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WO2024121796A1 - Procédés de génération de cellules tueuses induites par des cytokines génétiquement modifiées (cik) - Google Patents

Procédés de génération de cellules tueuses induites par des cytokines génétiquement modifiées (cik) Download PDF

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Publication number
WO2024121796A1
WO2024121796A1 PCT/IB2023/062377 IB2023062377W WO2024121796A1 WO 2024121796 A1 WO2024121796 A1 WO 2024121796A1 IB 2023062377 W IB2023062377 W IB 2023062377W WO 2024121796 A1 WO2024121796 A1 WO 2024121796A1
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cells
cik
cell
committed
receptor
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PCT/IB2023/062377
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Irina Tcherepanova
Melissa Adams
John KRISKO
Mark Debenedette
Charles Nicolette
Sarah Tettamanti
Giuseppe GAIPA
Andrea Biondi
Laura CONTRERAS-RUIZ
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Fondazione Matilde Tettamanti E Menotti De Marchi Onlus
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Publication of WO2024121796A1 publication Critical patent/WO2024121796A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0636—T lymphocytes
    • C12N5/0638—Cytotoxic T lymphocytes [CTL] or lymphokine activated killer cells [LAK]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4613—Natural-killer cells [NK or NK-T]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/463—Cellular immunotherapy characterised by recombinant expression
    • A61K39/4631—Chimeric Antigen Receptors [CAR]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/463—Cellular immunotherapy characterised by recombinant expression
    • A61K39/4635—Cytokines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/464402—Receptors, cell surface antigens or cell surface determinants
    • A61K39/464411—Immunoglobulin superfamily
    • A61K39/464412—CD19 or B4
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/464402—Receptors, cell surface antigens or cell surface determinants
    • A61K39/464429—Molecules with a "CD" designation not provided for elsewhere
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0646—Natural killers cells [NK], NKT cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/10—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterized by the structure of the chimeric antigen receptor [CAR]
    • A61K2239/11—Antigen recognition domain
    • A61K2239/13—Antibody-based
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/10—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterized by the structure of the chimeric antigen receptor [CAR]
    • A61K2239/22—Intracellular domain
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the cancer treated
    • A61K2239/48—Blood cells, e.g. leukemia or lymphoma
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2510/00—Genetically modified cells

Definitions

  • the present disclosure provides methods of generating genetically modified cytokine induced killer (CIK) cells, comprising the sequential steps of: (a) culturing a population of mononuclear cells in culture medium comprising at least one differentiating agent to induce differentiation of the mononuclear cells in a cell culture into Committed CIK Precursor cells; (b) adding at least one stimulating agent and at least one expanding agent to the cell culture; and (c) expanding the cells from the cell culture to obtain a cell population comprising Committed CIK Precursor cells, transfecting the cell population comprising the Committed CIK Precursor cells with one or more nucleic acids to produce genetically modified Committed CIK Precursor cells, and expanding the genetically modified Committed CIK Precursor cells in culture medium to produce the genetically modified CIK cells; or (c) expanding the cells from the cell culture to obtain a cell population comprising Committed CIK Precursor cells and transfecting the cell population
  • Cytokine-induced killer (CIK) cells are cytotoxic T cells, which have both NK and T cell properties.
  • the qualifier “cytokine-induced killer” indicates that they are generated via administration of cytokines during in vitro culture (Arafar, A., Biomedical Research and Therapy; 1(2):71-77 (2014); Linn, Y.C., and Hui, K.M., J Biomed Biotechnol.; 2010: 435745 (2010)).
  • Cells which have the most potential effector function in CIK culture coexpress CD3 and CD56 surface molecules; possess a potent, HLA-unrestricted tumor- killing ability; and significantly reduced alloreactivity.
  • CIK cells are capable of killing a wide range of tumor cells, and hold great promise in the field of adoptive immunotherapy for cancer treatment and virus infections. [0004] Accordingly, there is a need to develop the methods of improved generation of genetically modified CIK cells in vitro, in order to obtain a population of effector cells with immunotherapeutic activity.
  • One aspect of the present disclosure is directed to methods of generating genetically modified cytokine induced killer (CIK) cells, comprising the sequential steps of: (a) culturing a population of mononuclear cells in culture medium comprising at least one differentiating agent to induce differentiation of the mononuclear cells in a cell culture into Committed CIK Precursor cells; (b) adding at least one stimulating agent and at least one expanding agent to the cell culture; and (c) expanding the cells from the cell culture to obtain a cell population comprising Committed CIK Precursor cells, transfecting the cell population comprising the Committed CIK Precursor cells with one or more nucleic acids to produce genetically modified Committed CIK Precursor cells, and expanding the genetically modified Committed CIK Precursor cells in culture medium to produce the genetically modified CIK cells; wherein steps (a), (b) and (c) are performed in the absence of non-irradiated or
  • One aspect of the present disclosure is directed to methods of generating genetically modified cytokine induced killer (CIK) cells, comprising the sequential steps of: (a) culturing a population of mononuclear cells in culture medium comprising at least one differentiating agent to induce differentiation of the mononuclear cells in a cell culture into Committed CIK Precursor cells; (b) adding at least one stimulating agent and at least one expanding agent to the cell culture; (c) expanding the cells from the cell culture to obtain a cell population comprising Committed CIK Precursor cells and transfecting the cell population comprising the Committed CIK Precursor cells with one or more nucleic acids; (d) formulating transfected cells comprising the Committed CIK Precursor cells for administration to a subject in need thereof; and (e) administering the transfected cells to the subject, wherein transfected Committed CIK Precursor cells are expanded to produce the genetically modified CIK cells in the subject; wherein steps (a), (b
  • One aspect of the present disclosure is directed to methods of generating genetically modified cytokine induced killer (CIK) cells, comprising the sequential steps of: (a) culturing peripheral blood mononuclear cells (PBMCs) in culture medium comprising at least one differentiating agent to induce differentiation of the PBMCs in a cell culture into Committed CIK Precursor cells; (b) adding at least one stimulating agent and at least one expanding agent to the cell culture; and (c) expanding the cells from the cell culture to obtain a cell population comprising Committed CIK Precursor cells, transfecting the cell population comprising the Committed CIK Precursor cells with one or more nucleic acids to produce genetically modified Committed CIK Precursor cells, and expanding the genetically modified Committed CIK Precursor cells in culture medium to produce the genetically modified CIK cells; wherein steps (a), (b) and (c) are performed in the absence of non-irradiated or irradiated feeder cells; and wherein the PBMCs
  • One aspect of the present disclosure is directed to methods of generating genetically modified cytokine induced killer (CIK) cells, comprising the sequential steps of: (a) culturing peripheral blood mononuclear cells (PBMCs) in culture medium comprising at least one differentiating agent to induce differentiation of the PBMCs in a cell culture into Committed CIK Precursor cells; (b) adding at least one stimulating agent and at least one expanding agent to the cell culture; (c) expanding the cells from the cell culture to obtain a cell population comprising Committed CIK Precursor cells and transfecting the cell population comprising the Committed CIK Precursor cells with one or more nucleic acids; (d) formulating transfected cells comprising the Committed CIK Precursor cells for administration to a subject in need thereof; and (e) administering the transfected cells to the subject, wherein transfected Committed CIK Precursor cells are expanded to produce the genetically modified CIK cells in the subject; wherein steps (a), (PBMCs
  • the stimulating agent and the expanding agent are added to the cell culture approximately 8 to 48 hours after initiating step (a). In some aspects, the stimulating agent and the expanding agent are added to the cell culture approximately 18 to 24 hours after initiating step (a). [0010] In some aspects, the Committed CIK Precursor cells are transfected with one or more nucleic acids approximately 18 to 48 hours after initiating step (b). In some aspects, the Committed CIK Precursor cells are transfected with one or more nucleic acids approximately 18 to 24 hours after initiating step (b).
  • the methods disclosed herein further comprise (d) replacing a portion of the culture medium with a fresh culture medium comprising at least one expanding agent approximately 8 to 96 hours after initiating step (c). In some aspects, the methods disclosed herein further comprise (d) replacing a portion of the culture medium with a fresh culture medium comprising at least one expanding agent approximately 18 to 24 hours after initiating step (c). [0012] In some aspects, the methods disclosed herein further comprise (e) passaging and culturing transfected cells in culture medium comprising at least one expanding agent approximately 8 to 96 hours after initiating step (d).
  • the methods disclosed herein further comprise (e) passaging and culturing transfected cells in culture medium [0013] comprising at least one expanding agent approximately 72 to 96 hours after initiating step (d). [0014] In some aspects, after step (e) the transfected cells in culture medium are transferred to a cell culture bag, a cell culture flask, or a culture device. In some aspects, the transfected cells in culture medium are transferred to the cell culture bag. In some aspects, the culture device is a bioreactor.
  • the transfected cells are expanded by culturing the transfected cells in culture medium comprising at least one expanding agent about every 2 to 3 days or about every 3 to 4 days until about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, about 15 days, about 16 days, about 17 days, about 18 days, about 19 days, about 20 days, about 21 days, about 22 days, about 23 days, about 24 days, about 25 days, about 26 days, about 27 days, or about 28 days after initiating step (a).
  • the transfected cells are expanded until about 10 days after initiating step (a). In some aspects, the transfected cells are expanded until about 14 days after initiating step (a).
  • the transfected cells are expanded until about 17 days after initiating step (a). In some aspects, the transfected cells are expanded until about 21 days after initiating step (a). In some aspects, the transfected cells are expanded until about 28 days after initiating step (a). [0017] In some aspects, the methods disclosed herein further comprise (f) isolating the cells from the cell culture to obtain a cell population comprising the genetically modified CIK cells. [0018] In some aspects, the transfected cells are cultured to obtain a cell population comprising the genetically modified CIK cells. [0019] In some aspects, the methods disclosed herein further comprise the step of freezing the genetically modified CIK cells.
  • the genetically modified CIK cells disclosed herein express one or more T cell receptors (TCR-CIK), chimeric antigen receptors (CAR-CIK), a genetically modified adhesion molecule, a genetically modified ligand, a genetically modified cytokine receptor, a genetically modified chemokine receptor, a genetically modified cytokine, a genetically modified chemokine, an enzyme, or a checkpoint inhibitor.
  • TCR-CIK T cell receptors
  • CAR-CIK chimeric antigen receptors
  • a genetically modified adhesion molecule a genetically modified ligand
  • a genetically modified cytokine receptor a genetically modified chemokine receptor
  • a genetically modified cytokine a genetically modified chemokine
  • an enzyme or a checkpoint inhibitor.
  • the genetically modified CIK cells disclosed herein express a prodrug converting enzyme, an IgG-degrading enzyme of S.
  • IdeS Intercellular Adhesion Molecule 1
  • IdeS Intercellular Adhesion Molecule 1
  • CXCR4 C-X-C chemokine receptor type 4
  • IL-15 IL-18, IL-21 IL-23 IL-33, IL-1a, IL-1b, matrix metalloproteinase (MMP), heparinase, an anti-PD-1 antibody or antigen binding fragment thereof, an anti-T cell immunoglobulin and mucin-domain containing-3 (TIM-3) antibody or antigen binding fragment thereof, IL-3 zetakine, or any combination thereof.
  • MMP matrix metalloproteinase
  • the population of mononuclear cells disclosed herein is selected from the group consisting of: umbilical cord blood derived mononuclear cells, peripheral blood mononuclear cells (PBMCs), bone marrow derived mononuclear cells, lymphocytes, monocytes, dendritic cells, macrophages, T cells, naive T cells, memory T cells, natural killer cells, hematopoietic stem cells, pluripotent embryonic stem cells, induced pluripotent stem cells, and any combination thereof.
  • the mononuclear cells are umbilical cord blood derived mononuclear cells.
  • the mononuclear cells are PBMCs.
  • the PBMCs were previously cryopreserved and thawed before culturing in step (a).
  • the mononuclear cells are from a human leukocyte antigen (HLA) matched donor.
  • the differentiating agent is selected from the group consisting of: IFN- ⁇ , IL-4, IL-5, IL-7, IFN- ⁇ , IL-10, IL-12, IL-13, IL-6, IL-15, IL-17, IL-18, IL-21, IL- 22, IL-23, IL-27, IL-1 ⁇ , TGF- ⁇ , GM-CSF, CCL3, CCL4, CCL5, CCL17, CCL21, and any combination thereof.
  • the differentiating agent is IFN- ⁇ .
  • the differentiating agent is added in an amount of about 10 U/ml to about 10000 U/ml.
  • the differentiating agent is added in an amount of about 1000 U/ml.
  • the stimulating agent is selected from the group consisting of: an anti-CD3 antibody, an anti-TCR antibody, an anti-CD28 antibody, an anti-CD137 antibody, an anti-CD134 antibody, an anti-CD27 antibody, an anti-ICAM-1 antibody, an anti- CD3/CD28-coated beads, a superantigen, phytohaemaglutinin (PHA), phorbol 12- myristate 13-acetate (PMA), ionomycin, and any combination thereof.
  • the stimulating agent is an anti-CD3 antibody.
  • the anti-CD3 antibody is an OKT3 antibody.
  • the stimulating agent is added in an amount of about 5 ng/ml, about 6 ng/ml, about 7 ng/ml, about 8 ng/ml, about 9 ng/ml, about 10 ng/ml, about 11 ng/ml, about 12 ng/ml, about 13 ng/ml, about 14 ng/ml, about 15 ng/ml, about 16 ng/ml, about 17 ng/ml, about 18 ng/ml, about 19 ng/ml, about 20 ng/ml, about 21 ng/ml, about 22 ng/ml, about 23 ng/ml, about 24 ng/ml, about 25 ng/ml, about 26 ng/ml, about 27 ng/ml, about 28 ng/ml, about 29 ng/ml, about 30 ng/ml, about 31 ng/ml, about 32 ng/ml, about 33 ng/ml, about 34 ng/ml, about 10 ng/
  • the stimulating agent is added in an amount of about 50 ng/ml.
  • the expanding agent is selected from the group consisting of: IL- 2, IL-4, IL-7, IL-9, IL-15, IL-18, IL-21, and any combination thereof.
  • the expanding agent is IL-2.
  • the expanding agent is added in an amount of about 10 U/ml to about 1000 U/ml. In some aspects, the expanding agent is added in an amount of about 300 U/ml.
  • the cell population comprising the Committed CIK Precursor cells is transfected using electroporation.
  • the electroporation in performed in an isotonic buffer In some aspects, the electroporation in performed in CoStorSol, Opti-MEM, or Lonza isotonic buffer.
  • the transfection is selected from the group consisting of: a non- viral transfer of one or more nucleic acids encoding an antigen receptor, a chimeric antigen receptor, a T cell receptor, a suicide gene, a gene encoding an inducible caspase 9 system, and any combination thereof into the cell population comprising the Committed CIK Precursor cells in the cell culture.
  • the non-viral transfer of nucleic acids comprises the use of the group consisting of: a transposon-based integration system, Zn-finger nucleases, integrases, transcription activator-like effectors, clustered regularly interspaced short palindromic repeats (CRISPR), sequence-specific recombinase systems able to integrate nucleic acids by recombination between attachment sites, and any combination thereof.
  • the transposon-based system is a Sleeping Beauty (SB) transposon- based system.
  • SB transposon-based system comprises the use of Sleeping Beauty transposase SB100X.
  • one or more nucleic acids encode T cell receptors, chimeric antigen receptors, a cell adhesion molecule, a ligand, a cytokine receptor, a chemokine receptor, a cytokine, a chemokine, an enzyme, or a checkpoint inhibitor.
  • one or more nucleic acids encode a prodrug converting enzyme, an IgG-degrading enzyme of S.
  • IdeS Intercellular Adhesion Molecule 1
  • IdeS Intercellular Adhesion Molecule 1
  • CXCR4 Intercellular Adhesion Molecule 1
  • IL-15 CCR5, CCR4, CD25, CD122, CD132, C-X-C chemokine receptor type 4 C-X-C chemokine receptor type 4 (CXCR4), IL-15, IL-18, IL-21 IL-23 IL-33, IL-1a, IL-1b, matrix metalloproteinase (MMP), heparinase, an anti-PD-1 antibody or antigen binding fragment thereof, an anti-T cell immunoglobulin and mucin-domain containing-3 (TIM-3) antibody or antigen binding fragment thereof, IL-3 zetakine, , or any combination thereof.
  • MMP matrix metalloproteinase
  • the prodrug converting enzyme is carboxypeptidase G2 (CPG2) or ⁇ -lactamase.
  • CPG2 carboxypeptidase G2
  • chimeric antigen receptors are specific for CD19, CD123, TIM- 3, C-type lectin-like molecule-1 (CLL-1), CD70, mucin 1 (MUC-1), CD20, CD22, B-cell activating factor receptor (BAFFR), CD23, cytokine receptor like factor 2 (CRLF2), CD79b, CD79d, CD7, CD43, CD5, CD25, Lewis Y (LeY), natural killer group 2 member D (NKG2D), receptor tyrosine kinase like orphan receptor 1 (ROR1), receptor tyrosine kinase like orphan receptor 2 (ROR2), Wilms' tumor 1 (WT1), CD44 variant 6 (CD44v6), CD33, CD38, human epidermal growth factor receptor 2 (Her2), epidermal growth factor receptor (EG
  • the chimeric antigen receptor is specific for CD19.
  • one or more nucleic acids are DNA and/or RNA.
  • one or more nucleic acids are RNA.
  • the Committed CIK Precursor cells are transfected with an RNA encoding SB100X transposase and a DNA encoding a Sleeping Beauty compatible chimeric antigen receptor (CAR) transposon.
  • the transfected cells are administered with a genetically modified IL-2.
  • One aspect of the present disclosure is directed to genetically modified cytokine induced killer (CIK) cells obtained by the methods disclosed herein.
  • One aspect of the present disclosure is directed to the compositions comprising the genetically modified CIK cells disclosed herein.
  • the genetically modified CIK cells or the compositions disclosed herein are for use in the prevention or treatment of cancers, tumors, viral infections, inflammatory diseases and disorders, autoimmune diseases and disorders, or any combination thereof.
  • Other features and advantages of the present disclosure will be apparent from the following detailed description and examples which should not be construed as limiting. The contents of all cited references, including scientific articles, newspaper reports, GenBank entries, patents and patent applications cited throughout this application are expressly incorporated herein by reference. BRIEF DESCRIPTION OF THE DRAWINGS [0050] FIG.
  • FIGs. 2A-2D show cell expansion parameters during the CD33.CAR-CIK cells generation process.
  • FIGs.2A and 2B show cell number (x10 ⁇ 6) and glucose levels (mg/dl) during the day 2 feeder free CIK generation process (day 2 electroporation without feeder cells; plating the cells immediately after electroporation at 1 x 10 6 cells/cm 2 cell density ("10 ⁇ 6") (FIG.2A) or 2 x 10 6 cells/cm 2 cell density (“2x10 ⁇ 6”) (FIG.2B); expanding the cells in the 6-well plates until day 7).
  • FIG.2A cell number of cells/cm 2 cell density
  • FIG.2B shows cell number (x10 ⁇ 6) and glucose levels (mg/dl) during the day 2 feeder free CIK generation process (day 2 electroporation without feeder cells; plating the cells immediately after electroporation at 1 x 10 6 cells/cm 2 cell density ("10 ⁇ 6") (FIG.2A) or 2 x 10 6 cells/cm 2 cell density (“2x10 ⁇ 6”) (FIG.2B); expanding the cells in the 6-well plates
  • FIG. 2C shows cell number (x10 ⁇ 6) and glucose levels (mg/dl) during the Gas Permeable Rapid Expansion (G-Rex) CIK generation process where the cells were transferred to G-Rex for expansion on day 7 (10 x 6 cells/cm 2 ) (“DAY 7”).
