required for optimal transfection using the methods disclosed herein. For example, traditional manufacturing processes require up to about 300 million cells, while the manufacturing process disclosed herein works efficiently with about 3 million cells. This difference can be attributed to the freshness of the apheresis product.

[0092] In some embodiments, an apheresis sample (e.g., a white blood cell apheresis sample) is collected from a subject and transported to a cell manufacturing facility as a fresh product (e.g., an unfrozen product). For example, a cell sorter (such as a CliniMACS device) is used to select the desired cells (e.g., immune cells, CD4 + T cells and / or CD8+ T cells). Then the enriched cells (e.g., immune cells, CD4 + T cells and / or CD8 + T cells) are inoculated for CART manufacturing using the methods described herein.

[0093] In some embodiments, an apheresis sample (e.g., a leukapheresis sample) is collected from a subject and shipped to a cell manufacturing facility as a frozen sample (e.g., a cryopreserved sample). The frozen apheresis sample is then thawed, and the desired cells (e.g., immune cells, CD4 T cells and / or CD8 + T cells) are selected from the apheresis sample, for example, using a cell sorter (such as + T cells). Then the enriched cells (e.g., immune cells, CD4 + T cells and / or CD8 + T cells) are inoculated for CART manufacturing using the methods described herein. In some embodiments, at the end of the manufacturing process, CAR T cells are harvested and cryopreserved, then thawed and administered to the subject. In some embodiments, the enriched cells (e.g., CD4 + T cells and / or CD8 + T cells) undergo one or more freeze-thaw cycles before being inoculated for CAR T manufacturing.

[0094] In some embodiments, an apheresis sample (e.g., a leukapheresis sample) is collected from a subject. The desired cells (e.g., immune cells, CD4 T cells and / or CD8 + T cells) are selected from the apheresis sample, for example, using a cell sorter (such as CliniMACS + T cells). Then the enriched cells (e.g., immune cells, CD4 + T cells and / or CD8 + T cells) are shipped to a cell manufacturing facility as a frozen sample (e.g., a cryopreserved sample). Then the enriched cells (e.g., immune cells, CD4 + T cells and / or CD8 + T cells) are subsequently thawed and inoculated for CART manufacturing using the methods described herein.

[0095] In some embodiments, after inoculating cells (e.g., T cells), one or more cytokines and one or more modifiers (e.g., a vector encoding a CAR) are added to the cells. In this embodiment, the one or more cytokines can be selected from IL-2, IL-7, IL-15, hetIL-15 (IL15 / sIL-15Ra), IL-21, or IL-6 (e.g., IL-6 / sIL-6R). After incubating for at least about 5 - 72 hours, the cells are harvested, washed, and formulated for storage (e.g., cryopreservation) or administration.

[0096] One aspect of the present disclosure provides a method for manufacturing an engineered population of immune cells, the method comprising: enriching a population of lymphocytes, immune cells, or CD4 + and CD8 + cell population from blood obtained from a subject; mixing the population of lymphocytes, immune cells, or CD4 + and CD8 + cell population with one or more buffer solutions; transfecting the population of lymphocytes, immune cells, or CD4 + and CD8 + cell population with an effective dose of a modifier; culturing and expanding the transfected population of lymphocytes, immune cells, or CD4 + and CD8 + cell population; and harvesting the engineered lymphocytes, immune cells, or CD4 + and CD8 + cells; thereby generating a modified population of lymphocytes, a modified population of immune cells, or a modified population of CD4 + and CD8 + cell population. In some embodiments, prior to transfection, the population of immune cells or CD4 + and CD8 + cell population is not stimulated and / or activated. In some embodiments, the transfected cells are cultured and expanded in the presence of one or more stimulants described herein.

[0097] Another aspect of the present disclosure provides a method for manufacturing an engineered population of immune cells, the method comprising: (1) enriching a population of lymphocytes, immune cells, or CD4 + and CD8 + cell population from a donor leukapheresis; (2) mixing the population of lymphocytes, immune cells, or CD4 + and CD8 + cell population with one or more buffer solutions; and (3) transfecting the population of lymphocytes, immune cells, or CD4 + and CD8 +A cell population; thereby generating a modified lymphocyte population, a modified immune cell population, or a modified CD4 + and CD8 + cell population.

[0098] Another aspect of the present disclosure provides a method for manufacturing an engineered immune cell population, the method comprising: (1) enriching a lymphocyte population, an immune cell population, or a CD4 + and CD8 + cell population from a donor leukapheresis product; (2) mixing the lymphocyte population, the immune cell population, or the CD4 + and CD8 + cell population with one or more buffer solutions; (3) transfecting the lymphocyte population, the immune cell population, or the CD4 + and CD8 + cell population with an effective dose of a modifier; (4) culturing and expanding the transfected lymphocyte population, the immune cell population, or the CD4 + and CD8 + cell population; and (5) harvesting the engineered lymphocytes, immune cells, or CD4 + and CD8 + cells; thereby generating a modified lymphocyte population, a modified immune cell population, or a modified CD4 + and CD8 + cell population. In some embodiments, the transfected cells are cultured and expanded in the presence of one or more stimulators as described herein. In some embodiments, the lymphocyte population, the immune cell population, or the CD4 + and CD8 + cell population is not stimulated and / or activated prior to transfection.

[0099] In some embodiments, the apheresis product (e.g., a lymphocyte population, an immune cell population, or a eukaryotic donor cell population) is selected from mononuclear cells, lymphocyte-rich cells, B lymphocytes, T lymphocytes, CD4 + T lymphocytes, CD8 + T lymphocytes, dendritic cells, monocytes, natural killer (NK) cells, natural killer T (NKT) cells, regulatory T cells, CD4 + helper T cells, CD8 + cytotoxic T lymphocytes (CTLs), CD62L + cells, CD27 + cells, CCR7 + cells, CD45RO - cells, CD45RA +Cells, neutrophils, basophils, eosinophils, megakaryocytes, stem cells, hematopoietic stem cells (HSCs), hematopoietic progenitor cells (HPCs), CD34 + Cells, CD34 + Peripheral blood stem cells, lymphokine-activated killer cells (LAKs), tumor-infiltrating lymphocytes (TILs), mesenchymal stem cells, mast cells, monocytes, macrophages, neutrophils, basophils, eosinophils, dendritic cells, megakaryocytes, and combinations thereof.

[0100] Another aspect of the present disclosure provides a method for manufacturing an engineered eukaryotic cell population, the method comprising: (1) obtaining a population of eukaryotic donor cells (e.g., from a subject or a cell line); (2) mixing the population of eukaryotic donor cells with one or more buffer solutions; and (3) transfecting the population of eukaryotic donor cells with an effective dose of a modifier to produce a modified population of eukaryotic donor cells. In some embodiments, the transfected cells are cultured and expanded in the presence of one or more stimulators.

[0101] Another aspect of the present disclosure provides a method for manufacturing an engineered eukaryotic cell population, the method comprising: (1) obtaining a population of eukaryotic donor cells from a subject; (2) mixing the population of eukaryotic donor cells with one or more buffer solutions; (3) transfecting the population of eukaryotic donor cells with an effective dose of a modifier; (4) culturing and expanding the transfected population of eukaryotic donor cells; and (5) harvesting the engineered eukaryotic cells to produce a modified population of eukaryotic donor cells. In some embodiments, the transfected cells are cultured and expanded in the presence of one or more stimulators. In some embodiments, the population of eukaryotic donor cells is not activated and / or stimulated prior to transfection.

[0102] In some embodiments, the methods disclosed herein can produce a modified population of lymphocytes, a modified population of immune cells, or a modified population of CD4 + and CD8 + populations expressing a modifier (such as a CAR) in less than about 24 hours, in about 24 hours, or in about 24 hours. In some embodiments, the methods disclosed herein can produce a modified population of lymphocytes, a modified population of immune cells, or a modified population of CD4 + and CD8 + populations expressing a modifier (such as a CAR) in about 24 hours. In some embodiments, the methods disclosed herein can produce a modified population of lymphocytes, a modified population of immune cells, or a modified population of CD4 + and CD8 +Population. In some embodiments, the methods disclosed herein can generate a modified lymphocyte population, a modified immune cell population, or a modified CD4 expressing a modifier (such as a CAR) within about 72 hours or less. + and CD8 + population. B. Source of Immune Cells

[0103] The methods disclosed herein for generating engineered immune cell populations include obtaining immune cells from a subject for ex vivo manipulation. Sources of target cells for ex vivo manipulation can also include, for example, autologous or allogeneic donor blood, cord blood, or bone marrow. For example, the source of immune cells can be from a subject to be treated with the modified immune cells of the present invention, such as the subject's blood, the subject's cord blood, or the subject's bone marrow. Non-limiting examples of subjects include humans, dogs, cats, mice, rats, and their transgenic species. Preferably, the subject is human.

[0104] Target cells can be obtained from a variety of sources, including blood, peripheral blood mononuclear cells, bone marrow, lymph node tissue, spleen tissue, umbilical cord, lymph, or lymphoid organs. Immune cells are cells of the immune system, such as cells of innate or adaptive immunity (myeloid or lymphoid cells, including lymphocytes, typically T cells and / or NK cells). In some aspects, the cells are human cells. With respect to the subject to be treated, the cells can be allogeneic and / or autologous. The cells are typically primary cells, such as those directly isolated from the subject and / or isolated from the subject and cryopreserved. 1. Immune Cells

[0105] In certain embodiments, the target cells are immune cells, T cells (e.g., CD8 + T cells, CD8 + naive T cells, central memory T cells, or effector memory T cells, CD4 + T cells, natural killer T cells (NKT cells), regulatory T cells (Tregs), stem cell memory T cells), lymphoid progenitors, hematopoietic stem cells, natural killer cells (NK cells), or dendritic cells. In some embodiments, the cells are monocytes or granulocytes (e.g., myeloid cells, macrophages, neutrophils, dendritic cells, mast cells, eosinophils, and / or basophils).

[0106] In some embodiments, the cells include one or more subgroups of T cells or other cell types, such as the entire T cell population, CD4 + cells, CD8 +Cells and their subsets, such as those defined by: function, activation state, maturity, differentiation potential, expansion, recirculation, localization and / or persistence, antigen specificity, antigen receptor type, presence in a particular organ or compartment, marker or cytokine secretion profile, and / or degree of differentiation. T cells and / or CD4 + and / or CD8 + Subtypes and subsets of T cells include naive T (TN) cells, effector T cells (TEFF), memory T cells and their subtypes (such as stem cell memory T (TSCM), central memory T (TCM), effector memory T (TEM) or terminally differentiated effector memory T cells), tumor-infiltrating lymphocytes (TIL), immature T cells, mature T cells, helper T cells, cytotoxic T cells, mucosa-associated invariant T (MAIT) cells, naturally occurring and adaptive regulatory T (Treg) cells, helper T cells (such as TH1 cells, TH2 cells, TH3 cells, TH17 cells, TH9 cells, TH22 cells, follicular helper T cells), α / β T cells and δ / γ T cells. In certain embodiments, any number of T cell lines available in the art can be used. 2. Stem cells

[0107] Other exemplary cells that can be engineered using the manufacturing processes of the present disclosure include stem cells, such as pluripotent and multipotent stem cells (including induced pluripotent stem cells (iPSCs)). In an embodiment, the target cells are induced pluripotent (iPS) cells or cells derived from iPS cells, such as iPS cells generated from a subject. In some embodiments, the iPS cells are manipulated to alter (e.g., induce mutations) or to induce the expression of one or more target genes. In some embodiments, the iPS cells are differentiated into T cells, CD8 + T cells (e.g., CD8 + naive T cells, central memory T cells or effector memory T cells), CD4 + T cells, stem cell memory T cells, lymphoid progenitors or hematopoietic stem cells. 3. Cell isolation

[0108] In some embodiments, the manufacturing process of the present disclosure includes separating target cells (e.g., immune cells; enriched apheresis products) from a subject, preparing, processing, optionally culturing, and / or transfecting them. In some embodiments, the preparation of engineered cells includes one or more culturing and / or preparation steps. Cells for engineering as described can be isolated from a sample (such as a biological sample, e.g., a biological sample obtained from or derived from a subject). In some embodiments, the subject from whom the cells are isolated is a subject suffering from a disease or disorder, in need of cell therapy, or to whom cell therapy will be administered. In some embodiments, the subject is a human in need of a specific therapeutic intervention (such as adoptive cell therapy, separating, processing, and / or engineering cells for said adoptive cell therapy). Thus, in some embodiments, the cells are primary cells (e.g., primary human cells). Samples include tissues, fluids, and other samples taken directly from a subject, as well as samples from one or more processing steps (such as separation, centrifugation, genetic engineering (e.g., transduction with a viral vector), washing, and / or incubation). A biological sample can be a sample obtained directly from a biological source or a processed sample. Biological samples include, but are not limited to, body fluids (e.g., blood, plasma, serum, cerebrospinal fluid, synovial fluid, urine, and sweat), tissue and organ samples, and processed samples derived from their sources.

[0109] In certain aspects, the sample from which immune cells are sourced or isolated is blood, a blood-derived sample, or an apheresis or leukapheresis product. Exemplary samples include whole blood, peripheral blood mononuclear cells (PBMCs), white blood cells, bone marrow, thymus, tissue biopsies, tumors, leukemias, lymphomas, lymph nodes, gut-associated lymphoid tissue, mucosa-associated lymphoid tissue, spleen, other lymphoid tissues, liver, lung, stomach, intestine, colon, kidney, pancreas, breast, bone, prostate, cervix, testis, ovary, tonsils, or other organs and / or cells derived from their sources. In the context of cell therapy (e.g., adoptive cell therapy), the sample can be from autologous and allogeneic sources.

[0110] In some embodiments, the isolation of cells includes one or more preparation and / or non-affinity-based cell separation steps. In some embodiments, cells are washed, centrifuged, and / or incubated in the presence of one or more reagents (e.g., to remove unwanted components), enrich desired components, lyse, or remove cells sensitive to a particular reagent. In some embodiments, cells are separated based on one or more characteristics (such as density, adhesion properties, size, sensitivity, and / or resistance to specific components).

[0111] In some embodiments, cells of the circulating blood from a subject are obtained by apheresis. In certain aspects, the sample contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and / or platelets, and in certain aspects contains cells other than red blood cells and platelets. In some embodiments, the blood cells collected from the subject are washed to remove the plasma fraction, and / or the cells are placed in a suitable buffer or medium for subsequent processing steps. In some embodiments, the cells are washed with phosphate buffered saline (PBS). In some embodiments, the washing step is accomplished by tangential flow filtration (TFF) according to the manufacturer's instructions. In some embodiments, the cells are resuspended in a variety of biocompatible buffers after washing. In some embodiments, components of the blood cell sample are removed and the cells are directly resuspended in a medium. In some embodiments, the method includes a density-based cell separation method, such as preparing white blood cells from peripheral blood by lysing red blood cells and centrifuging through a Percoll or Ficoll gradient.

[0112] In one embodiment, immune cells are obtained from the circulating blood of an individual, by apheresis or leukapheresis. The apheresis product typically contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets. The cells collected by apheresis can be washed to remove the plasma fraction and the cells can be placed in a suitable buffer or medium (such as phosphate buffered saline (PBS); or a wash solution that lacks calcium and may lack magnesium or may lack many (if not all) divalent cations) for subsequent processing steps. As will be readily appreciated by one of ordinary skill in the art, the washing step can be accomplished by methods known to those of skill in the art, such as by using a semi-automated "flow-through" centrifuge (e.g., Cobe 2991 cell processor, Baxter CytoMate, or Haemonetics Cell Saver 5) according to the manufacturer's instructions. After washing, the cells can be resuspended in a variety of biocompatible buffers (such as Ca 2+ -free, Mg 2+ -free PBS, PlasmaLyte A, or another saline solution with or without a buffer). In some embodiments, undesired components of the apheresis sample can be removed and the cells can be directly resuspended in a medium.

[0113] In some embodiments of the manufacturing process described herein, cells are obtained from the circulating blood of a subject by apheresis or leukapheresis using an in vitro apheresis system. In some embodiments, cell separation and transfection are performed on the same day. 4. In Vitro Apheresis

[0114] In some embodiments of the manufacturing processes described herein, standard apheresis equipment (such as Spectra, Spectra Fenwal RM or equivalent equipment) is used to collect cells. In some embodiments, cells from the circulating blood of a subject are obtained by red blood cell apheresis, thromboapheresis, thrombocyte apheresis, leukapheresis, stem cell harvest, plasma apheresis, or platelet apheresis. The leukapheresis process typically yields approximately 200 - 400 mL of apheresis product from a patient (i.e., the subject). The apheresis product is subjected to the manufacturing process on-site (e.g., at the point of care).

[0115] In some embodiments, the enriched apheresis product is a "leukapheresis" product. As used herein, the term "leukapheresis" refers to the presence of a large number of mononuclear cells in the blood, i.e., the separation and collection of white blood cells (WBC) from plasma and red blood cells.

[0116] In some embodiments, the enriched apheresis product contains from about 5% to about 25% of the total peripheral blood mononuclear cell fraction. In some embodiments, the enriched apheresis product is a lymphoid cell population or lymphoid cells. In this embodiment, the lymphoid cells are selected from T cells, B cells, natural killer (NK) cells, CD8 + T cells, CD4 + T cells, cytotoxic T lymphocytes, regulatory T cells, and any combination thereof.

[0117] In some embodiments, the enriched apheresis product is a myeloid cell population or myeloid cells. In this embodiment, the myeloid cells can be selected from monocytes, macrophages, neutrophils, basophils, eosinophils, dendritic cells, megakaryocytes, and any combination thereof.

[0118] In some embodiments, the enriched apheresis product has a predetermined volume and / or a predetermined hematocrit, regardless of the number of mononuclear cell collection cycles performed by the apheresis system and / or the amount of precursor product used to generate the enriched apheresis product.

[0119] In some embodiments, the predetermined volume is from about 120 ml to about 400 mL. In some embodiments, the predetermined volume is about 120 ml, about 150 ml, about 175 ml, about 180 ml, about 200 ml, about 225 ml, about 250 ml, about 275 ml, about 300 ml, about 325 ml, about 350 ml, about 375 ml, or about 400 ml or less. In some embodiments, the apheresis is configured to have a specific target yield of mononuclear cells to be collected and processed. The specific target yield of mononuclear cells to be collected and processed can be evaluated by the apheresis system and / or by inputting the expected number of mononuclear cells of the subject. Based on the target mononuclear cell yield and the number of mononuclear cells collected during each mononuclear cell collection cycle, the controller of the apheresis system can determine the number of mononuclear cell collection cycles to be performed. As an example, in some embodiments, if the target mononuclear cell yield is about 5×10 9 mononuclear cells and the apheresis system collects about 1×10 9 mononuclear cells per mononuclear cell collection cycle, the controller will determine that it is appropriate to perform five mononuclear cell collection cycles.

[0120] In some embodiments, the target mononuclear cell yield is at least about 0.7×10 7 , at least about 0.8×10 7 , at least about 0.9×10 7 , at least about 1×10 7 , at least about 2×10 7 , at least about 4×10 7 , at least about 6×10 7 , at least about 8×10 7 , at least about 1×10 8 , or at least about 5×10 8 cells / mL. In some embodiments, the target mononuclear cell yield is from about 0.5×10 6 cells / mL to about 4×10 6 cells / mL. In some embodiments, the target mononuclear cell yield is from about 0.5×10 6 cells / mL to about 1×10 8 cells / mL. In some embodiments, the target mononuclear cell yield is from about 4.0×10 6 cells / mL to about 1×10 8 cells / mL.

[0121] In some embodiments, the predetermined hematocrit is from about 0% to about 10%. In another embodiment, the predetermined hematocrit is about 2%. In some embodiments, the predetermined volume is about 200 mL and the predetermined hematocrit is about 2%. The predetermined volume and / or the predetermined hematocrit can vary without departing from the scope of the present disclosure. 5. Cell Enrichment

[0122] In some embodiments, the manufacturing processes disclosed herein include selecting specific cells to improve the enrichment of desired immune effector cells suitable for CAR expression. Systems or devices for cell enrichment and purification purposes include, for example, the BAXTER ISOLEX 300I RM and the Miltenyi CLINIMACS RM , which enrich peripheral blood progenitor cells (PBPCs) based on specific ligands on the cell surface (e.g., CD34 or CD133).

[0123] In some embodiments, the selection includes positive selection, such as selecting the desired immune effector cells. In some embodiments, the selection includes negative selection, such as selecting unwanted cells, e.g., removing unwanted cells. In some embodiments, the positive or negative selection methods described herein are performed under flow conditions using a flow-through device or a cell processing system to further enrich the cell preparation for the desired immune effector cells. Negative T cell selection is performed by removing unwanted cells via a combination of Miltenyi beads and column technology using CD19, CD14, and CD26 ( system, Plus or CliniMACS ). Positive T cell selection can be performed using a combination of Miltenyi beads and column technology using CD4 and CD8 ( system, Plus or ). Alternatively, a column-free technology using releasable CD3 beads (GE Healthcare) can be used. Additionally, bead-free technologies such as the ThermoGenesis X series devices can also be utilized. Additional exemplary cell separation and bead removal methods are known to those skilled in the art, such as those shown in WO 2017 / 117112.

[0124] In some embodiments, the enriched apheresis product is enriched for one or more target cell types selected from: B lymphocytes, T lymphocytes, CD4 and CD8 T lymphocytes, dendritic cells, monocytes, natural killer (NK) cells, NKT cells, regulatory T cells, CD4 helper T cells, CD8 cytotoxic T lymphocytes (CTL), NKT cells, neutrophils, basophils, eosinophils, megakaryocytes, hematopoietic stem cells (HSC), hematopoietic progenitor cells (HPC), lymphokine-activated killer cells (LAK), tumor-infiltrating lymphocytes (TIL), mesenchymal stem cells, mast cells, subgroups of such cells, and combinations thereof.

[0125] In some embodiments, the enriched apheresis product is enriched for one or more target lymphocyte or myeloid cell populations. In some embodiments, the enriched apheresis product is enriched for lymphoid cells selected from: T cells, B cells, natural killer (NK) cells, CD8 + T cells, CD4 + T cells, cytotoxic T lymphocytes, regulatory T cells, and any combination thereof.

[0126] In some embodiments, the enriched apheresis product is enriched for myeloid cells selected from: monocytes, macrophages, neutrophils, basophils, eosinophils, dendritic cells, megakaryocytes, and any combination thereof.

[0127] In some embodiments, one or more markers, one or more "cell surface determinants" or "cell surface markers" are used to enrich the target cell population. In some embodiments, one or more markers or cell surface determinants are selected from: CD19 and / or CD20 for B cells; CD3, CD56 - , CD4 and / or CD8 for T cells; CD25 and / or CD69 for activated and / or regulatory T cells; CD1c, CD83, CD141, CD209, MHC II, and / or CD11c for dendritic cells; CD3-, CD16, and / or CD56 for NK cells; CD34, CD90, and / or CD135 for hematopoietic stem cells or hematopoietic progenitor cells; CD11b, CD68, CD163, and / or CD33 for macrophages; CD14, CD16, and / or CD64 for monocytes; CD15, CD16, and / or CD49d for neutrophils - ; 2D7 antigen, CD117-, CD123, CD203c, and / or FcεRIa for basophils; or CD11b, CD193, EMR1, and / or Siglec-8 for eosinophils.

[0128] Techniques for enriching the enriched apheresis product are known to those skilled in the art and include, but are not limited to, magnetic separation, filtration, immunoaffinity separation, gravity separation, density gradient separation, elutriation, and any combination thereof. The cell separation module can employ any of these or other methods known in the art for further enriching and / or obtaining a population of target nucleated blood cells from a patient. For example, after cell separation, binding to one or more selection agents or affinity agents (such as antibodies attached to degradable buoyancy beads or magnetic beads or microbubbles) can be used to enrich for specific target cell types or classes.

[0129] In some embodiments, magnetic beads coated with antibodies against one or more specific cell surface antigens are used to enrich a target cell population from the enriched apheresis product. This results in cells expressing the target antigen attaching to the magnetic beads. When exposed to a strong magnetic field, the cells attached to the beads (expressing the cell surface marker) remain on the column or in the sample tube, while other cells (not expressing the cell surface marker) flow through or remain in suspension. Using this method, cells can be selected as positive or negative for a specific cell surface marker, or using a combination of positive and negative selection. In some embodiments, the antibody conjugated to the microbeads remains bound to the cells during transfection. In some embodiments, the antibody conjugated to the microbeads is uncoupled from the cells prior to transfection.

[0130] In some embodiments, one or more methods known in the art (including but not limited to antigen capture) are used to enrich target cells. In some embodiments, antigen capture is selected from filters, beads, magnetic beads, fluorescence-activated cell sorting, microfluidics, solid support affinity, acoustics, bioluminescence, antibody labeling, and enzyme substrates. In some embodiments, suitable solid supports selected from ferromagnetic and density-modified particles are used to enrich target cells. In some embodiments, the solid support comprises an affinity molecule, such as an antibody domain that binds a given cell surface marker and can be obtained, for example, from Miltenyi Biotec and Dynal. Methods that can be used to release the captured cells include competition with an excess ligand, enzymatic digestion, pH change, ionic strength change, removal of the magnetic field, and / or physical agitation.

[0131] In some embodiments, the separation method includes separating different cell types based on the expression or presence of one or more specific molecules (such as surface markers, surface proteins, intracellular markers, or nucleic acids) in cells. In some embodiments, any known method for separation based on such markers can be used. In some embodiments, the separation is an affinity or immunoaffinity-based separation. For example, the separation can include separating cells and cell populations based on the expression or expression level of one or more markers of the cells. Typically, the cell surface markers are incubated with an antibody or binding partner that specifically binds to the marker. This incubation step is followed by a washing step, and the cells that have bound the antibody or binding partner are purified from those cells that have not been bound by the antibody or binding partner. Such purification steps can be based on positive selection (wherein the cells that have bound the reagent are retained for further use) and / or negative selection (wherein the cells that have not bound to the antibody or binding partner are retained). In some examples, both fractions are retained for further use.

[0132] In some embodiments, negative selection can be particularly useful in cases where antibodies that specifically identify the cell types in a heterogeneous population are not available, such that separation is preferably based on markers expressed by cells other than the desired population. The separation does not need to result in 100% enrichment or depletion of a particular cell population or cells expressing a particular marker. For example, positive selection or enrichment for a particular type of cell (e.g., those expressing a marker) can increase the number or percentage of such cells, but does not need to result in the complete absence of cells that do not express the marker. Similarly, negative selection, removal, or depletion of a particular type of cell (e.g., those expressing a marker) can reduce the number or percentage of such cells, but does not need to result in the complete removal of all such cells. In certain exemplary embodiments, multiple rounds of separation steps can be performed, wherein the positive or negative selection fractions from one step are subjected to another separation step, such as a subsequent positive or negative selection. In certain exemplary embodiments, a single separation step can simultaneously deplete cells expressing multiple markers, such as by incubating the cells with multiple antibodies or binding partners (each antibody or binding partner being specific for a marker targeted for negative selection). Similarly, multiple cell types can be positively selected simultaneously by incubating the cells with multiple antibodies or binding partners expressed on various cell types.

[0133] Enrichment of a T cell population by negative selection can be accomplished using a combination of antibodies against surface markers unique to the cells targeted for negative selection. Exemplary methods are cell sorting and / or selection via negative magnetic immunoadhesion or flow cytometry using a cocktail of monoclonal antibodies against cell surface markers present on the cells targeted for negative selection. For example, to enrich CD4 by negative selection +Cells, monoclonal antibody mixtures typically contain antibodies against CD14, CD20, CD11b, CD16, HLA-DR, and CD8.

[0134] For isolating the desired cell population by positive or negative selection, the concentration of cells and surfaces (e.g., particles such as beads) can be varied. In certain embodiments, it may be desirable to significantly reduce the volume in which the beads are mixed with the cells (i.e., increase the cell concentration) to ensure maximum contact between the cells and the beads. For example, in one embodiment, a concentration of 2 billion cells / ml is used. In one embodiment, a concentration of 1 billion cells / ml is used. In another embodiment, a concentration greater than about 100 million cells / ml is used. In another embodiment, cell concentrations of about 1 million, about 2 million, about 3 million, about 4 million, about 5 million, about 6 million, about 7 million, about 8 million, about 9 million, about 10 million, about 15 million, about 20 million, about 25 million, about 30 million, about 35 million, about 40 million, about 45 million, or about 50 million cells / ml are used. In yet another embodiment, cell concentrations of about 75 million, about 80 million, about 85 million, about 90 million, about 95 million, or about 100 million cells / ml are used. In additional embodiments, concentrations of about 125 million or about 150 million cells / ml can be used. Using high concentrations can result in increased cell yield, cell activation, and cell expansion.

[0135] In some embodiments, a cell sorter (such as CliniMACS ) is used to enrich different cell types. For example, a cell sorter (such as CliniMACS device) can be used to select T cells (e.g., CD4 + T cells and / or CD8 + T cells) from apheresis products. The selected T cells (e.g., CD4 + T cells and / or CD8 + T cells) are then washed and transfected to produce engineered T cells as described herein.