  • FIG. 2D shows comparison of cell number (x10 ⁇ 6) within the different culture conditions in feeder free, Day 2 electroporation CIK generation process process where cells were expanded in Flasks ("Flask”) and the G-Rex expansion process described in FIG.2A (“10 ⁇ 6"), FIG.2B (“2x10 ⁇ 6”), and FIG.2C (“DAY 7").
  • FIG.3A shows comparison of CAR expression levels (% CD3 + CAR + cells) during the G-Rex CIK generation process (day 2 electroporation without feeder cells; plating the cells immediately after electroporation at 1 x 10 6 cells/cm 2 cell density ("10 ⁇ 6") or 2 x 10 6 cells/cm 2 cell density (“2x10 ⁇ 6”); expanding the cells in the 6-well plates until day 7; transferring to G-Rex for expansion on day 7 (10 x 6 cells/cm 2 ) ("DAY 7") or expanding in Flasks ("Flask”)).
  • FIGs.3B-3E show comparison of memory phenotype (T na ⁇ ve (Tn) cells, T central memory cells (Tcm), effector memory cells (Tem), and terminally differentiated effector memory T cells (Temra)) during the Day 2 feeder free CIK generation process (day 2 electroporation without feeder cells; plating the cells immediately after electroporation at 1 x 10 6 cells/cm 2 cell density ("10 ⁇ 6") (FIG.3B) or 2 x 10 6 cells/cm 2 cell density (“2x10 ⁇ 6”) (FIG.3C); expanding the cells in the 6-well plates until day 7; transferring to G-Rex for expansion on day 7 (10 x 6 cells/cm 2 ) ("DAY 7") (FIG.3D) or expanding in Flasks ("Flask”) (FIG.3E)).
  • FIGs.3F-3I show comparison of CD4-CD8 phenotype during the day 2 feeder free CIK generation process (day 2 electroporation without feeder cells; plating the cells immediately after electroporation at 1 x 10 6 cells/cm 2 cell density (“10 ⁇ 6") (FIG.3F) or 2 x 10 6 cells/cm 2 cell density (“2x10 ⁇ 6") (FIG.3G); expanding the cells in the 6-well plates until day 7; transferring to G-Rex for expansion on day 7 (10 x 6 cells/cm 2 ) ("DAY 7") (FIG. 3H) or expanding in Flasks ("Flask”) (FIG.3I)).
  • FIG.3F shows comparison of CD4-CD8 phenotype during the day 2 feeder free CIK generation process (day 2 electroporation without feeder cells; plating the cells immediately after electroporation at 1 x 10 6 cells/cm 2 cell density (“10 ⁇ 6") (FIG.3F) or 2 x 10 6 cells/c
  • 33 CAR1 CAR-CIK-CD33 cells generated using the day 2 feeder free process, in the condition of plating the cells immediately after electroporation at 10 6 cells /cm 2 cell density.
  • 33 CAR 2 CAR-CIK- CD33 cells generated using the day 2 feeder free process, in the condition of plating the cells immediately after electroporation at 2x10 ⁇ 6 cells /cm 2 cell density.
  • 33 CAR-FLASK CAR-CIK-CD33 cells generated using the original CIK generation process (day 0 electroporation + ⁇ irradiated feeder cells and expanded in Flasks).
  • FIG.4B shows the proliferation ability of CAR-CIK cells (% Ki-67 positive cells) evaluated after co-culture with the Cell Tracker-labeled targets THP-1 (“THP-1”) or without THP-1 ("Alone").
  • THP-1 Cell Tracker-labeled targets
  • Alone THP-1
  • No DNA CIK cells generated in flasks without electroporation with genetic material.
  • 33 CAR1 CAR-CIK-CD33 cells generated using the day 2 feeder free process, in the condition of plating the cells immediately after electroporation at 10 6 cells /cm 2 cell density.
  • 33 CAR 2 CAR-CIK- CD33 cells generated using the day 2 feeder free process, in the condition of plating the cells immediately after electroporation at 2x10 ⁇ 6 cells /cm 2 cell density.
  • FIGs. 4C-4D show secretion profile (IL-2 production (FIG. 4C) and IFN- ⁇ production (FIG.4D)) in cells generated using the original CIK generation process (day 0 electroporation + ⁇ irradiated feeder cells and expanded in Flasks) or the day 2 feeder free G-Rex process after co-culture with the Cell Tracker-labeled targets THP-1 ("THP-1") or without THP-1 ("Alone").
  • FIG.4E Gating strategy to detect CD3+ (PerCp) CAR+ (FC-APC) cells.
  • FIG. 4F Gating strategy to detect CAR+ (FC-APC) cells (under the first gate) and IL-2.
  • FIG. 4G Gating strategy to detect CAR+ (FC-APC) cells (under the first gate) and IFN- ⁇ .
  • FIGs.5A-5B show representative dot plots of the memory phenotype analysis.
  • FIG. 5A CAR-CIK Donor A, expanded in flasks or in the G-REX device.
  • FIG.5B CAR-CIK Donor B, expanded in flasks or in the G-REX device. Day 2/feeder free protocol was used.
  • FIG. 6A shows distribution of the memory phenotype in the final products produced in good manufacturing practices (GMP) for the clinical study, using the standard protocol at day 0 + feeder cells and culturing the cells in Flasks.
  • FIG. 6B shows distribution of the memory phenotype in the G-REX CAR-CIK cultures, by using the day 2 feeder free methods and transferring the cells at day 7 in G- REX device.
  • FIG.6C shows table representing the mean and standard deviation calculations on the batches of FIGs.6A-6B.
  • FIG.7A shows total number of cells recovered at the end of the cell cultures (flasks and G-REX-day 7).
  • FIG.7B shows percentage of CAR expression at the end of the cell cultures (flasks and G-REX-day 7).
  • FIG. 7C shows percentage of CD3+CD56+ cells obtained at the end of the cell cultures (flasks and G-REX-day 7).
  • FIGs.7D-7G show Day 0 (+feeder) and Day 2 (feeder free) comparison by using pT4 transposon and SB100X DNA.
  • FIG.7D total number of cells recovered at the end of the cell cultures (flasks and G-REX-day 7), both using the Day 0 and the Day 2 gene transfer methods.
  • FIG. 7E percentage of CAR expression at the end of the cell cultures (flasks and G-REX-day 7) both using the Day 0 and the Day 2 gene transfer methods.
  • FIG. 7F percentage of CD3+CD56+ cells obtained at the end of the cell cultures (flasks and G- REX-day 7).
  • FIG. 7G percentage of CD3+CD4+ and CD3+CD8+ cells obtained at the end of the cell cultures (flasks and G-REX-day 7). [0068] FIG.
  • FIG. 8A shows complete immunophenotype (CD3+CD4+, CD3+CD8+, CD3+CD56+, Tn, Tcm, Tem, Temra, CAR+) of CD19.CAR-CIK cells produced in G- REX-day 7, with the SB plasmids ratio of 7.5+1.
  • FIG.8B shows fold increase of CD19.CAR-CIK cells cultured both in Flasks and G-REX. Day 2/feeder free protocol was used.
  • FIG.8C shows vector copy number (VCN) of CD19.CAR-CIK cells cultured in G- REX. Day 2/feeder free protocol was used.
  • FIG. 8D shows SB100X detection of CD19.CAR-CIK cells cultured in G-REX. Day 2/feeder free protocol was used.
  • FIG. 8E shows in vitro cytotoxicity of CD19.CAR-CIK cells cultured in G-REX and Flasks against the CD19+ cell line REH. Day 2/feeder free protocol used.
  • FIGs. 9A-9C show CD33.CAR CIK cells produced in Flasks and G-REX, by the day 2 feeder free method.
  • FIG.9A total cell numbers of CD33.CAR-CIK cells cultured both in Flasks and G-REX.
  • FIG.9B percentage of CD3+CAR+ CIK cells obtained at the end of the cell cultures (flasks and G-REX-day 7).
  • FIG.9C Complete immunophenotype of CD33.CAR-CIK cells.
  • FIGs.9D-9E show evolution of the memory phenotype (Tn, Tcm, Tem, Temra) of CD33.CAR-CIK cells produced in Flasks (FIG.9D) and G-REX-day 7 (FIG.9E) by using the day 2 feeder free method.
  • FIG. 10B shows proliferation of CD33.CARCIK cells generated by the Day 2 feeder free (either produced in Flasks or G-REX) evaluated after co-culture with the Cell Tracker-labeled targets THP-1 ("THP-1") or without THP-1 ("Alone”).
  • THP-1 THP-1
  • Alone THP-1
  • 10C-10D shows cytokine production (IL-2 (FIG. 10C) and IFN- ⁇ (FIG. 10D)) by CD33.CARCIK cells generated by the Day 2 feeder free (either produced in Flasks or G-REX) evaluated after co-culture with the Cell Tracker-labeled targets THP-1 ("THP-1") or without THP-1 ("Alone”).
  • THP-1 THP-1
  • Alone THP-1
  • No DNA CIK cells generated in flasks without electroporation with genetic material.
  • FIG.11A shows absolute quantification of CD19+ DAUDI cells in mice untreated (Daudi only) and treated with of 5x10 ⁇ 6 (G-REX 5) or 10x10 ⁇ 6 (G-REX 10) CAR+cells/mouse during days 12, 20, 33, 40, 50, 61, 76, and 90.
  • FIG. 11B shows absolute quantification of CD3+ CAR-CIK cells in mice treated with of 5x10 ⁇ 6 (G-REX 5) and 10x10 ⁇ 6 (G-REX 10) CAR+cells/mouse during days 12, 20, 33, 40, 50, 61, 76, and 90.
  • FIG.11C shows survival curve analysis in mice untreated (Daudi only) and treated with of 5x10 ⁇ 6 (G-REX 5) or 10x10 ⁇ 6 (G-REX 10) CAR+cells/mouse.
  • FIGs. 11D-11F show representative dot plot flow cytometric analysis of in vivo specimens.
  • FIG.11D Gating strategy to select human CD45+ cells out of mouse CD45+ cells.
  • FIG.11E Dot plot of CD19+CD10+ DAUDI cells in the bone marrow.
  • FIG.11F Dot plot to detect CD3+ CAR-CIK cells and CD19+ DAUDI cells.
  • FIG.12 shows composition of CAR-CIK-CD19 cultures throughout the production process.
  • FIGs. 13A-13M show representative gating strategy to identify CD25, CD69, CD137 and HLA-DR positive cells on day 0. Single cells are identified by gating on FSC- H and FSC-A (FIG.13A). Viable cells are shown in the Aquadye-negative gate (FIG.13B). Viable CD3+ cells for day 0 PBMCs (FIG.13C), gated CD3+ CD4+ cells (FIG.13D) and CD8+ cells (FIG. 13E) are shown.
  • FIGs. 14A-14M show representative gating strategy to identify CD25, CD69, CD137 and HLA-DR positive cells on day 2. Single cells are identified by gating on FSC- H and FSC-A (FIG.14A). Viable cells are shown in the Aquadye-negative gate (FIG.14B).
  • FIG.15A-15B show activation marker upregulated on day 2 cells.
  • FIG. 15A shows time course of CAR expression during the 21 day culture period after electroporation with pT4-CD19-CAR plasmid DNA and pCMV-SB100X plasmid DNA.
  • the percentage of CAR positive, CD3+ cells is shown for days 7, 14 and 21 of the cell culture period.
  • FIGs. 17A-17J show CAR positivity post electroporation with pT4-CD19-CAR plasmid DNA and pCMV-SB100X plasmid DNA.
  • Day 2 activated cells were electroporated with pT4-CD19CAR plasmid DNA and pCMV-SB100X plasmid DNA in the four electroporation buffers tested, BioRad, CoStorSol, Lonza and OptiMEM.
  • Representative pseudo-color dot plots showing the percentage of CAR positive cells (CAR+ positivity) are shown for days 14 (BioRad (FIG. 17B), CoStorSol (FIG. 17C), Lonza (FIG. 17D) and OptiMEM (FIG. 17E)) and 21 (BioRad (FIG.
  • FIG.17G shows time course of CAR expression during the 21-day culture period after electroporation with pT4-CD19CAR plasmid DNA and Capped-SB100X RNA.
  • FIGs.19A-19J show CAR positivity post electroporation with pT4-CD19CAR plasmid DNA and Capped-SB100X RNA.
  • Day 2 activated cells were electroporated with pT4- CD19CAR plasmid DNA and SB100X transposase RNA in the four electroporation buffers tested, BioRad, CoStorSol, Lonza and OptiMEM.
  • Representative pseudo-color dot plots showing the percentage of CAR positive cells (CAR+ positivity) are shown for days 14 (BioRad (FIG.19B), CoStorSol (FIG.19C), Lonza (FIG.19D) and OptiMEM (FIG.19E)) and 21 (BioRad (FIG. 19G), CoStorSol (FIG. 19H), Lonza (FIG.
  • FIGs. 20A-20C show T cell enrichment, T cell activation, and electroporation efficiency and CAR expression in lots initiated from cryopreserved PBMCs (frozen) versus fresh PBMCs (fresh).
  • FIG.20A shows the % of T cells in the culture on day 2 (% T cells (d2)).
  • FIG.20B shows T cell activation by evaluating the expression of CD25 activation marker on T cells (% CD25+ T cells).
  • FIGs.21A-21B show the effect of PBMC cryopreservation (frozen v. fresh PBMCs) on the fold expansion and memory phenotype of CARCIK-1918 cells.
  • FIG. 21A shows a fold expansion calculated from day 2 (electroporation) to day 17 (D2-D17).
  • FIG. 21A shows a fold expansion calculated from day 2 (electroporation) to day 17 (D2-D17).
  • FIG.23 shows the representative plots of the expression of CD25 activation marker in viable T cells manufactured from fresh or frozen PBMCs.
  • FIG. 24 shows a diagram of bicistronic CAR transgenes linked to IL-18 gene. Plasmids A and B contain a 3 rd generation CAR transgene encoding the FMC63 scFv domain linked to the human IL-18 gene. Plasmids C and D contain a 2 nd generation CAR transgene encoding the SJ25C1 scFv domain linked to the human IL-18 gene. Plasmids B and D contain a CD28 signaling domain with the amino acid substitution YMNM ⁇ YSNV.
  • FIG. 25 shows a schematic representation of study design in NSG/Raji survival model.
  • FIG.26 shows bioluminescence imaging (BLI) of mice treated with CARCIK-1918 cells. Mice were either left untreated or treated with Arm A (3 rd gen pT4-CD19CAR- IL18), Arm B (3 rd gen pT4-CD19CARYSNV-IL18), Arm C (2 nd gen pT4-1928z-IL18), and Arm D (2 nd gen pT4-1928YSNVz-18) CARCIK-1918 cells.
  • Arm A (3 rd gen pT4-CD19CAR- IL18
  • Arm B (3 rd gen pT4-CD19CARYSNV-IL18
  • Arm C (2 nd gen pT4-1928z-IL18
  • Arm D (2 nd gen pT4-1928YSNVz-18
  • FIG.27 shows an average radiance of tumor burden in mice treated with CARCIK- 1918 cells. Quantification of BLI measured as average radiance (p/s/cm3/sr) for Region of Interest (ROI) for untreated mice and mice treated with Arm A (3 rd gen pT4-CD19CAR- IL18), Arm B (3 rd gen pT4-CD19CARYSNV-IL18), Arm C (2 nd gen pT4-1928z-IL18), and Arm D (2 nd gen pT4-1928YSNVz-18) CARCIK-1918 cells.
  • FIG.27 shows an average radiance of tumor burden in mice treated with CARCIK- 1918 cells. Quantification of BLI measured as average radiance (p/s/cm3/sr) for Region of Interest (ROI) for untreated mice and mice treated with Arm A (3 rd gen pT4-CD19CAR- IL18), Arm B (3 rd gen pT4-CD19
  • FIG.29 shows measurement of animal body weight (grams) recorded at least twice weekly throughout the study until mice succumbed to disease or reached the humane endpoint (day (D) 0, D3, D7, D10, D14, D17, D22, D25, D28, D35, D38, D42, D45, D49, D52, D57, D59, D63, D66, D70, D73, D77, D84, and D86) for untreated mice and mice treated with Arm A (3 rd gen pT4-CD19CAR-IL18), Arm B (3 rd gen pT4- CD19CARYSNV-IL18), Arm C (2 nd gen pT4-1928z-IL18), and Arm D (2 nd gen pT4- 1928YSNVz-18) CARCIK-1918 cells.
  • Arm A (3 rd gen pT4-CD19CAR-IL18
  • Arm B (3 rd gen pT4- CD19CARYSNV-IL18
  • FIGs.30A-30C show an exploratory analysis of CARCIK-1918 cell persistence in vivo.
  • FIG.30A shows numbers of human CD4+CD3+ cells and CD8+CD3+ cells present in the peripheral blood for sacrificed mice on day 22 either untreated or treated with Arm A (3rd gen pT4-CD19CAR-IL18) and Arm B CARCIK-1918 cells (3rd gen pT4- CD19CARYSNV-IL18).
  • Arm A 3rd gen pT4-CD19CAR-IL18
  • Arm B CARCIK-1918 cells
  • FIG. 30B shows numbers of human CD4+CD3+ cells and CD8+CD3+ cells present in the bone marrow for sacrificed animals on day 22 either untreated or treated with Arm A (3rd gen pT4-CD19CAR-IL18) and Arm B (3rd gen pT4- CD19CARYSNV-IL18) CARCIK-1918 cells.
  • FIG. 30C shows numbers of human CD4+CD3+ cells and CD8+CD3+ cells present in the peripheral blood for one mouse sacrificed on day 46 treated with Arm D (2nd gen pT4-1928YSNVz-18) CARCIK-1918 cells. The number of CD4 and CD8 CD3 cells is reported from the viable cell gate as number of cells/mL of blood. [0101] FIGs.
  • 31A-31C show cytokine measurement in peripheral blood of mice treated with CARCIK-1918 cells.
  • Peripheral blood was collected from untreated mice and mice treated with Arm A (3 rd gen pT4-CD19CAR-IL18), Arm B (3 rd gen pT4- CD19CARYSNV-IL18), Arm C (2 nd gen pT4-1928z-IL18), and Arm D (2 nd gen pT4- 1928YSNVz-18) CARCIK-1918 cells on day 8.
  • Cytokine levels for GM-CSF (FIG.31A), IFN- ⁇ (FIG. 31B) and TNF- ⁇ (FIG.
  • FIG.32 shows bioluminescence imaging of mice treated with CARCIK-1918 cells. Mice were either left untreated or treated with Arm A (3 rd gen pT4-CD19CAR-IL18), Arm B (3 rd gen pT4-CD19CARYSNV-IL18), Arm C (2 nd gen pT4-1928z-IL18), and Arm D (2 nd gen pT4-1928YSNVz-18) CARCIK-1918 cells. BLI measurements were taken at weekly intervals, starting at week 2, and at weeks 3, 4 and 5.
  • FIG.33 shows an average radiance of tumor burden in mice treated with CARCIK- 1918. Quantification of BLI measured as average radiance (p/s/cm3/sr) for ROI for untreated mice and mice treated with Arm A (3 rd gen pT4-CD19CAR-IL18), Arm B (3 rd gen pT4-CD19CARYSNV-IL18), Arm C (2 nd gen pT4-1928z-IL18), and Arm D (2 nd gen pT4-1928YSNVz-18) CARCIK-1918 cells.
  • FIG.34 shows an average radiance of tumor burden in individual mice treated with CARCIK-1918 cells, day 14.
  • FIG.35 shows animal survival (probability of survival) using Kaplan–Meier curves for untreated mice and mice treated with Arm A (3rd gen pT4-CD19CAR-IL18), Arm B (3rd gen pT4-CD19CARYSNV-IL18), Arm C (2nd gen pT4-1928z-IL18), and Arm D (2nd gen pT4-1928YSNVz-18) CARCIK-1918 cells.
  • Arm A (3rd gen pT4-CD19CAR-IL18
  • Arm B (3rd gen pT4-CD19CARYSNV-IL18)
  • Arm C (2nd gen pT4-1928z-IL18
  • Arm D (2nd gen pT4-1928YSNVz-18) CARCIK-1918 cells.
  • FIG.36 shows measurement of animal body weight (grams) recorded at least twice weekly throughout the study until mice succumbed to disease or reached the humane endpoint (day (D) 2, D5, D8, D12, D15, D19, D22, D26, D29, D33, D40, D44, D47, and D51) for untreated mice and mice treated with Arm A (3 rd gen pT4-CD19CAR-IL18), Arm B (3 rd gen pT4-CD19CARYSNV-IL18), Arm C (2 nd gen pT4-1928z-IL18), and Arm D (2 nd gen pT4-1928YSNVz-18) CARCIK-1918 cells.
  • Arm A (3 rd gen pT4-CD19CAR-IL18
  • Arm B (3 rd gen pT4-CD19CARYSNV-IL18
  • Arm C (2 nd gen pT4-1928z-IL18
  • Arm D (2 nd gen pT
  • FIGs. 37A-37B show an exploratory analysis of tumor cell engraftment in mice treated with CARCIK-1918 cells.
  • Peripheral blood was collected on day 8 for 3 mice from either untreated or treaded with Arm A (3 rd gen pT4-CD19CAR-IL18), Arm B (3 rd gen pT4-CD19CARYSNV-IL18), Arm C (2 nd gen pT4-1928z-IL18), and Arm D (2 nd gen pT4- 1928YSNVz-18) CARCIK-1918 cells and the numbers (cells/ml) (FIG.
  • FIGs. 38A-38B show an exploratory analysis of CARCIK-1918 cell persistence.
  • Peripheral blood was collected on day 8 for 3 mice from either untreated or treaded with Arm A (3 rd gen pT4-CD19CAR-IL18), Arm B (3 rd gen pT4-CD19CARYSNV-IL18), Arm C (2 nd gen pT4-1928z-IL18), and Arm D (2 nd gen pT4-1928YSNVz-18) CARCIK- 1918 cells and the numbers (cells/ml) (FIG. 37A) and frequency (% of the viable cells) (FIG.37B) of human CD3+ cells present in the peripheral blood was determined by flow cytometry. Data is reported as cells/mL of human CD3 positive cells in the viable cell gate.
  • FIGs.39A-39B show an exploratory analysis of CARCIK-1918 cell persistence in vivo in mice treated with CARCIK-1918 cells.
  • FIG. 39A shows numbers of human CD4+CD3+ cells and CD8+CD3+ cells present in the peripheral blood collected on day 8 from animals untreated or treated with Arm A (3 rd gen pT4-CD19CAR-IL18), Arm B (3 rd gen pT4-CD19CARYSNV-IL18), Arm C (2 nd gen pT4-1928z-IL18), and Arm D (2 nd gen pT4-1928YSNVz-18) CARCIK-1918 cells.
  • Arm A (3 rd gen pT4-CD19CAR-IL18
  • Arm B (3 rd gen pT4-CD19CARYSNV-IL18
  • Arm C (2 nd gen pT4-1928z-IL18
  • Arm D (2 nd gen pT4-1928YSNVz-18
  • FIGs. 40A-40C show cytokine measurement in peripheral blood of mice treated with CARCIK-1918 cells.
  • Peripheral blood was collected from untreated mice and mice treated with Arm A (3 rd gen pT4-CD19CAR-IL18), Arm B (3 rd gen pT4- CD19CARYSNV-IL18), Arm C (2 nd gen pT4-1928z-IL18), and Arm D (2 nd gen pT4- 1928YSNVz-18) CARCIK-1918 cells.
  • Cytokine levels for GM-CFS (FIG. 40A), IFN- ⁇ (FIG. 40B) and TNF- ⁇ (FIG. 40C) in the peripheral blood were measured by cytokine bead array and reported as pg/mL of blood.
  • FIG.41 shows a schematic representation of study design in NSG/Daudi survival model.
  • FIG.42 shows the mean concentration of hCD45+/hCD19+ cells/mL of blood for each experimental group (DAUDI only, CARCIK-19185x10 ⁇ 6, CARCIK-191810x10 ⁇ 6) was plotted versus sampling day (days 10, 20, 30, 40, 50, 60, 70, 80, and 90) to monitor cell expansion over the course of the study.
  • FIG. 43 shows results of flow cytometry analysis of peripheral blood collected across the course of the study. CARCIK-1918 cells were identified by staining for the human CD45 and human CD3 cell surface markers.
  • FIG. 44 shows mean body weight (g) of the mice in each experimental group (DAUDI only, CARCIK-19185x10 ⁇ 6, CARCIK-191810x10 ⁇ 6) monitored at least twice weekly until the animals succumbed to disease or reached the humane endpoint (days 0, 5, 9, 14, 20, 23, 26, 33, 37, 43, 49, 54, 58, 62, 65, 67, 72, 76, 83, and 90).
  • FIG.45 shows an estimated animal survival (probability of survival) curves in each experimental group (DAUDI only, IL18 CAR 5x10 ⁇ 6, IL18 CAR 10x10 ⁇ 6) based on the Kaplan-Meier method.
  • FIGs. 46A-46D show percentage (%) of hCD45+/hCD19+ (filled circles, Daudi) and hCD45+/hCD3+ (open circles, hCD3+) cells in the bone marrow (BM) (FIG.
  • FIG.46A peripheral blood (PB)
  • FIG.46B peripheral blood
  • FIG.46C spleen
  • FIG.46D kidney
  • CIK cytokine induced killer cells
  • methods of generating genetically modified cytokine induced killer (CIK) cells comprising the sequential steps of: (a) culturing a population of mononuclear cells in culture medium comprising at least one differentiating agent to induce differentiation of the mononuclear cells in a cell culture into Committed CIK Precursor cells; (b) adding at least one stimulating agent and at least one expanding agent to the cell culture; and (c) expanding the cells from the cell culture to obtain a cell population comprising Committed CIK Precursor cells, transfecting the cell population comprising the Committed CIK Precursor cells with one or more nucleic acids to produce genetically modified Committed CIK Precursor cells, and expanding the genetically modified Committed CIK Precursor cells in culture medium to produce the genetically modified CIK cells; or (c) expanding the cells from the cell culture to obtain a cell population comprising Committed CIK Precursor cells
  • the mononuclear cells are PBMCs.
  • the PBMCs were previously cryopreserved and thawed before culturing in step (a).
  • the terms “comprises”, “comprising”, “includes”, “including”, “having,” and their conjugates mean “including but not limited to.”
  • the term “consisting of” means “including and limited to.”
  • the term “consisting essentially of” means the specified material of a composition, or the specified steps of a method, and those additional materials or steps that do not materially affect the basic characteristics of the material or method.
  • the term "at least" prior to a number or series of numbers is understood to include the number adjacent to the term “at least,” and all subsequent numbers or integers that could logically be included, as clear from context.
  • the number of nucleotides in a nucleic acid molecule must be an integer.
  • "at least 18 nucleotides of a 21-nucleotide nucleic acid molecule” means that 18, 19, 20, or 21 nucleotides have the indicated property.
  • At least is also not limited to integers (e.g., “at least 5%” includes 5.0%, 5.1%, 5.18% without consideration of the number of significant figures).
  • “no more than” or “less than” is understood as the value adjacent to the phrase and logical lower values or integers, as logical from context, to zero. When “no more than” is present before a series of numbers or a range, it is understood that “no more than” can modify each of the numbers in the series or range.
  • the term “approximately,” as applied to one or more values of interest refers to a value that is similar to a stated reference value.
  • the term "approximately,” like the term, “about,” refers to a range of values that fall within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value).
  • any concentration range, percentage range, ratio range, or integer range is to be understood to include the value of any integer within the recited range and, when appropriate, fractions thereof (such as one tenth and one hundredth of an integer), unless otherwise indicated.
  • antibody and “antibodies” are terms of art and can be used interchangeably herein and refer to an immunoglobulin molecule that recognizes and specifically binds to a target, such as a protein, polypeptide, peptide, carbohydrate, polynucleotide, lipid, or combinations of the foregoing through at least one antigen recognition site within the variable region of the immunoglobulin molecule.
  • a target such as a protein, polypeptide, peptide, carbohydrate, polynucleotide, lipid, or combinations of the foregoing through at least one antigen recognition site within the variable region of the immunoglobulin molecule.
  • antibody encompasses intact polyclonal antibodies, intact monoclonal antibodies, chimeric antibodies, humanized antibodies, human antibodies, fusion proteins comprising an antibody, and any other modified immunoglobulin molecule so long as the antibodies exhibit the desired biological activity.
  • An antibody can be of any the five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, or subclasses (isotypes) thereof (e.g. IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2), based on the identity of their heavy-chain constant domains referred to as alpha, delta, epsilon, gamma, and mu, respectively.
  • the different classes of immunoglobulins have different and well-known subunit structures and three-dimensional configurations.
  • Antibodies can be naked or conjugated to other molecules such as toxins, radioisotopes, etc.
  • antibody fragment refers to a portion of an intact antibody.
  • An “antigen- binding fragment,” “antigen-binding domain,” or “antigen-binding region,” refers to a portion of an intact antibody that binds to an antigen.
  • An antigen-binding fragment can contain an antigen recognition site of an intact antibody (e.g., complementarity determining regions (CDRs) sufficient to bind antigen).
  • CDRs complementarity determining regions
  • antigen-binding fragments of antibodies include, but are not limited to Fab, Fab’, F(ab’)2, and Fv fragments, linear antibodies, and single chain antibodies.
  • an antigen-binding fragment of an antibody can be derived from any animal species, such as rodents (e.g., mouse, rat, or hamster) and humans or can be artificially produced.
  • the term "antigen" is well understood in the art and includes substances which are immunogenic, i.e., immunogen. It will be appreciated that the use of any antigen is envisioned for use in the present disclosure and thus includes, but is not limited to a self- antigen (whether normal or disease-related), an infectious antigen (e.g., a microbial antigen, viral antigen, etc.), or some other foreign antigen (e.g., a food component, pollen, etc.).
  • the term "antigen” or alternatively, "immunogen” applies to collections of more than one immunogen, so that immune responses to multiple immunogens can be modulated simultaneously.
  • the term includes any of a variety of different formulations of immunogen or antigen.
  • the antigen can be from a cancer cell (e.g., a renal cancer cell, a multiple myeloma cell, and a melanoma cell) or a pathogen (e.g., HIV and HCV).
  • the antigen can be delivered to the antigen presenting cell (APC) in the form of RNA isolated or derived from a cancer cell or a pathogen.
  • APC antigen presenting cell
  • a "native" or “natural” or “wild-type” antigen is a polypeptide, protein or a fragment which contains an epitope, which has been isolated from a natural biological source, and which can specifically bind to an antigen receptor, when presented as an HLA/peptide complex, in particular a T cell antigen receptor (TCR), in a subject.
  • TCR T cell antigen receptor
  • an "epitope” is a term in the art and refers to a localized region of an antigen to which an antibody can specifically bind.
  • An epitope can be, for example, contiguous amino acids of a polypeptide (linear or contiguous epitope) or an epitope can, for example, come together from two or more non-contiguous regions of a polypeptide or polypeptides (conformational, non- linear, discontinuous, or non-contiguous epitope). Epitopes formed from contiguous amino acids are typically, but not always, retained on exposure to denaturing solvents, whereas epitopes formed by tertiary folding are typically lost on treatment with denaturing solvents.
  • An epitope typically includes at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 20 amino acids in a unique spatial conformation.
  • Methods for determining what epitopes are bound by a given antibody i.e., epitope mapping
  • epitope mapping include, for example, immunoblotting and immunoprecipitation assays, wherein overlapping or contiguous peptides from (e.g., CD16) are tested for reactivity with a given antibody (e.g., anti-human CD16 antibody).
  • Methods of determining spatial conformation of epitopes include techniques in the art and those described herein, for example, x-ray crystallography, 2-dimensional nuclear magnetic resonance and HDX-MS (see, e.g., Epitope Mapping Protocols in Methods in Molecular Biology, Vol.66, G. E. Morris, Ed. (1996)).
  • the epitope to which an antibody binds can be determined by, e.g., NMR spectroscopy, X-ray diffraction crystallography studies, ELISA assays, hydrogen/deuterium exchange coupled with mass spectrometry (e.g., liquid chromatography electrospray mass spectrometry), array-based oligo-peptide scanning assays, and/or mutagenesis mapping (e.g., site-directed mutagenesis mapping).
  • crystallization can be accomplished using any of the known methods in the art (e.g., Giege R et al., (1994) Acta Crystallogr D Biol Crystallogr 50(Pt 4): 339-350; McPherson A (1990) Eur J Biochem 189: 1-23; Chayen NE (1997) Structure 5: 1269-1274; McPherson A (1976) J Biol Chem 251 : 6300-6303).
  • Antibody:antigen crystals can be studied using well known X-ray diffraction techniques and can be refined using computer software such as X-PLOR (Yale University, 1992, distributed by Molecular Simulations, Inc.; see, e.g., Meth Enzymol (1985) volumes 114 & 115, eds Wyckoff HW et al.,; U.S.
  • epitope mapping refers to the process of identification of the molecular determinants for antibody-antigen recognition.
  • MHC major histocompatibility complex
  • the MHC is also known as the "human leukocyte antigen” or "HLA” complex.
  • the proteins encoded by the MHC are known as “MHC molecules” and are classified into Class I and Class II MHC molecules.
  • Class I MHC molecules include membrane heterodimeric proteins made up of an ⁇ chain encoded in the MHC noncovalently linked with the ⁇ 2-microglobulin.
  • Class I MHC molecules are expressed by nearly all nucleated cells and have been shown to function in antigen presentation to CD8 + T cells.
  • Class I molecules include HLA-A, B, and C in humans.
  • Class II MHC molecules also include membrane heterodimeric proteins consisting of noncovalently associated ⁇ and ⁇ chains.
  • Class II MHC molecules are known to function in CD4 + T cells and, in humans, include HLA-DP, -DQ, and -DR. [0140]
  • the term "culturing” as used herein refers to the controlled growth of cells ex vivo and/or in vitro.
  • culturing includes the growth of cells, e.g., one or more Committed CIK Precursor cell disclosed herein, during cell expansion, or cell engineering (e.g., transfecting the cell population comprising the Committed CIK Precursor cells with one or more nucleic acids (e.g., co-electroporation with non-viral DNA construct encoding CAR (e.g., pT4-CD19-CAR) and mRNA encoding transposase (e.g., mRNA encoding SB100X transposase)).
  • CAR e.g., pT4-CD19-CAR
  • mRNA encoding transposase e.g., mRNA encoding SB100X transposase
  • the cultured cells are obtained from a subject, e.g., a human subject/patient (e.g., mononuclear cells are isolated from a human leukocyte antigen (HLA) matched donor).
  • the cultured cells comprise cells obtained from a human subject.
  • the cultured cells comprise one or more engineered cell disclosed herein (e.g., transfected cells comprising the Committed CIK Precursor cells).
  • the cultured cells comprise PBMCs obtained from a human subject/patient.
  • the expansion process can lead to an increase in the proportion or the total number of desired cells, e.g., an increase in the proportion or total number of cells in a population of cultured cells, after the cells are stimulated or activated and cultured. Expansion does not require that all cell types in a population of cultured cells are increased in number. Rather, in some aspects, only a subset of cells in a population of cultured cells are increased in number during expansion, while the number of other cell types may not change or may decrease. [0142] As used herein, the term “yield” refers to the total number of cells following a culture method or a portion thereof.
  • the term “yield” refers to a particular population of cells, e.g., Committed CIK Precursor cells in a population cultured cells or genetically modified Committed CIK Precursor cells in a population cultured cells. The yield can be determined using any methods, including, but not limited to, estimating the yield based on a representative sample.
  • the term “immune cell” refers to a cell of the immune system. In some aspects, the immune cell is selected from a T lymphocyte ("T cell”), B lymphocyte ("B cell”), natural killer (NK) cell, natural killer T lymphocytes (NKT cells), macrophage, eosinophil, mast cell, dendritic cell or neutrophil.
  • a "population" of cells refers to a collection of more than one cell, e.g., a plurality of cells.
  • the population of cells comprises more than one immune cell, e.g., a plurality of immune cells.
  • the population of cells is comprises a heterogeneous mixture of cells, comprising multiple types of cells, e.g., a heterogeneous mixture of immune cells and non-immune cells.
  • the population of cells comprises a population of mononuclear cells.
  • the population of cells comprises a population comprising Committed CIK Precursor cells.
  • the population of cells comprises a population comprising genetically modified CIK cells.
  • T cell and "T lymphocyte” are interchangeable and refer to any lymphocytes produced or processed by the thymus gland.
  • Non-limiting classes of T cells include effector T cells and T helper (Th) cells (such as CD4 + or CD8 + T cells).
  • the T cell is a Th1 cell.
  • the T cell is a Th2 cell.
  • the T cell is a Tc17 cell.
  • the T cell is a Th17 cell.
  • the T cell is a Treg cell.
  • the T cell is a tumor-infiltrating cell (TIL).
  • TIL tumor-infiltrating cell
  • memory T cells refers to T cells that have previously encountered and responded to their cognate antigen (e.g., in vivo, in vitro, or ex vivo) or which have been stimulated, e.g., with an anti-CD3 antibody (e.g., in vitro or ex vivo).
  • Immune cells having a "memory-like" phenotype upon secondary exposure, such memory T cells can reproduce to mount a faster and strong immune response than during the primary exposure.
  • memory T cells comprise central memory T cells (TCM cells), effector memory T cells (TEM cells), tissue resident memory T cells (TRM cells), stem cell- like memory T cells (T SCM cells), or any combination thereof.
  • T memory stem cells refers to memory T cells that express CD95, CD45RA, CCR7, and CD62L and are endowed with the stem cell-like ability to self-renew and the multipotent capacity to reconstitute the entire spectrum of memory and effector T cell subsets.
  • central memory T cells or “TCM cells” refers to memory T cells that express CD45RO, CCR7, and CD62L. Central memory T cells are generally found within the lymph nodes and in peripheral circulation.
  • effector memory T cells or “TEM cells” refers to memory T cells that express CD45RO but lack expression of CCR7 and CD62L. Because effector memory T cells lack lymph node-homing receptors (e.g., CCR7 and CD62L), these cells are typically found in peripheral circulation and in non-lymphoid tissues.
  • TEMRA terminally differentiated effector memory T cells
  • Temra refers to CD8 T cells which constitute a preformed effector population with an enhanced expression of effector molecules that can be efficiently activated using TCR stimulation alone or in combination with common-gamma chain cytokines.
  • tissue resident memory T cells refers to memory T cells that do not circulate and remain resident in peripheral tissues, such as skin, lung, and gastrointestinal tract. In some aspects, tissue resident memory T cells are also effector memory T cells.
  • tissue resident memory T cells are also effector memory T cells.
  • TN cells refers to T cells that express CD45RA, CCR7, and CD62L, but which do not express CD95.