[0136] In some embodiments, cells that are positive for one or more specific markers (such as surface markers) (marker + ) or express high levels of one or more specific markers (such as surface markers) (marker 高 ), or cells that are negative for one or more markers (marker - ) or express relatively low levels of one or more markers (marker 低) enrich or deplete one or more T cell populations. For example, in some aspects, specific subsets of T cells are isolated by positive or negative selection techniques, such as cells that are positive for one or more surface markers or express high levels of one or more surface markers (e.g., CD28 + , CD62L + , CCR7 + , CD27 + , CD127 + , CD4 + , CD8 + , CD45RA + and / or CD45RO + T cells). In some cases, such markers are those that are absent or expressed at relatively low levels on certain T cell populations (such as non-memory cells), but are present or expressed at relatively high levels on certain other T cell populations (such as memory cells). In one embodiment, cells are enriched (i.e., positively selected) for cells that are positive for or express high surface levels of CD45RO, CCR7, CD28, CD27, CD44, CD127, and / or CD62L, such as CD8 + cells or T cells, e.g., CD3 + cells, and / or depleted (e.g., negatively selected) for cells that are positive for or express high levels of CD45RA, such as CD8 + cells or T cells, e.g., CD3 + cells. In some embodiments, cells are enriched or depleted for cells that are positive for or express high surface levels of CD122, CD95, CD25, CD27, and / or IL7-Ra (CD127). In some embodiments of the methods disclosed herein, the enrichment step of the apheresis product includes CD25 + cell depletion. In some embodiments of the methods disclosed herein, the enrichment step of the apheresis product does not include CD25 + cell depletion. See, e.g., WO 2016 / 109410. CD25 + depletion can improve lentiviral transduction efficiency, which can ultimately improve the therapeutic efficacy of CAR T therapy. However, this step may not be crucial for manufacturing the electroporated CAR T cells described herein.

[0137] In certain exemplary embodiments, CD8 is enriched for cells that are positive for CD45RO (or negative for CD45RA) and positive for CD62L+ T cells. For example, CD3 / CD28-conjugated magnetic beads (e.g., M-450 CD3 / CD28 T cell expander) can be used for positive selection of CD3 + , CD28 + T cells.

[0138] In some embodiments, T cells are isolated from peripheral blood mononuclear cell (PBMC) samples by negative selection of markers (such as CD14) expressed on non-T cells (such as B cells, monocytes or other white blood cells). In certain aspects, CD4 + or CD8 + is used in the selection step to isolate CD4 + helper T cells and CD8 + cytotoxic T cells. By positive or negative selection of markers expressed on or expressed to a relatively high degree on one or more naive, memory, and / or effector T cell subsets, such CD4 + and CD8 + populations can be further classified into subsets. In some embodiments, such as by positive or negative selection based on surface antigens associated with the respective subsets, CD8 + cells are further enriched or depleted for naive, central memory, effector memory, and / or central memory stem cells. In some embodiments, enrichment is performed for central memory T (TCM) cells to improve efficacy, such as to improve long-term survival, expansion, and / or engraftment after administration, which is particularly robust in certain aspects in such subsets.

[0139] In some embodiments, combining CD8 + TCM-enriched T cells with CD4 + T cells further improves efficacy. In some embodiments, memory T cells are present in CD8 + CD62L of peripheral blood lymphocytes + and CD62L - in two subgroups. CD62L-CD8 + and / or CD62L + CD8 + fractions of PBMC can be enriched or depleted, such as using anti-CD8 and anti-CD62L antibodies. In some embodiments, CD4 + T cell populations and / or CD8 +T cell populations. In some embodiments, enrichment of central memory T (TCM) cells is based on positive or high surface expression of CD45RO, CD62L, CCR7, CD28, CD8, and / or CD127. In some embodiments, enrichment can be based on negative selection of cells expressing or highly expressing CD45RA and / or granzyme B. In some embodiments, depletion of cells expressing CD4, CD14, CD45RA and positive selection or enrichment of cells expressing CD62L are used to perform CD8 + isolation of populations. In some embodiments, enrichment of central memory T (TCM) cells begins with a negative cell fraction selected based on CD4 expression, the negative cell fraction undergoes negative selection based on expression of CD14 and CD45RA, and positive selection based on CD62L. Such selection is simultaneous in some respects and sequential in any order in other respects. In some embodiments, the same CD4-expression-based selection steps used to prepare CD8 + cell populations or subsets are used to generate CD4 + cell populations or subsets such that both the positive and negative fractions from the CD4-based isolation are retained and used in subsequent steps of the method, optionally after one or more other positive or negative selection steps.

[0140] CD4 + helper T cells are classified as naive, central memory, and effector cells by identifying cell populations with cell surface antigens. CD4 + lymphocytes can be obtained by standard methods. In some embodiments, naive CD4 + T lymphocytes are CD45RO - , CD45RA + , CD62L + , CD4 + T cells. In some embodiments, central memory CD4 + cells are CD62L + and CD45RO + . In some embodiments, effector CD4 + cells are CD62L- and CD45RO. In one example, to enrich CD4 + cells by negative selection, a monoclonal antibody mixture typically contains antibodies against CD14, CD20, CD11b, CD16, HLA-DR, and CDS. In some embodiments, the antibody or binding partner is conjugated to a solid support or matrix (such as magnetic beads or paramagnetic beads) to allow isolation of cells for positive and / or negative selection.

[0141] In some embodiments, cells are incubated and / or cultured before or in combination with genetic engineering. The incubation step can include culturing, nurturing, stimulating, activating, and / or propagating. In some embodiments, the composition or cells are incubated in the presence of a stimulating condition or stimulant. Such conditions include those designed for: inducing the proliferation, expansion, activation, and / or survival of cells in a population, mimicking antigen exposure, and / or priming cells for genetic engineering (such as for introducing a recombinant antigen receptor). Conditions can include one or more of the following: a specific culture medium, temperature, oxygen content, carbon dioxide content, time, agents (e.g., nutrients, amino acids, antibiotics, ions, and / or stimulating factors such as cytokines, chemokines, antigens, binding partners, fusion proteins, recombinant soluble receptors, and any other agents designed to activate cells). In some embodiments, the stimulating condition or agent includes one or more agents. A ligand can be capable of activating the intracellular signaling domain of the TCR complex. In some embodiments, the agent turns on or initiates the TCR / CD3 intracellular signaling cascade in T cells. Such agents can include antibodies (such as those specific for TCR components (e.g., anti-CD3, anti-CD28)), co-stimulatory receptors, and / or one or more cytokines. The agent can be bound to a solid support such as a bead. Optionally, the amplification method can further include the step of adding anti-CD3 and / or anti-CD28 antibodies to the culture medium (e.g., at a concentration of at least about 0.5 ng / ml). In some embodiments, the stimulant includes IL-2 and / or IL-15, such as IL-2 at a concentration of at least about 10 units / ml. In some embodiments, the stimulant includes IL-7 and / or IL-15. In some embodiments, the stimulant includes IL-2, IL-7, and / or IL-15. In some embodiments, the stimulant includes IL-2, IL-15, and / or IL-15Ra. In some embodiments, the stimulant includes IL-2 and / or the heterodimeric IL-15 (i.e., a polypeptide comprising IL-15 and the IL-15 receptor α chain).

[0142] In another embodiment, T cells are isolated from peripheral blood by lysing red blood cells and depleting monocytes (e.g., by RM gradient centrifugation via PERCOLL). Alternatively, T cells can be isolated from umbilical cord. In any case, specific T cell subsets can be further isolated by positive or negative selection techniques. Cells expressing certain antigens (including but not limited to CD34, CD8, CD14, CD19, and CD56) can be depleted from the so-isolated cord blood mononuclear cells. These cell depletions can be accomplished using isolated antibodies, biological samples containing antibodies (such as ascites), antibodies bound to a physical support, and cell-bound antibodies. C. Activation before transfection

[0143] One aspect of the method disclosed herein does not require stimulation and / or activation of the enriched apheresis product (e.g., immune cell population, CD4 + and CD8 + cell population or eukaryotic donor cell population) with one or more stimulants prior to the transfection step. However,

[0144] In some embodiments, the method may further comprise stimulating and / or activating the enriched apheresis product (e.g., immune cell population, CD4 + and CD8 + cell population or eukaryotic donor cell population) with one or more stimulants prior to the transfection step. The stimulants described herein (e.g., CD3, CD28, cytokines, and / or growth factors) can promote efficient transduction and / or electroporation of primary human immune cells (e.g., T cells), and supplementing the culture medium with cytokines selected from the following can significantly enhance the expansion of transfected cells: IL-7, IL-15, IL-15Ra, IL-7 and IL-15 and / or the heterodimeric IL-15 (i.e., a polypeptide comprising IL-15 and the IL-15 receptor α chain). In addition, stimulation and / or activation before and / or after transfection can preserve undifferentiated T cells during CAR T manufacturing, which can extend the lifespan of the manufactured T cells, thereby improving the therapeutic efficacy of CAR T therapy.

[0145] In some embodiments, one or more stimulants are selected from agonistic antibodies, cytokines, recombinant costimulatory molecules, anti-CD3 antibodies or fragments thereof, anti-CD28 antibodies or fragments, small molecule inhibitors, and / or combinations thereof. In some embodiments, one or more stimulants are anti-CD3 and anti-CD28 antibodies or fragments thereof. In some embodiments, one or more stimulants are anti-CD3 and anti-CD28 antibodies or fragments thereof and one or more cytokines. 1. CD3 / TCR Complex

[0146] In some embodiments, the enriched apheresis product (e.g., immune cell population, CD4 + and CD8 + cell population or eukaryotic donor cell population) is stimulated and / or activated with an agent that stimulates the CD3 / TCR complex and / or an agent that stimulates co-stimulatory molecules and / or growth factor receptors on the cell surface. In some embodiments, the agent that stimulates the CD3 / TCR complex is an agent that stimulates CD3. In some embodiments, the agent that stimulates the CD3 / TCR complex is selected from antibodies (e.g., single domain antibodies, heavy chain variable domain antibodies, peptibodies, Fab fragments, or scFvs), small molecules, or ligands (e.g., naturally occurring, recombinant, or chimeric ligands).

[0147] In some embodiments, the agent that stimulates the CD3 / TCR complex does not constitute a bead. In some embodiments, the agent that stimulates a co-stimulatory molecule and / or a growth factor receptor does not constitute a bead. In some embodiments, the agent that stimulates the CD3 / TCR complex comprises an anti-CD3 antibody. In some embodiments, the agent that stimulates the CD3 / TCR complex comprises an anti-CD3 antibody covalently attached to a colloidal polymeric nanomatrix. In some embodiments, the agent that stimulates CD3 comprises one or more of the following: a CD3 or TCR antigen-binding domain (e.g., an anti-CD3 or anti-TCR antibody or antibody fragment), the antigen-binding domain comprising one or more of its CDRs, heavy chain, and / or light chain, as known to those skilled in the art.

[0148] In some embodiments, the agent that stimulates the CD3 / TCR complex and the agent that stimulates a co-stimulatory molecule and / or a growth factor receptor comprise T cell Trans Act RM . In some embodiments, the agent that stimulates the CD3 / TCR complex and the agent that stimulates a co-stimulatory molecule and / or a growth factor receptor are included in a multispecific binding molecule. In some embodiments, the multispecific binding molecule comprises a CD3 antigen-binding domain and a CD28 or CD2 antigen-binding domain. In some embodiments, the multispecific binding molecule comprises one or more heavy chains and / or light chains. In some embodiments, the multispecific binding molecule comprises a bispecific antibody. In some embodiments, one or more of a plurality of bispecific antibodies are conjugated together to form a multimer. 2. Co-stimulatory molecule

[0149] In some embodiments, the agent that stimulates a co-stimulatory molecule and / or a growth factor receptor is an agent that stimulates: CD28, ICOS, CD27, HVEM, LIGHT, CD40, 4-1BB, 0X40, DR3, GITR, CD30, TIM1, CD2, CD226, or any combination thereof. In some embodiments, the agent that stimulates a co-stimulatory molecule and / or a growth factor receptor is an agent that stimulates CD28. In some embodiments, the agent that stimulates a co-stimulatory molecule and / or a growth factor receptor is selected from an antibody (e.g., a single-domain antibody (e.g., a heavy chain variable domain antibody), peptibody, Fab fragment, or scFv), a small molecule, or a ligand (e.g., a naturally occurring, recombinant, or chimeric ligand).

[0150] In some embodiments, agents that stimulate co-stimulatory molecules and / or growth factor receptors include anti-CD28 antibodies. In some embodiments, agents that stimulate co-stimulatory molecules and / or growth factor receptors include anti-CD28 antibodies covalently attached to a colloidal polymeric nanomatrix. In some embodiments, agents that stimulate co-stimulatory molecules and / or growth factor receptors are agents that stimulate: CD28, ICOS, CD27, CD25, 4-1BB, IL6RA, IL6RB, or CD2. In some embodiments, agents that stimulate co-stimulatory molecules and / or growth factor receptors comprise one or more of the following: CD28, ICOS, CD27, CD25, 4-1BB, IL6RB, and / or CD2 antigen-binding domains. For example, the agent can be an anti-CD28, anti-ICOS, anti-CD27, anti-CD25, anti-4-IBB, anti-IL6RA, anti-IL6RB, anti-CD2 antibody, or an antibody fragment comprising one or more of its CDRs, heavy chains, and / or light chains, as known to those of skill in the art.

[0151] In some embodiments, prior to the transfection step, an enriched apheresis product (e.g., a lymphocyte population, an immune cell population, a CD4 + and CD8 + cell population, or a eukaryotic donor cell population) is stimulated and / or activated in vitro with an agent that stimulates the CD3 / TCR complex (e.g., an anti-CD3 antibody) and / or an agent that stimulates co-stimulatory molecules and / or growth factor receptors on the cell surface (e.g., an anti-CD28 antibody). + and CD8 + In some embodiments, the enriched apheresis product (e.g., a lymphocyte population, an immune cell population, a CD4

[0152] and CD8 + cell population, or a eukaryotic donor cell population) can be stimulated and / or activated for about less than or equal to about 1 hour, about 1.5 hours, about 2 hours, about 2.5 hours, about 3 hours, about 3.5 hours, about 4 hours, about 4.5 hours, or about 5 hours. + In some embodiments, the enriched apheresis product (e.g., a lymphocyte population, an immune cell population, a CD4

[0153] In some embodiments, an agent that stimulates the CD3 / TCR complex and an agent that stimulates a co-stimulatory molecule and / or a growth factor receptor are included in a multispecific binding molecule. The multispecific binding molecule can include an agent that stimulates the CD3 / TCR complex and an agent that stimulates a co-stimulatory molecule and / or a growth factor receptor. For example, the multispecific binding molecule can include a CD3 antigen-binding domain and one or more of the following: CD28, ICOS, CD27, CD25, 4-1BB, IL6RA, IL6RB, and / or a CD2 antigen-binding domain. In some embodiments, the multispecific binding molecule includes a CD3 antigen-binding domain and a CD28 or CD2 antigen-binding domain. 3. Cytokines

[0154] In some embodiments, one or more stimulants are cytokines selected from the group consisting of interleukin-2 (IL-2), interleukin-3 (IL-3), interleukin-6 (IL-6), interleukin-7 (IL-7), interleukin-7 receptor (IL-7R), interleukin-11 (IL-11), interleukin-12 (IL-12), interleukin-15 (IL-15), interleukin-15 receptor (IL-15R), heterodimeric IL-15 (i.e., a polypeptide comprising IL-15 and the IL-15 receptor alpha chain), interleukin-18 (IL-18), interleukin-18 receptor (IL-18R), interleukin-21 (IL-21), granulocyte macrophage colony-stimulating factor, alpha, beta, or gamma interferon, erythropoietin, and any combination thereof.

[0155] Cytokines can be selected from IL-2, IL-7, IL-6, IL-15, IL-15Ra, heterodimeric IL-15 (i.e., a polypeptide comprising IL-15 and the IL-15 receptor alpha chain; hetIL-15), or IL-21. IL-2 is the most commonly used cytokine for generating lymphocytes for adoptive immunotherapy. IL-2 promotes T cell survival and expansion and enhances the tumor-killing ability of T cells. IL-2 significantly increases the accumulation of CAR-T cells and their cytotoxic ability, but CAR-T cells exposed to IL-2 exhibit poor anti-tumor immunity in vivo after adoptive transfer. CAR-T cells exposed to IL-2 also display a relatively mature phenotype with low expression of CD62L, CCR7, CD27, and CD28, which have low persistence in vivo. The adoptive transfer of less differentiated T cells is associated with better tumor regression, which supports the finding that CAR-T cells exposed to IL-2 are less effective than other groups (Gattinoni et al., Nat Med, 2011, 17:1290-7; and Markley et al., Blood, 2010, 115:3508-19).

[0156] IL-15 exhibits properties similar to IL-2 in stimulating the expansion and tumor lysis function of CAR-T cells, and shows better anti-tumor immunity in animal models. In addition, IL-15 induces a less differentiated phenotype (higher expression of CD27 and CD28). Therefore, IL-15 can support the persistence of CAR-T cells in vivo. IL-7 similarly promotes the in vitro expansion of CAR-T cells.

[0157] IL-7 also induces a higher level of CD62L expression and exhibits the highest proportion of CAR-Tscm cells in an antigen-free environment. Ex vivo exposure of T cells or CAR T cells to IL-7 without antigen stimulation improves the anti-tumor efficacy of CAR-T cells. However, when compared with IL-2, CAR-T cells exposed to IL-7 do not produce better anti-tumor efficacy in vivo. Due to less expansion of CAR-T cells upon antigen stimulation, the efficacy of IL-7 is also inferior to that of IL-15. The combination of IL-7 and IL-15 promotes the generation of Tscm, which is beneficial for the generation of more "young" CAR-T cells. Therefore, combining IL-7 with IL-15 can promote CAR-T cell expansion and induce the T cell phenotype most effective for therapeutic treatment.

[0158] IL-21 can induce the expansion of less differentiated CAR-T cells, with a phenotype of high expression of CD62L, CCR7, CD27, and CD28, even in the presence of antigen stimulation. Therefore, CAR-T cells exposed to IL-21 show the best persistence in animal models and in vivo IL-21 injection, and also exhibit better efficacy than other cytokine groups except IL-15 in promoting tumor eradication. See, for example, WO 2016 / 109410.

[0159] Therefore, in some embodiments, the cytokine can also be selected from IL-15 and IL-7; IL-7 and IL-21; IL-7 and IL-2; IL-15 and IL-2; IL-7, IL-15, and IL-21; IL-15 and IL-15Ra; or IL-7, IL-15, and IL-15Ra. In some embodiments, the cytokine is IL-2. In some embodiments, the cytokine is IL-15 (e.g., hetIL-15 (IL15 / sIL-15Ra)). In some embodiments, the cytokine is IL-6 (e.g., IL-6 / sIL-6Ra). In some embodiments, the cytokine is IL-7. In some embodiments, the cytokine is IL-7 and IL-15.

[0160] In some embodiments, enriched apheresis products (e.g., lymphocyte populations, immune cell populations, CD4 + and CD8 + cell populations or eukaryotic donor cell populations) are stimulated and / or activated with the following amounts of IL-2: about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 100, about 110, about 120, about 130, about 140, about 150, about 160, about 170, about 180, about 190, about 200, about 210, about 220, about 230, about 240, about 250, about 260, about 270, about 280, about 290, or about 300 U / ml (or any amount between these values). In some embodiments, enriched apheresis products (e.g., immune cell populations, CD4 + and CD8 + cell populations or eukaryotic donor cell populations) are stimulated and / or activated with the following amounts of IL-7: about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 ng / ml (or any amount between these values). In some embodiments, enriched apheresis products (e.g., immune cell populations, CD4 + and CD8 + cell populations or eukaryotic donor cell populations) are stimulated and / or activated with the following amounts of IL-15: about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 ng / ml (or any amount between these values). Cytokine stimulation preserves or increases the undifferentiated phenotype of T cells during the manufacture of CAR T (electroporated CAR T cells) disclosed herein and produces CAR T cells (electroporated CAR T cells) that persist longer in a subject after administration. Supplementing the culture medium with cytokines selected from the following significantly enhances the expansion of transduced cells by at least 200-fold over a 14-day period: IL-7, IL-15, IL-15Ra, IL-7 and IL-15 and / or the heterodimeric IL-15 (i.e., a polypeptide comprising IL-15 and the IL-15 receptor alpha chain). See, e.g., WO 2016 / 109410. 4. Nanostructures

[0161] In some embodiments, one or more stimulants are conjugated to beads or nanostructures. In some embodiments, the nanostructure is a nanomatrix. The nanomatrix can comprise a matrix of mobile polymer chains and anti-CD3 and anti-CD28 antibodies or fragments thereof. The size of the nanomatrix can be from about 1 to about 500 nm (or any size between these two values). In some embodiments, one or more stimulants are the nanomatrices described herein and one or more cytokines.

[0162] In some embodiments, the nanomatrix can comprise polymeric, biodegradable, or biocompatible inert materials. The inert materials can be non-toxic to cells. In some embodiments, the nanomatrix can be composed of hydrophilic polymer chains that acquire maximum mobility in aqueous solution due to hydration of the chains. In some embodiments, the mobile nanomatrix can have a collagen, purified protein, purified peptide, polysaccharide, glycosaminoglycan, or extracellular matrix composition. Polysaccharides can include, for example, cellulose ethers, starch, gum arabic, agarose, dextran, chitosan, hyaluronic acid, pectin, xanthan gum, guar gum, or alginate. Other polymers can include polyesters, polyethers, polyacrylates, polyacrylamides, polyamines, polyethyleneimine, polyquaternary ammonium polymers, polyphosphazenes, polyvinyl alcohol, polyvinyl acetate, polyvinylpyrrolidone, block copolymers, or polyurethanes. In some embodiments, the mobile nanomatrix is a polymer of dextran.

[0163] Another aspect of the present disclosure provides a method for manufacturing an engineered population of eukaryotic cells, the method comprising: (1) obtaining a population of eukaryotic donor cells from a subject; (2) mixing the population of eukaryotic donor cells with one or more buffer solutions; (3) stimulating the population of eukaryotic donor cells with one or more stimulants; (4) transfecting the stimulated population of eukaryotic donor cells with an effective dose of a modifier; (5) culturing and expanding the transfected population of eukaryotic donor cells; and (6) harvesting the engineered eukaryotic cells, thereby producing a modified population of eukaryotic donor cells. In some embodiments, the transfected cells are cultured and expanded in the presence of one or more stimulants.

[0164] Another aspect of the present disclosure provides a method for manufacturing an engineered population of immune cells, the method comprising: (1) enriching a population of lymphocytes, immune cells, or CD4 + and CD8 + cell population from a donor leukapheresis; (2) mixing the population of lymphocytes, immune cells, or CD4 + and CD8 + cell population with one or more buffer solutions; (3) stimulating the population of eukaryotic donor cells with one or more stimulants; (4) transfecting the population of lymphocytes, immune cells, or CD4 + and CD8 +Population of cells; (5) culturing and expanding the transfected population of lymphocytes, population of immune cells, or CD4 + and CD8 + population of cells; and (6) harvesting the engineered lymphocytes, immune cells, or CD4 + and CD8 + cells; thereby generating a modified population of lymphocytes, modified population of immune cells, or modified CD4 + and CD8 + population of cells. In some embodiments, the transfected cells are cultured and expanded in the presence of one or more stimulants.

[0165] One aspect of the present disclosure provides a method for manufacturing a population of engineered immune cells, the method comprising: (1) enriching a population of lymphocytes, population of immune cells, or CD4 + and CD8 + population of cells obtained from a subject; (2) mixing the population of lymphocytes, population of immune cells, or CD4 + and CD8 + population of cells with one or more buffer solutions; (3) stimulating the population of lymphocytes, population of immune cells, or CD4 + and CD8 + population of cells with one or more stimulants; (4) transfecting the population of lymphocytes, population of immune cells, or CD4 + and CD8 + population of cells with an effective dose of a modifying agent; (5) culturing and expanding the transfected population of lymphocytes, population of immune cells, or CD4 + and CD8 + population of cells; and (6) harvesting the engineered lymphocytes, immune cells, or CD4 + and CD8 + cells; thereby generating a modified population of lymphocytes, modified population of immune cells, or modified CD4 + and CD8 + population of cells. In some embodiments, the transfected cells are cultured and expanded in the presence of one or more stimulants as described herein.

[0166] In some embodiments, the enriched apheresis product (e.g., population of lymphocytes, population of immune cells, CD4 + and CD8 +A population of cells or a population of eukaryotic donor cells) is stimulated and / or activated with anti-CD3 and anti-CD28 antibodies for about 12 hours, after which it is transfected with a modifying agent (e.g., transduced and / or electroporated with a vector or lentiviral vector encoding a CAR, engineered TCR, or polypeptide or functional derivative thereof that enhances immune cell function). Then, at about 24 hours after the start of stimulation, the cells are washed and formulated for storage or administration. In other aspects, at about 12 hours, about 22 hours, about 30 hours, about 40 hours, about 45 hours, about 50 hours, about 60 hours, or about 72 hours after the start of stimulation, the cells are washed and formulated for storage or administration. D. Methods for introducing viral vectors into cells

[0167] Methods for introducing a modifying agent (e.g., an expression vector, viral vector, polynucleotide, or nucleic acid) into a cell include physical, biological, chemical methods, and combinations thereof. An expression vector (including the viral vectors or expression vectors of the present disclosure) can be introduced into a host cell by any means known to those skilled in the art. If desired, the expression vector can contain viral sequences for transfection. Alternatively, the expression vector can be introduced by fusion, electroporation, biolistics (e.g., gene gun), transfection, liposome transfection (e.g., cationic liposomes), polymer encapsulation, etc. The host cells (e.g., immune cells) can be grown and expanded in culture before introducing the expression vector, and then appropriately treated to introduce and integrate the vector. Then the host cells (e.g., immune cells) can be expanded and screened by markers present in the vector. Methods for generating cells containing a vector and / or exogenous nucleic acid are well known in the art. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York (2001).

[0168] In some embodiments, the enriched apheresis product and / or the enriched target cell population can be modified using any method known in the art (such as activation, amplification, induction of apoptosis, gene manipulation, induction of antigen specificity). In some embodiments, the enriched apheresis product and / or the enriched target cell population can be modified by adding cytokines, crosslinking specific receptors, adding antigens, introducing nucleic acid molecules (DNA, RNA, and / or their modified forms), protein agents, adding drugs or small molecules, or any combination thereof. In some embodiments, introducing a modifier (e.g., an expression vector, a viral vector, an exogenous nucleic acid molecule, a polynucleotide, or a nucleic acid) includes viral transfection (transduction), non-viral transfection, electroporation, liposome transfection, cationic lipid-mediated transfection using liposomes, polymer encapsulation, peptide-mediated transfection, or a biolistic particle delivery system (such as a “gene gun”) (see, e.g., Nishikawa et al., Hum Gene Ther., 12(8):861-70 (2001)). 1. Biological methods

[0169] Biological methods for introducing a modifier of interest into a host cell (e.g., an immune cell) include using an expression vector (e.g., a viral vector, an exogenous nucleic acid molecule, a polynucleotide, or a nucleic acid (DNA and RNA)). Viral vectors (especially retroviral vectors) (viral transfection) have become the most widely used method for inserting genes into mammals (e.g., human cells). Viral vectors can be derived from lentiviruses, poxviruses, herpes simplex virus I, adenoviruses, adeno-associated viruses, etc. See, e.g., U.S. Patent Nos. 5,350,674 and 5,585,362.

[0170] In some embodiments, nucleic acids encoding the subject CAR, subject engineered TCR, subject KIR, subject antigen-binding polypeptide, subject cell surface receptor ligand, subject tumor antigen, subject switch receptor, subject dominant negative receptor, and / or subject polypeptide that enhances immune function (e.g., T cell priming or T cell infiltration) can be introduced into cells using an expression vector (viral transfection). Provided herein are expression vectors (e.g., lentiviral vectors or retroviral vectors) comprising nucleic acids encoding the subject CAR, subject engineered TCR, subject KIR, subject antigen-binding polypeptide, subject cell surface receptor ligand, subject tumor antigen, subject switch receptor, subject dominant negative receptor, and / or subject polypeptide that enhances immune function (e.g., T cell priming or T cell infiltration). Suitable expression vectors include lentiviral vectors, gamma retroviral vectors, foamy virus vectors, adeno-associated virus (AAV) vectors, adenovirus vectors, engineered hybrid viruses, naked DNA, including but not limited to transposon-mediated vectors such as Sleeping Beauty, Piggyback, and integrases (e.g., Phi31). Some other suitable expression vectors include herpes simplex virus (HSV) and retroviral expression vectors.

[0171] Adenovirus expression vectors are based on adenovirus and have a low ability to integrate into genomic DNA but a high efficiency of transfecting host cells. Adenovirus expression vectors contain adenovirus sequences that are sufficient to: (a) support packaging of the expression vector and (b) ultimately express in a host cell the subject CAR, subject engineered TCR, subject KIR, subject antigen-binding polypeptide, subject cell surface receptor ligand, subject tumor antigen, subject switch receptor, subject dominant negative receptor, and / or subject polypeptide that enhances immune function (e.g., T cell priming or T cell infiltration). In some embodiments, the adenovirus genome is a 36 kb linear double-stranded DNA with foreign DNA sequences. For example, nucleic acids encoding the subject CAR, subject engineered TCR, subject KIR, subject antigen-binding polypeptide, subject cell surface receptor ligand, subject tumor antigen, subject switch receptor, subject dominant negative receptor, and / or subject polypeptide that enhances immune function (e.g., T cell priming or T cell infiltration) can be inserted to replace large segments of adenovirus DNA to prepare the expression vectors of the invention.

[0172] Another expression vector is based on adeno-associated virus and utilizes an adenovirus coupling system. This AAV expression vector has a high frequency of integration into the host genome. It can infect non-dividing cells, thus making it useful for delivering genes into mammalian cells, e.g., in tissue culture or in vivo. AAV vectors are infectious for a wide range of hosts. Details regarding the production and use of AAV vectors are described in U.S. Patent Nos. 5,139,941 and 4,797,368.