  • TN cells represent the most undifferentiated cell in the T cell lineage.
  • the interaction between a T N cell and an antigen presenting cell (APC) induces differentiation of the T N cell towards an activated TEFF cell and an immune response.
  • APC antigen presenting cell
  • cytokine refers to refers to small, secreted proteins released by cells that have a specific effect on the interactions and communications between cells.
  • cytokines can possess one or more of the following properties: ability to mediate and/or regulate immune defense functions by acting as messengers between the various immune cells; functioning over short distances with a brief half-life; produced by a variety of cells types; ability to act on diverse cell targets within the immune system and/or on organs; ability to stimulate and/or inhibit growth; and/or directly or indirectly causeing a cytokine cascade.
  • cytokines can include interleukins, interferons, colony stimulating factors and tumour necrosis factor.
  • Non-limiting examples of cytokines which can be used alone or in combination in the practice of the present disclosure include, tnterleukin-1 alpha (IL-1 alpha or IL-1a), interleukin-1 beta (IL-1 beta or IL-1b), interleukin-2 (IL-2), stem cell factor (SCF), interleukin-3 (IL-3), interleukin-4 (IL-4), interleukin-5 (IL-5), interleukin-6 (IL-6), interleukin-7 (IL-7), interleukin-10 (IL- 10), interleukin-11 (IL-11), interleukin-12 (IL-12), interleukin-13 (IL-13), interleukin-15 (IL-15), interleukin-17 (IL-17), interleukin-18 (IL-18), interleukin-21 (IL-21), interleukin- 22 (IL-22), interleukin-23 (IL-23), interleukin-27 (IL-27), interleukin-33 (IL-33), granulocyte-colony stimulating factor (G-C
  • Cytokines are commercially available from several vendors such as, for example, Genzyme (Framingham, Mass.), Genentech (South San Francisco, Calif.), Amgen (Thousand Oaks, Calif.), R&D Systems (Minneapolis, Minn.) and Immunex (Seattle, Wash.). It is intended, although not always explicitly stated, that molecules having similar biological activity as wild-type or purified cytokines (e.g., recombinantly produced or muteins thereof) are intended to be used within the spirit and scope of the disclosure.
  • cytokine receptor refers to the cell-surface glycoproteins that bind specifically to cytokines and transduce their signals.
  • cytokine receptors which can be used alone or in combination in the practice of the present disclosure include, type I cytokine receptors (e.g., type 1 interleukin receptors (e.g., IL-15 receptor), erythropoietin receptor, GM-CSF receptor, G-CSF receptor, growth hormone receptor, prolactin receptor, oncostatin M receptor, eukemia inhibitory factor receptor); type II cytokine receptors (e.g., type II interleukin receptors, interferon-alpha/beta receptor, interferon-gamma receptor); members of the immunoglobulin superfamily (e.g., interleukin-1 receptor, CSF1, C-kit receptor, interleukin-18 receptor); tumor necrosis factor receptor family (e.g., CD27, CD30, CD40, CD120, lymphotoxin beta receptor); TGF
  • the cytokine receptor is interleukin-2 receptor alpha chain (also called CD25). In some aspects, the cytokine receptor is interleukin-2 receptor subunit beta (also called CD122; IL15RB; P70-75). In some aspects, the cytokine receptor is interleukin-2 receptor subunit gamma (also called CD122; IL-2RG).
  • chemokine or "chemotactic cytokine” refers to a family of small cytokines or signaling proteins secreted by cells that induce directional movement of leukocytes, as well as other cell types, including endothelial and epithelial cells.
  • chemokines are important for biological processes, including morphogenesis and wound healing, as well as in the pathogenesis of diseases like cancers. Chemokines have been classified into four main subfamilies: CXC, CC, CX3C and C. All of these proteins exert their biological effects by interacting with G protein-linked transmembrane receptors called chemokine receptors, that are selectively found on the surfaces of their target cells.
  • Non-limiting examples of cytokine receptors which can be used alone or in combination in the practice of the present disclosure include, CCL1, CCL2, CCL3, CCL4, CCL5, CCL6, CCL7, CCL8, CCL9/CCL10, CCL11, CCL12, CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCL17, XCL1, and XCL2, CX3CL1.
  • chemokine receptor refers to cytokine receptors found on the surface of certain cells that interact with a type of cytokine called a chemokine. Chemokine receptors are divided into different families, CXC chemokine receptors, CC chemokine receptors, CX3C chemokine receptors and XC chemokine receptors that correspond to the 4 distinct subfamilies of chemokines they bind. Four families of chemokine receptors differ in spacing of cysteine residues near N-terminal of the receptor.
  • Non-limiting examples of chemokine receptors which can be used alone or in combination in the practice of the present disclosure include, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CCR11, XCR1, and CX3CR1.
  • CAM refers to a subset of cell surface proteins that are involved in the binding of cells with other cells or with the extracellular matrix (ECM), in a process called cell adhesion. In essence, CAMs help cells stick to each other and to their surroundings.
  • CAMs are crucial components in maintaining tissue structure and function. In fully developed animals, these molecules play an integral role in generating force and movement and consequently ensuring that organs are able to execute their functions normally. In addition to serving as "molecular glue,” CAMs play important roles in the cellular mechanisms of growth, contact inhibition, and apoptosis. Aberrant expression of CAMs can result in a wide range of pathologies, ranging from frostbite to cancer.
  • Non-limiting examples of the cell adhesion molecules which can be used alone or in combination in the practice of the present disclosure include, (i) the immunoglobulin super family of cell adhesion molecules (IgCAMs) (e.g., neural cell adhesion molecule (N-CAM), intercellular adhesion molecule (ICAM)-1, ICAM-2, ICAM- 3, ICAM-4, ICAM-5, vascular cell adhesion molecule-1 (VCAM-1), platelet endothelial cell adhesion molecule (PE-CAM-1), L1 family (e.g., L1-CAM, neuronal cell adhesion molecule (NRCAM), neurofascin (NFASC), neural cell adhesion molecule L1-like protein also known as close homolog of L1 (CHL1)), Nectin (e.g., poliovirus receptor-related 1 (PVRL1), PVRL2, PVRL3, cell adhesion molecule 1 (CADM1), CADM3, CD155)); (ii) Integrins (e.g
  • the term "ligand,” refers to any molecule or atom that irreversibly binds to a receiving protein molecule, otherwise known as a receptor. When a ligand binds to its respective receptor, the shape and/or activity of the ligand is altered to initiate several different types of cellular responses.
  • the ligand is a costimulatory ligand.
  • the costimulatory ligand binds to the tumor necrosis factor receptor (TNFR), including OX40 (CD134), 4-1BB (CD137), CD27, glucocorticoid-induced TNFR (GITR; CD357), or CD27.
  • the costimulatory ligand is OX40L, CD137L, CD70, or any combination thereof.
  • the ligand is IL-3 zetakine.
  • the term "enzyme,” refers to the protein that act as biological catalysts by accelerating chemical reactions. The molecules upon which enzymes can act are called substrates, and the enzyme converts the substrates into different molecules known as products.
  • Non-limiting examples of enzymes that can be used in the present methods comprise a prodrug converting enzyme, an IgG-degrading enzyme of S. pyogenes (IdeS), a consensus variant 1 (CV1) protein, matrix metalloproteinase (MMP), heparinase, or any combination thereof.
  • checkpoint inhibitor refers to the inhibitor that blocks signaling through the particular immune checkpoint pathway.
  • immune checkpoint inhibitors that can be used in the present methods comprise a CTLA-4 antagonist (e.g., anti-CTLA-4 antibody or antigen binding fragment thereof), PD-1 antagonist (e.g., anti-PD-1 antibody or antigen binding fragment thereof, anti-PD-L1 antibody or antigen binding fragment thereof), TIM-3 antagonist (e.g., anti-TIM-3 antibody or antigen binding fragment thereof), or combinations thereof.
  • the checkpoint inhibitor is a PD-1 antagonist.
  • the checkpoint inhibitor is an anti-PD-1 antibody.
  • the checkpoint inhibitor is an anti-PD-L1 antibody or antigen binding fragment thereof. In some aspects, the checkpoint inhibitor is a TIM-3 antagonist. In some aspects, the checkpoint inhibitor is an anti-TIM-3 antibody or antigen binding fragment thereof. [0161] As used herein, the term "differentiating agent,” refers to an agent that induces differentiation of the mononuclear cells in a cell culture into Committed CIK Precursor cells.
  • Non-limiting examples of differentiating agents that can be used in the present methods comprise IFN- ⁇ , IL-4, IL-5, IL-7, IFN- ⁇ , IL-10, IL-12, IL-13, IL-6, IL-15, IL-17, IL-18, IL-21, IL-22, IL-23, IL-27, IL-1 ⁇ , TGF- ⁇ , GM-CSF, CCL3, CCL4, CCL5, CCL17, CCL21, or any combination thereof.
  • the differentiating agent is IFN- ⁇ .
  • the term "stimulating agent,” refers to an agent that supports the stimulation of T cells (e.g., CD3+ cells), before, during or after the transfer of nucleic acids.
  • Non-limiting examples of stimulating agents that can be used in the present methods comprise an anti-CD3 antibody, an anti-TCR antibody, an anti-CD28 antibody, an anti- CD137 antibody, an anti-CD134 antibody, an anti-CD27 antibody, an anti-ICAM-1 antibody, an anti-CD3/CD28-coated beads, a superantigen, phytohaemaglutinin (PHA), phorbol 12-myristate 13-acetate (PMA), ionomycin, or any combination thereof.
  • the stimulating agent is an anti-CD3 antibody.
  • the anti-CD3 antibody is an OKT3 antibody.
  • the term "expanding agent,” refers to an agent that expands cells (e.g., mononuclear cells, such as for example human peripheral blood mononuclear cells (PBMCs)) to generate cells and/or cell populations comprising, for example, the genetically modified CIK cells that express one or more T cell receptors (CIK-TCR), chimeric antigen receptors (CAR-CIK), a genetically modified cell adhesion molecule, a genetically modified ligand, a genetically modified cytokine receptor, a genetically modified chemokine receptor, a genetically modified cytokine, a genetically modified chemokine, an enzyme, or a checkpoint inhibitor.
  • CIK-TCR T cell receptors
  • CAR-CIK chimeric antigen receptors
  • a genetically modified cell adhesion molecule e.g., a genetically modified ligand, a genetically modified cytokine receptor, a genetically modified chemokine receptor, a genetic
  • Non-limiting examples of expanding agents that can be used in the present methods IL-2, IL-4, IL-7, IL-9, IL-15, IL-18, IL-21, or any combination thereof.
  • the expanding agent is IL-2.
  • the terms "mononuclear cell,” “mononuclear cells,” and “MNCs” refer to a mixture of different types of cells and contain most of the different stem cells within this component of the marrow, but principally contain a number of immature and mature cell types of different myeloid, lymphoid, and erythroid lineages.
  • Non-limiting examples of mononuclear cells that can be used in the present methods comprise umbilical cord blood derived mononuclear cells, peripheral blood mononuclear cells (PBMCs), bone marrow derived mononuclear cells, lymphocytes, monocytes, dendritic cells, macrophages, T cells, naive T cells, memory T cells, natural killer cells, hematopoietic stem cells, pluripotent embryonic stem cells, induced pluripotent stem cells, or any combination thereof.
  • the mononuclear cells are umbilical cord blood derived mononuclear cells.
  • the mononuclear cells are PBMCs.
  • the PBMCs were previously cryopreserved and thawed before culturing in step (a).
  • the mononuclear cells are from a human leukocyte antigen (HLA) matched donor.
  • HLA human leukocyte antigen
  • APCs antigen presenting cells
  • APCs can be intact whole cells such as macrophages, B-cells, endothelial cells, activated T-cells, and dendritic cells; or other molecules, naturally occurring or synthetic, such as purified MHC Class I molecules complexed to ⁇ 2-microglobulin. While many types of cells may be capable of presenting antigens on their cell surface for T-cell recognition, only dendritic cells have the capacity to present antigens in an efficient amount to activate naive T-cells for cytotoxic T-lymphocyte (CTL) responses.
  • CTL cytotoxic T-lymphocyte
  • immune effector cells refers to cells capable of binding an antigen and which mediate an immune response.
  • a "na ⁇ ve" immune effector cell is an immune effector cell that has never been exposed to an antigen capable of activating that cell. Activation of naive immune effector cells requires both recognition of the peptide:HLA complex and the simultaneous delivery of a costimulatory signal by a professional APC in order to proliferate and differentiate into antigen-specific armed effector T cells.
  • an "immune response” is as understood in the art, and generally refers to a biological response within a vertebrate against foreign agents or abnormal, e.g., cancerous cells, which response protects the organism against these agents and diseases caused by them.
  • An immune response is mediated by the action of one or more cells of the immune system (for example, a T lymphocyte, B lymphocyte, natural killer (NK) cell, macrophage, eosinophil, mast cell, dendritic cell or neutrophil) and soluble macromolecules produced by any of these cells or the liver (including antibodies, cytokines, and complement) that results in selective targeting, binding to, damage to, destruction of, and/or elimination from the vertebrate's body of invading pathogens, cells or tissues infected with pathogens, cancerous or other abnormal cells, or, in cases of autoimmunity or pathological inflammation, normal human cells or tissues.
  • a T lymphocyte, B lymphocyte, natural killer (NK) cell for example, a T lymphocyte, B lymphocyte, natural killer (NK) cell, macrophage, eosinophil, mast cell, dendritic cell or neutrophil
  • soluble macromolecules produced by any of these cells or the liver (including antibodies, cytokines, and complement) that results
  • An immune reaction includes, e.g., activation or inhibition of a T cell, e.g., an effector T cell, a Th cell, a CD4 + cell, a CD8 + T cell, or a Treg cell, or activation or inhibition of any other cell of the immune system, e.g., NK cell.
  • a T cell e.g., an effector T cell, a Th cell, a CD4 + cell, a CD8 + T cell, or a Treg cell
  • a Treg cell e.g., a T cell
  • the term "educated, antigen-specific immune effector cell” is an immune effector cell as defined above, which has previously encountered an antigen. In contrast to its naive counterpart, activation of an educated, antigen specific immune effector cell does not require a costimulatory signal. Recognition of the peptide:HLA complex is sufficient.
  • Immunotherapy refers to the treatment of a subject afflicted with, or at risk of contracting or suffering a recurrence of, a disease by a method comprising inducing, enhancing, suppressing or otherwise modifying the immune system or an immune response.
  • Immuno stimulating therapy or “immuno stimulatory therapy” refers to a therapy that results in increasing (inducing or enhancing) an immune response in a subject for, e.g., treating cancer.
  • An increased ability to stimulate an immune response or the immune system can result from an enhanced agonist activity of T cell co-stimulatory receptors and/or an enhanced antagonist activity of inhibitory receptors.
  • T cell-mediated response refers to a response mediated by T cells, including effector T cells (e.g., CD8+ cells) and helper T cells (e.g., CD4+ cells).
  • T cell mediated responses include, for example, T cell cytotoxicity and proliferation.
  • CTL cytotoxic T lymphocyte
  • autologous refers to any material derived from the same individual to which it is later to be re-introduced.
  • the tumor antigen that is autologous to the patient comprises administering to a subject the tumor antigen that was isolated from the same subject.
  • a “cancer” refers to a broad group of various diseases characterized by the uncontrolled growth of abnormal cells in the body.
  • Unregulated cell division and growth results in the formation of malignant tumors that invade neighboring tissues and can also metastasize to distant parts of the body through the lymphatic system or bloodstream.
  • An example of a cancer that can be treated by the methods of the present disclosure includes, but is not limited to, renal cell cancer.
  • the methods of the present disclosure can be used to reduce the tumor size of a tumor derived from, for example, renal cancer, breast cancer, pancreatic cancer, brain cancer (e.g., astrocytoma, glioblastoma multiforme), bone cancer, prostate cancer, colon cancer, lung cancer, cutaneous or intraocular malignant melanoma, skin cancer, cancer of the head or neck, cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, testicular cancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, Waldenström macroglobulinaemia, Hodgkin's Disease, non-Hodgkin's lymphoma (NHL), primary mediastinal large B cell lymphoma (PMBC), diffuse large B cell lymphoma (DLBCL), follicular lymph
  • NHL
  • tumor refers to any mass of tissue that results from excessive cell growth or proliferation, either benign (non-cancerous) or malignant (cancerous), including pre-cancerous lesions.
  • inflammatory disease or "inflammatory disorder” refers to a medical condition at least partially characterised by inappropriate secretion of inflammatory mediators (e.g. highly toxic reactive oxygen intermediates (ROIs) or granule enzymes or cytokines) from granulocytes into an affected tissue.
  • ROIs reactive oxygen intermediates
  • cytokines granule enzymes or cytokines
  • Such conditions include, but are not limited to, rheumatoid arthritis, Behcet's disease, Anti-Neutrophil Cytoplasmic Antibody (ANCA)-associated vasculitis, systemic vasculitis, cystic fibrosis, asthma, Crohn's Disease, multiple sclerosis, autoimmune tyroiditis, diabetes mellitus (Juvenile onset diabetes), autoimmune uveoretinitis, myasthenia gravis, systemic lupus erythematosus (SLE), Sjögren's syndrome, celiac disease, alopecia, irritable bowel syndrome (IBS), psoriasis, and any combination thereof.
  • ANCA Anti-Neutrophil Cytoplasmic Antibody
  • autoimmune disease refers to diseases and disorders that occur as a result of the immune system attacking the body’s own organs, tissues, and cells.
  • Autoimmune diseases in mammals can generally be classified in one of two different categories: cell-mediated disease (i.e., T-cell) or antibody-mediated disorders.
  • cell-mediated autoimmune diseases include multiple sclerosis, rheumatoid arthritis, autoimmune tyroiditis, diabetes mellitus (Juvenile onset diabetes), and autoimmune uveoretinitis.
  • Antibody-mediated autoimmune disorders include myasthenia gravis and systemic lupus erythematosus (or SLE).
  • viral infection refers to an infection from e.g., Classes I through V viruses.
  • Class I-V viruses refers to the different classes of virus identified by genome composition and strategy for mRNA synthesis, as described in Lodish, H. et al., Molecular Cell Biology, Fourth Edition, W.H. Freeman and Company (2000).
  • Class I-V viruses are identified as follows: Class I viruses contain a single molecule of double-stranded DNA; Class II viruses contain a single molecule of single-stranded DNA; Class III viruses contain double-stranded genomic RNA; Class IV viruses contain a single strand of viral mRNA (also known as a positive/plus strand of genomic RNA), wherein the viral mRNA encodes proteins and is infectious by itself; and Class V viruses contain a single strand of an RNA sequence that is complimentary to the genomic viral mRNA (also known as a negative/minus strand of genomic RNA), wherein the genomic RNA acts as a template for synthesis of mRNA but does not itself encode proteins.
  • Non-limiting examples of viruses that can treated by the present invention include arboviruses (including but not limited to dengue virus, yellow fever, etc.); adenoviruses (acute respiratory disease, pneumonia, conjunctivitis, gastroenteritis, pharynx) Inflammation, acute hemorrhagic cystitis, African swine fever, swine circovirus, swine adenovirus type A, type B, and type C); herpes virus [herpes simplex virus, varicella-zoster virus (Including, but not limited to, chicken pox and shingles), Epstein-Barr virus; human papillomavirus (including but not limited to HPV types 1-65); parvovirus (parvovirus B19, canine parvovirus) Including but not limited to: reovirus (orbivirus, rotavirus) Including, but not limited to, aquareovirus, cortivirus; picornavirus (including but not limited to enterovirus,
  • composition refers to a combination of active agent and another compound or composition, inert (for example, a detectable agent or label) or active, such as an adjuvant.
  • pharmaceutical composition refers to the combination of an active agent with a carrier, inert or active, making the composition suitable for diagnostic or therapeutic use in vitro, in vivo or ex vivo.
  • the term "pharmaceutically acceptable carrier” encompasses any of the standard pharmaceutical carriers, such as a phosphate buffered saline solution, water, and emulsions, such as an oil/water or water/oil emulsion, and various types of wetting agents.
  • the compositions also can include stabilizers and preservatives.
  • stabilizers and adjuvants see Martin REMINGTON'S PHARM. SCI., 18th Ed. (Mack Publ. Co., Easton (1990)).
  • the term “formulating” refers to formulating transfected cells comprising the Committed CIK Precursor cells, as described herein, that are recovered post electroporation. In some aspects, transfected cells comprising the Committed CIK Precursor cells are for administration to the subject. In some aspects, transfected cells comprising the Committed CIK Precursor cells are formulated in cryopreservation media.
  • administering refers to the physical introduction of an agent to a subject, using any of the various methods and delivery systems known to those skilled in the art.
  • Exemplary routes of administration for the formulations disclosed herein include intravenous, intramuscular, subcutaneous, intraperitoneal, spinal or other parenteral routes of administration, for example by injection or infusion.
  • parenteral administration means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion, as well as in vivo electroporation.
  • the formulation is administered via a non-parenteral route, e.g., orally.
  • non-parenteral routes include a topical, epidermal or mucosal route of administration, for example, intranasally, vaginally, rectally, sublingually or topically.
  • Administering can also be performed, for example, once, a plurality of times, and/or over one or more extended periods.
  • a "subject” includes any human or nonhuman animal.
  • the term "nonhuman animal” includes, but is not limited to, vertebrates such as nonhuman primates, sheep, dogs, and rodents such as mice, rats and guinea pigs.
  • the subject is a human.
  • subject refers to subjects, such as mammalian subjects, that would benefit, e.g., from administration of formulated transfected cells comprising the Committed CIK Precursor cells cultured using the methods provided herein, as described herein.
  • therapeutically effective amount refers to an amount of an agent (e.g., formulated transfected cells comprising the Committed CIK Precursor cells cultured as described herein) that provides the desired biological, therapeutic, and/or prophylactic result.
  • an effective amount comprises an amount sufficient to cause a tumor to shrink and/or to decrease the growth rate of the tumor (such as to suppress tumor growth) or to prevent or delay other unwanted cell proliferation.
  • an effective amount is an amount sufficient to delay tumor development.
  • an effective amount is an amount sufficient to prevent or delay tumor recurrence.
  • An effective amount can be administered in one or more administrations.
  • the effective amount of the composition can, for example, (i) reduce the number of cancer cells; (ii) reduce tumor size; (iii) inhibit, delay, slow to some extent and can stop cancer cell infiltration into peripheral organs; (iv) inhibit (i.e., slow to some extent and can stop tumor metastasis); (v) inhibit tumor growth; (vi) prevent or delay occurrence and/or recurrence of tumor; and/or (vii) relieve to some extent one or more of the symptoms associated with the cancer.
  • a "therapeutically effective amount” is the amount of a composition disclosed herein (e.g., formulated transfected cells comprising the Committed CIK Precursor cells cultured as described herein), which is clinically proven to effect a significant decrease in cancer or slowing of progression (regression) of cancer, such as an advanced solid tumor.
  • a therapeutic agent of the present disclosure e.g., formulated transfected cells comprising the Committed CIK Precursor cells cultured as described herein
  • to promote disease regression can be evaluated using a variety of methods known to the skilled practitioner, such as in human subjects during clinical trials, in animal model systems predictive of efficacy in humans, or by assaying the activity of the agent in in vitro assays.
  • the terms "effective” and “effectiveness” with regard to a treatment include both pharmacological effectiveness and physiological safety.
  • Pharmacological effectiveness refers to the ability of a composition disclosed herein (e.g., immune cells modified and cultured as described herein) to promote cancer regression in the patient.
  • Physiological safety refers to the level of toxicity, or other adverse physiological effects at the cellular, organ, and/or organism level (adverse effects) resulting from administration of a composition disclosed herein (e.g., immune cells modified and cultured as described herein).
  • cytokine-induced killer cell refers to multi functional cells that can express both T cell and natural killer T (NKT) cells markers (e.g., CD3+CD56+).
  • TAT natural killer T
  • the terminally differentiated CD3- and CD56-positive subset of the CIK cells primarily exert the direct HLA-unrestricted tumor killing activity.
  • the tems “committed cytokine-induced killer precursor cells,” “committed cytokine induced killer precursor cells,” and “committed CIK precursor cells” refer to activated day 2 cells derived from peripheral blood mononuclear cells (PBMCs) by the sequential addition of one or more cytokines, such as IFN- ⁇ on day 0, and then stimulation through the CD3 receptor with the addition of IL-2 on day 1. Based on the fact that activated day 2 cells still have high CD28 and CCR7 together with the fact that the cells did not yet upregulate CD56 and NKG2D markers, they cannot be called cytokine induced killer (CIK) cells yet.
  • PBMCs peripheral blood mononuclear cells
  • chimeric antigen receptor and "CAR,” as used herein, refer to a set of polypeptides, typically two in the simplest form, which when in an immune effector cell, provides the cell with specificity for a target cell, typically a cancer cell, and with intracellular signal generation.
  • a CAR comprises at least an extracellular antigen-binding domain, a transmembrane domain and a cytoplasmic signaling domain (also referred to herein as "an intracellular signaling domain") comprising a functional signaling domain derived from a stimulatory molecule and/or costimulatory molecule as defined below.
  • the set of polypeptides are in the same polypeptide chain, e.g., comprise a chimeric fusion protein.
  • the set of polypeptides are not contiguous with each other, e.g., are in different polypeptide chains.
  • the set of polypeptides include a dimerization switch that, upon the presence of a dimerization molecule, can couple the polypeptides to one another, e.g., can couple an antigen-binding domain to an intracellular signaling domain.
  • the stimulatory molecule of the CAR is the zeta chain associated with the T cell receptor complex (e.g., CD3 zeta).
  • the cytoplasmic signaling domain comprises a primary signaling domain (e.g., a primary signaling domain of CD3-zeta).
  • the cytoplasmic signaling domain further comprises one or more functional signaling domains derived from at least one costimulatory molecule as defined below.
  • the costimulatory molecule is chosen from the costimulatory molecules described herein, e.g., 4-1BB (i.e., CD137), CD27, and/or CD28.
  • the CAR comprises a chimeric fusion protein comprising an antigen-binding domain, a transmembrane domain, and an intracellular signaling domain comprising a functional signaling domain derived from a stimulatory molecule, wherein the antigen-binding domain and the transmembrane domain are linked by a CAR spacer.
  • the CAR comprises a chimeric fusion protein comprising an antigen-binding domain linked to a transmembrane domain via a CAR spacer and an intracellular signaling domain comprising a functional signaling domain derived from a costimulatory molecule and a functional signaling domain derived from a stimulatory molecule.
  • the CAR comprises a chimeric fusion protein comprising an antigen-binding domain linked to a transmembrane domain via a CAR spacer and an intracellular signaling domain comprising two functional signaling domains derived from one or more costimulatory molecule(s) and a functional signaling domain derived from a stimulatory molecule.
  • the CAR comprises a chimeric fusion protein comprising an antigen-binding domain linked to a transmembrane domain via a CAR spacer and an intracellular signaling domain comprising at least two functional signaling domains derived from one or more costimulatory molecule(s) and a functional signaling domain derived from a stimulatory molecule.
  • the CAR comprises an optional leader sequence at the amino- terminus (N-terminus) of the CAR.
  • the CAR further comprises a leader sequence at the N-terminus of the antigen-binding domain, wherein the leader sequence is optionally cleaved from the antigen-binding domain (e.g., a scFv) during cellular processing and localization of the CAR to the cellular membrane.
  • the antigen-specific extracellular domain of a chimeric antigen receptor recognizes and specifically binds an antigen, typically a surface-expressed antigen of a malignancy.
  • An antigen-specific extracellular domain specifically binds an antigen when, for example, it binds the antigen with an affinity constant or affinity of interaction (K D ) between about 0.1 pM to about 10 ⁇ M, for example, about 0.1 pM to about 1 ⁇ M or about 0.1 pM to about 100 nM.
  • K D affinity constant or affinity of interaction
  • An antigen-specific extracellular domain suitable for use in a CAR of the present disclosure can be any antigen-binding polypeptide, a wide variety of which are known in the art.
  • the antigen-binding domain is a single chain Fv (scFv).
  • T cell receptor (TCR) based recognition domains such as single chain TCR (scTv, i.e., single chain two-domain TCR containing V ⁇ V ⁇ ) are also suitable for use in the chimeric binding proteins of the present disclosure.
  • T cell receptor refers to a heterodimer composed of 2 different transmembrane polypeptide chains: an ⁇ chain and a ⁇ chain, each consisting of a constant region, which anchors the chain inside the T-cell surface membrane, and a variable region, which recognizes and binds to the antigen presented by HLAs.
  • the TCR complex is associated with 6 polypeptides forming 2 heterodimers, CD3 ⁇ and CD3 ⁇ , and 1 homodimer ⁇ , which together forms the CD3 complex.
  • T-cell receptor- engineered T-cell therapy utilizes the modification of T cells that retain these complexes to specifically target the antigens expressed by particular tumor cells.
  • TCR includes naturally occurring TCRs and engineered TCRs.
  • a "TCR mimic” or a “TCRm” refers to a type of antibody that recognize epitopes comprising both the peptide and the MHC-I molecule, similar to the recognition of such complexes by the TCR on T cells.
  • the term “genetically modified” refers to containing and/or expressing a foreign gene or nucleic acid sequence which in turn, modifies the genotype or phenotype of the cell or its progeny. In other words, it refers to any addition, deletion or disruption to a cell's endogenous nucleotides.
  • nucleic acids can be used interchangeably and refer to the phosphate ester polymeric form of ribonucleosides (adenosine, guanosine, uridine or cytidine; "RNA molecules”) or deoxyribonucleosides (deoxyadenosine, deoxyguanosine, deoxythymidine, or deoxycytidine; "DNA molecules”), or any phosphoester analogs thereof, such as phosphorothioates and thioesters, in either single stranded form, or a double-stranded helix.
  • Single stranded nucleic acid sequences refer to single-stranded DNA (ssDNA) or single- stranded RNA (ssRNA). Double stranded DNA-DNA, DNA-RNA and RNA-RNA helices are possible.
  • nucleic acid molecule and in particular DNA or RNA molecule, refers only to the primary and secondary structure of the molecule, and does not limit it to any particular tertiary forms. Thus, this term includes double-stranded DNA found, inter alia, in linear or circular DNA molecules (e.g., restriction fragments), plasmids, supercoiled DNA and chromosomes.
  • a "recombinant DNA molecule” is a DNA molecule that has undergone a molecular biological manipulation.
  • DNA includes, but is not limited to, cDNA, genomic DNA, plasmid DNA, synthetic DNA, and semi- synthetic DNA.
  • a "nucleic acid composition" of the disclosure comprises one or more nucleic acids as described herein.
  • a polynucleotide of the present disclosure can comprise a single nucleotide sequence encoding a single protein.
  • a polynucleotide of the present disclosure is polycistronic (i.e., comprises two or more cistrons).
  • each of the cistrons of a polycistronic polynucleotide can encode for a protein disclosed herein.
  • each of the cistrons can be translated independently of one another.
  • polypeptide encompasses both peptides and proteins, unless indicated otherwise.
  • Polypeptides include gene products, naturally occurring polypeptides, synthetic polypeptides, homologs, orthologs, paralogs, fragments and other equivalents, variants, and analogs of the foregoing.
  • a polypeptide can be a single polypeptide or can be a multi-molecular complex such as a dimer, trimer or tetramer. They can also comprise single chain or multichain polypeptides. Most commonly disulfide linkages are found in multichain polypeptides.
  • the term polypeptide can also apply to amino acid polymers in which one or more amino acid residues are an artificial chemical analogue of a corresponding naturally occurring amino acid.
  • a "peptide” can be less than or equal to 50 amino acids long, e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids long.
  • fragment of a polypeptide refers to an amino acid sequence of a polypeptide that is shorter than the naturally-occurring sequence, N- and/or C-terminally deleted or any part of the polypeptide deleted in comparison to the naturally occurring polypeptide. Thus, a fragment does not necessary need to have only N- and/or C- terminal amino acids deleted. A polypeptide in which internal amino acids have been deleted with respect to the naturally occurring sequence is also considered a fragment.
  • a functional fragment refers to a polypeptide fragment that retains polypeptide function. Accordingly, in some aspects, a functional fragment of an Ig hinge, retains the ability to position an antigen-binding domain (e.g., an scFv) in a chimeric binding protein at a distance from a target epitope (e.g., a tumor antigen) such that the antigen-binding domain (e.g., an scFv) can effectively interact with the target epitope (e.g., a tumor antigen).
  • a "recombinant" polypeptide or protein refers to a polypeptide or protein produced via recombinant DNA technology.
  • polypeptides and proteins expressed in engineered host cells are considered isolated for the purpose of the disclosure, as are native or recombinant polypeptides which have been separated, fractionated, or partially or substantially purified by any suitable technique.
  • the polypeptides encoded by the polynucleotides disclosed herein can be recombinantly produced using methods known in the art.
  • the polypeptides encoded by the polynucleotides of the present disclosure are produced by cells, e.g., T cells, following transfection or modification with at least one polynucleotide or vector encoding the polypeptides described here.
  • a "coding region,” “coding sequence,” or “translatable sequence” is a portion of polynucleotide which consists of codons translatable into amino acids.
  • a “stop codon” (TAG, TGA, or TAA) is typically not translated into an amino acid, it can be considered to be part of a coding region, but any flanking sequences, for example promoters, ribosome binding sites, transcriptional terminators, introns, and the like, are not part of a coding region.
  • a coding region typically determined by a start codon at the 5' terminus, encoding the amino terminus of the resultant polypeptide, and a translation stop codon at the 3' terminus, encoding the carboxyl terminus of the resulting polypeptide.
  • the terms "complementary” and “complementarity” refer to two or more oligomers (i.e., each comprising a nucleobase sequence), or between an oligomer and a target gene, that are related with one another by Watson-Crick base-pairing rules.
  • nucleobase sequence "T-G-A (5' to 3'),” is complementary to the nucleobase sequence "A- C-T (3' to 5').”
  • Complementarity can be “partial,” in which less than all of the nucleobases of a given nucleobase sequence are matched to the other nucleobase sequence according to base pairing rules.
  • complementarity between a given nucleobase sequence and the other nucleobase sequence can be about 70%, about 75%, about 80%, about 85%, about 90%, or about 95%.
  • the term "complementary” refers to at least about 80%, at least about 85%, 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%, or at least about 99% match or complementarity to a target nucleic acid sequence (e.g., miR-485 nucleic acid sequence). Or, there can be “complete” or “perfect” (100%) complementarity between a given nucleobase sequence and the other nucleobase sequence to continue the example. In some aspects, the degree of complementarity between nucleobase sequences has significant effects on the efficiency and strength of hybridization between the sequences.
  • telomere a gene product
  • mRNA messenger RNA
  • expression produces a "gene product.”
  • a gene product can be either a nucleic acid, e.g., a messenger RNA produced by transcription of a gene, or a polypeptide which is translated from a transcript.
  • Gene products described herein further include nucleic acids with post transcriptional modifications, e.g., polyadenylation or splicing, or polypeptides with post translational modifications, e.g., methylation, glycosylation, the addition of lipids, association with other protein subunits, or proteolytic cleavage.
  • Gene delivery e.g., polyadenylation or splicing, or polypeptides with post translational modifications, e.g., methylation, glycosylation, the addition of lipids, association with other protein subunits, or proteolytic cleavage.
  • Transfection refers to delivery of any nucleic acid to the interior of a cell.
  • Gene delivery refers to the delivery of a nucleic acid that can be integrated into the host cell's genome, or that can replicate independently of the host cell genome.
  • Transfection methods include a variety of techniques such as electroporation, protein-based, lipid-based and cationic ion based nucleic acid delivery complexes, viral vectors, “gene gun” delivery and various other techniques known to those of skill in the art.
  • the introduced polynucleotide can be stably maintained in the host cell or can be transiently expressed.
  • Stable maintenance typically requires that the introduced polynucleotide either contains an origin of replication compatible with the host cell or integrates into a replicon of the host cell such as an extrachromosomal replicon (e.g., a plasmid) or a nuclear or mitochondrial chromosome.
  • a replicon of the host cell such as an extrachromosomal replicon (e.g., a plasmid) or a nuclear or mitochondrial chromosome.
  • a number of vectors are capable of mediating transfer of genes to mammalian cells, as is known in the art.
  • electroroporation refers to a physical transfection method that permeabilizes the cell membrane by applying an electrical pulse and moves molecules via the electrical field into the cell.
  • identity refers to the overall monomer conservation between polymeric molecules, e.g., between polynucleotide molecules.
  • identity without any additional qualifiers, e.g., polynucleotide A is identical to polynucleotide B, implies the polynucleotide sequences are 100% identical (100% sequence identity).
  • Calculation of the percent identity of two polypeptide or polynucleotide sequences can be performed by aligning the two sequences for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second polypeptide or polynucleotide sequences for optimal alignment and non-identical sequences can be disregarded for comparison purposes).
  • the length of a sequence aligned for comparison purposes is at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or about 100% of the length of the reference sequence.
  • the amino acids at corresponding amino acid positions, or bases in the case of polynucleotides are then compared.
  • a position in the first sequence is occupied by the same amino acid or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position.
  • the percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which needs to be introduced for optimal alignment of the two sequences.
  • the comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm.
  • Suitable software programs that can be used to align different sequences are available from various sources.
  • Bl2seq performs a comparison between two sequences using either the BLASTN or BLASTP algorithm.
  • BLASTN is used to compare nucleic acid sequences
  • BLASTP is used to compare amino acid sequences.
  • Other suitable programs are, e.g., Needle, Stretcher, Water, or Matcher, part of the EMBOSS suite of bioinformatics programs and also available from the European Bioinformatics Institute (EBI) at ebi.ac.uk/Tools/psa.
  • Sequence alignments can be conducted using methods known in the art such as MAFFT, Clustal (ClustalW, Clustal X or Clustal Omega), MUSCLE, etc.
  • Different regions within a single polynucleotide or polypeptide target sequence that aligns with a polynucleotide or polypeptide reference sequence can each have their own percent sequence identity. It is noted that the percent sequence identity value is rounded to the nearest tenth. For example, 80.11, 80.12, 80.13, and 80.14 are rounded down to 80.1, while 80.15, 80.16, 80.17, 80.18, and 80.19 are rounded up to 80.2. It also is noted that the length value will always be an integer.
  • sequence alignments can be generated by integrating sequence data with data from heterogeneous sources such as structural data (e.g., crystallographic protein structures), functional data (e.g., location of mutations), or phylogenetic data.
  • a suitable program that integrates heterogeneous data to generate a multiple sequence alignment is T-Coffee, available at tcoffee.org, and alternatively available, e.g., from the EBI. It will also be appreciated that the final alignment used to calculate percent sequence identity can be curated either automatically or manually.