[0173] Retroviral expression vectors are capable of integrating into the host genome, delivering large amounts of foreign genetic material, infecting a wide range of species and cell types, and are packaged in special cell lines. Retroviral vectors are constructed by inserting nucleic acids (e.g., nucleic acids encoding a subject CAR, subject engineered TCR, subject KIR, subject antigen-binding polypeptide, subject cell surface receptor ligand, subject tumor antigen, subject switch receptor, subject dominant negative receptor, and / or a subject polypeptide that enhances immune function (e.g., T cell priming or T cell infiltration)) into certain positions in the viral genome to produce replication-defective viruses. Although retroviral vectors are capable of infecting a variety of cell types, integration and stable expression of a subject CAR, subject engineered TCR, subject KIR, subject antigen-binding polypeptide, subject cell surface receptor ligand, subject tumor antigen, subject switch receptor, subject dominant negative receptor, and / or a subject polypeptide that enhances immune function (e.g., T cell priming or T cell infiltration) requires division of the host cell.

[0174] Lentiviral vectors are derived from lentiviruses, which are complex retroviruses that contain additional genes with regulatory or structural functions in addition to the common retroviral genes gag, pol, and env. See, for example, U.S. Patent Nos. 6,013,516 and 5,994,136. Some examples of lentiviruses include human immunodeficiency virus (HTV-1, HTV-2) and simian immunodeficiency virus (SIV). Lentiviral vectors have been generated by multiple attenuation of HIV virulence genes, such as deletion of the genes env, vif, vpr, vpu, and nef, such that the vectors are biologically safe. Lentiviral vectors are capable of infecting non-dividing cells and can be used for in vivo and ex vivo gene transfer and expression of nucleic acids encoding a subject CAR, subject engineered TCR, subject KIR, subject antigen-binding polypeptide, subject cell surface receptor ligand, subject tumor antigen, subject switch receptor, subject dominant negative receptor, and / or a subject polypeptide that enhances immune function (e.g., T cell priming or T cell infiltration). See, for example, U.S. Patent No. 5,994,136.

[0175] In some embodiments, nucleic acids encoding the subject CAR, subject engineered TCR, subject KIR, subject antigen-binding polypeptide, subject cell surface receptor ligand, subject tumor antigen, subject switch receptor, subject dominant negative receptor, and / or subject polypeptide that enhances immune function (e.g., T cell priming or T cell infiltration) are introduced into immune cells by viral transduction. In some embodiments, viral transduction comprises contacting immune cells with a viral vector comprising one or more nucleic acids. In some embodiments, the viral vector is selected from retroviral vectors, Sendai virus vectors, adenoviral vectors, adeno-associated virus vectors, and lentiviral vectors. A variety of markers that can be used are known in the art and can include hprt, neomycin resistance, thymidine kinase, hygromycin resistance, and the like.

[0176] The modified and enriched apheresis products (e.g., immune cells) of the invention (e.g., comprising nucleic acids encoding the subject CAR, subject engineered TCR, subject KIR, subject antigen-binding polypeptide, subject cell surface receptor ligand, subject tumor antigen, subject switch receptor, subject dominant negative receptor, and / or subject polypeptide that enhances immune function (e.g., T cell priming or T cell infiltration)) can be produced by stably transfecting a host cell (e.g., an immune cell) with an expression vector comprising a nucleic acid of the present disclosure.

[0177] The transfected cells (i.e., immune cells) can be expanded ex vivo, and the transfected cells express nucleic acids encoding the subject CAR, KIR, TCR, KIR, antigen-binding polypeptide, cell surface receptor ligand, tumor antigen, subject switch receptor, subject dominant negative receptor, and / or subject polypeptide that enhances immune function (e.g., T cell priming or T cell infiltration) of the present disclosure. In some embodiments, the transfected cells (i.e., immune cells) are not expanded ex vivo, and the transfected cells express nucleic acids encoding the subject CAR, KIR, TCR, KIR, antigen-binding polypeptide, cell surface receptor ligand, tumor antigen, subject switch receptor, subject dominant negative receptor, and / or subject polypeptide that enhances immune function (e.g., T cell priming or T cell infiltration) of the present disclosure.

[0178] Additional methods for producing the modified cells of the present disclosure include, but are not limited to, chemical transformation methods (e.g., using calcium phosphate, dendrimers, liposomes, and / or cationic polymers), non-chemical transformation methods (e.g., electroporation, optical transformation, gene electrotransfer, and / or hydrodynamic delivery), and / or particle-based methods (e.g., impalefection, using a gene gun, and / or magnetofection). 2. Physical methods

[0179] Physical methods for introducing polynucleotides (RNA or DNA) or expression vectors into host cells include liposome transfection, particle bombardment, microinjection, electroporation, etc. Commercially available methods can be used to introduce expression vectors or polynucleotides into target cells, and the methods include electroporation, such as 4D-Nucleofector RM technology (Lonza Bioscience, Walkersville, Maryland), Amaxa Nucleofector-II (Amaxa Biosystems, Cologne, Germany), ECM 830 (BTX) (Harvard Instruments, Boston, Massachusetts), Gene Pulser II (BioRad, Denver, Colorado) or Multiporator (Eppendorf, Hamburg, Germany). a. Electroporation

[0180] In some embodiments, the enriched apheresis product and / or the enriched target cell population are transfected. In some embodiments, the enriched apheresis product and / or the enriched target cell population are electroporated. In some embodiments, the cell transfection device comprises a flow-through electroporation chamber. For example, the chamber or system is described in U.S. Patent Nos. 5,720,921, 6,074,605, 7,141,425, 7,521,224 and 8,673,623, and U.S. Patent Application Publication Nos. 2007 / 0128708A1, 2008 / 0182251A1, 2013 / 0196441, 2017 / 0233716A1, and Kim et al., Biosens Bioelectron 2008 23(9):1353-60.

[0181] Electroporation applies an electric field to the cells to introduce pores (electropores) into the cell membrane, and (usually charged) macromolecules or agents can flow into the cells through the pores. Removal of the electric field allows the pores to reseal, and the introduced molecules are inside the cells. Important parameters for successful electroporation include the maximum voltage applied and the duration of the current pulse. The voltage and capacitance settings should also be optimized for each cell type, and the resistance of the electroporation buffer is important for selecting the initial instrument settings. Optimal stable and transient transformation occurs at approximately the same instrument settings, so transient expression can be used to optimize conditions when adapting to a new cell type.

[0182] Accordingly, electroporation-mediated administration of nucleic acids, including expression constructs, into cells presents a means for delivering RNA of interest to target cells. Electroporation-mediated administration can utilize any of a number of available devices and electroporation systems known to those of skill in the art. Exemplary formulations of nucleic acid constructs and exemplary methods of electroporating nucleic acid constructs into mammalian cells are taught in US2004 / 0014645, US2005 / 0052630, US2005 / 0070841, US2004 / 0059285, US2004 / 0092907, and US2007 / 0128708. The various parameters (including electric field strength) required for electroporation of any known cell type are generally known in the relevant research literature as well as in many patents and applications in the art. See, for example, U.S. Patent Nos. 6,678,556, 7,171,264, and 7,173,116.

[0183] In some embodiments, the cell transfection device is a commercially available device for therapeutic electroporation, selected from, but not limited to, the MedPulser RM DNA Electroporation Therapy System (Inovio / Genetronics, San Diego, Calif.), and is described in patents such as U.S. Patent Nos. 6,567,694, 6,516,223, 5,993,434, 6,181,964, 6,241,701, and 6,233,482.

[0184] In some embodiments, the cells are not activated prior to the transfection step. In some embodiments, prior to transfection, cells can generally be activated and expanded using methods described, for example, in the following patents: U.S. Patent Nos. 6,352,694; 6,534,055; 6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681; 7,144,575; 7,067,318; 7,172,869; 7,232,566; 7,175,843; 5,883,223; 6,905,874; 6,797,514; 6,867,041; and U.S.2006 / 0121005.

[0185] In some embodiments, when compared to a population of stimulated CAR T cells, most unstimulated CAR T cells generated by lentiviral electroporation (e.g., nucleofection) can maintain a less differentiated phenotype. For example, more than at least about 30%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70% of the electroporated CAR T cells can be naive CAR T cells. In some embodiments, electroporation of the CAR lentiviral vector can result in efficient integration of the CAR transgene into the T cell genome. In some embodiments, the vector copy number / cell of the electroporated CAR T cells can be substantially similar to the copy number / cell of conventional (transduced) CAR T cells. In some embodiments, the electroporated CAR transgene can be expressed within about 0.5 hour, about 0.75 hour, about 1 hour, about 1.5 hours, about 2.0 hours, about 2.5 hours, about 3.0 hours, about 3.5 hours, about 3.5 hours or at least about 5.0 hours after nucleofection.

[0186] In some embodiments, an enriched apheresis product and / or an enriched population of target cells can be modified using a suitable electroporation device, which can be, for example, from 4D-Nucleofector RM technology (Lonza Bioscience, Walkersville, Maryland), Amaxa NUCLEOFECTOR RM -II (Amaxa Biosystems (Cologne, Germany)), ECM830 (BTX; Harvard Instruments, Boston, Massachusetts), Gene Pulser II or Gene PulserMXCELL RM (BioRad, Denver, Colorado), Multiporator (Eppendort, Hamburg, Germany) or FLOW technology (MaxCyte). In some embodiments, the transfection device is an ECM830 Electro Square Wave Porator (Harvard Apparatus BTX), and cells are electroporated in a 2 mm cuvette (Harvard Apparatus BTX, Holliston, Massachusetts, USA).

[0187] One of ordinary skill in the art will understand that the pulse type, pulse duration, voltage, and frequency of use depend on the instrument type and cell type; and optimization of transfection efficiency can be adjusted based on the pulse type, pulse duration, voltage, frequency of use, and concentration of the nucleic acid or particle to be electroporated (e.g., DNA, RNA, expression vector, lentiviral vector or lentiviral particle).

[0188] Electroporation can be carried out in one of the following two ways: batch electroporation or flow-through electroporation. i. Batch electroporation

[0189] Most commonly, in batch form, in a relatively small volume (about 1 ml, and typically about 1×10 6 cells), electroporation is carried out by placing a suspension of the cells and macromolecules to be introduced into a small chamber containing two electrodes, which are connected to a pulse generator and arranged to deliver an electric current through the suspension. In batch form, one or more electric field pulses are applied to the cells, and the treated cells are typically transferred to a culture medium to allow the cells to recover.

[0190] In one embodiment, the batch processing mode can be used in the methods and systems described herein. In this embodiment, cells are electroporated by shunting a suspension of the washed and enriched apheresis product or enriched target cell population into an electroporation chamber when a given concentration of target cells (e.g., as detected by a detector) is reached in the cell separation module, and adding a cell modifier or cell customization agent (such as an agent for generating CART cells (e.g., a lentiviral vector, lentiviral particles, an expression vector, DNA, RNA, or protein)) to the electroporation device chamber, and applying one or more electrical pulses from a pulse generator to the cell suspension. When electroporating each batch of cells, the electroporated cells can be reintroduced into the patient, or if continuous electroporation is utilized, they can be introduced continuously. ii. Flow-through electroporation

[0191] In some embodiments, a flow-through or continuous flow electroporation system can be used. In this embodiment, washed cells (e.g., enriched apheresis product or enriched target cells) and a modifier (e.g., lentiviral particles, an expression vector, a lentiviral vector, DNA, or RNA) are passed through an electroporation unit, and a voltage is continuously applied across the electroporation unit. In some embodiments, the flow-through or continuous flow electroporation system can achieve processing of up to 20 ml of cell suspension per minute, with a transfection efficiency of up to 75%. In some embodiments, the flow-through or continuous flow electroporation system can achieve processing of up to 1 to 10 10 cells / second, 10 4 to 10 7 cells / second, 10 5 to 10 8 cells / second, or 10 6 to 10 9 cells / second, or in a single transformation procedure, processing in the range from 1 cell to 10 10a cell batch of cells with an efficiency range of up to about 70%, about 75%, about 80%, about 85%, about 90% or about 95% or higher.

[0192] In some embodiments, the transfection devices disclosed herein incorporate fluid channels having a constant depth and / or a variable width along the length of the channel such that some portions are narrow while other portions are wider. The electric field at any point depends on the width of the channel, with the narrower portions having a stronger electric field than the wider portions. The width and current are selected such that the electric field only exceeds the transmembrane potential that permits electroporation at the narrow points, and the alternating narrow and wide segments along the length of the channel provide an effect similar to a pulsed electric field without the need for a pulsed electric field generator. The flow rate through the channel and the corresponding lengths of the wide and narrow segments can be adjusted to adjust the time that the cells are exposed to a current strong enough to electroporate them. Systems for continuous flow electroporation are described, for example, in Wei and Li, Methods Mol. Biol. 1121:99-110 (2014), Geng et al., J. Controlled Release 144:91-100 (2010), U.S. Patent Nos. 10,253,316, 6,617,154, 6,673,669, 7,029,916, 7,771,984, 9,546,350 or 10,253,316.

[0193] In some embodiments, the flow-through electroporation system within the transfection device employs a fluid system fabricated from polydimethylsiloxane (PDMS) on a glass substrate and incorporates a channel having alternating wide extension segments (10,000 - 5,000 μm, e.g., about 7,500 μm) and narrow extension segments (500 - 700 μm, e.g., about 500 μm). The inlet of the device can be connected to a conduit or tubing through which cells are delivered from a buffer exchange device to the transfection device. Wire electrodes inserted into the inlet and outlet of the flow-through electroporation system can be connected to a constant voltage power supply. Cells suspended in an electroporation buffer and containing a lentiviral vector and / or nucleic acid encoding a CAR or TRC are pumped through the fluid channel.

[0194] In some embodiments, the apheresis product is transfected with about 0.5 μl, about 1 μl, about 1.5 μl, about 2 μl, about 2.5 μl, about 3 μl, about 3.5 μl, about 4 μl, about 5 μl, about 6 μl, about 7 μl, about 8 μl, about 9 μl, about 10 μl, about 15 μl or about 20 μl of lentiviral vector. In some embodiments, the effective dose of the lentiviral vector comprises a multiplicity of infection (MOI) of about 0.01, about 0.02, about 0.03, about 0.04, about 0.05, about 0.06, about 0.07, about 0.08, about 0.09, about 0.1, about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1.0, about 1.25, about 1.5, about 2.0, about 3.0, about 4.0 or about 5.0, with the MOI being from about 0.01 to about 5.0. In some embodiments, the apheresis product is transfected with about 2 μl of lentiviral vector at an MOI of about 0.08, 5 μl of lentiviral vector at an MOI of about 0.2 or 10 μl of lentiviral vector at an MOI of about 0.4.

[0195] In some embodiments, the electroporation module comprises a flow-through electroporation chamber. For example, the chamber or system is described in U.S. Patent Nos. 5,720,921, 6,074,605 and 7,141,425.

[0196] Another aspect of the invention provides a novel method of electroporating cells.

[0197] Typically, during electroporation, cells are contacted with an effective dose of a lentiviral vector before applying electricity to a well containing the cells to be transfected. However, the inventors have found that electroporation can be toxic to the expression vector, thereby reducing transfection efficiency. Thus, to improve the efficiency of electroporation, the inventors electroporated the cells in the absence of the expression vector and observed an increase in transfection efficiency. Accordingly, in some embodiments of the present disclosure, cells can be contacted with an effective dose of a lentiviral vector before electroporation. In alternative embodiments, cells can be contacted with an effective dose of a lentiviral vector for up to about 4 hours after electroporation (e.g., application of electricity). For example, after electroporation, cells can be contacted with an effective dose of a lentiviral vector for at least about 5 - 30 minutes, at least about 25 - 50 minutes, at least about 5 - 60 minutes, at least about 5 - 12 minutes, at least about 60 - 120 minutes, at least about 120 - 240 minutes. Alternatively, after electroporation, cells can be contacted with an effective dose of a lentiviral vector for at least about 1 minute, at least about 2 minutes, at least about 5 minutes, at least about 10 minutes, at least about 20 minutes, at least about 30 minutes, at least about 45 minutes, at least about 60 minutes, at least about 75 minutes, at least about 90 minutes, at least about 100 minutes, at least about 110 minutes, at least about 120 minutes, at least about 150 minutes, at least about 160 minutes, at least about 170 minutes, at least about 180 minutes, at least about 190 minutes, at least about 200 minutes, at least about 220 minutes, or at least about 240 minutes.

[0198] Adding the lentiviral vector to the cells up to 4 hours after electroporation (e.g., 1 minute to 2 hours or 1 minute to 4 hours) can reduce the amount of lentiviral particles killed by electroporation, which can be toxic to lentiviral particles. Thus, adding the lentiviral vector to the cells up to 4 hours after electroporation can enhance CAR transfection. For example, when compared to a conventional electroporation process (e.g., adding the lentivirus to the cells before electroporation), adding the lentiviral vector to the cells up to 4 hours after electroporation can increase CAR expression by about 10% - 15%.

[0199] Thus, in some embodiments of the manufacturing processes disclosed herein, transfecting cells comprises electroporating the cells with a lentiviral vector and / or particles. In some embodiments, electroporation comprises adding the lentiviral vector to the cells before, simultaneously with, or after applying an electric current to the cells. In some embodiments, an electric current is applied to the cells after adding the lentiviral vector. In some embodiments of the manufacturing processes disclosed herein, an electric current is applied to the cells before adding the lentiviral vector. For example, before adding the lentiviral vector, an electric current is applied to the cells for at least about 5 - 30 minutes, at least about 25 - 50 minutes, at least about 5 - 60 minutes, at least about 5 - 12 minutes, at least about 60 - 120 minutes, at least about 120 - 240 minutes. Alternatively, before adding the lentiviral vector, an electric current is applied to the cells for at least about 1 minute, at least about 2 minutes, at least about 5 minutes, at least about 10 minutes, at least about 20 minutes, at least about 30 minutes, at least about 45 minutes, at least about 60 minutes, at least about 75 minutes, at least about 90 minutes, at least about 100 minutes, at least about 110 minutes, at least about 120 minutes, at least about 150 minutes, at least about 160 minutes, at least about 170 minutes, at least about 180 minutes, at least about 190 minutes, at least about 200 minutes, at least about 220 minutes, or at least about 240 minutes. b. Cell extrusion microfluidics

[0200] In some embodiments, an effective dose of a lentiviral vector is introduced into the enriched apheresis product using cell extrusion microfluidics. In some embodiments, the lentiviral vector is introduced into the mononuclear cells by forcing the cells through a constriction of a smaller diameter under pressure. Pulses of rapid stretching, rapid compression, or high shear rate cause molecules to be taken up from the surrounding cell culture medium into the cytoplasm of the cells. This so-called "cell extrusion" microfluidic technique is applicable to a variety of cell types and is well-suited for introducing materials into mononuclear cells. The cell extrusion microfluidic technique is described, for example, in WO 2013 / 059343 and US2014 / 287509. 3. Chemical methods

[0201] Chemical methods for introducing an expression vector into a host cell include colloidal dispersion systems such as macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems including water-in-oil emulsions, micelles, mixed micelles, and liposomes. Chemical means for introducing a polynucleotide into a host cell include colloidal dispersion systems such as macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems including water-in-oil emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system used as an in vitro and in vivo delivery vehicle is a liposome (e.g., an artificial membrane vesicle).

[0202] Regardless of the method used to introduce exogenous nucleic acids into host cells or otherwise expose cells to the inhibitors of the present invention, in order to confirm the presence of nucleic acids in host cells, a variety of assays can be performed. Such assays include, for example, molecular biological assays well known to those skilled in the art, such as Southern blotting and Northern blotting, RT-PCR and PCR; biochemical assays, such as detecting the presence or absence of specific peptides, for example, by immunological means (ELISA and Western blotting) or by the assays described herein to identify agents falling within the scope of the present invention.

[0203] In addition, nucleic acids can be introduced by any means, such as transducing amplified host cells (e.g., immune cells; enriched apheresis products), transfecting amplified host cells (e.g., immune cells; enriched apheresis products), and electroporating amplified host cells (e.g., immune cells; enriched apheresis products). One nucleic acid can be introduced by one method, and another nucleic acid can be introduced into host cells (e.g., immune cells; enriched apheresis products) by a different method. In some embodiments, viral transfection and chemical or physical transfection can be used to introduce expression systems such as lentiviral or retroviral particles. For example, lentiviral or retroviral particles can be transfected into cells using electroporation. 4. Lentiviral Vector Transfection

[0204] In some embodiments, the methods described herein include transfecting stimulated and / or unstimulated apheresis or blood products or enriched apheresis or blood products with one or more modifiers. In some embodiments, the one or more modifiers are selected from small molecule agents, biological agents, therapeutic agents, proteins, peptides, protein therapeutics, peptide therapeutics, nucleic acids, DNA, RNA, mRNA, chimeric antigen receptors, heterologous T cell receptors, expression vectors, viral vectors, vectors, retroviral vectors, lentiviral vectors, adenoviral vectors, and adeno-associated viral vectors. The modifier can include a virus not permissive to human or eukaryotic cells (e.g., a virus that cannot naturally infect or enter human or eukaryotic cells). In some embodiments, the modifier can also be selected from retroviral vectors or lentiviral vectors. In some embodiments, the modifier can be a retroviral vector, a lentiviral vector, an adenoviral vector, or an adeno-associated viral vector. In one embodiment, the modifier is a lentiviral vector or a retroviral vector. In some embodiments, the lentiviral vector is a lentiviral particle.

[0205] In some embodiments, transfection is viral transfection (e.g., viral transduction), or transfection is electroporation of nucleic acids or electroporation of lentiviral vectors, the nucleic acids encoding chimeric antigen receptors (CARs), engineered T cell receptors (TCRs), and / or polypeptides or functional derivatives thereof that enhance immune cell function, the lentiviral vectors comprising nucleic acid sequences encoding chimeric antigen receptors (CARs), engineered T cell receptors (TCRs), and / or polypeptides or functional derivatives thereof that enhance immune cell function.

[0206] In some embodiments, transfection is selected from viral transfection (e.g., viral transduction), non-viral transfection, and / or a mixture of viral and non-viral transfection. In some embodiments, transfection is selected from electroporation, laser beam, gene injection, spinoculation, sonoporation, magnetofection, metal-coated nanoparticles, magnetically conjugated adeno-associated virus, particle / nanoparticle-mediated transfection, liposome transfection, lipid-based transfection, anionic liposomes, cationic liposome-mediated transfection, cationic polymers, polymer encapsulation, peptide-mediated transfection, calcium phosphate, dendrimers, flow transfection, photoporation, solvent poration, transient cell membrane disruption, deformation, extrusion, stretching, constriction, weakening, elongation, thinning, biolistic particle delivery systems, and combinations thereof.

[0207] In some embodiments of the above methods, cells are transduced by spinoculation. For example, transducing apheresis cell products with a viral vector includes subjecting the apheresis cell products and the viral vector to centrifugal force to enhance cellular uptake of viral particles, thereby increasing transduction efficiency.

[0208] In some embodiments, apheresis products (e.g., immune cell populations, CD4 + and CD8 + cell populations, or eukaryotic donor cell populations) are transfected by electroporation of viral particles (i.e., a mixture of viral and non-viral transfection). In some embodiments, apheresis products (e.g., immune cell populations, CD4 + and CD8 + cell populations, or eukaryotic donor cell populations) are transfected by electroporation and / or viral transfection (transduction). In some embodiments, apheresis products (e.g., immune cell populations, CD4 + and CD8 + cell populations, or eukaryotic donor cell populations) are transfected by viral transfection and / or lipid-based transfection. In some embodiments, apheresis products (e.g., immune cell populations, CD4 + and CD8 + cell populations, or eukaryotic donor cell populations) are transfected by viral transfection and liposome-based transfection.

[0209] In some embodiments, transfection of an apheresis product population (e.g., an immune cell population, a CD4 + and CD8 + cell population or a eukaryotic donor cell population) with one or more modifiers (e.g., nucleic acid sequences encoding chimeric antigen receptors (CARs), engineered T cell receptors (TCRs), and / or polypeptides that enhance immune cell function or functional derivatives thereof) occurs concurrently with stimulation and / or activation of the apheresis product population with one or more stimulants as described above (e.g., cytokines, recombinant costimulatory molecules, anti-CD3 antibodies or fragments thereof, anti-CD28 antibodies or fragments, small molecule inhibitors, and / or combinations thereof).

[0210] In some embodiments, transfection of an apheresis product population (e.g., an immune cell population, a CD4 + and CD8 + cell population or a eukaryotic donor cell population) with one or more modifiers (e.g., nucleic acid sequences encoding chimeric antigen receptors (CARs), engineered T cell receptors (TCRs), and / or polypeptides that enhance immune cell function or functional derivatives thereof) occurs no later than 1 hour, 1.5 hours, 2 hours, 2.5 hours, 3 hours, 3.5 hours, 4 hours, 4.5 hours, 5 hours, 5.5 hours, 6 hours, 6.5 hours, 7 hours, 7.5 hours, 8 hours, 8.5 hours, 9 hours, 9.5 hours, or 10 hours after the start of stimulation and / or activation of the apheresis product with one or more stimulants as described above.

[0211] In some embodiments, transfection of an apheresis product population (e.g., an immune cell population, a CD4 + and CD8 + cell population or a eukaryotic donor cell population) with one or more modifiers (e.g., nucleic acid sequences encoding chimeric antigen receptors (CARs), engineered T cell receptors (TCRs), and / or polypeptides that enhance immune cell function or functional derivatives thereof) occurs no later than 5 hours after the start of stimulation and / or activation of the apheresis product with one or more of the stimulants described above.

[0212] In some embodiments, transfection of an apheresis product population (e.g., an immune cell population, a CD4 + and CD8 + cell population or a eukaryotic donor cell population) with one or more modifiers (e.g., nucleic acid sequences encoding chimeric antigen receptors (CARs), engineered T cell receptors (TCRs), and / or polypeptides that enhance immune cell function or functional derivatives thereof) occurs no later than 4 hours after the start of stimulation and / or activation of the apheresis product with one or more of the stimulants described above.

[0213] In some embodiments, transfection of the apheresis product population (e.g., a population of immune cells, a population of CD4 + and CD8 + cells or a population of eukaryotic donor cells) with one or more modifiers (e.g., nucleic acid sequences encoding chimeric antigen receptors (CARs), engineered T cell receptors (TCRs), and / or polypeptides that enhance immune cell function or functional derivatives thereof) occurs no later than 3 hours after initiation of stimulation and / or activation of the apheresis product with one or more of the above stimulants.

[0214] In some embodiments, transfection of the apheresis product population (e.g., a population of immune cells, a population of CD4 + and CD8 + cells or a population of eukaryotic donor cells) with one or more modifiers (e.g., nucleic acid sequences encoding chimeric antigen receptors (CARs), engineered T cell receptors (TCRs), and / or polypeptides that enhance immune cell function or functional derivatives thereof) occurs no later than 2 hours after initiation of stimulation and / or activation of the apheresis product with one or more of the above stimulants.

[0215] In some embodiments, transfection of the apheresis product population (e.g., a population of immune cells, a population of CD4 + and CD8 + cells or a population of eukaryotic donor cells) with one or more modifiers (e.g., nucleic acid sequences encoding chimeric antigen receptors (CARs), engineered T cell receptors (TCRs), and / or polypeptides that enhance immune cell function or functional derivatives thereof) occurs no later than 1 hour after initiation of stimulation and / or activation of the apheresis product with one or more of the above stimulants.

[0216] In some embodiments, transfection of the apheresis product population (e.g., a population of immune cells, a population of CD4 + and CD8 + cells or a population of eukaryotic donor cells) with one or more modifiers (e.g., nucleic acid sequences encoding chimeric antigen receptors (CARs), engineered T cell receptors (TCRs), and / or polypeptides that enhance immune cell function or functional derivatives thereof) occurs without stimulation and / or activation of the apheresis product with one or more of the above stimulants.

[0217] In some embodiments, the transfected apheresis product (e.g., a population of immune cells or a population of eukaryotic donor cells) is selected from mononuclear cells, lymphocyte-rich cells, B lymphocytes, T lymphocytes, CD4 + T lymphocytes, CD8 + T lymphocytes, dendritic cells, monocytes, natural killer (NK) cells, natural killer T (NKT) cells, regulatory T cells, CD4 + helper T cells, CD8 +Cytotoxic T lymphocytes (CTL), CD62L + cells, CD27 + cells, CCR7 + cells, CD45RO - cells, CD45RA + cells, neutrophils, basophils, eosinophils, megakaryocytes, stem cells, hematopoietic stem cells (HSC), hematopoietic progenitor cells (HPC), CD34 + cells, CD34 + Peripheral blood stem cells, lymphokine-activated killer cells (LAK), tumor-infiltrating lymphocytes (TIL), mesenchymal stem cells, mast cells, monocytes, macrophages, neutrophils, basophils, eosinophils, dendritic cells, megakaryocytes, and combinations thereof.

[0218] In some embodiments, the concentration of the transfected apheresis product (e.g., a population of immune cells, CD4 + and CD8 + cell population or a population of eukaryotic donor cells) can be at least about 0.7×10 7 at least about 0.8×10 7 at least about 0.9×10 7 at least about 1×10 7 at least about 2×10 7 at least about 4×10 7 at least about 6×10 7 at least about 8×10 7 at least about 1×10 8 or at least about 5×10 8 cells / mL. In some embodiments, the concentration of the apheresis product (e.g., a population of immune cells, CD4 + and CD8 + cell population or a population of eukaryotic donor cells) can be from about 0.5×10 6 cells / mL to about 4×10 6 cells / mL. The concentration of the apheresis product (e.g., a population of immune cells, CD4 + and CD8 + cell population or a population of eukaryotic donor cells) can also be from about 0.5×10 6 cells / mL to about 1×10 8 cells / mL. In some embodiments, the concentration of the apheresis product can also be from about 4.0×10 6 cells / mL to about 1×10 8 cells / mL.