  • an isolated composition has no detectable undesired activity or, alternatively, the level or amount of the undesired activity is at or below an acceptable level or amount. In some aspects, an isolated composition has an amount and/or concentration of desired composition of the present disclosure, at or above an acceptable amount and/or concentration and/or activity.
  • the isolated composition is enriched as compared to the starting material from which the composition is obtained. This enrichment can be by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.9%, at least about 99.99%, at least about 99.999%, at least about 99.9999%, or greater than 99.9999% as compared to the starting material.
  • isolated preparations are substantially free of residual biological products.
  • the isolated preparations are 100% free, at least about 99% free, at least about 98% free, at least about 97% free, at least about 96% free, at least about 95% free, at least about 94% free, at least about 93% free, at least about 92% free, at least about 91% free, or at least about 90% free of any contaminating biological matter.
  • Residual biological products can include abiotic materials (including chemicals) or unwanted nucleic acids, proteins, lipids, or metabolites.
  • the term "linked" as used herein refers to a first amino acid sequence or polynucleotide sequence covalently or non-covalently joined to a second amino acid sequence or polynucleotide sequence, respectively.
  • the first amino acid or polynucleotide sequence can be directly joined or juxtaposed to the second amino acid or polynucleotide sequence or alternatively an intervening sequence can covalently join the first sequence to the second sequence.
  • the term "linked" means not only a fusion of a first polynucleotide sequence to a second polynucleotide sequence at the 5'-end or the 3'-end, but also includes insertion of the whole first polynucleotide sequence (or the second polynucleotide sequence) into any two nucleotides in the second polynucleotide sequence (or the first polynucleotide sequence, respectively).
  • the first polynucleotide sequence can be linked to a second polynucleotide sequence by a phosphodiester bond or a linker.
  • the linker can be, e.g., a polynucleotide.
  • Exemplary routes of administration include intravenous, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, intrasterna, oral, rectal, topical, epidermal, mucosal, intranasal, vaginal, rectal, sublingual administration, and combinations thereof.
  • Administering can also be performed, for example, once, a plurality of times, and/or over one or more extended periods.
  • Treatment or “therapy” (including any grammatical derivatives thereof) of a subject refers to any type of intervention or process performed on, or the administration of an active agent to, a subject with the objective of reversing, alleviating, ameliorating, inhibiting, slowing down, or preventing the onset, progression, development, severity, or recurrence of a symptom, complication, condition, or biochemical indicia associated with a disease.
  • the term refers to inducing an immune response in a subject against an antigen.
  • prevent refers partially or completely delaying onset of an disease, disorder and/or condition; partially or completely delaying onset of one or more symptoms, features, or clinical manifestations of a particular disease, disorder, and/or condition; partially or completely delaying onset of one or more symptoms, features, or manifestations of a particular disease, disorder, and/or condition; partially or completely delaying progression from a particular disease, disorder and/or condition; and/or decreasing the risk of developing pathology associated with the disease, disorder, and/or condition. In some aspects, preventing an outcome is achieved through prophylactic treatment.
  • the term "therapeutically effective amount” is the amount of reagent or pharmaceutical compound comprising a composition disclosed herein (e.g., modified immune cell described herein) that is sufficient to a produce a desired therapeutic effect, pharmacologic and/or physiologic effect on a subject in need thereof.
  • a therapeutically effective amount can be a "prophylactically effective amount” as prophylaxis can be considered therapy.
  • prophylaxis can be considered therapy.
  • prophylactic refers to a therapeutic or course of action used to prevent the onset of a disease or condition, or to prevent or delay a symptom associated with a disease or condition.
  • a “prophylaxis” refers to a measure taken to maintain health and prevent the onset of a disease or condition, or to prevent or delay a symptom associated with a disease or condition.
  • the term “promoter” refers to DNA sequence capable of controlling the expression of a coding sequence or functional RNA. In general, a coding sequence is located 3' to a promoter sequence. Promoters can be derived in their entirety from a native gene, or be composed of different elements derived from different promoters found in nature, or even comprise synthetic DNA segments.
  • promoters can direct the expression of a gene in different tissues or cell types, or at different stages of development, or in response to different environmental or physiological conditions. Promoters that cause a gene to be expressed in most cell types at most times are commonly referred to as “constitutive promoters.” Promoters that cause a gene to be expressed in a specific cell type are commonly referred to as “cell-specific promoters” or “tissue-specific promoters.” Promoters that cause a gene to be expressed at a specific stage of development or cell differentiation are commonly referred to as “developmentally-specific promoters” or “cell differentiation-specific promoters.” Promoters that are induced and cause a gene to be expressed following exposure or treatment of the cell with an agent, biological molecule, chemical, ligand, light, or the like that induces the promoter are commonly referred to as “inducible promoters” or “regulatable promoters.” It is further recognized that since in most cases the exact boundaries of regulatory sequences have not been completely defined
  • a solution e.g., buffer
  • ug uM
  • ⁇ g ⁇
  • ⁇ ⁇
  • the present disclosure is directed to methods of generating genetically modified cytokine induced killer (CIK) cells, comprising the sequential steps of: (a) culturing a population of mononuclear cells in culture medium comprising at least one differentiating agent to induce differentiation of the mononuclear cells in a cell culture into Committed CIK Precursor cells; (b) adding at least one stimulating agent and at least one expanding agent to the cell culture; and (c) expanding the cells from the cell culture to obtain a cell population comprising Committed CIK Precursor cells, transfecting the cell population comprising the Committed CIK Precursor cells with one or more nucleic acids to produce genetically modified Committed CIK Precursor cells, and expanding the genetically modified Committed CIK Precursor cells in culture medium to produce the genetically modified CIK cells; wherein steps (a), (b) and (c) are performed in the absence of non-irradiated or irradiated feeder cells.
  • the present disclosure is also directed methods of of generating genetically modified cytokine induced killer (CIK) cells, comprising the sequential steps of: (a) culturing a population of mononuclear cells in culture medium comprising at least one differentiating agent to induce differentiation of the mononuclear cells in a cell culture into Committed CIK Precursor cells; (b) adding at least one stimulating agent and at least one expanding agent to the cell culture; (c) expanding the cells from the cell culture to obtain a cell population comprising Committed CIK Precursor cells and transfecting the cell population comprising the Committed CIK Precursor cells with one or more nucleic acids; (d) formulating transfected cells comprising the Committed CIK Precursor cells for administration to a subject in need thereof; and (e) administering the transfected cells to the subject, wherein transfected Committed CIK Precursor cells are expanded to produce the genetically modified CIK cells in the subject; wherein steps (a), (b) and
  • the present disclosure is directed to methods of generating genetically modified cytokine induced killer (CIK) cells, comprising the sequential steps of: (a) culturing peripheral blood mononuclear cells (PBMCs) in culture medium comprising at least one differentiating agent to induce differentiation of the PBMCs in a cell culture into Committed CIK Precursor cells; (b) adding at least one stimulating agent and at least one expanding agent to the cell culture; and (c) expanding the cells from the cell culture to obtain a cell population comprising Committed CIK Precursor cells, transfecting the cell population comprising the Committed CIK Precursor cells with one or more nucleic acids to produce genetically modified Committed CIK Precursor cells, and expanding the genetically modified Committed CIK Precursor cells in culture medium to produce the genetically modified CIK cells; wherein steps (a), (b) and (c) are performed in the absence of non-irradiated or irradiated feeder cells; and wherein the PBMCs
  • the present disclosure is also directed methods of of generating genetically modified cytokine induced killer (CIK) cells, comprising the sequential steps of: (a) culturing peripheral blood mononuclear cells (PBMCs) in culture medium comprising at least one differentiating agent to induce differentiation of the PBMCs in a cell culture into Committed CIK Precursor cells; (b) adding at least one stimulating agent and at least one expanding agent to the cell culture; (c) expanding the cells from the cell culture to obtain a cell population comprising Committed CIK Precursor cells and transfecting the cell population comprising the Committed CIK Precursor cells with one or more nucleic acids; (d) formulating transfected cells comprising the Committed CIK Precursor cells for administration to a subject in need thereof; and (e) administering the transfected cells to the subject, wherein transfected Committed CIK Precursor cells are expanded to produce the genetically modified CIK cells in the subject; wherein steps (a), (b)
  • the differentiating agent is selected from the group consisting of: IFN- ⁇ , IL-4, IL-5, IL-7, IFN- ⁇ , IL-10, IL-12, IL-13, IL-6, IL-15, IL-17, IL-18, IL-21, IL- 22, IL-23, IL-27, IL-1 ⁇ , TGF- ⁇ , GM-CSF, CCL3, CCL4, CCL5, CCL17, CCL21, and any combination thereof.
  • the differentiating agent is IFN- ⁇ .
  • the differentiating agent is added in an amount of about 10 U/ml to about 10000 U/ml.
  • the differentiating agent is added in an amount of about 10 U/ml, about 20 U/ml, about 30 U/ml, about 40 U/ml, about 50 U/ml, about 60 U/ml, about 70 U/ml, about 80 U/ml, about 90 U/ml, about 100 U/ml, about 200 U/ml, about 300 U/ml, about 400 U/ml, about 500 U/ml, about 600 U/ml, about 700 U/ml, about 800 U/ml, about 900 U/ml, about 1000 U/ml, about 2000 U/ml, about 3000 U/ml, about 4000 U/ml, about 5000 U/ml, about 6000 U/ml, about 7000 U/ml, about 8000 U/ml, about 9000 U/ml, or about 10000 U/ml.
  • the differentiating agent is added in an amount of about 1000 U/ml. II.B Expanding and Stimulating Agents
  • the stimulating agent is selected from the group consisting of: an anti-CD3 antibody, an anti-TCR antibody, an anti-CD28 antibody, an anti-CD137 antibody, an anti-CD134 antibody, an anti-CD27 antibody, an anti-ICAM-1 antibody, an anti- CD3/CD28-coated beads, a superantigen, phytohaemaglutinin (PHA), phorbol 12- myristate 13-acetate (PMA), ionomycin, and any combination thereof.
  • the stimulating agent is an anti-CD3 antibody.
  • the anti-CD3 antibody is an OKT3 antibody.
  • the stimulating agent is added in an amount of about 5 ng/ml, about 6 ng/ml, about 7 ng/ml, about 8 ng/ml, about 9 ng/ml, about 10 ng/ml, about 11 ng/ml, about 12 ng/ml, about 13 ng/ml, about 14 ng/ml, about 15 ng/ml, about 16 ng/ml, about 17 ng/ml, about 18 ng/ml, about 19 ng/ml, about 20 ng/ml, about 21 ng/ml, about 22 ng/ml, about 23 ng/ml, about 24 ng/ml, about 25 ng/ml, about 26 ng/ml, about 27 ng/ml, about 28 ng/ml, about 29 ng/ml, about 30 ng/ml, about 31 ng/ml, about 32 ng/
  • the stimulating agent is added in an amount of about 50 ng/ml.
  • the expanding agent is selected from the group consisting of: IL- 2, IL-4, IL-7, IL-9, IL-15, IL-18, IL-21, and any combination thereof.
  • the expanding agent is IL-2.
  • the expanding agent is added in an amount of about 10 U/ml to about 1000 U/ml.
  • the expanding agent is added in an amount of about 300 U/ml.
  • the stimulating agent and the expanding agent are added to the cell culture approximately 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, or 48 hours after initiating step (a). [0253] In some aspects, the stimulating agent and the expanding agent are added to the cell culture approximately 18 hours after initiating step (a). [0254] In some aspects, the stimulating agent and the expanding agent are added to the cell culture 18 hours after initiating step (a). [0255] In some aspects, the stimulating agent and the expanding agent are added to the cell culture approximately 19 hours after initiating step (a).
  • the stimulating agent and the expanding agent are added to the cell culture 19 hours after initiating step (a). [0257] In some aspects, the stimulating agent and the expanding agent are added to the cell culture approximately 20 hours after initiating step (a). [0258] In some aspects, the stimulating agent and the expanding agent are added to the cell culture 20 hours after initiating step (a). [0259] In some aspects, the stimulating agent and the expanding agent are added to the cell culture approximately 21 hours after initiating step (a). [0260] In some aspects, the stimulating agent and the expanding agent are added to the cell culture 21 hours after initiating step (a). [0261] In some aspects, the stimulating agent and the expanding agent are added to the cell culture approximately 22 hours after initiating step (a).
  • the stimulating agent and the expanding agent are added to the cell culture 22 hours after initiating step (a). [0263] In some aspects, the stimulating agent and the expanding agent are added to the cell culture approximately 23 hours after initiating step (a). [0264] In some aspects, the stimulating agent and the expanding agent are added to the cell culture 23 hours after initiating step (a). [0265] In some aspects, the stimulating agent and the expanding agent are added to the cell culture approximately 24 hours after initiating step (a). [0266] In some aspects, the stimulating agent and the expanding agent are added to the cell culture 24 hours after initiating step (a).
  • the Committed CIK Precursor cells are transfected with one or more nucleic acids approximately 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, or 48 hours after initiating step (b).
  • the Committed CIK Precursor cells are transfected with one or more nucleic acids approximately 18 hours after initiating step (b).
  • the Committed CIK Precursor cells are transfected with one or more nucleic acids approximately 19 hours after initiating step (b).
  • the Committed CIK Precursor cells are transfected with one or more nucleic acids approximately 20 hours after initiating step (b). [0271] In some aspects, the Committed CIK Precursor cells are transfected with one or more nucleic acids approximately 21 hours after initiating step (b). [0272] In some aspects, the Committed CIK Precursor cells are transfected with one or more nucleic acids approximately 22 hours after initiating step (b). [0273] In some aspects, the Committed CIK Precursor cells are transfected with one or more nucleic acids approximately 23 hours after initiating step (b).
  • the Committed CIK Precursor cells are transfected with one or more nucleic acids approximately 24 hours after initiating step (b).
  • the cell population comprising the Committed CIK Precursor cells is transfected using electroporation.
  • the electroporation in performed in an isotonic buffer.
  • the electroporation in performed in Opti-MEM In some aspects, the electroporation in performed in Lonza isotonic buffer (cat# PBP3-02250).
  • the transfection is selected from the group consisting of: a non- viral transfer of one or more nucleic acids encoding an antigen receptor, a chimeric antigen receptor, a T cell receptor, a suicide gene, a gene encoding an inducible caspase 9 system, and any combination thereof into the cell population comprising the Committed CIK Precursor cells in the cell culture.
  • the non-viral transfer of nucleic acids comprises the use of the group consisting of: a transposon-based integration system, Zn-finger nucleases, integrases, transcription activator-like effectors, clustered regularly interspaced short palindromic repeats (CRISPR), sequence-specific recombinase systems able to integrate nucleic acids by recombination between attachment sites, and any combination thereof.
  • the transposon-based system is a Sleeping Beauty (SB) transposon- based system.
  • the SB transposon-based system comprises the use of Sleeping Beauty transposase SB100X (see e.g., Hudecek et al., Crit. Rev. in Biochem and Mol Bio 52(4):355–380 (2017)).
  • the Committed CIK Precursor cells are transfected with an RNA encoding SB100X transposase and a DNA encoding a Sleeping Beauty compatible chimeric antigen receptor (CAR) transposon.
  • CAR Sleeping Beauty compatible chimeric antigen receptor
  • one or more nucleic acids encode T cell receptors, chimeric antigen receptors, an adhesion molecule, a costimulatory ligand, a cytokine receptor, a chemokine receptor, a cytokine, a chemokine, an enzyme, a secreted drug, a checkpoint inhibitor, or a ligand.
  • one or more nucleic acids encode a prodrug converting enzyme, an IgG-degrading enzyme of S.
  • IdeS Intercellular Adhesion Molecule 1
  • IdeS Intercellular Adhesion Molecule 1
  • CXCR4 Intercellular Adhesion Molecule 1
  • IL-15 CCR5, CCR4, CD25, CD122, CD132, C-X-C chemokine receptor type 4 C-X-C chemokine receptor type 4 (CXCR4), IL-15, IL-18, IL-21 IL-23 IL-33, IL-1a, IL-1b, matrix metalloproteinase (MMP), heparinase, an anti-PD-1 antibody or antigen binding fragment thereof, an anti-T cell immunoglobulin and mucin-domain containing-3 (TIM-3) antibody or antigen binding fragment thereof, IL-3 zetakine, or any combination thereof.
  • MMP matrix metalloproteinase
  • the prodrug converting enzyme is carboxypeptidase G2 (CPG2) or ⁇ -lactamase.
  • CPG2 carboxypeptidase G2
  • chimeric antigen receptors are specific for CD19, CD123, TIM- 3, C-type lectin-like molecule-1 (CLL-1), CD70, mucin 1 (MUC-1), CD20, CD22, B-cell activating factor receptor (BAFFR), CD23, cytokine receptor like factor 2 (CRLF2), CD79b, CD79d, CD7, CD43, CD5, CD25, Lewis Y (LeY), natural killer group 2 member D (NKG2D), receptor tyrosine kinase like orphan receptor 1 (ROR1), receptor tyrosine kinase like orphan receptor 2 (ROR2), Wilms' tumor 1 (WT1), CD44 variant 6 (CD44v6), CD33, CD38, human epidermal growth factor receptor 2 (Her2), epidermal growth factor receptor (EG
  • the chimeric antigen receptor is specific for CD19.
  • one or more nucleic acids are DNA and/or RNA.
  • one or more nucleic acids are RNA.
  • the methods disclosed herein further comprise (d) replacing a portion of the culture medium with a fresh culture medium comprising at least one expanding agent approximately 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85
  • the methods disclosed herein further comprise (d) replacing a portion of the culture medium with a fresh culture medium comprising at least one expanding agent approximately 18 hours after initiating step (c). [0294] In some aspects, the methods disclosed herein further comprise (d) replacing a portion of the culture medium with a fresh culture medium comprising at least one expanding agent approximately 19 hours after initiating step (c). [0295] In some aspects, the methods disclosed herein further comprise (d) replacing a portion of the culture medium with a fresh culture medium comprising at least one expanding agent approximately 20 hours after initiating step (c).
  • the methods disclosed herein further comprise (d) replacing a portion of the culture medium with a fresh culture medium comprising at least one expanding agent approximately 21 hours after initiating step (c). [0297] In some aspects, the methods disclosed herein further comprise (d) replacing a portion of the culture medium with a fresh culture medium comprising at least one expanding agent approximately 22 hours after initiating step (c). [0298] In some aspects, the methods disclosed herein further comprise (d) replacing a portion of the culture medium with a fresh culture medium comprising at least one expanding agent approximately 23 hours after initiating step (c).
  • the methods disclosed herein further comprise (d) replacing a portion of the culture medium with a fresh culture medium comprising at least one expanding agent approximately 24 hours after initiating step (c).
  • the methods disclosed herein further comprise (e) passaging and culturing transfected cells in culture medium comprising at least one expanding agent approximately8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90
  • the methods disclosed herein further comprise (e) passaging and culturing transfected cells in culture medium comprising at least one expanding agent approximately 72 hours after initiating step (d). [0302] In some aspects, the methods disclosed herein further comprise (e) passaging and culturing transfected cells in culture medium comprising at least one expanding agent approximately 73 hours after initiating step (d). [0303] In some aspects, the methods disclosed herein further comprise (e) passaging and culturing transfected cells in culture medium comprising at least one expanding agent approximately 74 hours after initiating step (d).
  • the methods disclosed herein further comprise (e) passaging and culturing transfected cells in culture medium comprising at least one expanding agent approximately 75 hours after initiating step (d). [0305] In some aspects, the methods disclosed herein further comprise (e) passaging and culturing transfected cells in culture medium comprising at least one expanding agent approximately 76 hours after initiating step (d). [0306] In some aspects, the methods disclosed herein further comprise (e) passaging and culturing transfected cells in culture medium comprising at least one expanding agent approximately 77 hours after initiating step (d).
  • the methods disclosed herein further comprise (e) passaging and culturing transfected cells in culture medium comprising at least one expanding agent approximately 78 hours after initiating step (d). [0308] In some aspects, the methods disclosed herein further comprise (e) passaging and culturing transfected cells in culture medium comprising at least one expanding agent approximately 79 hours after initiating step (d). [0309] In some aspects, the methods disclosed herein further comprise (e) passaging and culturing transfected cells in culture medium comprising at least one expanding agent approximately 80 hours after initiating step (d).
  • the methods disclosed herein further comprise (e) passaging and culturing transfected cells in culture medium comprising at least one expanding agent approximately 81 hours after initiating step (d). [0311] In some aspects, the methods disclosed herein further comprise (e) passaging and culturing transfected cells in culture medium comprising at least one expanding agent approximately 82 hours after initiating step (d). [0312] In some aspects, the methods disclosed herein further comprise (e) passaging and culturing transfected cells in culture medium comprising at least one expanding agent approximately 83 hours after initiating step (d).
  • the methods disclosed herein further comprise (e) passaging and culturing transfected cells in culture medium comprising at least one expanding agent approximately 84 hours after initiating step (d). [0314] In some aspects, the methods disclosed herein further comprise (e) passaging and culturing transfected cells in culture medium comprising at least one expanding agent approximately 85 hours after initiating step (d). [0315] In some aspects, the methods disclosed herein further comprise (e) passaging and culturing transfected cells in culture medium comprising at least one expanding agent approximately 86 hours after initiating step (d).
  • the methods disclosed herein further comprise (e) passaging and culturing transfected cells in culture medium comprising at least one expanding agent approximately 87 hours after initiating step (d). [0317] In some aspects, the methods disclosed herein further comprise (e) passaging and culturing transfected cells in culture medium comprising at least one expanding agent approximately 88 hours after initiating step (d). [0318] In some aspects, the methods disclosed herein further comprise (e) passaging and culturing transfected cells in culture medium comprising at least one expanding agent approximately 89 hours after initiating step (d).
  • the methods disclosed herein further comprise (e) passaging and culturing transfected cells in culture medium comprising at least one expanding agent approximately 90 hours after initiating step (d). [0320] In some aspects, the methods disclosed herein further comprise (e) passaging and culturing transfected cells in culture medium comprising at least one expanding agent approximately 91 hours after initiating step (d). [0321] In some aspects, the methods disclosed herein further comprise (e) passaging and culturing transfected cells in culture medium comprising at least one expanding agent approximately 92 hours after initiating step (d).
  • the methods disclosed herein further comprise (e) passaging and culturing transfected cells in culture medium comprising at least one expanding agent approximately 93 hours after initiating step (d). [0323] In some aspects, the methods disclosed herein further comprise (e) passaging and culturing transfected cells in culture medium comprising at least one expanding agent approximately 94 hours after initiating step (d). [0324] In some aspects, the methods disclosed herein further comprise (e) passaging and culturing transfected cells in culture medium comprising at least one expanding agent approximately 95 hours after initiating step (d).
  • the methods disclosed herein further comprise (e) passaging and culturing transfected cells in culture medium comprising at least one expanding agent approximately 96 hours after initiating step (d). [0326] In some aspects, after step (e) the transfected cells in culture medium are transferred to a cell culture bag, a cell culture flask, or a culture device. [0327] In some aspects, the transfected cells in culture medium are transferred to the cell culture bag. [0328] In some aspects, the culture device is a bioreactor.
  • the transfected cells are expanded by culturing the transfected cells in culture medium comprising at least one expanding agent about every 2 to 3 days or about every 3 to 4 days until about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, about 15 days, about 16 days, about 17 days, about 18 days, about 19 days, about 20 days, about 21 days, about 22 days, about 23 days, about 24 days, about 25 days, about 26 days, about 27 days, or about 28 days after initiating step (a). [0330] In some aspects, the transfected cells are expanded until about 10 days after initiating step (a). [0331] In some aspects, the transfected cells are expanded until about 14 days after initiating step (a).
  • the transfected cells are expanded until about 17 days after initiating step (a). [0333] In some aspects, the transfected cells are expanded until about 21 days after initiating step (a). [0334] In some aspects, the transfected cells are expanded until about 28 days after initiating step (a). [0335] In some aspects, the methods disclosed herein further comprise (f) isolating the cells from the cell culture to obtain a cell population comprising the genetically modified CIK cells. [0336] In some aspects, the transfected cells are cultured to obtain a cell population comprising the genetically modified CIK cells. [0337] In some aspects, the methods disclosed herein further comprise the step of freezing the genetically modified CIK cells. III.
  • the genetically modified CIK cells disclosed herein express one or more T cell receptors (TCR-CIK), chimeric antigen receptors (CAR-CIK), a genetically modified adhesion molecule, a genetically modified costimulatory ligand, a genetically modified cytokine receptor, a genetically modified chemokine receptor, a genetically modified cytokine, a genetically modified chemokine, an enzyme, a secreted drug, a checkpoint inhibitor, or a ligand.
  • TCR-CIK T cell receptors
  • CAR-CIK chimeric antigen receptors
  • a genetically modified adhesion molecule a genetically modified costimulatory ligand
  • a genetically modified cytokine receptor a genetically modified chemokine receptor
  • a genetically modified cytokine a genetically modified chemokine
  • an enzyme a secreted drug
  • a checkpoint inhibitor or a ligand.
  • the genetically modified CIK cells
  • IdeS Intercellular Adhesion Molecule 1
  • IdeS Intercellular Adhesion Molecule 1
  • CD137L OX40L
  • CD70 IL-15 receptor
  • CCR5 CCR4, CD25
  • CD122 CD132
  • CX-C chemokine receptor type 4 CXCR4
  • IL-15 IL-18
  • IL-21 IL-23 IL-33 IL-1a
  • IL-1b matrix metalloproteinase
  • MMP matrix metalloproteinase
  • ECM enzyme for extracellular matrix
  • ECM enzyme for extracellular matrix
  • ECM enzyme for extracellular matrix
  • heparinase an anti-PD-1 antibody or fragment thereof
  • TIM-3 anti-T cell immunoglobulin and mucin-domain containing-3
  • TIM-3 zetakine
  • ADR alloimmune defense receptor
  • the cell e.g., the genetically modified CIK cell
  • comprises a CAR e.g., a CAR-CIK cell
  • the CAR is designed as a standard CAR, a split CAR, an off-switch CAR, an on-switch CAR, a first-generation CAR, a second-generation CAR, a third- generation CAR, or a fourth-generation CAR.
  • the CAR comprises antigen- binding domain, a transmembrane domain, a costimulatory domain, an intracellular signaling domain, or any combinations thereof.
  • the CAR specifically binds (i.e., target) one or more antigens expressed on a tumor cell, such as a malignant B cell, a malignant T cell, or a malignant plasma cell.
  • the chimeric antigen receptor (CAR) is specific for CD19, CD123, TIM-3, C-type lectin-like molecule-1 (CLL-1), CD70, mucin 1 (MUC-1), CD20, CD22, B-cell activating factor receptor (BAFFR), CD23, cytokine receptor like factor 2 (CRLF2), CD79b, CD79d, CD7, CD43, CD5, CD25, Lewis Y (LeY), natural killer group 2 member D (NKG2D), receptor tyrosine kinase like orphan receptor 1 (ROR1), receptor tyrosine kinase like orphan receptor 2 (ROR2), Wilms' tumor 1 (WT1), CD44 variant 6 (CD44v6)
  • the chimeric antigen receptor is specific for CD19.
  • the costimulatory domain comprises a costimulatory domain of an interleukin-2 receptor (IL-2R), interleukin-12 receptor (IL-12R), IL-7, IL-21, IL-23, IL-15, CD2, CD3, CD4, CD7, CD8, CD27, CD28, CD30, CD40, 4-1BB/CD137, ICOS, lymphocyte function-associated antigen-1 (LFA-1), LIGHT, NKG2C, OX40, DAP10, or any combination thereof.
  • the costimulatory domain comprises a 4- 1BB/CD137 costimulatory domain.
  • the transmembrane domain comprises a transmembrane domain of KIRDS2, OX40, CD2, CD27, LFA-1 (CD11a, CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD160, CD19, IL2R beta, IL2R gamma, IL7R ⁇ , ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1 (CD226), SLAMF4 (CD244,
  • the transmembrane domain comprises a CD28 transmembrane domain.
  • the intracellular signaling domain comprises an intracellular signaling domain derived from CD3 zeta, FcR gamma, common FcR gamma (FCER1G), Fc gamma RIIa, FcR beta (Fc Epsilon Rib), CD3 gamma, CD3 delta, CD3 epsilon, CD22, CD79a, CD79b, CD278 (“ICOS”), Fc ⁇ RI, CD66d, CD32, DAP10, DAP12, or any combination thereof.
  • the intracellular signaling domain comprises a CD3 zeta intracellular signaling domain.
  • an immune cell e.g., the genetically modified CIK cell, disclosed herein comprises a T cell receptor (TCR), e.g., an engineered TCR.
  • TCR T cell receptor
  • the TCR specifically binds to a tumor antigen.
  • engineered TCR or "engineered T-cell receptor” refers to a T-cell receptor (TCR) engineered to specifically bind with a desired affinity to HLA/peptide target antigen that is selected, cloned, and/or subsequently introduced into a population of immune cells, e.g., T cells, NK cells, and/or TILs.
  • the TCR specifically binds (i.e., targets) one or more antigens expressed on a tumor cell, such as a malignant B cell, a malignant T cell, or a malignant plasma cell. In some aspects, the TCR specifically binds a tumor antigen/HLA complex.
  • the tumor antigen is, or is derived from, AFP, CD19, BCMA, CLL-1, CS1, CD38, CD19, TSHR, CD123, CD22, CD30, CD171, CD33, EGFRvIII, GD2, GD3, Tn Ag, PSMA, ROR1, ROR2, GPC1, GPC2, FLT3, FAP, TAG72, CD44v6, CEA, EPCAM, B7H3, KIT, IL- 13Ra2, mesothelin, IL-l lRa, PSCA, PRSS21, VEGFR2, LewisY, CD24, PDGFR- beta, SSEA-4, CD20, folate receptor alpha, ERBB2 (Her2/neu), MUC1, MUC16, EGFR, NCAM, prostase, PAP, ELF2M, Ephrin B2, IGF-I receptor, CAIX, LMP2, gplOO, bcr-abl, tyrosinase, EphA2,
  • an engineered cell of the present disclosure can express a T cell receptor (TCR) targeting an antigen.
  • TCR engineered cells can target main types: shared tumor-associated antigens (shared TAAs) and unique tumor-associated antigens (unique TAAs), or tumor-specific antigens.
  • the former can include, without any limitation, cancer-testis (CT) antigens, overexpressed antigens, and differentiation antigens, while the latter can include, without any limitation, neoantigens and oncoviral antigens.
  • CTR cancer-testis
  • HPV Human papillomavirus
  • HPV E6 protein and HPV E7 protein belong to the category of oncoviral antigens.
  • the TCR engineered cells can target a CT antigen, e.g., melanoma- associated antigen (MAGE) including, but not limited to, MAGE-A1, MAGE-A2, MAGE- A3, MAGE-A4, MAGE-A6, MAGE-A8, MAGE-A9.23, MAGE-A10, and MAGE-A12.
  • MAGE melanoma- associated antigen
  • the TCR engineered cells can target glycoprotein (gp100), melanoma antigen recognized by T cells (MART-1), and/or tyrosinase, which are mainly found in melanomas and normal melanocytes.
  • the TCR engineered cells can target Wilms tumor 1 (WT1), i.e., one kind of overexpressed antigen that is highly expressed in most acute myeloid leukemia (AML), acute lymphoid leukemia, almost every type of solid tumor and several critical tissues, such as heart tissues.
  • WT1 Wilms tumor 1
  • the TCR engineered cells can target mesothelin, another kind of overexpressed antigen that is highly expressed in mesothelioma but is also present on mesothelial cells of several tissues, including trachea.
  • the TCR engineered cells can target any neoantigen, which can be formed by random somatic mutations specific to individual tumors.
  • the TCR specifically binds to (i.e., targets) a cancer antigen selected from the group consisting of AFP, Braf, CD19, TRAC, TCR ⁇ , BCMA, CLL-1, CS1, CD38, CD19, TSHR, CD123, CD22, CD30, CD171, CD33, EGFRvIII, GD2, GD3, Tn Ag, PSMA, ROR1, ROR2, GPC1, GPC2, FLT3, FAP, TAG72, CD44v6, CEA, EPCAM, B7H3, KIT, IL- 13Ra2, mesothelin, IL-l lRa, PSCA, PRSS21, VEGFR2, LewisY, CD24, PDGFR-beta, SSEA-4, CD20, folate receptor alpha, ERBB2 (Her2/neu), MUC1, MUC16, EGFR, NCAM, prostase, PAP, ELF2M, Ephrin B2, IGF-I receptor, CAIX, L
  • the TCR specifically binds (i.e., targets) hTERT. In some aspects, the TCR specifically binds (i.e., targets) KRAS. In some aspects, the TCR specifically binds (i.e., targets) Braf. In some aspects, the TCR specifically binds (i.e., targets) TGF ⁇ RII. In some aspects, the TCR specifically binds (i.e., targets) MAGE A10/A4. In some aspects, the TCR specifically binds (i.e., targets) AFP. In some aspects, the TCR specifically binds (i.e., targets) PRAME. In some aspects, the TCR specifically binds (i.e., targets) MAGE A1.
  • the TCR specifically binds (i.e., targets) WT-1. In some aspects, the TCR specifically binds (i.e., targets) NY-ESO. In some aspects, the TCR specifically binds (i.e., targets) PRAME. In some aspects, the TCR specifically binds (i.e., targets) NY-ESO. In some aspects, the TCR specifically binds (i.e., targets) CD19. [0354] In some aspects, the TCR comprises an intracellular gamma/delta domain.
  • the TCR is an antibody-T-cell receptor (AbTCR) (see, e.g., Xu et al., Cell Discovery 4:62 (2016), which is incorporated by reference herein in its entirety.
  • AbTCR antibody-T-cell receptor
  • Pharmaceutical compositions suitable for administration to human patients are typically formulated for parenteral administration, e.g., in a liquid carrier, or suitable for reconstitution into liquid solution or suspension for intravenous administration.
  • such compositions typically comprise a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable means approved by a government regulatory agency or listed in the U.S. Pharmacopeia or another generally recognized pharmacopeia for use in animals, particularly in humans.
  • carrier refers to a diluent, adjuvant, excipient, or vehicle with which the compound is administered.
  • Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, glycerol polyethylene glycol ricinoleate, and the like.
  • Water or aqueous solution saline and aqueous dextrose and glycerol solutions can be employed as carriers, particularly for injectable solutions.
  • Liquid compositions for parenteral administration can be formulated for administration by injection or continuous infusion. Routes of administration by injection or infusion include intravenous, intraperitoneal, intramuscular, intrathecal and subcutaneous.
  • the genetically modified cytokine induced killer (CIK) cells obtained by the methods disclosed herein are present in a pharmaceutical composition.
  • some aspects of the present disclosure are directed to a pharmaceutical composition comprising the genetically modified CIK cells obtained by the methods disclosed herein with a pharmaceutically acceptable carrier, diluent, solubilizer, emulsifier, preservative and/or adjuvant.
  • acceptable formulation materials preferably are nontoxic to recipients at the dosages and concentrations employed.
  • the formulation material(s) are for s.c. and/or I.V. administration.
  • the pharmaceutical composition comprises formulation materials for modifying, maintaining or preserving, for example, the pH, osmolality, viscosity, clarity, color, isotonicity, odor, sterility, stability, rate of dissolution or release, adsorption or penetration of the composition.
  • suitable formulation materials include, but are not limited to, amino acids (such as glycine, glutamine, asparagine, arginine or lysine); antimicrobials; antioxidants (such as ascorbic acid, sodium sulfite or sodium hydrogen- sulfite); buffers (such as borate, bicarbonate, Tris- HCl, citrates, phosphates or other organic acids); bulking agents (such as mannitol or glycine); chelating agents (such as ethylenediamine tetraacetic acid (EDTA)); complexing agents (such as caffeine, polyvinylpyrrolidone, beta-cyclodextrin or hydroxypropyl-beta- cyclodextrin); fillers; monosaccharides; disaccharides; and other carbohydrates (such as glucose, mannose or dextrins); proteins (such as serum albumin, gelatin or immunoglobulins); coloring, flavoring and diluting agents; emulsifying amino acids (
  • the optimal pharmaceutical composition will be determined by one skilled in the art depending upon, for example, the intended route of administration, delivery format and desired dosage. See, for example, Remington's Pharmaceutical Sciences, supra.
  • the primary vehicle or carrier in a pharmaceutical composition is either aqueous or non-aqueous in nature.
  • a suitable vehicle or carrier is water for injection, physiological saline solution or artificial cerebrospinal fluid, possibly supplemented with other materials common in compositions for parenteral administration.
  • the saline comprises isotonic phosphate-buffered saline.
  • neutral buffered saline or saline mixed with serum albumin are further exemplary vehicles.
  • pharmaceutical compositions comprise Tris buffer of about pH 7.0-8.5, or acetate buffer of about pH 4.0-5.5.
  • the pharmaceutical compositon further comprises sorbitol or a suitable substitute therefore.
  • a composition comprising the genetically modified CIK cells is prepared for storage by mixing the selected composition having the desired degree of purity with optional formulation agents (Remington's Pharmaceutical Sciences, supra) in the form of a lyophilized cake or an aqueous solution.
  • the pharmaceutical composition is selected for parenteral delivery.
  • the formulation components are present in concentrations that are acceptable to the site of administration.
  • buffers are used to maintain the composition at physiological pH or at a slightly lower pH, typically within a pH range of from about 5 to about 8.
  • a therapeutic composition is in the form of a pyrogen-free, parenterally acceptable aqueous solution comprising the genetically modified CIK cells obtained by the methods of the present disclosure, in a pharmaceutically acceptable vehicle.
  • a vehicle for parenteral injection is sterile distilled water in which the genetically modified CIK cells obtained by the methods disclosed herein formulated as a sterile, isotonic solution, and properly preserved.
  • the preparation involves the formulation of the desired molecule with an agent, such as injectable microspheres, bio-erodible particles, polymeric compounds (such as polylactic acid or polyglycolic acid), beads or liposomes, that can provide for the controlled or sustained release of the product which can then be delivered via a depot injection.
  • hyaluronic acid is also used. Hyaluronic acid, when present, can have the effect of promoting sustained duration in the circulation.
  • implantable drug delivery devices are used to introduce the desired molecule.
  • a pharmaceutical composition involves the genetically modified CIK cells obtained by the methods of the present disclosure in a mixture with non-toxic excipients which are suitable for the manufacture of tablets.
  • suitable excipients include, but are not limited to, inert diluents, such as calcium carbonate, sodium carbonate or bicarbonate, lactose, or calcium phosphate; or binding agents, such as starch, gelatin, or acacia; or lubricating agents such as magnesium stearate, stearic acid, or talc.
  • sustained- or controlled-delivery formulations include formulations involving the genetically modified CIK cells obtained by the methods of the present disclosure in sustained- or controlled-delivery formulations.