[0219] In some embodiments of the methods disclosed herein, the methods further comprise adding an adjuvant or a transfection enhancer reagent to the cell culture medium to increase transfection (e.g., transduction) efficiency. In some embodiments, the adjuvant or transduction enhancer reagent comprises a cationic polymer. In some embodiments, the adjuvant or transduction enhancer reagent is selected from: LentiBOOST TM (Sirion Biotech), vectofusin-1, F108 (Poloxamer 338 or F-38), protamine sulfate, heparin sulfate (polybrene), PEA, Pluronic F68, Pluronic F127, Poloxamer (Synperonic) or LentiTrans TM . In some embodiments, the transduction enhancer reagent is LentiBOOST TM (Sirion Biotech). In some embodiments, the transduction enhancer reagent is F108 (Poloxamer 338 or F-38).

[0220] The manufacturing processes (e.g., electroporating CAR T cells) disclosed herein are made possible by a new strategy of transducing immune cells with lentiviral vectors that contain nucleic acids encoding CARs, TCRs, and / or polypeptides or functional derivatives thereof that enhance the function of immune cells.

[0221] The CAR T cell manufacturing method relies on a hybrid transfection method that combines biological transfection (virus-based transduction) and physical transfection (such as electroporation) as described in Example 1 below. In particular, lentiviral particles are electroporated into immune cells or T cells. Electroporation of lentiviral particles into cells accelerates viral transfection / transduction and allows for the manufacture of CAR T cells (e.g., electroporated CAR T cells) in 1 day without post-transfection culture and / or expansion. As Figures 5 - 8 and Figure 10 shown, electroporated CAR T cells can be harvested within a few hours. As further discussed in Examples 3 and 4, and as Figure 11 and shown in Tables 4 - 6, such CAR T cells (e.g., electroporated CAR T cells) efficiently kill target cells.

[0222] Thus, in some embodiments of the manufacturing processes disclosed herein, the apheresis product (e.g., a population of immune cells, CD4 + and CD8 +a population of cells or a population of eukaryotic donor cells). The lentiviral vector or retroviral vector may comprise a nucleic acid sequence encoding a chimeric antigen receptor (CAR), an engineered T cell receptor (TCR), and / or a polypeptide or a functional derivative thereof that enhances the function of immune cells.

[0223] In some embodiments, the manufacturing process envisioned herein includes transfecting an enriched apheresis product with an effective dose of a lentiviral vector or a retroviral vector, the effective dose of the lentiviral vector or retroviral vector comprising a multiplicity of infection (MOI) of about 0.01, about 0.02, about 0.03, about 0.04, about 0.05, about 0.06, about 0.07, about 0.08, about 0.09, about 0.1, about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1.0, about 1.25, about 1.5, about 2.0, about 3.0, about 4.0, or about 5. In some embodiments, the manufacturing process envisioned herein includes transfecting an enriched apheresis product with a lentiviral vector or a retroviral vector at an MOI of about 10 or 20. In a preferred embodiment, the manufacturing process envisioned herein includes transfecting an enriched apheresis product with a lentiviral vector or a retroviral vector at an MOI of about 0.08, 0.2, or 0.4.

[0224] In some embodiments, the manufacturing process envisioned herein includes transfecting an enriched apheresis product with about 0.5, about 1, about 1.5, about 2, about 2.5, about 3, about 3.5, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 15, or about 20 μl of a lentiviral vector at a multiplicity of infection (MOI) of 0.01 to about 20.0.

[0225] In some embodiments, the manufacturing process envisioned herein includes transfecting an enriched apheresis product with 2 μl of a lentiviral vector or a retroviral vector at an MOI of about 0.08, 5 μl of a lentiviral vector or a retroviral vector at an MOI of about 0.2, or 10 μl of a lentiviral vector or a retroviral vector at an MOI of about 0.4.

[0226] Lentiviral vectors can be based on viruses selected from the following: retroviruses, alpharetroviruses, betaretroviruses, gammaretroviruses, deltaretroviruses, and epsilonretroviruses. For example, lentiviral vectors can be based on human immunodeficiency virus (HIV), equine infectious anemia virus (EIAV), visna-maedi virus (VMV), caprine arthritis-encephalitis virus (CAEV), feline immunodeficiency virus (FIV), bovine immunodeficiency virus (BIV), visna virus, and simian immunodeficiency virus (SIV). In some embodiments, lentiviral vectors can be pseudotyped with envelope glycoproteins (Env) from viruses selected from the following: murine leukemia virus (MLV), vesicular stomatitis virus (VSV) Indiana strain, VSV New Jersey strain, Kokobera virus, Kindred virus, Briese virus, spring viremia of carp virus (SVCV), sigma virus, infectious hematopoietic necrosis virus (IHNV), Mokola virus, rabies virus CVS virus, Isfahan virus, Araguari virus, Calchaqui virus, Jurona virus, La Jolla virus, Maraba virus, feline endogenous retrovirus (RD114) envelope protein, Perinet virus, Yougo Bogdanovac virus, prototype foamy virus (PFV), and gibbon ape leukemia virus (GaLV). In some embodiments, lentiviral vectors can be pseudotyped with envelope glycoproteins (Env) selected from the following: VSV Indiana strain, VSV New Jersey strain, and Kokobera virus.

[0227] In some embodiments, the manufacturing processes contemplated herein include transfecting enriched apheresis products with 2 μl of lentiviral vector or retroviral vector at an MOI of about 0.08, 5 μl of lentiviral vector or retroviral vector at an MOI of about 0.2, or 10 μl of lentiviral vector or retroviral vector at an MOI of about 0.4.

[0228] In some embodiments, the lentiviral vector comprises a heterologous viral envelope protein (Env) selected from the following: VSV-G of the Indiana strain, VSV-G of the New Jersey strain, Kokobera virus envelope protein, Isfahan virus envelope protein, Kindred virus envelope protein, Briese virus envelope protein, murine leukemia virus (MLV) envelope glycoprotein, SVCV virus envelope protein, and variants thereof.

[0229] In some embodiments, the lentiviral vector comprises a nucleotide sequence encoding a VSV-G envelope protein or a variant of the VSV G protein.

[0230] In some embodiments of the manufacturing processes disclosed herein, apheresis products (e.g., populations of immune cells, CD4 + and CD8 +a cell population or a eukaryotic donor cell population). E. Ex vivo culture after transfection: activation and stimulation

[0231] In another aspect, the methods disclosed herein further comprise the step of stimulating and activating the transfected cell population (e.g., a modified lymphocyte population, a modified immune cell population, a modified CD4 + and CD8 + cell population or a modified eukaryotic donor cell population) with one or more stimulants to produce an activated cell population (e.g., an activated modified immune cell population, an activated modified CD4 + and CD8 + cell population or an activated modified eukaryotic cell population).

[0232] In yet another aspect, the methods disclosed herein further comprise the step of culturing and / or expanding an activated modified immune cell population, an activated modified mononuclear cell population, an activated modified CD4 + and CD8 + cell population or an activated modified eukaryotic donor cell population for a predetermined time to produce an engineered cell population or an engineered CD4 + and CD8 + cell population.

[0233] In some embodiments, the expansion step is carried out under shaking conditions or rotational conditions. In some embodiments, the expansion step is carried out in a closed system. In some embodiments, the expansion step is carried out using a serum-free medium and / or in the presence of one or more stimulants described herein. In some embodiments, the expansion step is carried out in the presence of one or more stimulants described herein.

[0234] In some embodiments, the activated apheresis product population (e.g., an activated modified immune cell population, an activated modified CD4 + and CD8 + cell population and / or an activated modified eukaryotic donor cell population) is expanded by at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold or at least about 25-fold compared to the cell population before or immediately after transfection.

[0235] In some embodiments, the cell population is expanded by no more than about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, or about 60% compared to the cell population before transfection or immediately after transfection, e.g., as assessed by the number of live cells. In some embodiments, the cell population is expanded by no more than about 5%, no more than about 10%, no more than about 15%, no more than about 20%, no more than about 25%, no more than about 30%, no more than about 35%, or no more than about 40% compared to the cell population before transfection or immediately after transfection, e.g., as assessed by the number of live cells.

[0236] In some embodiments, the cell population is expanded for no more than about 1, about 1.5, about 2, about 2.5, about 3, about 3.5, about 4, about 4.5, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 16, about 20, about 24, about 36, about 48, about 55, about 60, about 65, about 70, about 72, about 80, about 90, or about 96 hours, as assessed by the number of live cells.

[0237] In some embodiments, during the culturing and expansion steps, the transfected cell population is contacted in vitro with an agent that stimulates the CD3 / TCR complex (e.g., an anti-CD3 antibody) and / or an agent that stimulates a co-stimulatory molecule and / or a growth factor receptor on the cell surface (e.g., an anti-CD28 antibody). In some embodiments, the transfected cell population is stimulated throughout the expansion. In some embodiments, the transfected cell population is stimulated for at least about 20 hours, at least about 21 hours, at least about 22 hours, at least about 23 hours, at least about 24 hours, at least about 25 hours, at least about 26 hours, at least about 27 hours, or at least about 28 hours. In some embodiments, the transfected cell population is cultured and expanded in a medium containing no more than about 0%, about 0.5%, about 1%, about 1.5%, 2%, about 2.5%, about 3%, about 3.5%, about 4%, about 4.5%, about 5%, about 5.5%, about 6%, about 6.5%, about 7%, 7.5%, or 8% serum. In some embodiments, the cytokine processes provided herein are performed in a cell culture medium containing an LSD1 inhibitor, a MALT1 inhibitor, or a combination thereof.

[0238] In some embodiments, a cell population (i.e., apheresis product) made by the methods disclosed herein shows a higher percentage of naive immune cells among the CAR-expressing cells. For example, compared to cells prepared by other conventional CAR T cell manufacturing methods, the percentage of naive immune cells among the CAR-expressing cells can be at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 11%, at least about 12%, at least about 13%, at least about 14%, at least about 15%, at least about 16%, at least about 17%, at least about 18%, at least about 19%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, or at least about 60% higher.

[0239] In some embodiments of the methods disclosed herein, after transfection, the transfected population (e.g., a population of modified lymphocytes, a population of modified immune cells, a population of modified CD4 + and CD8 + cell population, or a population of modified eukaryotic donor cells) is not further cultured. In some embodiments, after transfection, the transfected cell population (e.g., a population of modified immune cells, a population of modified CD4 + and CD8 + cell population, or a population of modified eukaryotic donor cells) is not activated with one or more stimulants. In this embodiment, after transfection, the transfected cell population (e.g., a population of modified immune cells, a population of modified CD4 + and CD8 + cell population, or a population of modified eukaryotic donor cells) is also not ex vivo expanded. In this embodiment, the transfected cell population is harvested within 24 hours of transfection. In this embodiment, prior to the transfection step (c), before transfection, the enriched cell population (e.g., a population of immune cells, CD4 + and CD8 + cell population, or a population of eukaryotic donor cells) can be stimulated and / or activated with one or more stimulants. F. Harvest

[0240] In another aspect, the methods disclosed herein further comprise the step of: harvesting a population of modified apheresis products (e.g., a population of modified lymphocytes, a population of modified immune cells, a population of modified CD4 + and CD8 + cell population, or a population of modified eukaryotic donor cells) for cryopreservation or administration.

[0241] In some embodiments, harvesting comprises selecting and enriching engineered lymphocytes, engineered immune cells, engineered CD4 + and CD8 +Cells or engineered donor eukaryotic cells. In some embodiments, harvesting further comprises formulating engineered lymphocytes, engineered immune cells, engineered CD4 + and CD8 + cells or engineered donor eukaryotic cells for cryopreservation or administration to a subject in need.

[0242] In some embodiments, when the transfected apheresis product (e.g., a population of modified lymphocytes, a population of modified immune cells, a population of modified CD4 + and CD8 + cells or a population of modified eukaryotic donor cells) is further cultured and expanded ex vivo, the transfected apheresis product (e.g., a population of modified lymphocytes, a population of modified immune cells, a population of modified CD4 + and CD8 + cells or a population of engineered eukaryotic donor cells) can be cultured for a predetermined time before harvesting the engineered desired cell population (e.g., a population of engineered lymphocytes, a population of engineered immune cells, a population of engineered CD4 + and CD8 + cells or a population of engineered eukaryotic donor cells).

[0243] In some embodiments, the predetermined time for expansion can be less than or equal to about 24 hours; less than or equal to about 30 hours; less than or equal to about 48 hours; less than or equal to about 72 hours; less than or equal to about 96 hours; or less than or equal to about 120 hours. In some embodiments, the predetermined time for expansion can be less than about 0.5 hours, less than about 1 hour, less than about 2 hours, less than about 3 hours, less than about 4 hours, less than about 5 hours, less than about 6 hours, less than about 7 hours, less than about 8 hours, less than about 9 hours, less than about 10 hours, less than about 11 hours, less than about 12 hours, less than about 13 hours, less than about 14 hours, less than about 15 hours, less than about 16 hours, less than about 17 hours, less than about 18 hours, less than about 19 hours, less than about 20 hours, less than about 21 hours, less than about 22 hours or less than about 23 hours.

[0244] In some embodiments, the predetermined time for expansion can be about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days or more days.

[0245] In some embodiments of the methods disclosed herein for manufacturing a population of engineered immune cells, from enriching and / or obtaining an apheresis product (e.g., a population of immune cells, CD4 + and CD8 +a population of cells or a population of eukaryotic donor cells) to harvesting the engineered cells (e.g., engineered lymphocytes, engineered immune cells, engineered CD4 + and CD8 + cells or engineered eukaryotic donor cells) can be about 12 hours or less, about 18 hours or less, about 20 hours or less, about 22 hours or less, about 24 hours or less, about 26 hours or less, about 28 hours or less, about 30 hours or less, about 32 hours or less, about 36 hours or less, about 40 hours or less, about 45 hours or less, about 48 hours or less, about 50 hours or less, about 55 hours or less, about 60 hours or less, about 65 hours or less, about 70 hours or less or about 72 hours or less.

[0246] In some embodiments, the time from enriching and / or obtaining an apheresis product (e.g., a population of immune cells, CD4 + and CD8 + cells or a population of eukaryotic donor cells) to harvesting the engineered cells (e.g., engineered lymphocytes, engineered immune cells, engineered CD4 + and CD8 + cells or engineered eukaryotic donor cells) is from about 18 hours to about 72 hours, from about 18 hours to about 36 hours, from about 18 hours to about 24 hours, from about 24 hours to about 72 hours, from about 24 hours to about 36 hours or from about 36 hours to about 72 hours.

[0247] In some embodiments, the time from enriching and / or obtaining an apheresis product (e.g., a population of immune cells, CD4 + and CD8 + cells or a population of eukaryotic donor cells) to harvesting the engineered cells (e.g., engineered lymphocytes, engineered immune cells, engineered CD4 + and CD8 +The time from enrichment and / or acquisition of the apheresis product (e.g., immune cell population, CD4

[0248] and CD8 + cell population or eukaryotic donor cell population) to harvesting of the engineered cells (e.g., engineered lymphocytes, engineered immune cells, engineered CD4 + and CD8 + cells or engineered eukaryotic donor cells) can be less than about 2 hours, less than about 3 hours, less than about 4 hours, less than about 5 hours, less than about 6 hours, less than about 7 hours, less than about 8 hours, less than about 9 hours, less than about 10 hours, less than about 11 hours, less than about 12 hours, less than about 13 hours, less than about 14 hours, less than about 15 hours, less than about 16 hours, less than about 17 hours, less than about 18 hours, less than about 19 hours, less than about 20 hours, less than about 21 hours, less than about 22 hours, less than about 23 hours, less than about 24 hours, less than about 30 hours, less than about 35 hours, less than about 40 hours, less than about 45 hours, less than about 50 hours, less than about 55 hours, less than about 60 hours, less than about 65 hours, less than about 70 hours or less than about 72 hours. + In some embodiments, the time from enrichment and / or acquisition of the apheresis product (e.g., immune cell population, CD4 + and CD8 + cell population or eukaryotic donor cell population) to harvesting of the engineered cells (e.g., engineered lymphocytes, engineered immune cells, engineered CD4 + and CD8 + cells or engineered eukaryotic donor cells) is about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days or more days. In some embodiments, the time from enrichment and / or acquisition of the apheresis product (e.g., immune cell population, CD4

[0249] and CD8 In some embodiments, the electroporation step, activation step, and / or amplification step are performed in a closed system, semi-closed, and / or functionally closed system. The manufacturing processes disclosed herein can occur in a closed system where the possibility of contamination is minimal due to limited manual manipulation. Thus, a closed system can minimize the risk of contamination (e.g., environmental contamination). In some embodiments, T cell isolation, activation, transduction, incubation, and washing are all performed in a closed system. In some embodiments of the methods disclosed herein, the method is performed in separate devices. In some embodiments, T cell isolation, activation, transduction, incubation, and washing are performed in separate devices. In some embodiments, the closed system is selected from a closed bag system, an automated closed cell sample processing system, and a bioreactor (e.g., such as XuriTM Cell expansion system W25 - Girgin Ltd (or any WAVE Bioreactor of GE Healthcare TM technology).

[0250] In certain embodiments, the closed system is a closed - bag culture system using any suitable cell culture bag (e.g., Mitenyi Biotec GMP cell differentiation bag, Origen Biomedical PermaLife TM cell culture bag, or Origen PermaLife TM PL240 bag). In some embodiments, the cell culture bag in the closed - bag culture system is coated with recombinant human fibronectin during the transduction step. In certain embodiments, the cell culture bag in the closed - bag culture system is coated with recombinant human fibronectin fragments during the transduction step. The recombinant human fibronectin fragments can comprise three functional domains: a central cell - binding domain, a heparin - binding domain II, and a CS1 sequence. Recombinant human fibronectin or its fragments can be used to increase the gene efficiency of retroviral transduction of immune cells by helping the co - localization of target cells and viral vectors. In certain embodiments, the recombinant human fibronectin fragment is (Takara Bio, Japan). In certain embodiments, the cell culture bag can be coated with recombinant human fibronectin fragments at a concentration of about 1 - 60 μg / mL, preferably 1 - 40 μg / mL. In certain embodiments, the cell culture bag can be coated with recombinant human fibronectin fragments at a concentration of about 1 - 20 μg / mL, 20 - 40 μg / mL, or 40 - 60 μg / mL.

[0251] In some embodiments of the methods disclosed herein, enriched apheresis products (e.g., T cells) are stimulated and / or activated and transfected in a cell culture flask that contains an air - permeable membrane at the bottom to support a large volume of medium with substantially no impairment of gas exchange. In some embodiments, cell growth is achieved by providing access to nutrients (substantially uninterrupted access) via convection. III. Lentiviral vectors

[0252] One aspect of the present disclosure provides the lentiviral vectors described herein.

[0253] Another aspect of the present disclosure provides a lentiviral vector comprising a polynucleotide sequence encoding at least one heterologous viral envelope protein derived from a virus; a polynucleotide sequence encoding at least one viral rev protein; a polynucleotide sequence encoding at least one viral gag protein and at least one viral pol protein; and / or a polynucleotide sequence encoding a chimeric antigen receptor or an engineered T cell receptor (TCR).

[0254] The lentiviral vector can be based on a virus selected from the group consisting of retroviruses, alpharetroviruses, betaretroviruses, gammaretroviruses, deltaretroviruses, and epsilonretroviruses. For example, the lentiviral vector can be based on human immunodeficiency virus (HIV), equine infectious anemia virus (EIAV), visna-maedi virus (VMV), caprine arthritis-encephalitis virus (CAEV), feline immunodeficiency virus (FIV), bovine immunodeficiency virus (BIV), visna virus, and simian immunodeficiency virus (SIV). In some embodiments, the lentiviral vector can be pseudotyped with an envelope glycoprotein (Env) from a virus selected from the group consisting of murine leukemia virus (MLV), vesicular stomatitis virus (VSV) Indiana strain, VSV New Jersey strain, Kokobera virus, Kindia G virus, Pollivirus, spring viremia of carp virus (SVCV), Sigma virus, infectious hematopoietic necrosis virus (IHNV), Mokola virus, rabies virus CVS virus, Isfahan virus, Araguari virus, Calchaqui virus, Jurona virus, La Jolla virus, Maraba virus, feline endogenous retrovirus (RD114) envelope protein, Perinet virus, Yougo Bogdanovac virus, prototype foamy virus (PFV), and gibbon ape leukemia virus (GaLV). In some embodiments, the lentiviral vector can be pseudotyped with an envelope glycoprotein (Env) selected from the group consisting of vesicular stomatitis virus (VSV) Indiana strain, VSV New Jersey strain, and Kokobera virus.

[0255] In some embodiments of the lentiviral vectors described herein, the viral envelope protein (Env) includes a VSV-G glycoprotein selected from the group consisting of VSV-G of the Indiana strain, VSV-G of the New Jersey strain, Kokobera virus envelope protein, Isfahan virus envelope protein, Kindia G virus envelope protein, Pollivirus envelope protein, murine leukemia virus (MLV) envelope glycoprotein, SVCV virus envelope protein, and variants thereof. The lentiviral vector can also comprise a nucleotide sequence encoding a heterologous VSV-G envelope protein.

[0256] Codon optimization can be performed for the expression of the heterologous VSV G envelope protein in humans. Alternatively, the heterologous VSV G envelope protein can be a VSV G protein variant.

[0257] In some embodiments, the lentiviral vector comprises a nucleotide sequence encoding a VSV-G envelope protein or a variant of the VSV G protein.

[0258] In some embodiments of the lentiviral vectors described herein, the heterologous envelope protein can be under the control of a transcriptional regulatory element. The transcriptional regulatory element can be a promoter selected from: a eukaryotic promoter or a constitutive promoter.

[0259] The lentiviral vectors described herein can further comprise a transcriptional regulatory element, and the transcriptional regulatory element can be upstream of the heterologous envelope glycoprotein (i.e., in the 5' direction of the nucleotide sequence encoding the heterologous envelope glycoprotein). For example, the transcriptional regulatory element can control the expression (i.e., transcription and optionally translation accordingly) of the nucleic acid encoding the heterologous envelope glycoprotein. In some embodiments, the transcriptional regulatory element is constitutively active, or is a constitutive promoter. In an exemplary embodiment, the constitutively active transcriptional regulatory element or constitutive promoter can be the cytomegalovirus (CMV) promoter (such as the CMV major immediate early promoter (CMVIE1)), murine stem cell virus promoter, elongation factor-1α promoter (EF-1α), viral simian virus 40 (SV40) promoter (e.g., early or late), Moloney murine leukemia virus (MoMLV) promoter, ubiquitin C promoter, phosphoglycerate kinase (PGK) promoter, Rous sarcoma virus (RSV) promoter, or herpes simplex virus (HSV) (thymidine kinase) promoter.

[0260] In other embodiments, the activity of the transcriptional regulatory element can be inducible, or the promoter can be an inducible promoter. In some embodiments, the transcriptional regulatory element can be a eukaryotic promoter, such as the phosphoglycerate kinase promoter. Other transcriptional regulatory elements (including prokaryotic and eukaryotic, constitutive and inducible promoters, and origins of replication) can be found in, for example, MOLECULAR CLONING: A LABORATORY MANUAL (edited by Joseph F. Sambrook and David W. Russell; 3rd edition; volumes 1, 2, and 3; Cold Spring Harbor Laboratory Press; 2001) and MOLECULAR CLONING: A LABORATORY MANUAL (edited by Michael R. Green and Joseph F. Sambrook; 4th edition; volumes 1, 2, and 3; Cold Spring Harbor Laboratory Press; 2012).

[0261] In some embodiments, the lentiviral vectors described herein can be constructed and arranged such that the expression of the proteins, enzymes, and viral elements necessary for the production of retroviral particles (i.e., cis - acting and trans - acting genes) is under the control of transcriptional regulatory elements. In a preferred embodiment, the lentiviral vector can further comprise a transcriptional regulatory element, and the transcriptional regulatory element is upstream (i.e., in the 5' direction) of the proteins, enzymes, and viral elements necessary for the production of retroviral particles (i.e., cis - acting and trans - acting genes), and optionally, the transcriptional regulatory element controls the expression (i.e., transcription or translation) of the nucleic acid encoding the proteins, enzymes, and viral elements necessary for the production of retroviral particles (i.e., cis - acting and trans - acting genes). In some embodiments, the transcriptional regulatory element can be constitutively active or can be a constitutive promoter.

[0262] In some embodiments, the lentiviral vectors described herein and the nucleic acids encoding heterologous envelope proteins can be amplified or produced before introduction into the producer cells and accordingly before the production of viral particles. In some embodiments, the lentiviral vector and the nucleic acids encoding other proteins, enzymes, and elements necessary for the production of retroviral particles can be amplified or produced before introduction into the producer cells and accordingly before the production of retroviral proteins.

[0263] In some embodiments, the lentiviral vector and the nucleic acid encoding a heterologous envelope protein can be constructed and arranged such that the transcriptional control element drives the transcription of the heterologous envelope protein in the producer cells and thus drives translation to facilitate the production of lentiviral particles. In some embodiments, the lentiviral vector and the nucleic acids encoding the proteins, enzymes, viral elements necessary for the production of retroviral particles (i.e., cis - acting and trans - acting genes, including rev and gag / pol) can be constructed and arranged such that the transcriptional control element can drive the transcription of the proteins, enzymes, viral elements (i.e., cis - acting and trans - acting genes, including rev and gag / pol) in the producer cells and thus drive translation such that the producer cells produce retroviral particles.

[0264] In some embodiments of the manufacturing processes disclosed herein, apheresis products (e.g., populations of immune cells, CD4 + and CD8 + cell populations or populations of eukaryotic donor cells) are transfected with an effective dose of the lentiviral vector or retroviral vector comprising a chimeric antigen receptor (CAR) or an engineered TCR. In some embodiments, the lentiviral vector is a lentiviral vector particle. A. Lentivirus

[0265] Vectors derived from retroviruses, such as lentiviruses, are suitable tools for achieving long-term gene transfer because they allow for the long-term stable integration of transgenes and their transmission in progeny cells. Lentiviral vectors have additional advantages over vectors derived from oncoretroviruses, such as murine leukemia virus, because they can transduce non-proliferating cells, such as hepatocytes. They also have the additional advantage of low immunogenicity. Retroviral vectors can also be, for example, gammaretroviral vectors. Gammaretroviral vectors can contain, for example, a promoter, a packaging signal (ψ), a primer binding site (PBS), one or more (e.g., two) long terminal repeats (LTRs), and a transgene of interest (e.g., a gene encoding a CAR). Gammaretroviral vectors can lack viral structural genes, such as gag, pol, and env. Exemplary gammaretroviral vectors include murine leukemia virus (MLV), spleen focus-forming virus (SFFV), and myeloproliferative sarcoma virus (MPSV), and vectors derived therefrom. Other gammaretroviral vectors are described, for example, in Tobias Maetzig et al., "Gammaretroviral Vectors: Biology, Technology and Application" Viruses. June 2011; 3(6):677-713.

[0266] Retroviruses provide a convenient platform for gene delivery systems. The selected gene can be inserted into the vector and packaged into retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to the cells of a subject in vivo or ex vivo. A variety of retroviral systems are known in the art. In some embodiments, adenoviral vectors are used. A variety of adenoviral vectors are known in the art. In one embodiment, lentiviral vectors are used.

[0267] In some embodiments, the lentiviral vector is based on a virus selected from the group consisting of retroviral vectors, alpharetroviral vectors, betaretroviral vectors, gammaretroviral vectors, deltaretroviral vectors, and epsilonretroviral vectors. In some embodiments, the lentiviral vector is based on human immunodeficiency virus (HIV), equine infectious anemia virus (EIAV), visna-maedi virus (VMV), caprine arthritis-encephalitis virus (CAEV), feline immunodeficiency virus (FIV), bovine immunodeficiency virus (BIV), visna virus, and simian immunodeficiency virus (SIV). In one embodiment, the viral vector is derived from EIAV. EIAV has the simplest genomic structure among lentiviruses.

[0268] The feline immunodeficiency virus (FIV) RNA encapsidation determinants have been shown to be discrete and discontinuous, comprising a region (R-U5) at the 5' end of the genomic mRNA and another region within the 311 nt proximal to gag.

[0269] In some embodiments of the manufacturing process disclosed herein, the lentiviral vector comprises a heterologous viral envelope protein (Env) selected from: VSV-G of the Indiana strain, VSV-G of the New Jersey strain, Cocal vesiculovirus envelope protein, Isfahan virus envelope protein, Jindivirus envelope protein, Bohlavirus envelope protein, murine leukemia virus (MLV) envelope glycoprotein, SVCV virus envelope protein, and variants thereof. B. Pseudotyped lentiviral vectors

[0270] The viral envelope protein (env) determines the range of host cells that can ultimately be infected and transformed by the recombinant retrovirus produced from the cell line. In the case of lentiviruses (such as FflV-1, FflV-2, SIV, FIV, and EIV), the env protein comprises gp41 and gp120. Preferably, the viral env protein expressed by the packaging cells of the present disclosure is encoded on a vector separate from the viral gag and pol genes.

[0271] Examples of retrovirus-derived env genes that can be used in the present disclosure include, but are not limited to, MLV envelope, 10A1 envelope, BAEV, FeLV-B, RD114, SSAV, Ebola virus, Sendai virus, FPV (fowl plague virus), and influenza virus envelope. In some embodiments, the retrovirus-derived env gene is selected from the genes encoding envelope proteins from RNA viruses selected from: Picornaviridae, Caliciviridae, Astroviridae, Togaviridae, Flaviviridae, Coronaviridae, Paramyxoviridae, Rhabdoviridae, Filoviridae, Orthomyxoviridae, Bunyaviridae, Arenaviridae, Reoviridae, Birnaviridae, Retroviridae. In some embodiments, the retrovirus-derived env gene is selected from the genes encoding envelope proteins from DNA viruses selected from: Hepadnaviridae, Circoviridae, Parvoviridae, Papovaviridae, Adenoviridae, Herpesviridae, Poxviridae, and Iridoviridae.

[0272] In some embodiments, the retrovirus-derived env gene is selected from alfalfa mosaic virus (AMV), human papillomavirus (HPV), white spot syndrome virus (WDSV), Semliki Forest virus (SFV), rabies virus, avian leukosis virus (ALV), bovine immunodeficiency virus (BIV), bovine leukemia virus (BLV), Epstein-Barr virus (EBV), squirrel monkey retrovirus (SMRV), equine infectious anemia virus (EIAV), feline leukemia virus (FeLV), caprine arthritis-encephalitis virus (CAEV), Sin Nombre virus (SNV), human T-lymphotropic virus (HTLV), simian T cell leukemia virus (STLV), Venezuelan equine encephalitis virus (VEEV), Mason-Pfizer monkey virus (M-PMV), avian carcinoma virus MH2, avian encephalomyelitis virus (AEV), V-crk sarcoma virus CT10, and respiratory syncytial virus (RSV).

[0273] In some embodiments, the envelope proteins for pseudotyping the lentiviruses of the present disclosure include, but are not limited to, any of the following viruses: influenza A virus (such as H1N1, H1N2, H3N2, and H5N1 (avian influenza)), influenza B virus, influenza C virus, hepatitis A virus, hepatitis B virus, hepatitis C virus, hepatitis D virus, hepatitis E virus, rotavirus, any virus of the Norwalk virus group, enteric adenovirus, parvovirus, dengue virus, monkeypox virus, Mononegavirales, Lyssavirus (such as rabies virus, Lagos bat virus, Mokola virus, Duvenhage virus, European bat virus 1 and 2, and Australian bat virus), Ephemerovirus, vesiculovirus, vesicular stomatitis virus (VSV), herpesvirus (such as herpes simplex virus type 1 and 2, varicella-zoster virus, cytomegalovirus, Epstein-Barr virus (EBV), human herpesvirus (HHV), human herpesvirus 6 and 8), human immunodeficiency virus (HIV), papillomavirus, murine gammaherpesvirus, arenavirus (such as Junín virus, Machupo virus, Lassa fever virus, lymphocytic choriomeningitis virus (LCMV)), Bunyaviridae (such as Crimean-Congo hemorrhagic fever virus, hantavirus, virus causing hemorrhagic fever with renal syndrome, Rift Valley fever virus), Filoviridae (filovirus) (including Ebola hemorrhagic fever virus and Marburg hemorrhagic fever virus), Flaviviridae (including Kyasanur Forest disease virus, Omsk hemorrhagic fever virus, virus causing tick-borne encephalitis), and Paramyxoviridae (such as Hendra virus and Nipah virus), variola major and variola minor (smallpox) virus, alphavirus (such as Venezuelan equine encephalitis virus, eastern equine encephalitis virus, western equine encephalitis virus), SARS-associated coronavirus (SARS-CoV), West Nile virus, any virus causing encephalitis.

[0274] In some embodiments, the lentiviral vector can be pseudotyped with any selected molecule. In some embodiments, the lentiviral vector of the present disclosure is pseudotyped with an envelope glycoprotein (Env) selected from: murine leukemia virus (MLV), a chimeric envelope glycoprotein variant derived from MLV, vesicular stomatitis virus G glycoprotein (VSV-G), a modified envelope of prototype foamy virus (PFV), and a chimeric envelope glycoprotein variant derived from gibbon ape leukemia virus (GaLV).

[0275] In some embodiments, the lentiviral vector is pseudotyped with an envelope glycoprotein (Env) selected from the group consisting of murine leukemia virus (MLV), vesicular stomatitis virus (VSV) Indiana strain, VSV New Jersey strain, Kokobera virus, Kindunya virus, Palyam virus, spring viremia of carp virus (SVCV), sigma virus, infectious hematopoietic necrosis virus (IHNV), Mokola virus, rabies virus CVS virus, Isfahan virus, Araguari virus, Calchaqui virus, Jurona virus, La Jolla virus, Malabar virus, Perinet virus, Yougga Bogdanovac virus, prototype foamy virus (PFV), and gibbon ape leukemia virus (GaLV).

[0276] In some embodiments, the Env protein can be a modified Env protein, such as a mutant or engineered Env protein. Modifications can be made or selected to introduce targeting capabilities or reduce toxicity or for another purpose. The Env protein can be a modified Env protein, such as a mutant or engineered Env protein. Modifications can be made or selected to introduce targeting capabilities or reduce toxicity or for another purpose. 1. VSV-G

[0277] The envelope glycoprotein (G) of the rhabdovirus vesicular stomatitis virus (VSV) is an envelope protein that has been shown to be able to pseudotype certain enveloped viruses and viral vector virions. The ability of VSV-G to pseudotype MoMLV-based retroviral vectors in the absence of any retroviral envelope protein is known in the art. Any retroviral vector can be successfully pseudotyped with VSV-G. These VSV-G pseudotyped vectors can be used to transduce a wide range of mammalian cells. Non-infectious retroviral particles can be made infectious by the addition of VSV-G. VSV-G pseudotyped vectors have been shown to infect not only mammalian cells but also cell lines derived from fish, reptiles, and insects. The VSV-G protein can be used to pseudotype certain retroviruses because its cytoplasmic tail can interact with the retroviral core.

[0278] The advantage of providing a non-retroviral pseudotyped envelope (such as the VSV-G protein) is that vector particles can be concentrated to high titers without loss of infectivity. In contrast, the VSV glycoprotein consists of a single unit. Pseudotyping with the VSV-G protein offers potential advantages both for efficient target cell infection / transduction and during production because the VSV glycoprotein consists of a single unit and can withstand the shear forces during ultracentrifugation. In contrast, retroviral envelope proteins apparently cannot withstand the shear forces during ultracentrifugation because they consist of two non-covalently linked subunits and the interaction between the subunits can be disrupted by centrifugation. WO 2000 / 52188 describes the generation of pseudotyped retroviral vectors from stable producer cell lines that have the vesicular stomatitis virus-G protein (VSV-G) as the membrane-associated viral envelope protein and provides the gene sequence of the VSV-G protein.

[0279] Pseudotyping can confer one or more advantages. For example, for lentiviral vectors, vectors based on the env gene product of HIV-I will restrict these vectors to infecting only cells that express a protein called CD4. However, if the env gene in these vectors has been replaced with an env sequence from another RNA virus, they can have a broader infection spectrum. 2. Cocal vesicular stomatitis virus envelope glycoprotein

[0280] Particles and the Cocal vesicular stomatitis virus envelope glycoprotein (Cocal-G) are less toxic to the cells that produce them (i.e., "producer cells") and have a higher transduction efficiency in the cells they infect (i.e., "target cells"). The Cocal vesicular stomatitis virus envelope glycoprotein has a higher titer of particles than compositions containing other viral particles. Thus, lentiviral vectors containing the Cocal vesicular stomatitis virus envelope glycoprotein can be produced at higher concentrations. In addition, when compared to envelope glycoproteins derived from non-Cocal vesicular stomatitis viruses, compositions containing the Cocal vesicular stomatitis virus envelope glycoprotein have a higher titer of mature and immature particles, a higher titer of infectious particles, and a higher titer of the genetic information carried within the particles (e.g., CAR). In some embodiments, the lentiviral vector contains a nucleotide sequence encoding the Cocal-G envelope protein. In some embodiments, the Cocal-G envelope protein is a Cocal-G protein variant. 3. Ross River virus

[0281] Ross River virus (RRV) is an endemic mosquito-borne alphavirus and is prevalent in tropical and temperate regions of Australia. Antibody rates in the normal population in temperate coastal areas tend to be low (6% to 15%), although seroprevalence in the Murray Valley water system plain reaches 27% to 37%. In 1979 to 1980, RRV was endemic in the Pacific Islands. The disease is not contagious between humans and is never fatal. The first symptom in about half of the patients is joint pain, accompanied by fatigue and lethargy (Fields Virology).

[0282] The Ross River virus envelope has been used to pseudotype non-primate lentiviral vectors (FIV), and after systemic administration, it mainly transduces the liver. It has been reported that the transduction efficiency of lentiviral vectors pseudotyped with the Ross River virus envelope is 20 times that obtained with vectors pseudotyped with VSV-G. In addition, lentiviral vectors pseudotyped with the Ross River virus envelope cause less cytotoxicity, as measured by serum levels of liver enzymes that indicate hepatotoxicity. 4. Baculovirus GP64

[0283] For viral vectors in high-titer viruses required for large-scale production for clinical and commercial applications, the baculovirus GP64 protein has been shown to be an attractive alternative to VSVG. Compared with VSVG, GP64 vectors have a similar broad tropism and a similar native titer. Since GP64 expression does not kill cells, 293T-based cell lines that constitutively express GP64 can be generated. In some embodiments, the lentiviral vector comprises a nucleotide sequence encoding the baculovirus GP64 protein. In some embodiments, the baculovirus GP64 protein is a variant baculovirus GP64 protein. 5. Other envelope glycoproteins

[0284] The lentiviral vectors of the present disclosure can be pseudotyped with at least a portion of the rabies virus G protein or its mutants, variants, homologs, or fragments. The teachings regarding the rabies virus G protein and its mutants can be found in WO 1999 / 61639; EP 0445625. Other envelopes that give reasonable titers when used to pseudotype EIAV include Mokola virus, rabies virus, Ebola virus, and LCMV (lymphocytic choriomeningitis virus). C. Lentiviral vectors

[0285] In some embodiments of the manufacturing processes, CAR T cell therapy methods, methods of introducing modifications into mononuclear cells, or lentiviral vectors of the present disclosure, the lentiviral vector is an infectious lentiviral vector or a lentiviral vector.

[0286] One aspect of the present disclosure provides a lentiviral vector comprising a polynucleotide sequence encoding at least one heterologous viral envelope protein derived from a virus; a polynucleotide sequence encoding at least one viral rev protein; a polynucleotide sequence encoding at least one viral gag protein and at least one viral pol protein; and a polynucleotide sequence encoding a chimeric antigen receptor or an engineered T cell receptor (TCR). In some embodiments, at least a portion of one or more regions essential for replication in the viral genome is mutated. In some embodiments, at least a portion of one or more regions essential for replication in the viral genome is selected from the rev gene, gag gene, pol gene, integrase gene, 5′ LTR, 3′ LTR, and combinations thereof. In some embodiments, the mutation is selected from a deletion, an insertion, or a substitution. 1. Non-replicating vector

[0287] In an exemplary retroviral vector of the present disclosure, at least a portion of one or more protein-coding regions essential for replication can be removed from the virus. For example, gag / pol and env can be absent or non-functional. This renders the viral vector replication-defective. A portion of the viral genome can also be replaced with a library encoding candidate nucleic acid binding sequences that are operably linked to regulatory control regions and a reporter gene in the vector genome to produce a vector containing the candidate nucleic acid binding sequences that is capable of transducing non-dividing target cells and / or integrating its genome into the host genome.

[0288] In the genome of a replication-defective lentiviral vector, the sequences of gag / pol and / or env can be mutated, absent, and / or non-functional. In a typical lentiviral vector, at least a portion of one or more coding regions of proteins essential for viral replication can be removed from the vector. This renders the viral vector replication-defective. A portion of the viral genome can also be replaced with a nucleotide of interest to produce a vector containing the nucleotide of interest that is capable of transducing non-dividing target cells and / or integrating its genome into the target cell genome.

[0289] In some embodiments, the lentiviral or retroviral vectors of the present disclosure are non-integrating vectors and / or non-replicating vectors. See, e.g., WO 2006 / 010834 and WO2007 / 071994. In one aspect of the present disclosure, the lentiviral or retroviral vectors of the present disclosure may be unable to autonomously replicate and specifically integrate in the transduced cells. In some embodiments, the lentiviral or retroviral vectors of the present disclosure comprise a recombinant genome that includes a lentiviral capsid psi sequence and an RNA nuclear export element, a transgene, and possibly a promoter and / or sequences that facilitate RNA nuclear import between the LTR 5′ and 3′ lentiviral sequences. In some embodiments, the lentiviral vector comprises a mutation in at least a portion of one or more regions necessary for replication in the viral genome. In this embodiment, the one or more regions are selected from the rev gene, gag gene, pol gene, integrase gene, 5′ LTR, 3′ LTR, and combinations thereof. The mutations are selected from deletions, insertions, or substitutions.

[0290] In some embodiments, the lentiviral or retroviral vector comprises and / or further comprises a mutated integrase that prevents integration of the retroviral or lentiviral genome into the host cell genome. In some embodiments, the lentiviral or retroviral vector comprises a modified pol sequence that produces a non-functional integrase.

[0291] In another embodiment, the lentiviral vector has the ability to deliver sequences that do not contain or lack viral RNA. In another embodiment, a heterologous binding domain (heterologous to gag) located on the RNA to be delivered and a homologous binding domain on Gag or Gag Pol can be used to ensure packaging of the RNA to be delivered. Both of these vectors are described in WO 2007 / 072056. In some embodiments, the recombinant retrovirus is replication-incompetent, meaning that the retrovirus cannot replicate once it leaves the packaging cell. 2. Self-inactivating vectors

[0292] In some embodiments, the lentiviral vector is a non-replicating, self-inactivating minimal lentiviral vector derived from human immunodeficiency virus (HIV) or equine infectious anemia virus (EIAV), which can be pseudotyped with an env selected from: VSV-G, Ebola virus, Flu-HA, Sendai virus envelope F or HN, baculovirus GP64, rabies virus G, Cocal virus envelope protein, or an alternative viral envelope protein.

[0293] One of ordinary skill in the art will understand how to modify the methods disclosed herein for use with different retroviruses. For example, in some embodiments, the HIV RRE and the polynucleotide region encoding HIV Rev can be replaced with an N-terminal RGG box RNA-binding motif and a polynucleotide region encoding ICP27. In some embodiments, the polynucleotide region encoding HIV Rev can be replaced with one or more polynucleotide regions encoding adenovirus E1B 55 kDa and E4 Orf6. In some embodiments, the recombinant retrovirus can be an adenovirus, an adeno-associated virus, a herpes virus, a cytomegalovirus, a poxvirus, a fowlpox virus, an influenza virus, a vesicular stomatitis virus (VSV), or a Sindbis virus.

[0294] In some embodiments, the retroviral vector or lentiviral vector disclosed herein is a self-inactivating vector. As used herein, the term "self-inactivating vector" refers to a vector in which the 3' LTR enhancer promoter region (U3 region) has been modified (e.g., by deletion or substitution). A self-inactivating vector can prevent viral transcription after the first round of viral replication. Thus, a self-inactivating vector can only be able to infect a host genome (e.g., a mammalian genome), then integrate into it once, and cannot be further transmitted. Thus, a self-inactivating vector can greatly reduce the risk of generating replication-competent virus.

[0295] In some embodiments, the viral particle can be self-inactivating. A self-inactivating viral particle can prevent viral transcription after the first round of viral replication. Thus, a self-inactivating particle can be able to infect a cell, and the genetic information therein can integrate into the host genome (e.g., a mammalian genome). This integration and transduction can only occur once and cannot be further transmitted. Thus, a self-inactivating particle can greatly reduce the risk of generating replication-competent virus.

[0296] A commonly used lentiviral vector system is the so-called third-generation self-inactivating system. The third-generation lentiviral vector system can comprise four plasmids. The "transfer plasmid" encodes the polynucleotide sequence that is delivered to the target cell by the lentiviral vector system. The transfer plasmid typically has one or more transgene sequences flanked by long terminal repeat (LTR) sequences, and the long terminal repeat sequences facilitate the integration of the transfer plasmid sequence into the host genome. For safety reasons, the transfer plasmid is typically designed such that the resulting vector is replication-incompetent. For example, the transfer plasmid lacks the gene elements necessary to produce infectious particles in the host cell. Additionally, the transfer plasmid can be designed to delete the 3' LTF, rendering the virus "self-inactivating" (SIN).

[0297] The third-generation systems generally also include two "packaging plasmids" and one "envelope plasmid". The "envelope plasmid" generally encodes an Env gene operably linked to a promoter. In at least one embodiment of the third-generation system, the Env gene is VSV-G, Ebola virus env, Flu-HA, Sendai virus envelope F, HN, baculovirus GP64, rabies virus G, Cocal virus envelope protein, or a derivative thereof (e.g., a variant as described herein), and the promoter is the CMV promoter. The third-generation system uses two packaging plasmids, one encoding Gag and Pol, and the other encoding Rev as another safety feature, an improvement over the single packaging plasmid of the so-called second-generation system. Although safer, the third-generation system may be more cumbersome to use and may result in lower virus titers due to the addition of an additional plasmid. Exemplary packaging plasmids include, but are not limited to, pMD2.G, pRSV-rev, pMDLG-pRRE, and pRRL-GOI.

[0298] In some embodiments, the lentiviral vector is a third-generation self-inactivating (SIN) vector and contains no viral proteins and is replication-incompetent. In this embodiment, cells that have been transduced and / or transfected with the vector do not produce infectious particles. 3. Regulatory elements

[0299] In some embodiments, the retroviral or lentiviral vectors described herein contain transcriptional regulatory elements. In some embodiments, the transcriptional regulatory element is a promoter selected from the group consisting of eukaryotic promoters or constitutive promoters. Physiological promoters (e.g., the EF-1α promoter) may be less likely to induce integration-mediated genotoxicity and may eliminate the ability of retroviral vectors to transform stem cells. Other physiological promoters suitable for retroviral or lentiviral vectors are known to those of skill in the art and may be incorporated into exemplary embodiments of the nucleic acid vector. In some embodiments, the promoter is the elongation factor-1-α promoter (EF-1α promoter). The use of the EF-1α promoter can increase the efficiency of downstream transgene (e.g., a nucleic acid sequence encoding a TCR and / or a CAR) expression.

[0300] In some embodiments, the lentiviral or retroviral vector further contains non-essential cis-acting sequences that can increase titer and gene expression. A non-limiting example of a non-essential cis-acting sequence is the central polypurine tract and central termination sequence (cPPT / CTS), which is important for efficient reverse transcription and nuclear import. Other non-essential cis-acting sequences are known to those of skill in the art and may be incorporated into lentiviral or retroviral vector particles.

[0301] In some embodiments, the lentiviral or retroviral vectors disclosed herein further comprise post-transcriptional regulatory elements. Post-transcriptional regulatory elements can improve RNA translation, enhance transgene expression, and stabilize RNA transcripts. An example of a post-transcriptional regulatory element is the woodchuck hepatitis virus post-transcriptional regulatory element (WPRE). Thus, in some embodiments, the nucleic acid vector further comprises a WPRE sequence. A variety of post-transcriptional regulatory elements are known to those skilled in the art and can be incorporated into lentiviral or retroviral vectors.

[0302] The lentiviral or retroviral vectors disclosed herein can further comprise additional elements such as the rev-responsive element (RRE) for RNA trafficking, packaging sequences, and 5' and 3' long terminal repeats (LTRs). The term "long terminal repeat" or "LTR" refers to a domain of base pairs located at the ends of retroviral DNA that contains U3, R, and U5 regions. LTRs generally provide functions required for retroviral gene expression (e.g., promotion, initiation, and polyadenylation of gene transcripts) and viral replication. In one embodiment, the lentiviral or retroviral vector comprises a 3'U3-deleted LTR, a non-functional LTR, and / or lacks a functional 3' or 5' LTR. Thus, the lentiviral or retroviral vectors disclosed herein can comprise any combination of the elements described herein to enhance the efficiency of functional transgene expression. For example, in addition to the nucleic acid encoding a TCR or CAR, the lentiviral or retroviral vector can further comprise a WPRE sequence, a cPPT sequence, an RRE sequence, a 5' LTR, a 3'U3-deleted LTR'. D. Lentivirus production

[0303] One aspect of the present disclosure provides a method for producing the lentiviral or retroviral vector particles described herein. Another aspect of the present disclosure provides a method for producing lentiviral or retroviral vector particles, the method comprising introducing the lentiviral or retroviral vector particles disclosed herein into a host cell.

[0304] The production of lentiviral vectors relies on the use of "packaging cell lines". Generally, a packaging cell line is a cell line that, when a transfer plasmid, one or more packaging plasmids, and an envelope plasmid are introduced into the cells, is capable of producing infectious lentiviral particles. Various methods for introducing plasmids into cells can be used, including transfection or electroporation. In some cases, the packaging cell line is suitable for efficiently packaging the lentiviral vector system into lentiviral particles. In one embodiment, the present disclosure provides a packaging cell for producing a recombinant retrovirus (e.g., lentivirus) pseudotyped with a VSV-G glycoprotein or a variant thereof as disclosed herein. To achieve a reasonable virus titer, it is generally necessary to produce virus particles on a large scale. Virus particles are produced by transfecting a transfer vector into a packaging cell line containing viral structural and / or accessory genes (e.g., gag, pol, env, tat, rev, vif, vpr, vpu, vpx, or nef genes or other retroviral genes). 1. Production of retroviral therapeutic vectors

[0305] In some embodiments, the retroviral vectors of the present disclosure can be produced by transiently transfecting HEK293T cells with four plasmids consisting of: (1) a recombinant retroviral vector genomic plasmid encoding one or more desired transgenes and a binding site capable of interacting with an RNA-binding protein; (2) a synthetic retroviral gag / pol expression plasmid; (3) an envelope (env) expression plasmid (e.g., VSV-G or a variant thereof); (4) an RNA-binding protein expression plasmid.

[0306] In some embodiments, the retroviral vectors of the present disclosure are HIV. In this embodiment, the retroviral vector can be produced by transiently transfecting HEK293T cells with five plasmids: (1) a recombinant HIV vector genomic plasmid encoding one or more desired transgenes, a binding site capable of interacting with an RNA-binding protein, and an RRE sequence; (2) a synthetic gag / pol expression plasmid; (3) an envelope (env) expression plasmid (e.g., VSV-G, Cochlear vesicular stomatitis virus, or a variant thereof); (4) an RNA-binding protein expression plasmid; (5) a REV expression plasmid.

[0307] In some embodiments, the retroviral vectors of the invention can be produced by using packaging cells that stably express: (1) gag / pol; (2) env (e.g., VSV-G or variants thereof); and (3) an RNA binding protein, and Rev for HIV vectors, and introducing a plasmid encoding the recombinant retroviral vector genome into such cells by transient transfection, the recombinant retroviral vector genome encoding one or more desired transgenes (e.g., CAR) and a binding site capable of interacting with the RNA binding protein, and for HIV vectors, containing the RRE sequence.

[0308] In some embodiments, the retroviral vectors of the present disclosure can be produced in producer cells that stably express: (1) gag / pol, (2) env (e.g., VSV-G or variants thereof), (3) an RNA binding protein, (4) a recombinant EIAV vector genome encoding one or more desired transgenes (e.g., CAR) and a binding site capable of interacting with the RNA binding protein.

[0309] In some embodiments, the lentiviral vector is an HIV lentiviral vector. In this embodiment, the HIV vector can be produced in producer cells that stably express: (1) gag / pol; (2) env (e.g., VSV-G or variants thereof); (3) an RNA binding protein; (4) a recombinant HIV vector genome encoding one or more desired transgenes (e.g., CAR), a binding site capable of interacting with the RNA binding protein, and the RRE sequence; and (5) REV. a. Codon optimization

[0310] In some embodiments, any polynucleotide used to produce lentiviral vectors in the present disclosure can be codon optimized. Different cells use specific codons differently. This codon bias corresponds to a bias in the relative abundance of specific tRNAs in the cell type. By changing the codons in the sequence so that they are tailored to match the relative abundance of the corresponding tRNAs, expression may be increased. For the same reason, expression may be decreased by deliberately selecting codons known to be rarely used by the corresponding tRNAs in a particular cell type. Thus, an additional degree of translational control is available.

[0311] Many viruses (including HIV and other lentiviruses) use a large number of rarely used codons, and by changing these codons to correspond to commonly used mammalian codons, increased expression of the gene of interest or packaging components in mammalian producer cells can be achieved. Codon usage tables for mammalian cells as well as for a variety of other organisms are known in the art.

[0312] Codon optimization of viral vector components has many other advantages. By altering its sequence, RNA instability sequences in the nucleotide sequence encoding the packaging components of viral particles required for assembling viral particles in producer cells / packaging cells are eliminated. At the same time, the amino acid coding sequences of the packaging components are retained, such that the viral components encoded by the sequences remain the same, or at least similar enough, such that the functions of the packaging components are not impaired. In lentiviral vectors, codon optimization also overcomes the need for Rev / RRE for export, rendering the optimized sequence Rev-independent. Codon optimization also reduces homologous recombination between different constructs within the vector system (e.g., between the overlapping regions of the gag-pol and env open reading frames). Thus, the overall effect of codon optimization is a significant increase in viral titer and an improvement in safety.

[0313] In one embodiment, only the codons associated with the instability sequences are codon-optimized. In a preferred embodiment, global codon optimization of the sequence is performed. In this embodiment, the sequence encompasses the frameshift site of gag-pol (see below). The gag-pol gene contains two overlapping reading frames encoding the gag-pol protein. The expression of both proteins depends on a frameshift during translation. This frameshift occurs as a result of the ribosome "slipping" during translation. This slippage is thought to be caused at least in part by ribosome-stalling RNA secondary structures. Such secondary structures are present downstream of the frameshift site in the gag-pol gene. For example, for the accommodation of convenient restriction sites, it can be derived from optimal codon usage, and conservative amino acid changes can be introduced into the Gag-Pol protein.

[0314] In one embodiment, the codon optimization is based on moderately expressed mammalian genes. Due to the degenerate nature of the genetic code, it will be understood that those skilled in the art can obtain many gag-pol sequences. Many retroviral variants are also described, which can be used as starting points for generating codon-optimized gag-pol sequences. The lentiviral genome can be quite variable. For example, there are many quasispecies of HIV-1 that are still functional. The same is true for EIAV. These variants can be used to enhance specific parts of the transduction process. Examples of HIV-1 variants can be found in the HIV database operated by Los Alamos National Security, LLC at hiv-web.lanl.gov. Details of EIAV clones can be found in the National Center for Biotechnology Information (NCBI) database located at ncbi.nlm.nih.gov.

[0315] The strategy of codon-optimized gag-pol sequences can be used for any retrovirus. This will apply to all lentiviruses, including EIAV, FIV, BIV, CAEV, VMR, SIV, HIV-1, and HIV-2. Additionally, the method can be used to increase the expression of genes from HTLV-1, HTLV-2, HFV, HSRV, and human endogenous retroviruses (HERV), MLV, and other retroviruses.

[0316] Codon optimization can render gag-pol expression independent of Rev. However, in order to be able to use anti-rev or RRE factors in lentiviral vectors, it will be necessary to make the viral vector production system completely independent of Rev / RRE. Therefore, the genome will also need to be modified. This is achieved by optimizing the vector genome components. Advantageously, these modifications also result in a safer system that is free of all additional proteins in both producer cells and transduced cells. b. Production of viral particles

[0317] The production of infectious viral particles and viral stock solutions can be carried out using conventional techniques as described herein. Recombinant viruses (e.g., retroviral or lentiviral vectors or particles as described herein) with titers in the millions of transduction units per milliliter (TU / mL) can be generated by known techniques. After ultracentrifugation, a concentrated stock solution with a titer of approximately 10 8 TU / mL, 10 9 TU / mL, 10 10 TU / mL, 10 11 TU / mL, or 10 TU / mL, or any intermediate titer can be obtained. Recombinant viruses (e.g., retroviral or lentiviral vectors or particles as described herein) can be delivered according to the viral titer (TU / mL), which can be measured, for example, by using a commercially available p24 titer assay, which is an ELISA for the p24 viral capsid protein.

[0318] In some embodiments, enriched apheresis products are transfected with a lentiviral vector or a retroviral vector, and the concentration of viral transduction units (TU) per enriched apheresis product (cells) contained in the lentiviral vector or retroviral vector is from about 1 x 10 8 to about 1 x 10 10 TU / 10 8 cells, from about 5 x 10 8 to about 5 x 10 9 TU / 10 8 cells, from about 1 x 10 9 to about 5 x 10 9 TU / 10 8 cells, from about 1 x 10 9 to about 4 x 109 TU / 10 8 cells, about 1x 10 9 to about 3x 10 9 TU / 10 8 cells, about 1x 10 9 to about 2x 10 9 TU / 10 8 cells or any intermediate TU.

[0319] In one embodiment, the manufacturing process contemplated herein includes transfecting an enriched apheresis product with a lentiviral vector or a retroviral vector, the lentiviral vector or retroviral vector having a concentration of about 1x 10 8 TU / 10 8 cells, about 5x 10 8 TU / 10 8 cells, about 6x 10 8 TU / 10 8 cells, about 7x 10 8 TU / 10 8 cells, about 8x 10 8 TU / 10 8 cells, about 9x 10 8 TU / 10 8 cells, about 1x 10 9 TU / 10 8 cells, about 2x 10 9 TU / 10 8 cells, about 3x 10 9 TU / 10 8 cells, about 4x 10 9 TU / 10 8 cells, about 5x 10 9 TU / 10 8 cells, about 6x 10 9 TU / 10 8 cells, about 7x10 9 TU / 10 8 cells, about 8x 10 9 TU / 10 8 cells, about 9x 10 9 TU / 10 8 cells or about 1x 10 10 TU / 10 8 cells or any intermediate TU. In certain embodiments, at a concentration of about 1x10 7 to about 2x 10 9 TU / 10 8Transfect the enriched apheresis product with a lentiviral vector or retroviral vector of a cell.

[0320] Virus particles and viral stock solutions can be produced using conventional techniques. Methods for preparing viral stock solutions are known in the art and are illustrated by, for example, Soneoka et al. (1995) Nucl. Acids Res. 23:628 - 633 and Landau et al. (1992) J. Virol. 66:5110 - 5113. Recombinant viruses with titers in the millions of transduction units per milliliter (TU / mL) can be produced by known techniques. After ultracentrifugation, a concentrated stock solution with approximately 10 8 TU / mL, 10 9 TU / mL, 10 10 TU / mL, 10 11 TU / mL or 10 12 TU / mL or any intermediate titer can be obtained. c.p24 titer determination

[0321] The virus can be delivered according to the virus titer (TU / mL), which can be measured, for example, by using a commercially available p24 titer assay. The p24 titer assay is an ELISA for the p24 viral capsid protein. Assuming that each lentiviral physical particle (PP) has approximately 2000 p24 molecules, the following formula can be used to calculate the pg / mL of p24: (2x10 3 ) x (24x10 3 Da of p24 / PP), 48x10 6 / Avogadro's constant = (48x10 6 ) / (6x10 23 ) = 8x10 17 g of p24 / PP, approximately 1PP / 1x10 16 g of p24, 1x10 4 PP / pg of p24. In some embodiments, a well-packaged VSV-G pseudotyped lentiviral vector has an infection index in the range of about 1 TU / 1000 physical particles (PP) to about 1 TU / 100 PP (or less). Thus, the range of p24 is approximately about 10 to about 100 TU / pg. It is through this conversion that TU / mL is obtained.

[0322] The lentiviral titer can also be determined by analyzing transduced human osteosarcoma (HOS) cells. Briefly, the transduced HOS cells are cultured for seven days in DMEM supplemented with 10% fetal bovine serum (FBS), after which genomic DNA is extracted by DNeasy (Qiagen, Venlo, Netherlands, catalog number 69506) and evaluated by quantitative PCR (qPCR). The primer / probe set for the qPCR protocol measures the vector copy number (VCN) of the transduced cells by determining the copy number of the lentiviral psi-gag region / copy number of endogenous human RNase P. The integrity of the provirus is evaluated by sequencing individual proviral inserts. In some embodiments, the HOS cell line assay is used to determine the viral titer. d. Host cell

[0323] As used herein, a "host cell" is a cell that is transfected with a nucleic acid vector to replicate and produce more of the nucleic acid vector itself (i.e., more plasmids). In some embodiments, supernatants containing lentiviral vectors (LVs) encoding CARs or TCRs disclosed herein are produced in HEK 293T cells. To produce the lentiviral vectors of the present disclosure, 293 cells are transiently transfected with the following 4 plasmids: a plasmid encoding HIV gag-pol, a plasmid encoding the VSV-G envelope protein, a plasmid encoding the HIV rev protein, and a lentiviral transfer vector encoding CAR.

[0324] Bacterial cells, yeast cells, and animal cells can be used to amplify or produce nucleic acids and vectors encoding heterologous envelope proteins or proteins, enzymes, viral elements (i.e., cis - acting and trans - acting genes, including rev and gag / pol) necessary for the production of retroviral particles. For amplification in bacterial cells, suitable promoters include, but are not limited to, lacI, lacZ, T3, T7, gpt, λP, and trc. For amplification in eukaryotic cells or expression in eukaryotic cells, suitable promoters include, but are not limited to, light chain or heavy chain immunoglobulin gene promoters and enhancer elements; cytomegalovirus immediate early promoter; herpes simplex virus thymidine kinase promoter; early and late SV40 promoters; promoters present in long terminal repeats from retroviruses; mouse metallothionein - I promoter; and various tissue - specific promoters known in the art. Suitable reversible promoters (including reversible inducible promoters) are known in the art. Such reversible promoters can be isolated from and derived from many organisms (e.g., eukaryotes and prokaryotes). Modifications of reversible promoters from a first organism (e.g., first prokaryote and second eukaryote, first eukaryote and second prokaryote, etc.) for use in a second organism are well - known in the art. Such reversible promoters and systems based on such reversible promoters but also containing additional control proteins include, but are not limited to, alcohol - regulated promoters (e.g., alcohol dehydrogenase I (alcA) gene promoter, promoter responsive to the alcohol trans - activator protein (A1cR), etc.), tetracycline - regulated promoters (e.g., promoter systems containing Tet activator, TetON, TetOFF, etc.), steroid - regulated promoters (e.g., rat glucocorticoid receptor promoter system, human estrogen receptor promoter system, retinoic acid promoter system, thyroid promoter system, ecdysone promoter system, mifepristone promoter system, etc.), metal - regulated promoters (e.g., metallothionein promoter system, etc.), pathogenesis - related regulated promoters (e.g., salicylic acid - regulated promoter, ethylene - regulated promoter, benzothiadiazole - regulated promoter, etc.), temperature - regulated promoters (e.g., heat - shock inducible promoters (e.g., HSP - 70, HSP - 90, soybean heat - shock promoter, etc.)), light - regulated promoters, synthetic inducible promoters, etc.

[0325] In some embodiments, the host cell and the production cell can be from the same cell line. In some embodiments, the host cell and the production cell are HEK293-T cells. Thus, in some embodiments, the promoter can be expressed ubiquitously in all cells, or selectively in the production cells, or specifically in the production cells. In some embodiments, the promoter is a CD8 cell-specific promoter, a CD4 cell-specific promoter, a neutrophil-specific promoter, or an NK-specific promoter. For example, the CD4 gene promoter can be used; see, for example, Salmon et al. Proc. Natl. Acad. Sci. USA (1993) 90:7739; and Marodon et al. (2003) Blood 101:3416. As another example, the CD8 gene promoter can be used. NK cell-specific expression can be achieved by using the NcrI (p46) promoter; see, for example, Eckelhart et al. Blood (2011) 117:1565. For expression in yeast host cells for amplification, suitable promoters are constitutive promoters such as the ADH1 promoter, the PGK1 promoter, the ENO promoter, the PYK1 promoter, etc.; or inducible promoters such as the GAL1 promoter, the GAL10 promoter, the ADH2 promoter, the PHOS promoter, the CUP1 promoter, the GALT promoter, the MET25 promoter, the MET3 promoter, the CYC1 promoter, the HIS3 promoter, the ADH1 promoter, the PGK promoter, the GAPDH promoter, the ADC1 promoter, the TRP1 promoter, the URA3 promoter, the LEU2 promoter, the ENO promoter, the TP1 promoter, and the AOX1 (e.g., for use in Pichia). The selection of appropriate vectors and promoters is well within the level of ordinary skill in the art.Suitable promoters for use in prokaryotic host cells include, but are not limited to, the bacteriophage T7 RNA polymerase promoter; the trp promoter; the lac operon promoter; hybrid promoters such as the lac / tac hybrid promoter, the tac / trc hybrid promoter, the trp / lac promoter, the T7 / lac promoter; the trc promoter; the tac promoter; etc.; the araBAD promoter; in vivo regulatable promoters such as the ssaG promoter or related promoters (US 2004 / 0131637), the pagC promoter, the nirB promoter, etc. (see, e.g., Dunstan et al., Infect. Immun. (1999) 67:5133-5141; McKelvie et al., Vaccine (2004) 22:3243-3255); the σ70 promoter, such as the consensus σ70 promoter (see, e.g., GenBank accession numbers AX798980, AX798961, and AX798183); stationary phase promoters such as the dps promoter, the spv promoter, etc.; promoters derived from the virulence island SPI-2; the actA promoter; the rpsM promoter; the tet promoter; the SP6 promoter; etc. Suitable strong promoters for use in prokaryotes such as Escherichia coli include, but are not limited to, Trc, Tac, T5, T7, and Pλ.

[0326] One aspect of the present disclosure provides a method for producing lentiviral vector particles, the method comprising introducing into a host cell a lentiviral vector as described herein.

[0327] Another aspect of the present disclosure provides a method for delivering a nucleic acid encoding a chimeric antigen receptor (CAR), an engineered T cell receptor, or a therapeutic protein to a cell, the method comprising introducing into the cell a transfer plasmid comprising: a polynucleotide sequence encoding at least one heterologous viral envelope protein engineered by the methods described herein; a polynucleotide sequence encoding at least one retroviral rev protein; a polynucleotide sequence encoding at least one retroviral gag protein and a retroviral pol protein; and / or a polynucleotide sequence encoding a chimeric antigen receptor, an engineered T cell receptor (TCR), or a therapeutic protein. In some embodiments, at least a portion of one or more regions necessary for replication in the retroviral genome is mutated as described herein.

[0328] Another aspect of the present disclosure provides lentiviral vector particles produced by the methods described herein.

[0329] Another aspect of the present disclosure provides a method of introducing a modification into a cell, the method comprising electroporating the cell with an effective dose of the lentiviral vector particles described herein, thereby producing a modified cell. In some embodiments, the cell is contacted with an effective dose of the lentiviral vector prior to electroporation (e.g., application of electricity). Alternatively, after electroporation (e.g., application of electricity), the cell can be contacted with an effective dose of the lentiviral vector for up to about 4 hours. For example, after electroporation, the cell can be contacted with an effective dose of the lentiviral vector for at least about 5 - 30 minutes, at least about 25 - 50 minutes, at least about 5 - 60 minutes, at least about 5 - 12 minutes, at least about 60 - 120 minutes, at least about 120 - 240 minutes. Alternatively, after electroporation, the cell can be contacted with an effective dose of the lentiviral vector for at least about 1 minute, at least about 2 minutes, at least about 5 minutes, at least about 10 minutes, at least about 20 minutes, at least about 30 minutes, at least about 45 minutes, at least about 60 minutes, at least about 75 minutes, at least about 90 minutes, at least about 100 minutes, at least about 110 minutes, at least about 120 minutes, at least about 150 minutes, at least about 160 minutes, at least about 170 minutes, at least about 180 minutes, at least about 190 minutes, at least about 200 minutes, at least about 220 minutes, or at least about 240 minutes.

[0330] Adding the lentiviral vector to the cells up to 4 hours after electroporation (e.g., 1 minute to 2 hours or 1 minute to 4 hours) can reduce the amount of lentiviral particles killed by electroporation, which can be toxic to lentiviral particles. Thus, adding the lentiviral vector to the cells up to 4 hours after electroporation can enhance CAR transfection. For example, when compared to a conventional electroporation procedure (e.g., adding the lentivirus to the cells prior to electroporation), adding the lentiviral vector to the cells up to 4 hours after electroporation can increase CAR expression by about 10% - 15%.

[0331] In some embodiments, the cells are selected from immune cells, eukaryotic donor cells, mononuclear cells, enriched lymphocytes, B lymphocytes, T lymphocytes, CD4 + T lymphocytes, CD8 + T lymphocytes, dendritic cells, monocytes, natural killer (NK) cells, natural killer T (NKT) cells, regulatory T cells, CD4 + helper T cells, CD8 + cytotoxic T lymphocytes (CTL), CD62L + cells, CD27 + cells, CCR7 + cells, CD45RO - cells, CD45RA +Cells, neutrophils, basophils, eosinophils, megakaryocytes, stem cells, hematopoietic stem cells (HSC), hematopoietic progenitor cells (HPC), CD34 + Cells, CD34 + Peripheral blood stem cells, lymphokine-activated killer cells (LAK), tumor-infiltrating lymphocytes (TIL), circulating tumor-specific T cells, mesenchymal stem cells, mast cells, monocytes, macrophages, neutrophils, basophils, eosinophils, dendritic cells, megakaryocytes, and combinations thereof.

[0332] In some embodiments, the cells can be lymphoid cells selected from: T cells, B cells, natural killer (NK) cells, CD8 + T cells, CD4 + T cells, cytotoxic T lymphocytes, regulatory T cells, and any combination thereof. In some embodiments, the cells can be myeloid cells selected from: monocytes, macrophages, neutrophils, basophils, eosinophils, dendritic cells, megakaryocytes, and any combination thereof. In some embodiments, the cells can be stem cells, hematopoietic stem cells, hematopoietic progenitor cells, CD34 + Cells or CD34 + Peripheral blood stem cells.

[0333] In some embodiments of the method of introducing a modification into a cell, the method comprises electroporating the cell with an effective dose of lentiviral vector particles, and the effective dose comprises about 0.5, about 1, about 1.5, about 2, about 2.5, about 3, about 3.5, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 15, or about 20 μl of lentiviral vector.

[0334] In some embodiments, the effective dose of lentiviral vector particles comprises a multiplicity of infection (MOI) of about 0.01, about 0.02, about 0.03, about 0.04, about 0.05, about 0.06, about 0.07, about 0.08, about 0.09, about 0.1, about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1.0, about 1.25, about 1.5, about 2.0, about 3.0, about 4.0, or about 5.0. In some embodiments, the effective dose of lentiviral vector particles comprises about 2 μl of lentiviral vector particles with an MOI of about 0.08. In some embodiments, the effective dose of lentiviral vector particles comprises about 5 μl of lentiviral vector particles with an MOI of about 0.2. In some embodiments, the effective dose of lentiviral vector particles comprises about 10 μl of lentiviral vector particles with an MOI of about 0.4. E. Method for producing a therapeutic protein

[0335] One aspect of the present disclosure provides a method for producing a therapeutic protein, the method comprising manufacturing an engineered population of immune cells or an engineered population of eukaryotic cells comprising the therapeutic protein using the methods described herein; harvesting the therapeutic protein; and isolating and purifying the therapeutic protein. In some embodiments, the therapeutic protein is selected from enzymes, regulatory proteins, receptors, peptides, peptide hormones, cytokines, membrane or transport proteins, vaccine antigens, antigen-binding proteins, immunostimulatory proteins, allergens, full-length antibodies or antibody fragments or derivatives, single-chain antibodies (scFv), Fab fragments, Fv fragments, single-domain antibodies (VH or VL fragments), domain antibodies, camelid single-domain antibodies (VHH), nanobodies, and combinations thereof. IV. Chimeric Receptors

[0336] One aspect of the present disclosure provides a method for manufacturing an engineered population of immune cells, the method comprising: enriching a population of lymphocytes, a population of immune cells, or a population of CD4 + and CD8 + cells from blood obtained from a subject; mixing the population of lymphocytes, the population of immune cells, or the population of CD4 + and CD8 + cells with one or more buffer solutions; and transfecting the population of lymphocytes, the population of immune cells, or the population of CD4 + and CD8 + cells with an effective dose of a modifier; thereby producing a modified population of lymphocytes, a modified population of immune cells, or a modified population of CD4 + and CD8 + cells.

[0337] Another aspect of the present disclosure provides a method for manufacturing an engineered population of immune cells, the method comprising: enriching a population of lymphocytes, a population of immune cells, or a population of CD4 + and CD8 + cells from a donor leukapheresis; mixing the population of lymphocytes, the population of immune cells, or the population of CD4 + and CD8 + cells with one or more buffer solutions; transfecting the population of lymphocytes, the population of immune cells, or the population of CD4 + and CD8 + cells with an effective dose of a modifier; thereby producing a modified population of lymphocytes, a modified population of immune cells, or a modified population of CD4 + and CD8 + cells.

[0338] Yet another aspect of the present disclosure provides a method for manufacturing an engineered eukaryotic cell population, the method comprising: obtaining a population of eukaryotic donor cells from a subject; mixing the population of eukaryotic donor cells with one or more buffer solutions; and transfecting the population of eukaryotic donor cells with an effective dose of a modifier to produce a modified population of eukaryotic donor cells. In some embodiments, the transfected cells are cultured and expanded in the presence of one or more stimulators.

[0339] In some embodiments, the cells are transfected with one or more modifiers selected from: small molecule agents, biologic agents, therapeutic agents, proteins, peptides, protein therapeutics, peptide therapeutics, nucleic acids, DNA, RNA, mRNA, chimeric antigen receptors, heterologous T cell receptors, retroviral vectors, lentiviral vectors, adenoviral vectors, and adeno-associated viral vectors.

[0340] In some embodiments, the lentiviral vector or retroviral vector comprises a nucleotide sequence encoding a chimeric antigen receptor (CAR), an engineered T cell receptor; and / or a nucleic acid sequence encoding a polypeptide that enhances immune cell function or a functional derivative thereof.

[0341] In some embodiments, the lentiviral vector or retroviral vector comprises a nucleic acid encoding a chimeric antigen receptor (CAR), an engineered T cell receptor (TCR), a killer cell immunoglobulin-like receptor (KIR), an antigen-binding polypeptide, a cell surface receptor ligand, or a tumor antigen. In some embodiments, the nucleic acid encodes a chimeric antigen receptor (CAR). In some embodiments, the nucleic acid encodes an antigen-binding polypeptide. In some embodiments, the nucleic acid encodes a killer cell immunoglobulin-like receptor (KIR). In additional embodiments, the exogenous nucleic acid encodes a cell surface receptor ligand or a tumor antigen.

[0342] In some embodiments, a retroviral vector or lentiviral vector can be used to introduce a TCR or CAR into an immune cell or its precursor (e.g., a T cell). In some embodiments, the retroviral vector or lentiviral vector particles can comprise additional elements that will facilitate the functional expression of the TCR or CAR encoded therein. In some embodiments, the expression vector comprising the nucleic acid encoding the TCR or CAR further comprises a mammalian promoter. A. Chimeric Antigen Receptor

[0343] The present invention provides engineered immune effector cells (e.g., T cells or NK cells), the engineered immune effector cells comprising one or more CARs that direct the immune effector cells to cancer. In some embodiments, the CAR comprises an antigen-binding domain, a transmembrane domain, a co-stimulatory domain, and an intracellular domain. The CAR can comprise any antigen-binding domain, any hinge, any transmembrane domain, any co-stimulatory domain, and any intracellular signaling domain described herein.

[0344] The antigen-binding domain can be operably linked to another domain of the CAR (such as the transmembrane domain or the intracellular domain, both described herein) for expression in any of the immune cells described herein. In one embodiment, a first nucleic acid sequence encoding the antigen-binding domain is operably linked to a second nucleic acid encoding the transmembrane domain and further operably linked to a third nucleic acid sequence encoding the intracellular domain.

[0345] The antigen-binding domains described herein can be combined with any of the transmembrane domains, any of the intracellular domains or cytoplasmic domains, or any other domains that can be included in the CARs of the present invention described herein. The subject CARs of the present invention can also comprise a spacer domain as described herein. In some embodiments, each of the antigen-binding domain, transmembrane domain, and intracellular domain is separated by a linker. 1. Antigen-binding domain

[0346] The antigen-binding domain of the CAR is the extracellular region of the CAR for binding to a specific target antigen, including proteins, carbohydrates, and glycolipids. In some embodiments, the CAR comprises an affinity for a target antigen (e.g., a tumor-associated antigen) on a target cell (e.g., a cancer cell). The target antigen can include any type of protein or epitope thereof associated with the target cell. For example, the CAR can comprise an affinity for a target antigen on a target cell, the target antigen indicating a particular state of the target cell.

[0347] As described herein, the CARs of the present disclosure having an affinity for a specific target antigen on a target cell can comprise a target-specific binding domain. In some embodiments, the target-specific binding domain is a murine target-specific binding domain, e.g., the target-specific binding domain is of murine origin. In some embodiments, the target-specific binding domain is a human target-specific binding domain, e.g., the target-specific binding domain is of human origin.

[0348] An antigen-binding domain can include any domain that binds to an antigen and can include, but is not limited to, monoclonal antibodies, polyclonal antibodies, synthetic antibodies, human antibodies, humanized antibodies, non-human antibodies, and any fragments thereof. Thus, in one embodiment, the antigen-binding domain portion includes a mammalian antibody or a fragment thereof. In some embodiments, the antigen-binding domain includes a full-length antibody. In some embodiments, the antigen-binding domain includes an antigen-binding fragment (Fab) (e.g., Fab, Fab’, F(ab’)2, monospecific Fab2, bispecific Fab2, trispecific Fab2), single-chain variable fragment (scFv), dAb, tandem scFv, VhH, V-NAR, camelid, diabody, minibody, triabody, or tetrabody. In some embodiments, the antigen-binding domain is selected from (a) a full-length antibody or an antigen-binding fragment thereof, (b) Fab, (c) single-chain variable fragment (scFv), and (d) single-domain antibody.

[0349] In some embodiments, the CAR of the present disclosure can have affinity for one or more target antigens on one or more target cells. In some embodiments, the CAR can have affinity for one or more target antigens on a single type of target cell. In such embodiments, the CAR is a bispecific CAR or a multispecific CAR. In some embodiments, the CAR includes one or more target-specific binding domains that confer affinity for one or more target antigens. In some embodiments, the CAR includes one or more target-specific binding domains that confer affinity for the same target antigen. For example, a CAR that includes one or more target-specific binding domains having affinity for the same target antigen can bind to different epitopes of the target antigen. When multiple target-specific binding domains are present in the CAR, the binding domains can be arranged in tandem and can be separated by a linker peptide. For example, in a CAR that includes two target-specific binding domains, the binding domains are covalently linked to each other on a single polypeptide chain by a polypeptide linker, an Fc hinge region, or a membrane hinge region.

[0350] In some cases, the antigen-binding domain can be derived from the same species in which the CAR will ultimately be used. For example, for use in humans, the antigen-binding domain of the CAR can include a human antibody or a fragment thereof as described elsewhere herein.

[0351] Thus, the CAR encoded by the lentiviral vector or retroviral vector of the present disclosure can target one of the following cancer-related antigens (tumor antigens): CD19; CD20; CD22 (Siglec 2); CD37; CD123; CD22; CD30; CD171; CS-1 (also known as CD2 subset 1, CRACC, SLAMF7, CD319, and 19A24); C-type lectin-like molecule-1 (CLL-1 or CLECL1); CD33; CD133; epidermal growth factor receptor (EGFR); epidermal growth factor receptor variant III (EGFRvIII); human epidermal growth factor receptor (HER1); ganglioside G2 (GD2); ganglioside GD3 (aNeu5Ac(2-8)aNeu5Ac(2-3)bDGalp(l-4)bDGlcp(l-l)Cer); TNF receptor family member B-cell maturation antigen (BCMA); Tn antigen ((Tn Ag) or (GalNAca-Ser / Thr)); prostate-specific membrane antigen (PSMA); receptor tyrosine kinase-like orphan receptor 1 (ROR1); Fms-like tyrosine kinase 3 (FLT3); tumor-associated glycoprotein 72 (TAG72); CD38; CD44v6; carcinoembryonic antigen (CEA); epithelial cell adhesion molecule (EPCAM); B7H3 (CD276); KIT (CD117); interleukin-13 receptor subunit alpha-2 (IL-13Ra2 or CD213A2); mesothelin; interleukin 11 receptor alpha (IL-llRa); prostate stem cell antigen (PSCA); serine protease 21 (Testisin or PRSS21); vascular endothelial growth factor receptor 2 (VEGFR2); Lewis (Y) antigen; CD24; platelet-derived growth factor receptor beta (PDGFR-β); stage-specific embryonic antigen-4 (SSEA-4); folate receptor alpha; receptor tyrosine protein kinase ERBB2 (Her2 / neu); cell surface-associated mucin 1 (MUC 1); GalNAca1-O-Ser / Thr (Tn) MUC 1 (TnMUC1); neural cell adhesion molecule (NCAM); prostate enzyme; prostate acid phosphatase (PAP); mutant elongation factor 2 (ELF2M); ephrin B2; fibroblast activation protein alpha (FAP); insulin-like growth factor 1 receptor (IGF-I receptor); carbonic anhydrase IX (CAIX); beta-type proteasome (precursor, macropain) subunit 9 (LMP2); glycoprotein 100 (gp100); oncogenic fusion protein composed of breakpoint cluster region (BCR) and Abelson murine leukemia virus oncogene homolog 1 (Abl) (bcr-abl); tyrosinase; ephrin type-A receptor 2 (EphA2); fucosyl GM1;Sialyl Lewis adhesion molecule (sLe); ganglioside GM3 (aNeu5Ac(2-3)bDGalp(l-4)bDGlcp(l-l)Cer); transglutaminase 5 (TGS5); high molecular weight melanoma-associated antigen (HMWMAA); o-acetyl-GD2 ganglioside (OAcGD2); folate receptor β; tumor endothelial marker 1 (TEM1 / CD248); tumor endothelial marker-related protein 7 (TEM7R); claudin 6 (CLDN6); thyroid-stimulating hormone receptor (TSHR); G protein-coupled receptor class C group 5 member D (GPRC5D); X chromosome open reading frame 61 (CXORF61); CD97; CD179a; anaplastic lymphoma kinase (ALK); polysialic acid; placenta-specific protein 1 (PLAC1); hexose moiety of globoH glycosphingolipid (GloboH); breast differentiation antigen (NY-BR-1); uroplakin 2 (UPK2); tyrosine protein kinase Met (c-Met); hepatitis A virus cellular receptor 1 (HAVCR1); adrenergic receptor β3 (ADRB3); pannexin 3 (PANX3); G protein-coupled receptor 20 (GPR20); lymphocyte antigen 6 complex locus K 9 (LY6K); olfactory receptor 51E2 (OR51E2); TCRγ alternative reading frame protein (TARP); Wilms tumor protein (WT1); cancer / testis antigen 1 (NY-ESO-1); cancer / testis antigen 2 (LAGE-la); melanoma-associated antigen 1 (MAGE-A1); ETS translocation variant gene 6 located on chromosome 12p (ETV6-AML); sperm protein 17 (SPA17); X antigen family member 1A (XAGEl); cell surface receptor 2 that binds angiopoietin (Tie 2); melanoma cancer testis antigen-1 (MAD-CT-1); melanoma cancer testis antigen-2 (MAD-CT-2); Fos-related antigen 1; tumor protein p53 (p53); p53 mutant; prostein; survivin; telomerase; prostate cancer tumor antigen-1 (PCTA-1 or galectin 8); melanoma antigen recognized by T cells 1 (MelanA or MARTI); rat sarcoma (Ras) mutant; human telomerase reverse transcriptase (hTERT); sarcoma translocation breakpoint; melanoma apoptosis inhibitor protein (ML-IAP); ERG (ETS fusion gene of transmembrane serine protease 2 (TMPRSS2)); N-acetylglucosaminyltransferase V (NA17); paired box protein Pax-3 (PAX3); androgen receptor; cyclin B1; v-myc avian myelocytomatosis viral oncogene neuroblastoma-derived homolog (MYCN); Ras homolog family member C (RhoC);Tyrosinase-related protein 2 (TRP-2); Cytochrome P450 1B1 (CYP1B1); CCCTC-binding factor (zinc finger protein)-like protein (BORIS or Brother of the Regulator of Imprinted Sites); Squamous cell carcinoma antigen recognized by T cells 3 (SART3); Paired box protein Pax-5 (PAX5); Acrosinogen-binding protein sp32 (OY-TES1); Lymphocyte-specific protein tyrosine kinase (LCK); A-kinase anchor protein 4 (AKAP-4); Synovial sarcoma X breakpoint 2 (SSX2); Receptor for advanced glycation end products 1 (RAGE-1); Renal ubiquitous protein 1 (RU1); Renal ubiquitous protein 2 (RU2); Podoplanin; Human papillomavirus E6 (HPV E6); Human papillomavirus E7 (HPV E7); Intestinal carboxylesterase; Mutated heat shock protein 70-2 (mut hsp70-2); CD79a; CD79b; CD72; Leukocyte-associated immunoglobulin-like receptor 1 (LAIR1); IgA Fc fragment receptor (FCAR or CD89); Leukocyte immunoglobulin-like receptor subfamily A member 2 (LILRA2); CD300 molecule-like family member f (CD300LF); C-type lectin domain family 12 member A (CLEC12A); Bone marrow stromal cell antigen 2 (BST2); EGF-like module-containing mucin-like hormone receptor-like 2 (EMR2); Lymphocyte antigen 75 (LY75); Glypican-2 (GPC2); Glypican-3 (GPC3); NKG2D; KRAS; GDNF family receptor alpha-4 (GFRa4); IL13Ra2; Fc receptor-like protein 5 (FCRL5); and Immunoglobulin lambda-like polypeptide 1 (IGLL1).;

[0352] In some embodiments, the CAR targets CD19, CD20, CD22, BCMA, CD37, mesothelin, PSMA, PSCA, Tn-MUC1, EGFR, EGFRvIII, c-Met, HER1, HER2, CD33, CD133, GD2, GPC2, GPC3, NKG2D, KRAS, or WT1. In some embodiments, the antigen-binding domain specifically binds to a target antigen selected from: CD4, CD19, CD20, CD22, BCMA, CD123, CD133, EGFR, EGFRvIII, mesothelin, Her2, PSMA, CEA, GD2, IL-13Ra2, glypican-3, GPC2, TnMuc1, CIAX, LI-CAM, CA 125, CTAG1B, mucin 1, and folate receptor-α. 2. Transmembrane domain

[0353] The CAR encoded by the lentiviral or retroviral vector of the present disclosure can be designed to include a transmembrane domain that links the antigen-binding domain of the CAR to the intracellular domain. The transmembrane domain of the subject CAR is a region capable of spanning the plasma membrane of a cell (e.g., an immune cell or its precursor). The transmembrane domain is used to insert into the cell membrane (e.g., a eukaryotic cell membrane). In some embodiments, the transmembrane domain is between the antigen-binding domain and the intracellular domain of the CAR.

[0354] In one embodiment, the transmembrane domain is naturally associated with one or more domains in the CAR. In some instances, the transmembrane domain can be selected or modified by amino acid substitution to avoid binding of such domains to the transmembrane domains of the same or different surface membrane proteins, to minimize interaction with other members of the receptor complex.

[0355] In some embodiments, the transmembrane domain can be derived from a natural source or a synthetic source. Where the source is natural, the domain can be derived from any membrane-bound or transmembrane protein (e.g., a type I transmembrane protein). Where the source is synthetic, the transmembrane domain can be any artificial sequence that promotes insertion of the CAR into the cell membrane, such as an artificial hydrophobic sequence. In some embodiments, transmembrane domains that are particularly useful in the present invention include, but are not limited to, transmembrane domains derived from the α, β, or ζ chains of the T cell receptor, CD28, CD2, CD3ε, CD45, CD4, CD5, CD7, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134 (OX-40), CD137 (4-1BB), CD154 (CD40L), CD278 (ICOS), CD357 (GITR), Toll-like receptor 1 (TLR1), TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, and killer cell immunoglobulin-like receptor (KIR).

[0356] In some embodiments, the transmembrane domain comprises at least the transmembrane region of a protein selected from the α, β, or ζ chains of the T cell receptor, CD28, CD2, CD3ε, CD45, CD4, CD5, CD7, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134 (OX-40), CD137 (4-1BB), CD154 (CD40L), CD278 (ICOS), CD357 (GITR), Toll-like receptor 1 (TLR1), TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, and killer cell immunoglobulin-like receptor (KIR).

[0357] In some embodiments, the transmembrane domain can be synthetic. In some embodiments, the synthetic transmembrane domain predominantly comprises hydrophobic residues such as leucine and valine. In certain exemplary embodiments, a triad of phenylalanine, tryptophan, and valine will be found at each end of the synthetic transmembrane domain.

[0358] The transmembrane domains described herein can be combined with any antigen-binding domain described herein, any co-stimulatory signaling domain described herein, any intracellular signaling domain described herein, or any other domain that can be included in the subject CAR described herein.

[0359] In one embodiment, the transmembrane domain comprises a CD8α transmembrane domain. In some embodiments, the transmembrane domain comprises a CD8α transmembrane domain comprising the amino acid sequence set forth in SEQ ID NO:33. In some embodiments, the transmembrane domain comprises the nucleotide sequence set forth in SEQ ID NO:34.

[0360] In some embodiments, the transmembrane domain comprises a CD28 transmembrane domain. In some embodiments, the CAR comprises a CD28 transmembrane domain comprising the amino acid sequence set forth in SEQ ID NO:37. In some embodiments, the CD28 transmembrane domain comprises the nucleotide sequence set forth in SEQ ID NO:38.

[0361] Permissible variations of the transmembrane domain and / or hinge domain will be known to those skilled in the art while retaining their intended function. In some embodiments, the transmembrane domain comprises an amino acid sequence having at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity to any of the amino acid sequences set forth in SEQ ID NO:33 and / or 37. In some embodiments, the transmembrane domain is encoded by a nucleic acid sequence comprising a nucleotide sequence having at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity to any of the nucleotide sequences set forth in SEQ ID NO:34 and / or 38. The transmembrane domain can be combined with any hinge domain, and / or can comprise one or more of the transmembrane domains described herein.

[0362] In some embodiments, the CAR comprises any transmembrane domain selected from the transmembrane domains of: the α, β, or ζ chain of the T cell receptor, CD28, CD2, CD3ε, CD45, CD4, CD5, CD7, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134 (OX-40), CD137 (4-1BB), CD154 (CD40L), CD278 (ICOS), CD357 (GITR), Toll-like receptor 1 (TLR1), TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, and killer cell immunoglobulin-like receptor (KIR); any co-stimulatory signaling domain and any intracellular domain or cytoplasmic domain described herein; or any other domain that can be included in a CAR as described herein; and optionally a hinge domain.

[0363] In some embodiments, the CAR further comprises a spacer domain between the extracellular domain and the transmembrane domain of the CAR or between the intracellular domain and the transmembrane domain of the CAR. In some embodiments, the spacer domain can be a short oligopeptide linker or a polypeptide linker, for example, having a length between about 2 and about 10 amino acids. For example, a glycine-serine doublet provides a particularly suitable linker between the transmembrane domain and the intracellular signaling domain of the subject CAR. Thus, the CARs of the present disclosure can comprise any of the transmembrane domains, hinge domains, or spacer domains described herein. 3. Hinge domain

[0364] In some embodiments, the CAR encoded by the lentiviral or retroviral vector of the present disclosure further comprises a hinge region. The hinge region of the CAR is a hydrophilic region located between the antigen-binding domain and the transmembrane domain. In some embodiments, the hinge domain facilitates proper protein folding of the CAR. In some embodiments, the hinge domain is an optional component of the CAR. In some embodiments, the hinge domain comprises a domain selected from the following: the Fc fragment of an antibody, the hinge region of an antibody, the CH2 region of an antibody, the CH3 region of an antibody, an artificial hinge sequence, or a combination thereof. In some embodiments, the hinge domain is selected from, but not limited to, the CD8a hinge, an artificial hinge made of a polypeptide, which polypeptide can be as small as three glycines (Gly). In some embodiments, the hinge region is a hinge region polypeptide derived from a receptor. In some embodiments, the hinge region is a CD8-derived hinge region. In one embodiment, the hinge domain comprises an amino acid sequence derived from human CD8 or a variant thereof. In some embodiments, the subject CAR comprises a CD8α hinge domain and a CD8α transmembrane domain. In some embodiments, the CD8α hinge domain comprises the amino acid sequence shown in SEQ ID NO:35. In some embodiments, the CD8α hinge domain comprises the nucleotide sequence shown in SEQ ID NO:36.

[0365] In some embodiments, the hinge domain comprises an amino acid sequence having at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity to any of the amino acid sequences shown in SEQ ID NO:35.

[0366] In some embodiments, the hinge domain is encoded by a nucleic acid sequence comprising a nucleotide sequence having at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity to any of the nucleotide sequences shown in SEQ ID NO:36.

[0367] In some embodiments, the hinge domain connects the antigen-binding domain to a transmembrane domain that is linked to an intracellular domain. In an exemplary embodiment, the hinge region is capable of supporting the antigen-binding domain to recognize and bind to a target antigen on a target cell. In some embodiments, the hinge region is a flexible domain, thus allowing the antigen-binding domain to have a structure and density for optimally recognizing a target antigen on a cell, such as a tumor cell. The flexibility of the hinge region allows the hinge region to adopt many different conformations.

[0368] In some embodiments, the length of the hinge domain is selected from about 4 to about 50, from about 4 to about 10, from about 10 to about 15, from about 15 to about 20, from about 20 to about 25, from about 25 to about 30, from about 30 to about 40, or from about 40 to about 50 amino acids. A suitable hinge region can be readily selected and can have any of a variety of suitable lengths, such as from about 1 amino acid (e.g., glycine (Gly)) to about 20 amino acids, from about 2 to about 15, from about 3 to about 12 amino acids, including about 4 to about 10, about 5 to about 9, about 6 to about 8, or about 7 to about 8 amino acids, and can be about 1, about 2, about 3, about 4, about 5, about 6, or about 7 amino acids.

[0369] In some embodiments, the amino acid is glycine (Gly). Glycine and glycine-serine polymers can be used; both Gly and Ser are relatively unstructured and can thus act as neutral tethers between components. Glycine polymers can be used; the -ψ space of glycine is even significantly larger than that of alanine and is much less restricted than residues with longer side chains. In some embodiments, the hinge region comprises a glycine polymer (G)n, a glycine-serine polymer. In some embodiments, the hinge region comprises a glycine-serine polymer selected from the following: (GS)n, (GSGGS)n, and (GGGS)n, where n is an integer of at least one. In some embodiments, the hinge domain comprises an amino acid sequence including but not limited to GGSG (SEQ ID NO:24), GGSGG (SEQ ID NO:25), GSGSG (SEQ ID NO:26), GSGGG (SEQ ID NO:27), GGGSG (SEQ ID NO:28), GSSSG (SEQ ID NO:29). In some embodiments, the hinge region comprises a glycine-alanine polymer, an alanine-serine polymer, or other flexible linkers known in the art.

[0370] ​In some embodiments, the hinge region is an immunoglobulin heavy chain hinge region. Amino acid sequences of immunoglobulin hinge regions are known in the art. In some embodiments, the immunoglobulin hinge domain comprises an amino acid sequence selected from: DKTHT (SEQ ID NO:39); CPPC (SEQ ID NO:40); CPEPKSCDTPPPCPR (SEQ ID NO:41) (see, e.g., Glaser et al., J. Biol. Chem. (2005) 280:41494-41503); ELKTPLGDTTHT (SEQ ID NO:42); KSCDKTHTCP (SEQ ID NO:43); KCCVDCP (SEQ ID NO:44); KYGPPCP (SEQ ID NO:45); EPKSCDKTHTCPPCP (SEQ ID NO:46) (human IgG1 hinge); ERKCCVECPPCP (SEQ ID NO:47) (human IgG2 hinge); ELKTPLGDTTHTCPRCP (SEQ ID NO:48) (human IgG3 hinge); SPNMVPHAHHAQ (SEQ ID NO:49) (human IgG4 hinge); etc.

[0371] In some embodiments, the hinge region is an immunoglobulin heavy chain hinge region. In some embodiments, the hinge is selected from the CH1 and CH3 domains of IgG (such as human IgG4). In some embodiments, the hinge domain comprises the amino acid sequence of the human IgG1, IgG2, IgG3, or IgG4 hinge domain. In some embodiments, the hinge region may comprise one or more amino acid substitutions and / or insertions and / or deletions compared to the wild-type (naturally occurring) hinge region. In some embodiments, histidine (His229) at position 229 of the human IgG1 hinge is replaced with tyrosine (Tyr). In some embodiments, the hinge domain comprises the amino acid sequence EPKSCDKTYTCPPCP (SEQ ID NO:46). 4. Intracellular domain

[0372] The CAR encoded by the lentiviral or retroviral vector of the present disclosure also includes an intracellular domain. The intracellular domain or otherwise cytoplasmic domain of the CAR is responsible for activating the cell expressing the CAR. Thus, the term "intracellular domain" is intended to include any portion of the intracellular domain sufficient to transduce an activating signal. In one embodiment, the intracellular domain includes a domain responsible for effector function. The term "effector function" refers to the specialized function of a cell. The effector function of a T cell can be, for example, cytolytic activity or helper activity, including the secretion of cytokines. In one embodiment, the intracellular domain of the CAR includes a domain responsible for signal activation and / or transduction. The intracellular domain can transmit signal activation via protein-protein interactions, biochemical changes, or other responses to alter the metabolism, shape, gene expression, or other cellular responses of the cell to the activation of the chimeric intracellular signaling molecule.

[0373] Examples of intracellular domains for use in the present invention include, but are not limited to, the cytoplasmic portion of the T cell receptor (TCR) and any co-stimulatory molecule or any molecule that collaborates with the TCR to initiate signal transduction in T cells upon antigen receptor engagement, as well as any derivatives or variants of these elements and any synthetic sequences having the same functional capabilities.

[0374] In certain embodiments, the intracellular domain comprises an intracellular signaling domain. Examples of intracellular domains include fragments or domains from one or more molecules or receptors including but not limited to the following: TCR, CD3ζ, CD3γ, CD3δ, CD3ε, CD86, common FcRγ, FcRβ (FcεRib), CD79a, CD79b, FcγR11a, DAP10, DAP12, T cell receptor (TCR), CD2, CD8, CD27, CD28, 4-1BB (CD137), OX9, OX40, CD30, CD40, PD-1, ICOS, KIR family proteins, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, ligand that specifically binds to CD83, CD5, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD127, CD160, CD19, CD4, CD8α, CD8β, IL2Rβ, IL2Rγ, IL7Rα, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD1Id, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD lib, ITGAX, CD11c, ITGBl, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, Toll-like receptor 1 (TLR1), TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, syk family tyrosine kinases (Syk, ZAP 70, etc.), src family tyrosine kinases (Lck, Fyn, Lyn, etc.), other co-stimulatory molecules described herein, any derivatives, variants or fragments thereof, any synthetic sequences of co-stimulatory molecules having the same functional capabilities, and any combinations thereof.

[0375] In some embodiments, the intracellular signaling domain comprises an intracellular domain selected from the following or a variant thereof: the intracellular domain of human CD2, CD3ζ chain (CD3ζ), FcγRIII, FcsRI, the cytoplasmic tail of an Fc receptor, a cytoplasmic receptor carrying an immunoreceptor tyrosine - based activation motif (ITAM), TCRζ, FcRγ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, and the cytoplasmic signaling domain of CD66d. In some embodiments, the intracellular signaling domain comprises the CD3ζ intracellular signaling domain.

[0376] Additional examples of intracellular domains include, but are not limited to, the intracellular signaling domains of various other immune signaling receptors of several types, including but not limited to first, second, and third generation T - cell signaling proteins, including CD3, B7 family co - stimulatory receptors, and tumor necrosis factor receptor (TNFR) superfamily receptors. Additionally, the intracellular signaling domain can include signaling domains used by NK and NKT cells, such as the signaling domains of NKp30 (B7 - H6) and DAP 12, NKG2D, NKp44, NKp46, DAP10, and CD3z.

[0377] The intracellular signaling domain of the CAR suitable for the present invention includes any desired signaling domain that transduces a signal in response to the activation of the CAR (i.e., activation by an antigen and a dimerizing agent). In some embodiments, unique and detectable signals include, for example, an increase in one or more cytokines produced by the cell; a change in the transcription of a target gene; a change in protein activity; a change in cell behavior (e.g., cell death); cell proliferation; cell differentiation; cell survival; and / or regulation of a cell signaling response. In some embodiments, the intracellular signaling domain comprises a DAP10 / CD28 - type signaling chain. In some embodiments, the intracellular signaling domain is not covalently attached to the membrane - bound CAR but diffuses within the cytoplasm.

[0378] The intracellular signaling domain of the CAR suitable for the present invention includes an intracellular signaling polypeptide containing an immunoreceptor tyrosine - based activation motif (ITAM). In some embodiments, the intracellular signaling domain comprises at least one, at least two, at least three, at least four, at least five, or at least six ITAM motifs as described below. In some embodiments, the ITAM motifs are repeated twice in the intracellular signaling domain, wherein the first and second ITAM motif instances are spaced 6 to 8 amino acids apart from each other. In one embodiment, the intracellular signaling domain of the subject CAR comprises 3 ITAM motifs. In some embodiments, the intracellular signaling domain includes the signaling domain of a human immunoglobulin receptor containing an immunoreceptor tyrosine - based activation motif (ITAM), such as but not limited to FcγRI, FcγRIIA, FcγRIIC, FcγRIIIA, FcRL5.

[0379] A suitable intracellular signaling domain can be an ITAM - containing portion derived from a polypeptide containing an ITAM motif. For example, a suitable intracellular signaling domain can be an ITAM - containing domain from any protein containing an ITAM motif. Thus, a suitable intracellular signaling domain does not need to contain the entire sequence of the whole protein from which it is derived. Examples of suitable polypeptides containing an ITAM motif include but are not limited to: DAP12, FCER1G (Fcε receptor I gamma chain), CD3D (CD3 delta), CD3E (CD3 epsilon), CD3G (CD3 gamma), CD3Z (CD3 zeta), and CD79A (antigen receptor complex - associated protein alpha chain).

[0380] In one embodiment, the intracellular signaling domain is derived from DAP12 (also known as TYROBP; TYRO protein tyrosine kinase binding protein; KARAP; PLOSL; DNAX-activating protein 12; KAR-associated protein; TYRO protein tyrosine kinase-binding protein; killer activating receptor-associated protein; killer-activating receptor-associated protein; etc.). In one embodiment, the intracellular signaling domain is derived from FCER1G (also known as FCRG; Fcε receptor I gamma chain; Fc receptor gamma chain; fc-εRI-γ; fcRγ; fceR1γ; high-affinity immunoglobulin epsilon receptor subunit gamma; immunoglobulin E receptor high-affinity gamma chain; etc.). In one embodiment, the intracellular signaling domain is derived from the T cell surface glycoprotein CD3 delta chain (also known as CD3D; CD3-δ; T3D; CD3 antigen delta subunit; CD3δ; CD3d antigen, delta polypeptide (TiT3 complex); OKT3δ chain; T cell receptor T3δ chain; T cell surface glycoprotein CD3δ chain; etc.). In one embodiment, the intracellular signaling domain is derived from the T cell surface glycoprotein CD3 epsilon chain (also known as CD3e, T cell surface antigen T3 / Leu-4 epsilon chain, T cell surface glycoprotein CD3 epsilon chain, AI504783, CD3, CD3ε, T3e, etc.). In one embodiment, the intracellular signaling domain is derived from the T cell surface glycoprotein CD3 gamma chain (also known as CD3G, T cell receptor T3 gamma chain, CD3-γ, T3G, gamma polypeptide (TiT3 complex), etc.). In one embodiment, the intracellular signaling domain is derived from the T cell surface glycoprotein CD3 zeta chain (also known as CD3Z, T cell receptor T3 zeta chain, CD247, CD3-ζ, CD3H, CD3Q, T3Z, TCRZ, etc.). In one embodiment, the intracellular signaling domain is derived from CD79A (also known as B cell antigen receptor complex-associated protein alpha chain; CD79a antigen (immunoglobulin-related alpha); MB-1 membrane glycoprotein; Ig-α; membrane-bound immunoglobulin-related protein; surface IgM-related protein; etc.). In one embodiment, the intracellular signaling domain of the CAR suitable for the present disclosure comprises a DAP10 / CD28-type signaling chain. In one embodiment, the intracellular signaling domain of the subject CAR suitable for the present disclosure comprises a ZAP70 polypeptide.In some embodiments, the intracellular signaling domain includes the cytoplasmic signaling domain of TCRζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, or CD66d. In one embodiment, the intracellular signaling domain in the CAR includes the cytoplasmic signaling domain of human CD3ζ.

[0381] While it is generally possible to use the entire intracellular signaling domain, it is not necessary to use the entire chain in many cases. In terms of using a truncated portion of the intracellular signaling domain, such a truncated portion can be used in place of the entire chain as long as it transduces an effector function signal. The intracellular signaling domain includes any truncated portion of the intracellular signaling domain that is sufficient to transduce an effector function signal.

[0382] The intracellular signaling domains described herein can be combined with any co-stimulatory signaling domain described herein, any antigen-binding domain described herein, any transmembrane domain described herein, or any other domain that can be included in a CAR described herein. In some embodiments, the intracellular domain of the CAR contains a dual signaling domain. The dual signaling domain can include fragments or domains from any of the molecules described herein. In some embodiments, the intracellular domain contains a 4-1BB co-stimulatory domain and a CD3ζ signaling domain; a CD28 co-stimulatory domain and a CD3ζ signaling domain; a CD2 co-stimulatory domain and a CD3ζ signaling domain. In some embodiments, the intracellular domain of the CAR includes any portion of a co-stimulatory molecule, such as at least one signaling domain from CD3, CD27, CD28, ICOS, 4-1BB, PD-1, T cell receptor (TCR), any derivative or variant thereof, any synthetic sequence having the same functional capacity, and any combination thereof.

[0383] In addition, variant intracellular signaling domains suitable for the subject CARs are known in the art. The YMFM motif is found in ICOS and is an SH2-binding motif that recruits both the p85 and p50α subunits of PI3K to enhance AKT signaling. In one embodiment, a CD28 intracellular domain variant can be generated to contain the YMFM motif.

[0384] In one embodiment, the intracellular domain of the subject CAR contains a CD3ζ intracellular signaling domain having the amino acid sequence shown in SEQ ID NO:50 or SEQ ID NO:51, and the amino acid sequence can be encoded by a nucleic acid sequence comprising the nucleotide sequence shown in SEQ ID NO:52 or SEQ ID NO:53, respectively.

[0385] Permissible variations of the intracellular domain will be known to those skilled in the art while retaining its specific activity. In some embodiments, the intracellular domain comprises an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to any of the amino acid sequences shown in SEQ ID NO: 50 or 51. In some embodiments, the intracellular domain is encoded by a nucleic acid sequence comprising a nucleotide sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to any of the nucleotide sequences shown in SEQ ID NO: 52 or 53. 5. Co-stimulatory domain

[0386] In some embodiments, the intracellular domain comprises a co-stimulatory signaling domain and an intracellular signaling domain. In certain embodiments, the intracellular domain comprises a co-stimulatory signaling domain. In one embodiment, the intracellular domain of the CAR comprises a co-stimulatory signaling domain selected from: portions of the signaling domains of proteins from the TNFR superfamily, CD27, CD28, 4-1BB (CD137), OX40 (CD134), PD-1, CD7, LIGHT, CD83L, DAP10, DAP12, CD27, CD2, CD5, ICAM-1, LFA-1, Lck, TNFR-I, TNFR-II, Fas, CD30, CD40, ICOS (CD278), NKG2C, B7-H3 (CD276), and the intracellular domain of the killer cell immunoglobulin-like receptor (KIR), any derivative or variant thereof, any synthetic sequence having the same functional capacity, and any combination thereof.

[0387] In some embodiments, the co-stimulatory domain comprises one or more of the following: the co-stimulatory domain of a protein selected from the proteins in the TNFR superfamily, CD28, 4-1BB (CD137), OX40 (CD134), PD-1, CD7, LIGHT, CD83L, DAP10, DAP12, CD27, CD2, CD5, ICAM-1, LFA-1, Lck, TNFR-I, TNFR-II, Fas, CD30, CD40, ICOS (CD278), NKG2C, B7-H3 (CD276), and the intracellular domain of a killer cell immunoglobulin-like receptor (KIR), or a variant thereof. In some embodiments, the co-stimulatory domain comprises one or more co-stimulatory domains of a protein selected from CD28, 4-1BB (CD137), OX40 (CD134), CD27, CD2, or a combination thereof. In some embodiments, the co-stimulatory signaling domain comprises the 4-1BB co-stimulatory domain. In some embodiments, the co-stimulatory signaling domain comprises the CD2 co-stimulatory domain. In some embodiments, the co-stimulatory signaling domain comprises the CD28 co-stimulatory domain.

[0388] In some embodiments, the co-stimulatory domain comprises an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to any of the amino acid sequences shown in SEQ ID NO: 54, 57, 59, 61, 64, 66, 68, or 70. In some embodiments, the intracellular domain is encoded by a nucleic acid sequence comprising a nucleotide sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to any of the nucleotide sequences shown in SEQ ID NO: 55, 56, 58, 60, 62, 63, 65, 67, 69, or 71.

[0389] In one embodiment, the intracellular domain of the subject CAR comprises an ICOS co-stimulatory domain and a CD3ζ intracellular signaling domain. In one embodiment, the intracellular domain of the subject CAR comprises a CD28 co-stimulatory domain and a CD3ζ intracellular signaling domain. In one embodiment, the intracellular domain of the subject CAR comprises a CD28 YMFM variant co-stimulatory domain and a CD3ζ intracellular signaling domain. In one embodiment, the intracellular domain of the subject CAR comprises a CD27 co-stimulatory domain and a CD3ζ intracellular signaling domain. In one embodiment, the intracellular domain of the subject CAR comprises an OX40 co-stimulatory domain and a CD3ζ intracellular signaling domain. In one exemplary embodiment, the intracellular domain of the subject CAR comprises a 4-1BB co-stimulatory domain and a CD3ζ intracellular signaling domain. In one exemplary embodiment, the intracellular domain of the subject CAR comprises a CD2 co-stimulatory domain and a CD3ζ intracellular signaling domain. B. Additional antigen-binding polypeptides

[0390] In some embodiments, the modified T cells express an antigen-binding polypeptide, a cell surface receptor ligand, or a polypeptide that binds a tumor antigen. In some instances, the antigen-binding domain comprises an antibody that recognizes a cell surface protein or receptor expressed on a tumor cell. In some instances, the antigen-binding domain comprises an antibody that recognizes a tumor antigen. In some instances, the antigen-binding domain comprises a full-length antibody or an antigen-binding fragment thereof, Fab, F(ab)2, monospecific Fab2, bispecific Fab2, trispecific Fab2, single-chain variable fragment (scFv), diabody, triabody, minibody, V-NAR, or VhH. C. Cell surface receptor ligands

[0391] In some embodiments, the lentiviral or retroviral vector of the present disclosure further comprises a nucleic acid encoding a cell surface receptor ligand. In some instances, the ligand binds to a cell surface receptor expressed on a tumor cell. In some cases, the ligand comprises a wild-type protein or a variant thereof that binds to a cell surface receptor. In some instances, the ligand comprises a full-length protein or a functional fragment thereof that binds to a cell surface receptor. In some cases, the functional fragment comprises about 90%, about 80%, about 70%, about 60%, about 50%, or about 40% of the length compared to the full-length form of the protein, but retains binding to the cell surface receptor. In some cases, the ligand is a de novo engineered protein that binds to a cell surface receptor. Exemplary ligands include, but are not limited to, epidermal growth factor (EGF), platelet-derived growth factor (PDGF), or Wnt3A. D. Tumor antigens

[0392] In some embodiments, the lentiviral or retroviral vectors of the present disclosure further comprise a nucleic acid encoding a polypeptide that binds to a tumor antigen. In some embodiments, the tumor antigen is associated with a hematologic malignancy. Exemplary tumor antigens include, but are not limited to, CD19, CD20, CD22, CD33 / IL3Ra, ROR1, mesothelin, c-Met, PSMA, PSCA, folate receptor alpha, folate receptor beta, EGFRvIII, GPC2, Tn-MUC1, GDNF family receptor alpha-4 (GFRa4), fibroblast activation protein (FAP), and IL13Ra2. In some instances, the tumor antigens include CD19, CD20, CD22, BCMA, CD37, mesothelin, PSMA, PSCA, Tn-MUC1, EGFR, EGFRvIII, c-Met, HER1, HER2, CD33, CD133, GD2, GPC2, GPC3, NKG2D, KRAS, or WT1. In some instances, the polypeptide is a ligand of the tumor antigen, such as a full-length protein that binds to the tumor antigen, a functional fragment thereof, or a de novo engineered ligand that binds to the tumor antigen. In some instances, the polypeptide is an antibody that binds to the tumor antigen. E. Engineered T cell receptors

[0393] In some embodiments, the antigen-binding domain of the CAR described herein can be transplanted into one or more constant domains of a T cell receptor (“TCR”) chain (e.g., TCRα or TCRβ chain) to generate a chimeric TCR. The chimeric TCR can signal through the TCR complex upon antigen binding. For example, an scFv as disclosed herein can be transplanted into the constant domain of a TCR chain, or at least a portion of the extracellular constant domain, transmembrane domain. As another example, an antibody fragment (e.g., a VL domain as described herein) can be transplanted into the constant domain of the TCRα chain. Such chimeric TCRs can be generated, for example, by methods known in the art (e.g., Aggen et al., Gene Ther. April 2012; 19(4):365-74). F. Switch receptors and dominant negative receptors

[0394] In one aspect, the lentiviral or retroviral vector of the present disclosure further comprises a nucleic acid encoding a dominant negative receptor, a switch receptor, or a combination thereof. In some embodiments, the lentiviral or retroviral vector described herein comprises a chimeric antigen receptor (CAR) and / or a dominant negative receptor. In some embodiments, the lentiviral or retroviral vector comprises a CAR and / or a switch receptor. In some embodiments, the lentiviral or retroviral vector described herein comprises an engineered TCR and a switch receptor. In some embodiments, the lentiviral or retroviral vector described herein comprises an engineered TCR and a dominant negative receptor. In some embodiments, the lentiviral or retroviral vector described herein comprises a KIR and a switch receptor. In some embodiments, the lentiviral or retroviral vector described herein further comprises a KIR and a dominant negative receptor. 1. Switch receptor

[0395] The present disclosure provides a rapid and efficient manufacturing process for engineering modified immune cells that comprise a CAR or an exogenous TCR and / or a switch receptor. In some embodiments, the CAR, TCR, and / or switch receptor is encoded by one or more nucleic acids. In some embodiments, the lentiviral or retroviral vector disclosed herein comprises one or more nucleic acid sequences encoding a CAR, a TCR, and / or a switch receptor. In some embodiments, the nucleic acid sequence encoding the CAR is operably linked to the nucleic acid sequence encoding the switch receptor. In some embodiments, the switch receptor can enhance the efficiency of the CAR or the cell expressing the CAR.

[0396] Tumor cells generate an immunosuppressive microenvironment that protects them from immune recognition and elimination. This immunosuppressive microenvironment can limit the effectiveness of immunosuppressive therapies such as CAR-T or TCR-T cell therapies. For example, the secreted cytokine transforming growth factor β (TGFβ) directly inhibits the function of cytotoxic T cells and additionally induces the formation of regulatory T cells to further suppress the immune response. In the context of prostate cancer, T cell immunosuppression due to TGFβ has been previously demonstrated. To reduce the immunosuppressive effects of TGF on immune cells, immune cells can be modified to express an engineered TGFβR that includes an extracellular ligand-binding domain of TGFβR fused to an intracellular signaling domain of, for example, interleukin-12 receptor (IL12R) (TGFβR-IL12R). Thus, modified immune cells containing a switch receptor can bind negative signal transduction molecules in the microenvironment of the modified immune cells and convert the negative signal transduction signal of inhibitory molecules that may be present on the modified immune cells into a positive signal that stimulates the modified immune cells. The switch receptors of the present disclosure can be designed to reduce the effects of negative signal transduction molecules by including an intracellular domain associated with a positive signal or to convert a negative signal into a positive signal.

[0397] As used herein, the term "switch receptor" refers to a molecule that is designed to reduce the effects of negative signal transduction molecules on the modified immune cells of the present invention. A switch receptor includes: a first domain derived from a first polypeptide associated with a negative signal (signal transduction that suppresses or inhibits cell or T cell activation); and a second domain derived from a second polypeptide associated with a positive signal (signal transduction signal that stimulates a cell or T cell). In some embodiments, the protein associated with the negative signal is selected from CTLA4, PD-1, TGFβRII, BTLA, VSIG3, VSIG8, and TIM-3. In some embodiments, the protein associated with the positive signal is selected from CD28, 4-1BB, IL12Rβ1, IL12Rβ2, CD2, ICOS, and CD27.

[0398] In one embodiment, the first domain comprises at least a portion of the extracellular domain of a first polypeptide associated with a negative signal, and the second domain comprises at least a portion of the intracellular domain of a second polypeptide associated with a positive signal. Thus, the switch receptor comprises an extracellular domain associated with a negative signal fused to an intracellular domain associated with a positive signal. In some embodiments, the switch receptor comprises an extracellular domain, a transmembrane domain, and an intracellular domain of a signaling protein associated with a positive signal, where the extracellular domain is associated with a negative signal. In some embodiments, the transmembrane domain of the switch receptor is selected from the transmembrane domain of a protein associated with a negative signal or the transmembrane domain of a protein associated with a negative signal. In some embodiments, the transmembrane domain of the switch receptor is selected from the transmembrane domains of proteins selected from CTLA4, PD-1, VSIG3, VSIG8, TGFβRII, BTLA, TIM-3, CD28, 4-1BB, IL12Rβ1, IL12Rβ2, CD2, ICOS, and CD27.

[0399] In some embodiments, the switch receptor is selected from PD-1-CD28, PD-1A132L-CD28, PD-1-CD27, PD-1A132L-CD27, PD-1-4-1BB, PD-1A132L-4-1BB, PD-1-ICOS, PD-1A132L-ICOS, PD-1-IL12Rβ1, PD-1A132L-IL12Rβ1, PD-1-IL12Rβ2, PD-1A132L-IL12Rβ2, VSIG3-CD28, VSIG8-CD28, VSIG3-CD27, VSIG8-CD2...

Claims

1. A method for manufacturing an engineered immune cell population, the method comprising: (a) Enriched lymphocyte populations, immune cell populations, or CD4 + and CD8 + cell populations from blood obtained from a subject; (b) Mix the lymphocyte population, the immune cell population, or the CD4 + and CD8 + cell population with one or more buffer solutions; and (c) Transfecting the lymphocyte population, the immune cell population, or the CD4 + and CD8 + cell population with a modifier in an effective dose; thereby generating a modified lymphocyte population, a modified immune cell population, or a modified CD4 + and CD8 + cell population; wherein steps 1(a)-(c) are carried out within 24 hours.

2. The method according to claim 1, wherein prior to said enriching said population of immune cells or said CD4 + and CD8 + cell population, said blood is separated by apheresis into a plasma fraction, a mononuclear cell-containing layer, a platelet layer, and red blood cells to produce an apheresis product selected from the group consisting of red blood cell apheresis, thrombus apheresis, coagulation cell apheresis, white blood cell apheresis, stem cells, plasma apheresis, and platelet apheresis.

3. The method according to claim 1 or 2, wherein the population of immune cells or the population of CD4 + and CD8 + cells is enriched by apheresis, elutriation or gradient centrifugation.

4. A method for manufacturing an engineered immune cell population, the method comprising: (a) Enrich lymphocyte populations, immune cell populations, or CD4 + and CD8 + cell populations from a donor leukapheresis product; (b) Mix the lymphocyte population, the immune cell population or the CD4 + and CD8 + cell population with one or more buffer solutions; and (c) Transfecting the lymphocyte population, the immune cell population, or the CD4 + and CD8 + cell population with a modifying agent in an effective dose, thereby generating a lymphocyte population, a modified immune cell population, or a modified CD4 + and CD8 + cell population wherein steps 1(a)-(c) are carried out within 24 hours.

5. A method for manufacturing an engineered eukaryotic cell population, the method comprising: (a) obtaining a population of eukaryotic donor cells from a subject; (b) mixing the population of eukaryotic donor cells with one or more buffer solutions; and (c) transfecting the population of eukaryotic donor cells with an effective dose of a modifier to produce a modified population of eukaryotic donor cells, wherein steps 1(a)-(c) are carried out on the same day.

6. The method according to any one of claims 1-6, wherein before the transfection step (c), the immune cell population, the CD4 + and CD8 + cell population or the eukaryotic donor cell population is stimulated and / or activated with one or more stimulants.

7. The method according to any one of claims 1-6, wherein the modifier is selected from small molecule agents, biological agents, therapeutic agents, proteins, peptides, protein therapeutic agents, peptide therapeutic agents, chimeric antigen receptors, heterologous T cell receptors,, viral vectors, vectors, retroviral vectors, lentiviral vectors, adenoviral vectors, and adeno-associated viral vectors.

8. The method according to any one of claims 1-7, wherein the modifier is: (a) selected from a retroviral vector, a lentiviral vector, an adenoviral vector, or an adeno-associated viral vector; (b) a lentiviral vector; or (c) a retroviral vector.

9. The method according to any one of claims 1-8, wherein the population of immune cells, the CD4 + and CD8 + cell population or the population of eukaryotic donor cells is transfected with an effective dose of a lentiviral vector or a retroviral vector.

10. The method according to claim 9, wherein the lentiviral vector or the retroviral vector comprises a nucleic acid sequence encoding a chimeric antigen receptor (CAR), an engineered T cell receptor (TCR), and / or a nucleic acid sequence encoding a polypeptide that enhances the function of immune cells or a functional derivative thereof or a polypeptide that produces a therapeutic protein.

11. The method according to any one of claims 1-10, wherein the population of immune cells or the population of eukaryotic donor cells is selected from mononuclear cells, lymphocyte-rich cells, B lymphocytes, T lymphocytes, CD4 + T lymphocytes, CD8 + T lymphocytes, dendritic cells, monocytes, natural killer (NK) cells, natural killer T (NKT) cells, regulatory T cells, CD4 + helper T cells, CD8 + cytotoxic T lymphocytes (CTL), CD62L + cells, CD27 + cells, CCR7 + cells, CD45RO - cells, CD45RA + cells, neutrophils, basophils, eosinophils, megakaryocytes, stem cells, hematopoietic stem cells (HSC), hematopoietic progenitor cells (HPC), CD34 + cells, CD34 + peripheral blood stem cells, lymphokine-activated killer cells (LAK), tumor-infiltrating lymphocytes (TIL), mesenchymal stem cells, mast cells, monocytes, macrophages, neutrophils, basophils, eosinophils, dendritic cells, megakaryocytes, and combinations thereof.

12. The method according to any one of claims 1-11, wherein the concentration of the immune cell population, the CD4 + and CD8 + cell population or the eukaryotic donor cell population is: (a) At least about 0.7×10 7 , at least about 0.8×10 7 , at least about 0.9×10 7 , at least about 1×10 7 , at least about 2×10 7 , at least about 4×10 7 , at least about 6×10 7 , at least about 8×10 7 , at least about 1×10 8 or at least about 5×10 8 cells / mL; (b) from about 0.5×10 6 cells / mL to about 4×10 6 cells / mL; (c) from about 0.5×10 6 cells / mL to about 1×10 8 cells / mL; or (d) From about 4.0×10 6 cells / mL to about 1×10 8 cells / mL.

13. The method according to any one of claims 1-12, wherein the transfection is: (a) selected from viral transfection, transduction, non-viral transfection, and a combination of viral and non-viral transfection; (b) selected from electroporation, laser beam, gene injection, sonoporation, magnetofection, metal-coated nanoparticles, magnetically conjugated adeno-associated virus, particle / microparticle-mediated transfection, liposome transfection, lipid-based transfection, anionic liposomes, cationic liposome-mediated transfection, cationic polymers, polymer encapsulation, peptide-mediated transfection, calcium phosphate, dendrimers, flow transfection, photoporation, solvent poration, transient cell membrane disruption, deformation, extrusion, stretching, constriction, weakening, elongation, thinning, and biolistic particle delivery systems and combinations thereof; (c) electroporation of viral particles; (d) electroporation and viral transfection (transduction); (e) viral transfection and lipid-based transfection; or (f) viral transfection and liposome-based transfection.

14. The method according to any one of claims 1-13, wherein: (a) Before or after transfection, not activating the modified immune cell population, the modified CD4 + and CD8 + cell population or the modified eukaryotic donor cell population with one or more stimulants; and (b) After transfection, the modified immune cell population, the modified CD4 + and CD8 + cell population or the modified eukaryotic donor cell population are not expanded ex vivo.

15. The method according to any one of claims 1-13, the method further comprising stimulating and activating the modified immune cell population, the modified CD4 + and CD8 + cell population or the modified eukaryotic donor cell population with one or more stimulants to produce an activated modified immune cell population, an activated modified CD4 + and CD8 + cell population or an activated modified eukaryotic cell population.

16. The method according to claim 15, the method further comprising expanding the activated lymphocyte population, the activated modified immune cell population, the activated modified mononuclear cell population, the activated modified CD4 + and CD8 + cell population or the activated modified eukaryotic donor cell population for a predetermined time to produce an engineered lymphocyte population, an engineered immune cell population, an engineered CD4 + and CD8 + cell population or an engineered eukaryotic donor cell population.

17. The method according to claim 16, wherein the amplification step is carried out under the following conditions: (a) under shaking conditions or rotating conditions; (b) in a closed system; (c) using a serum-free medium; and / or (d) in the presence of one or more stimulators.

18. The method according to 16 or 17, wherein the activated modified immune cell population, the activated modified CD4 + and CD8 + cell population or the activated modified eukaryotic donor cell population is expanded by at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold or at least about 25-fold.

19. The method according to any one of claims 1-18, the method further comprising harvesting the modified lymphocyte population, the modified immune cell population, the modified CD4 + and CD8 + cell population or the modified eukaryotic donor cell population for cryopreservation or administration.

20. The method according to claim 19, wherein harvesting comprises selecting and enriching the engineered lymphocytes, the engineered immune cells, the engineered CD4 + and CD8 + cells or the engineered donor eukaryotic cells.

21. The method according to claim 19 or 20, wherein harvesting further comprises formulating the engineered lymphocytes, the engineered immune cells, the engineered CD4 + and CD8 + cells or the engineered donor eukaryotic cells for cryopreservation or administration to a subject in need.

22. The method according to any one of claims 16 - 21, wherein the predetermined time is: (a) less than about 24 hours, less than about 30 hours, less than about 48 hours, less than about 72 hours, less than about 96 hours, or less than about 120 hours; (b) less than about 0.5 hours, less than about 1 hour, less than about 2 hours, less than about 3 hours, less than about 4 hours, less than about 5 hours, less than about 6 hours, less than about 7 hours, less than about 8 hours, less than about 9 hours, less than about 10 hours, less than about 11 hours, less than about 12 hours, less than about 13 hours, less than about 14 hours, less than about 15 hours, less than about 16 hours, less than about 17 hours, less than about 18 hours, less than about 19 hours, less than about 20 hours, less than about 21 hours, less than about 22 hours, or less than about 23 hours; or (c) about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, or more days.

23. The method according to any one of claims 1-22, wherein the time from enriching and / or obtaining the lymphocyte population, the immune cell population, the CD4 + and CD8 + cell population or the eukaryotic donor cell population to harvesting the engineered immune cells, the engineered CD4 + and CD8 + cells or the engineered eukaryotic donor cells is: (a) about 72 hours or less; (b) from about 18 hours to about 72 hours, from about 18 hours to about 36 hours, from about 18 hours to about 24 hours, from about 24 hours to about 72 hours, from about 24 hours to about 36 hours, or from about 36 hours to about 72 hours; (c) less than about 2 hours, less than about 3 hours, less than about 4 hours, less than about 5 hours, less than about 6 hours, less than about 7 hours, less than about 8 hours, less than about 9 hours, less than about 10 hours, less than about 11 hours, less than about 12 hours, less than about 13 hours, less than about 14 hours, less than about 15 hours, less than about 16 hours, less than about 17 hours, less than about 18 hours, less than about 19 hours, less than about 20 hours, less than about 21 hours, less than about 22 hours, or less than about 23 hours; (d) about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, or more days; or (e) about 1 day, about 3 days, about 4 days, about 5 days, or about 6 days.

24. The method according to any one of claims 1 - 23, wherein the electroporation step, the activation step, and / or the amplification step are carried out in a closed system, a semi - closed, and / or a functionally closed system.

25. The method according to claim 24, wherein the closed system is selected from a closed bag system, an automated closed cell sample processing system, and a bioreactor.

26. The method according to any one of claims 6 - 25, wherein: (a) the one or more stimulants are selected from agonistic antibodies, cytokines, recombinant costimulatory molecules, anti - CD3 antibodies or fragments thereof, anti - CD28 antibodies or fragments, small molecule inhibitors, and / or combinations thereof; (b) The one or more stimulants are cytokines selected from the following: interleukin-2 (IL-2), interleukin-3 (IL-3), interleukin-6 (IL-6), interleukin-7 (IL-7), interleukin-7 receptor (IL-7R), interleukin-11 (IL-11), interleukin-12 (IL-12), interleukin-15 (IL-15), interleukin-15 receptor (IL-15R), interleukin-18 (IL-18), interleukin-18 receptor (IL-18R), interleukin-21 (IL-21), granulocyte macrophage colony-stimulating factor, alpha, beta or gamma interferon, erythropoietin, and combinations thereof.

27. The method according to claim 26, wherein the one or more stimulants are conjugated to beads or nanostructures.

28. The method according to claim 26 or 27, wherein: (a) The one or more stimulants are anti-CD3 and anti-CD28 antibodies or fragments thereof; (b) The one or more stimulants are anti-CD3 and anti-CD28 antibodies or fragments thereof and one or more cytokines; (b) The nanostructure is a nanomatrix; (c) The cytokine is selected from IL-2, IL-7, IL-6, IL-15, IL-15Ra or IL-21; (d) The cytokine is selected from IL-15 and IL-7, IL-7 and IL-21, IL-7 and IL-2, IL-15 and IL-2, IL-7, IL-15 and IL-21, IL-15 and IL-15Ra, or IL-7, IL-15 and IL-15Ra; and / or (e) The one or more stimulants are a nanomatrix and one or more cytokines.

29. The method according to claim 28, wherein the nanomatrix (a) comprises a matrix with mobile polymer chains and anti-CD3 and anti-CD28 antibodies or fragments thereof; or (c) has a size of 1 to 500 nm.

30. The method according to any one of claims 9-29, wherein the effective dose of the retroviral vector or the lentiviral vector comprises an MOI of about 0.01, about 0.02, about 0.03, about 0.04, about 0.05, about 0.06, about 0.07, about 0.08, about 0.09, about 0.1, about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1.0, about 1.25, about 1.5, about 2.0, about 3.0, about 4.0 or about 5.

0.

31. The method according to any one of claims 9-30, wherein the effective dose of the retroviral vector or the lentiviral vector comprises: (a) about 2 ul of the lentiviral vector with an MOI of about 0.08; (b) about 5 ul of the lentiviral vector with an MOI of about 0.2; or (c) about 10 ul of the lentiviral vector with an MOI of about 0.

4.

32. The method according to any one of claims 9 - 31, wherein the lentiviral vector is based on a virus selected from the group consisting of: retrovirus, alpharetrovirus, betaretrovirus, gammaretrovirus, deltaretrovirus, and epsilonretrovirus.

33. The method according to any one of claims 9 - 32, wherein the lentiviral vector is based on human immunodeficiency virus (HIV), equine infectious anemia virus (EIAV), Visna - Maedi virus (VMV), caprine arthritis - encephalitis virus (CAEV), feline immunodeficiency virus (FIV), bovine immunodeficiency virus (BIV), Visna virus, and simian immunodeficiency virus (SIV).

34. The method according to any one of claims 9 - 33, wherein the lentiviral vector is pseudotyped with an envelope glycoprotein (Env) from a virus selected from the group consisting of: murine leukemia virus (MLV), vesicular stomatitis virus (VSV) Indiana strain, VSV New Jersey strain, Kokobera virus, Kindunya virus, Bouri virus, spring viremia of carp virus (SVCV), sigma virus, infectious hematopoietic necrosis virus (IHNV), Mokola virus, rabies virus CVS virus, Isfahan virus, Araguari virus, Calchaqui virus, Jurona virus, La Jolla virus, Maraba virus, feline endogenous retrovirus (RD114) envelope protein, Perinet virus, Yug Bogdanovac virus, prototype foamy virus (PFV), and gibbon ape leukemia virus (GaLV).

35. The method according to any one of claims 9 - 31, wherein the lentiviral vector is pseudotyped with an envelope glycoprotein (Env) selected from the group consisting of: VSV Indiana strain, VSV New Jersey strain, and Kokobera virus.

36. The method according to any one of claims 9 - 35, wherein the lentiviral vector comprises a heterologous viral envelope protein (Env) selected from the group consisting of: VSV - G of the Indiana strain, VSV - G of the New Jersey strain, Kokobera virus envelope protein, Isfahan virus envelope protein, Kindunya virus envelope protein, Bouri virus envelope protein, murine leukemia virus (MLV) envelope glycoprotein, SVCV virus envelope protein, and variants thereof.

37. The method according to any one of claims 9 - 36, wherein the lentiviral vector comprises a nucleotide sequence encoding the VSV - G envelope protein or a variant of the VSV G protein.

38. The method according to any one of claims 9 - 37, wherein the lentiviral vector is a lentiviral particle.

39. The method according to any one of claims 10 - 38, wherein the CAR comprises an antigen - binding domain, a transmembrane domain, a co - stimulatory domain, and an intracellular domain, and wherein the antigen - binding domain is selected from (a) a full - length antibody or an antigen - binding fragment thereof, (b) Fab, (c) single - chain variable fragment (scFv), and (d) single - domain antibody.

40. The method according to claim 39, wherein the antigen-binding domain specifically binds to a target antigen selected from the following: CD4, CD5, CD19, CD20, CD22, CD79b, CD79a, CD33, CD30, CD70, BCMA, GPC2, CD123, CD133, EGFR, EGFRvIII, mesothelin, HER2, PSMA, PSCA, FAP, CEA, GD2, IL-13Ra2, glypican-3, CIAX, LI-CAM, CA 125, CTAG1B, TnMUC1, mucin 1, and folate receptor alpha (FRa), GFRα-4, NYESO, WT1, (AFP) / HLA-A2, AXL, B7-H3, CA-IX, CD3, CD7, CD8, CD38, CD44v6, CD80, CD86, CD117, CD147, CD276, CEA, Claudin 18.2, c-Met, DLL3, DR5, EpCAM, EphA2, FAP, folate-binding protein (FBP), glycolipid F77, glypican-3 (GPC3), glypican-2, HLA-A2, ICAM1, IL3Ra, LAGE-I, Lewis Y, LMP1 (EBV), MAGE-A1, MAGE-A3, MAGE-A4, Melan A, MG7 (glycosylated CEA), MMP, MUC1, connexin 4 / FAP, NKG2D ligand, MIC-A, MIC-B, ULBP 1 to 6, NY-ESO-1, P16, PD-L1, ROR1, ROR2, TIM-3, TM4SF1, VEGFR2, and any combination thereof.

41. The method according to claim 39 or 40, wherein the CAR transmembrane domain is selected from artificial hydrophobic sequences; transmembrane domains of type I transmembrane proteins, the α, β, or ζ chains of the T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, OX40 (CD134), 4-1BB (CD137), ICOS (CD278), or CD154; and transmembrane domains derived from killer cell immunoglobulin-like receptors (KIR).

42. The method according to any one of claims 39-41, wherein the co-stimulatory domain is the intracellular domain of a protein selected from the following: TNFR superfamily proteins, CD27, CD28, 4-1BB (CD137), OX40 (CD134), PD-1, CD7, LIGHT, CD83L, DAP10, DAP12, CD27, CD2, CD5, ICAM-1, LFA-1, Lck, TNFR-I, TNFR-II, Fas, CD30, CD40, ICOS (CD278), NKG2C, B7-H3 (CD276), and killer cell immunoglobulin-like receptor (KIR).

43. The method according to any one of claims 39-42, wherein the intracellular signaling domain comprises an intracellular domain selected from the following: the intracellular domain of human CD3ζ chain (CD3ζ), FcγRIII, FcsRI, the cytoplasmic tail of an Fc receptor, a cytoplasmic receptor carrying an immunoreceptor tyrosine-based activation motif (ITAM), TCRζ, FcRγ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, and the cytoplasmic signaling domain of CD66d.

44. The method according to any one of claims 39-43, wherein the CAR further comprises a hinge region.

45. A method for delivering a nucleic acid encoding a chimeric antigen receptor (CAR), an engineered T cell receptor, or a therapeutic protein to a cell, the method comprising introducing a transfer plasmid into the cell, the transfer plasmid comprising: (a) a polynucleotide sequence encoding at least one heterologous viral envelope protein engineered by the method according to any one of claims 32-37; (b) a polynucleotide sequence encoding at least one retroviral rev protein; (c) a polynucleotide sequence encoding at least one retroviral gag protein and a retroviral pol protein; and / or (d) a polynucleotide sequence encoding the chimeric antigen receptor, the engineered T cell receptor (TCR), or the therapeutic protein, wherein at least a portion of one or more regions essential for replication in the retroviral genome is mutated.

46. A lentiviral vector particle produced by the method according to claim 45.

47. A method for introducing a modification into a cell, the method comprising electroporating the cell with an effective dose of the lentiviral vector particle according to claim 46 to produce a modified cell.

48. The method according to claim 47, wherein the cell is contacted with the effective dose of the lentiviral vector prior to electroporation.

49. The method according to claim 47, wherein the cell is contacted with the effective dose of the lentiviral vector for up to about 4 hours after electroporation.

50. The method according to claim 49, wherein the cell is contacted with the effective dose of the lentiviral vector: (a) At least about 5 - 30 minutes, at least about 25 - 50 minutes, at least about 5 - 60 minutes, at least about 5 - 12 minutes, at least about 60 - 120 minutes, at least about 120 - 240 minutes after electroporation; (b) At least about 1 minute, at least about 2 minutes, at least about 5 minutes, at least about 10 minutes, at least about 20 minutes, at least about 30 minutes, at least about 45 minutes, at least about 60 minutes, at least about 75 minutes, at least about 90 minutes, at least about 100 minutes, at least about 110 minutes, at least about 120 minutes, at least about 150 minutes, at least about 160 minutes, at least about 170 minutes, at least about 180 minutes, at least about 190 minutes, at least about 200 minutes, at least about 220 minutes or at least about 240 minutes after electroporation.

51. The method according to any one of claims 47 - 50, wherein the cells are selected from immune cells, eukaryotic donor cells, mononuclear cells, enriched lymphocytes, B lymphocytes, T lymphocytes, CD4 + T lymphocytes, CD8 + T lymphocytes, dendritic cells, monocytes, natural killer (NK) cells, natural killer T (NKT) cells, regulatory T cells, CD4 + helper T cells, CD8 + cytotoxic T lymphocytes (CTL), CD62L + cells, CD27 + cells, CCR7 + cells, CD45RO - cells, CD45RA + cells, neutrophils, basophils, eosinophils, megakaryocytes, stem cells, hematopoietic stem cells (HSC), hematopoietic progenitor cells (HPC), CD34 + cells, CD34 + peripheral blood stem cells, lymphokine - activated killer cells (LAK), tumor - infiltrating lymphocytes (TIL), circulating tumor - specific T cells, mesenchymal stem cells, mast cells, monocytes, macrophages, neutrophils, basophils, eosinophils, dendritic cells, megakaryocytes, and combinations thereof.

52. The method according to any one of claims 47 - 51, wherein the cell is: (a) Lymphoid cells selected from the following: T cells, B cells, natural killer (NK) cells, CD8 + T cells, CD4 + T cells, cytotoxic T lymphocytes, regulatory T cells, and any combination thereof; (b) Myeloid cells selected from the following: monocytes, macrophages, neutrophils, basophils, eosinophils, dendritic cells, megakaryocytes, and any combination thereof; (c) Stem cells, hematopoietic stem cells, hematopoietic progenitor cells, CD34 + cells or CD34 + peripheral blood stem cells.

53. The method according to any one of claims 47 - 52, wherein the effective dose of the lentiviral vector particles comprises about 0.5 ul, about 1 ul, about 1.5 ul, about 2 ul, about 2.5 ul, about 3 ul, about 3.5 ul, about 4 ul, about 5 ul, about 6 ul, about 7 ul, about 8 ul, about 9 ul, about 10 ul, about 15 ul or about 20 ul of the lentiviral vector.

54. The method according to any one of claims 47 - 53, wherein the effective dose of the lentiviral vector particles comprises an multiplicity of infection (MOI) of about 0.01, about 0.02, about 0.03, about 0.04, about 0.05, about 0.06, about 0.07, about 0.08, about 0.09, about 0.1, about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1.0, about 1.25, about 1.5, about 2.0, about 3.0, about 4.0 or about 5.

0.

55. The method according to any one of claims 47 - 54, wherein the effective dose of the lentiviral vector particles comprises: (a) About 2 ul of lentiviral vector particles with an MOI of about 0.08; (b) About 5 ul of lentiviral vector particles with an MOI of about 0.2; or (c) About 10 ul of lentiviral vector particles with an MOI of about 0.

4.

56. A modified cell, a modified immune cell, a modified CD4 + and CD8 + cell or a modified eukaryotic donor cell engineered by the method according to any one of claims 1-38.

57. A modified cell population, modified immune cell population, modified CD4 + and CD8 + cell population or modified eukaryotic donor cell population engineered by the method according to any one of claims 1-38.

58. A modified cell, modified immune cell, modified CD4 + and CD8 + cell, or modified eukaryotic donor cell comprising a lentiviral vector according to claim 46.

59. A modified cell population, modified immune cell population, modified CD4 + and CD8 + cell population or modified eukaryotic donor cell population comprising the lentiviral vector according to claim 46.

60. An engineered modified cell, modified immune cell, modified CD4 + and CD8 + cell or modified eukaryotic donor cell for use in producing a protein of interest.

61. The engineered modified cell, modified immune cell, modified CD4 + and CD8 + cells or modified eukaryotic donor cells, wherein the target protein is selected from industrial proteins or therapeutic proteins.

62. The engineered modified cell, modified immune cell, modified CD4 + and CD8 + cell or modified eukaryotic donor cell, wherein the protein of interest is selected from enzymes, regulatory proteins, receptors, peptides, peptide hormones, cytokines, membrane or transport proteins, vaccine antigens, antigen-binding proteins, immunostimulatory proteins, allergens, full-length antibodies or antibody fragments or derivatives, single-chain antibodies (scFv), Fab fragments, Fy fragments, single-domain antibodies (VH or VL fragments), domain antibodies, camelid single-domain antibodies (VHH), nanobodies, and combinations thereof.

63. A composition, the composition comprising: (a) A modified cell, modified immune cell, modified CD4 + and CD8 + cell or modified eukaryotic donor cell; (b) The modified cell population, modified immune cell population, modified CD4 + and CD8 + cell population or modified eukaryotic donor cell population according to claim 57 or 59; or (c) The lentiviral vector according to any one of claims 5 - 44.

64. The composition according to claim 63, the composition further comprising a pharmaceutically acceptable excipient.

65. A method of treating a disease or disorder in a subject, the method comprising administering to the subject in need thereof a therapeutically effective amount of: (a) A modified cell, modified immune cell, modified CD4 + and CD8 + cell or modified eukaryotic donor cell; (b) The modified cell population, modified immune cell population, modified CD4 + and CD8 + cell population or modified eukaryotic donor cell population according to claim 57 or 59; or (c) The composition according to claim 60 or 63, thereby treating the disease or disorder in the subject.

66. The method according to claim 65, wherein the disease or disorder is selected from viral infection, bacterial infection, parasitic infection, cancer, malignancy, non-cancerous disorder, autoimmune disease, fibrotic disease, Alzheimer's disease, protein deficiency disorder, and factor VIII deficiency.

67. The method according to claim 66, wherein the cancer is selected from breast cancer, triple-negative breast cancer, prostate cancer, ovarian cancer, glioma, glioblastoma, renal cell carcinoma, kidney cancer, mesothelioma, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, kidney cancer, liver cancer, lung cancer, lung adenocarcinoma, gallbladder cancer, colon cancer, cervical squamous cell carcinoma, non-small cell lung cancer, small cell lung cancer, Merkel cell carcinoma, hepatocellular carcinoma, esophageal cancer, brain cancer, melanoma, Hodgkin lymphoma, non-Hodgkin lymphoma, urothelial carcinoma, gastric cancer, blood cancer, lymphoma, leukemia, multiple myeloma, diffuse large B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, acute myeloid leukemia, B-cell acute lymphoblastic leukemia (ALL), pre-B ALL, and any combination thereof.

68. The method according to any one of claims 65 - 67, wherein the modified immune cell, the modified CD4 + and CD8 + cells or the modified eukaryotic donor cell: (a) autologous to the subject; (b) allogeneic to the subject; or (c) xenogeneic to the subject.

69. The method according to any one of claims 66 - 68, wherein the modified cell, the modified immune cell, the modified CD4 + and CD8 + cells or the modified eukaryotic donor cells are allogeneic to the subject.

70. The method according to any one of claims 66-69, wherein the subject is a human.

71. A method for producing a therapeutic protein, the method comprising: (a) using the method according to any one of claims 1-38 to produce an engineered population of immune cells or an engineered population of eukaryotic cells comprising the therapeutic protein; (b) harvesting the therapeutic protein; and (c) isolating and purifying the therapeutic protein.

72. The method according to claim 71, wherein the therapeutic protein is selected from enzymes, regulatory proteins, receptors, peptides, peptide hormones, cytokines, membrane or transport proteins, vaccine antigens, antigen-binding proteins, immunostimulatory proteins, allergens, full-length antibodies or antibody fragments or derivatives, single-chain antibodies (scFv), Fab fragments, Fv fragments, single-domain antibodies (VH or VL fragments), domain antibodies, camelid single-domain antibodies (VHH), nanobodies, and combinations thereof.

73. A kit, the kit comprising: (a) A population of modified immune cells or modified CD4 + and CD8 + cell population or populations engineered by the method according to any one of claims 1 - 38; or (b) the lentiviral vector according to claim 46.

Images