  • techniques for formulating a variety of other sustained- or controlled-delivery means such as liposome carriers, bio-erodible microparticles or porous beads and depot injections, are also known to those skilled in the art. See for example, PCT Application No. PCT/US93/00829 which describes the controlled release of porous polymeric microparticles for the delivery of pharmaceutical compositions.
  • sustained- release preparations can include semipermeable polymer matrices in the form of shaped articles, e.g. films, or microcapsules.
  • Sustained release matrices can include polyesters, hydrogels, polylactides (U.S. Pat. No. 3,773,919 and EP 058,481), copolymers of L- glutamic acid and gamma ethyl-L-glutamate (Sidman et al., Biopolymers, 22:547-556 (1983)), poly (2-hydroxyethyl-methacrylate) (Langer et al., J. Biomed. Mater. Res., 15: 167-277 (1981) and Langer, Chem.
  • sustained release compositions can also include liposomes, which can be prepared by any of several methods known in the art. See, e.g., Eppstein et al, Proc. Natl. Acad. Sci. USA, 82:3688-3692 (1985); EP 036,676; EP 088,046 and EP 143,949.
  • the pharmaceutical composition to be used for in vivo administration typically is sterile. In some aspects, this is accomplished by filtration through sterile filtration membranes.
  • compositions for parenteral administration are stored in lyophilized form or in a solution.
  • parenteral compositions generally are placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.
  • sterile access port for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.
  • the pharmaceutical composition once the pharmaceutical composition has been formulated, it is stored in sterile vials as a solution, suspension, gel, emulsion, solid, or as a dehydrated or lyophilized powder.
  • such formulations are stored either in a ready-to-use form or in a form (e.g., lyophilized) that is reconstituted prior to administration.
  • V. Methods of Treatment Some aspects of the present disclosure are directed to methods of treating a subject in need thereof comprising administering to the subject a population of the genetically modified CIK cells obtained by the methods of the present disclosure.
  • CAR-CIK cells were prepared by a continuous process using fresh donor-derived whole blood as the starting material without requiring irradiated or non-irradiated feeder cells. See e.g., Table 2 below. The manufacturing process can be divided into four distinct steps: donor peripheral blood mononuclear cells (PBMCs) isolation, culture and differentiation, committed CIK precursor cell stimulation and expansion, committed CIK precursor cell electroporation, and expansion of electroporated, committed precursor cells into CAR-CIK cells.
  • PBMCs peripheral blood mononuclear cells
  • Donor PBMC isolation, culture and differentiation [0373] On Day 0, a healthy donor whole blood was diluted 1:1 with phosphate buffered saline (PBS), layered over Histopaque®-1077 gradient (MilliporeSigma) and centrifuged. The PBMCs layer was collected and pooled. PBMCs were washed with PBS and resuspended in Advanced RPMI 1640 media supplemented with 10% heat-inactivated Fetal Bovine Serum (FBS), 2 mM L-glutamine and 1,000 IU/mL INF gamma-1b (Actimmune®) at 37 ⁇ 1 °C and 5 ⁇ 1 % CO 2 at density of 3x10 ⁇ 6 cells/mL.
  • FBS heat-inactivated Fetal Bovine Serum
  • Actimmune® INF gamma-1b
  • Committed CIK precursor cell stimulation and expansion [0374] The next day (day 1), cells were stimulated with 50 ng/mL GMP grade anti-CD3 monoclonal antibody (Clone OKT3; Takara Bio) and 300 IU/mL recombinant human IL-2 (Proleukin®). On Day 2, after overnight incubation with OKT3 and IL-2, the resulting Committed CIK precursor cells were collected by centrifugation, washed with Phosphate Buffered Saline (PBS) to prepare for electroporation.
  • PBS Phosphate Buffered Saline
  • Committed CIK precursor cell stimulation and expansion [0375] Committed CIK precursor cells were resuspended at a concentration of 50 million cells/ml in electroporation medium (CoStorSol®; Preservation Solutions, Inc, Elkhorn, WI, USA) for non-viral genetic modification.
  • CoStorSol® is a sterile, non-pyrogenic solution for hypothermic flushing and storage of organs and is used to cool cellular material and lower its metabolic requirements during the electroporation step.
  • Thirty million cells in 0.6 mL suspension were added to each electroporation cuvette (gap size 4 mm) for processing with the Gene Pulser XcellTM device (BioRad).
  • Each cuvette contained premixed 0.2 ⁇ g of SB100X RNA and 1.5 ⁇ g of 3 rd generation pT4-CD19-CAR plasmid per million cells.
  • Each cuvette was pulsed at ambient temperature with the defined Square Wave program listed in Table 1 below. Program settings were verified prior to proceeding with the electroporation. Electroporators were calibrated on a routine schedule. Table 1: Parameters for Square Wave Electroporation in Gene Pulser XcellTM Device Parameter Setting Voltage 500 volts Pulse Length 5 milliseconds Pulse Number 2 pulses Pulse Interval 0.1 second [0376] The electroporation step was monitored by Voltage and Droop readings on the device for each cuvette.
  • Cells were harvested. The harvested cells were characterized side by side for fold-expansion, cell viability and immunophenotype (see Table 3 below).
  • Cells cultured in G-Rex were seeded at density of 0.5x10 ⁇ 6 cells/cm 2 in Advanced RMPI supplemented with 10% FBS, 2 mM L-glutamine and 300 IU/mL IL-2. At day 14, 70% of medium was replaced. At day 17 (since the process start) cells were harvested by centrifugation and characterized.
  • Day +7 Cell count, phenotypic analysis and transfer to cell culture bags or flasks. Cell expansion with complete medium supplemented with rhIL-2 at the final concentration of 300 U/ml. On day +10: +14:expand by diluting cells to 500,000 cells/mL in complete medium supplemented with 300 U/mL rhIL-2. At the end of production (Day + 17 or 21): cell count, phenotypic analysis, execution of functional tests and freezing. The electroporation information is for 4 mm cuvettes. *In bold: conditions for a different size of cuvettes with 2 mm gap which can be used.
  • Table 3 Evaluation of cell phenotype in cultures expanded using flasks or culture bags* Seeding Day 21 D Day 21 E xpansion Density Overall Day 21 Day 21 ay 21 % % vector copy D onor Vessel on day 7, Fold % % number Expansion Via CD3+ CD3+ Cells/mL bility CD3+ CD56+ CAR+ (VCN) cp/cell Flasks 7.5 x 10 5 142 89 96.4 48.4 72.1 2.4 1 Bags 5.0 x 10 5 227 91 96.3 46.6 79.2 3.5 F lasks 7.5 x 105 172 82 98.8 47.4 41.6 Not d etermined 2 B ags 5.15 x 105 299 79 97.9 59.4 73.4 Not d etermined * CAR-CIK cells were engineered using the SB transposon system using 3 rd generation pT4-CD19CAR plasmid
  • Electroporation on day 2 eliminated the need for the use of feeder cells (e.g., gamma irradiated or non-irradiated same donor PBMCs).
  • feeder cells e.g., gamma irradiated or non-irradiated same donor PBMCs
  • feeder cells e.g., gamma irradiated or non-irradiated same donor PBMCs
  • it is more practical for commercial production of CAR-CIKs e.g., the need for irradiation increases the cost which will ultimately be reflected in the cost of a drug product
  • it decreases the risk of rejection of CAR-CIK cells e.g., if there are irradiated feeders in the final product present they could prime host against CAR-CIK reaction).
  • Tables 4-8 show evaluation of large-scale manufacturing process for genetically modified CAR-CIK cells in bags. [0386] At the end of this cellular production process, regardless of the harvest day, cells in the final preparation typify the CAR-CIK immuno-phenotype as indicated in Table 4. Summary data from 5 lots of CAR-CIK cells harvested on Day 17 and 5 lots harvested on Day 21 are presented. Table 4: Phenotype of CAR-CIK Cells* Parameter Target Mean ⁇ s.d. Mean ⁇ s.d.
  • CAR-CIK cells were engineered using the SB transposon system using 3 rd generation pT4 CD19CAR plasmid. [0387] As shown in Table 5, CAR-CIK cells exhibit the expected potency against target antigen-positive cancer cells in an in vitro cytotoxicity assay regardless of the day of harvest.
  • Cytotoxicity of CAR-CIK cells was evaluated with a 4-hour co-culture assay.
  • CARCIK-CD19 cells were co-cultured with CD19+ REH target cells at an effector to target ratio (E:T) of 5:1.
  • E:T effector to target ratio
  • the REH cells were stained with 0.15uM CFSE to distinguish them from CARCIK-CD19 cells.
  • CFSE staining was quenched after 7 minutes by adding FBS and holding for 3 minutes.
  • the REH cells were washed and resuspended in complete RPMI media at a final concentration of 4 x 10 5 cells/mL.
  • CARCIK-CD19 cells An equal volume of 2 x 10 6 CARCIK-CD19 cells were added to the stained REH cells and incubated for 4 hours at 37°C and 5% CO2. At the end of incubation, the cells were washed in PBS and resuspended in Annexin-V binding buffer and stained with Annexin V-EnzoGold for 15 minutes. Cells were immediately analyzed by flow cytometry by first gating on forward and side scatter which will include both CARCIK-CD19 and REH cells. Cells were further gated on CFSE positive cells to distinguish REH cells and then gated on Annexin+ cells.
  • the percentage of cytotoxicity was calculated as the % of Annexin-V+ cells in the co- culture normalized for the % Annexin-V+ cells in the REH only well.
  • Table 5 Potency of CAR-CIK Cells* in vitro Parameter Target Mean ⁇ s.d. Mean ⁇ s.d. Day 17 Harvest Day 21 Harvest Cytotoxicity ⁇ 25% lysis of target 66 ⁇ 21.3% 40 ⁇ 7.3 % cells 1 1 Lysis of CD19 + REH cells in a co-culture with CAR-CIK cells * CAR-CIK cells were engineered using the SB transposon system using 3 rd generation pT4 CD19CAR plasmid.
  • FIG. 1 shows schematic representation of experimental flow in experiments comparing feeder free CIK process to the original process with feeder cells.
  • Both work flows utilize two seeding densities of cells transferred in the G-REX at day 0/day2: one million or two million of cells per square centimeter as indicated in the schema. Both work flows consider the additional variable of keeping the cells in the 6-wells after electroporation and then transferring the cells in the G-REX at day 7 (at a cell density of 10x 6 /cm 2 ).
  • FIGs.2A-2D demonstrate that cell expansion parameters measured such as cell numbers and level of glucose in the media are similar within the different culture conditions in feeder free, Day 2 electroporation process.
  • FIG. 3A shows that it is possible to obtain a high CAR expression (>50%) in all the conditions tested, similarly to the classical cell culture in flasks.
  • FIG.8A and FIGs.9D and 9E show that the Day 2 feeder free CIK generation process enables production of healthy cells, with a good T cell fitness.
  • Displaying a more immature memory phenotype is recognized as one of the main variables implicated in the CAR T cell persistence in vivo. Therefore, enriching the memory compartments of Tcm and Tn represents a further optimization of the CAR-CIK product.
  • CD4-CD8 Phenotype Evolution [0397] FIGs.3F-3I show that in all the conditions tested both CD4+ and CD8+ cells were differentiated, with the Day 7 condition better mimicking the ratio of CD4:CD8 typical of CAR-CIK cells cultured in flasks.
  • FIG. 3J shows that the CD56 expression in the G-Rex conditions is lower as compared to the flask. This is due to the fact that the cells are less manipulated in the bioreactor, so they are less activated and differentiated (as also observed by the memory phenotype).
  • Short-Term Cytotoxicity Assay [0399] Target cells were labeled with PE-Cell Tracker. At the end of the incubation, target cell killing was measured through apoptosis detection by flow cytometry, after annexin V and 7-amino-actinomycin D (7-AAD) (AnnV-7AAD) staining, gating in the Cell Tracker PE+.
  • FIG. 4A shows that CAR-CIK cells generated by the Day 2 feeder free method and expanded in G-REX have similar lytic activity to the CAR-CIK-CD33 cells generated using the original CIK generation process (day 0 electroporation + ⁇ irradiated feeder cells and expanded in Flasks).
  • Proliferation Assay [0400] The proliferation ability of CAR-CIK cells was evaluated after co-culture with the Cell Tracker-labeled targets THP-1, irradiated at 100 Gy g-radiations at an E:T ratio of 1:1.
  • FIG. 5B shows CAR-CIK cells generated by the Day2 feeder free method and expanded in G-REX have similar proliferative ability, when encountering the target cells, to the cells generated using the original CIK generation process (day 0 electroporation + ⁇ irradiated feeder cells and expanded in Flasks).
  • Cytokine Secretion Profile Intracellular Cytokine Staining CAR-CIK cell ability to produce cytokines was evaluated following a stimulation with the target cell at an E:T ratio of 1:3. After a 2 hr and 30 min co-culture, BD GolgiStop was added. The co-culture was then maintained for an additional period of 2 hr and 30 min, after which the cells were collected and stained for anti-CD3 and anti-Fc surface molecule (CAR) detection. Finally, intracellular cytokine staining (ICS) for IL-2 and IFN- ⁇ was performed using the BD Cytofix/Cytoperm kit, according to the manufacturer’s protocol. Specimens were then analyzed by flow cytometry.
  • ICS intracellular cytokine staining
  • FIGs. 4E-4G show the cytokine production is CAR-specific in response to the target challenge.
  • FIGs. 5A and 5B show the difference of the memory state between flasks and G- REX, starting from the same donor source (Donor A (FIG.5A) and Donor B (FIG.5B), suggesting that the G-REX device allows the cells to be more immature in their memory penotype. See also FIGs.6A-6C. Day 0 Plasmid Titration [0403] These experiments were done to titrate the novel SB molecules of pT4 transposon (carrying the CD19-CAR) and SB100X DNA.
  • FIGs. 7A-7C show that a good production of CAR-CIK cells was achieved in all the conditions, with the 7.5+1 as the preferred one, in terms of using less pT4 and thus total amount of DNA and in terms of cell numbers and CAR positivity.
  • FIGs.7D-7G show Day 0 (+ feeder cells) and Day 2 (feeder free) comparison by using pT4 transposon and SB100X DNA (7.5+1).
  • the Day 2 feeder free process ensures the production of high numbers of CAR-CIK cells carrying a high expression of the CAR, both in Flasks and G-REX, that is comparable to the standard Day 0 (+ feeder cells) method.
  • Fold increase, vector copy number (VCN), SB100X detection, and in vitro cytotoxicity of CD19.CAR-CIK cells produced with the Day 2 feeder free method, with 7.5+1 SB plasmid ratio were tested.
  • Fold Increase [0406] The fold increase was calculated as the ratio between the number of cells expanded at the end of the culture and the number of cells stimulated at day 0.
  • Enzyme RNA was extracted from CD19.CAR-CIK cells using the QIAamp RNA mini kit (QIAGEN) and retrotrascribed with SuperScript IV VILO master mix (Invitrogen). Amplification by qPCRs of homologous regions within the SB100X transcripts was performed using TaqMan gene expression master mix (Applied Biosystems, Thermo Fisher Scientific) and FastStart universal probe master (Roche Diagnostic, Mannheim, Germany).
  • Primers were as follows: forward primer, 50 - AAGCCGAAGAACACCATCC-30; reverse primer, 50 -AGCACCCC CACAACATGA-30; UPL probe #87, 50 - CTGACTTGCCAAAACT -30.
  • the number of copies was quantified based on a seven- point standard curve of plasmidic DNA diltutions from 107 to 20 copies of transposase.
  • b- glucuronidase gene (GUS) was used for data normalization by quantitative RT-PCR using the Ipsogen GUS control gene kit (QIAGEN), and analyzing data in agreement with the statistical delta-delta CT (ddCT) method.
  • Short-Term Cytotoxicity Assays [0409] In the short-term cytotoxic assay, CIK cells were co-cultured for 4 h with the CD19+ REH target cells (previously labeled with PE-cell tracker) at an E:T ratio of 5:1. At the end of the incubation, target cell killing was measured through apoptosis detection by flow cytometry, after annexin V and 7-amino-actinomycin D (7-AAD) (AnnV-7AAD) staining, gating in the Cell Tracker PE+ cells.
  • 7-AAD 7-amino-actinomycin D
  • the percentage of killed cells was calculated according to the following formula: (% annexin V+ target cells + % annexin V+ 7AAD+ target cells) after co-culture with CIK cells) - (% annexin V+ target cells + % annexin V+ 7AAD+) target cells alone / 100- (% annexin V+ target cells + % annexin V+ 7AAD+ target cells alone).
  • FIGs.8B-8E show that the Day 2 feeder free method grants the production of high numbers of CD19.CAR-CIK cells, carrying a high CAR expression, being compliant with the release criteria of VCN and SB100X transposase detection thresholds, and displaying a high cytotoxic activity against a CD19+ cell line.
  • FIGs.9A-9C show that the day 2 feeder free method, either applied to the standard culturing using Flasks or using G-Rex device, grants high numbers of CAR+CIK cells, that display the typical phenotype of CIK cells in terms of CD4-CD8 ratio, CD56 positivity, and memory phenotype.
  • FIG.9A-9C show that the day 2 feeder free method, either applied to the standard culturing using Flasks or using G-Rex device, grants high numbers of CAR+CIK cells, that display the typical phenotype of CIK cells in terms of CD4-CD8 ratio, CD56 positivity, and memory phenotype.
  • FIG. 10A shows that CAR-CIK cells generated by the Day 2 feeder free (either produced in Flasks or G-REX) display high lytic activity against a CD33+ cell line.
  • FIG. 10B shows CD33.CAR-CIK cells generated by the Day2 feeder free (either produced in Flasks or G-REX) proliferate in a CAR-specific manner when challenged with a CD33+ cell line
  • FIGs.10C-10D show that CD33.CAR-CIK cells generated by the Day 2 feeder free process (either produced in Flasks or G-REX) produce IL-2 (FIG.10C) and IFN- ⁇ (FIG.
  • CD19.CAR-CIK cells produced with the Day 2 feeder free process and cultured in G-REX-day7 were tested in vivo, by exploiting a model of CD19+ DAUDI-engrafted in NOD scid gamma (NSG) mice (Jackson Laboratory).
  • NSG NOD scid gamma mice
  • mice were irradiated at sub-lethal doses of 200 rad and 0.5x10 ⁇ 5 DAUDI cells (ATCC (American Type Culture Collection); Manassas, VA)/mouse were infused. Two days after, CD19.CAR-CIK cells were infused at different cell doses, and mice were followed during the days.
  • FIG.11C shows that the dose of 10x10 ⁇ 6 CAR+cells/mouse showed a better tumor control as compared to the lowest dose of 5x10 ⁇ 6 cells/mouse.
  • FIG. 11E shows that the mouse was treated with the 10x10 ⁇ 6 CD19.CAR-CIK cell dose, and no DAUDI cells were detected at the endpoint sacrifice analysis.
  • FIG.11F shows presence of CD3+ cells at the endpoint sacrifice analysis.
  • mice treated with the highest dose (10x10 ⁇ 6 CAR+cells/mouse) showed a lower tumor burden, in parallel with a better expansion of CAR-CIK cells and a prolonged survival.
  • Example 3 Purity of CAR-CIK Cells [0417] CIK cell is a population of CD3 cells bearing CD56 and NKG2D markers. Other cell types remaining at the end of CAR-CIK-CD19 cell production are composed of B cells, NK cells and myeloid cells. To characterize the cellular make up of cultures during the manufacturing cycle and on the day of harvest, samples were taken on day 0 and throughout the culture period described in Example 1 above.
  • CD3 T cell marker
  • CD56/CD16 NK cell marker
  • CD19 B cell marker
  • CD14 CD15
  • Fluorochrome conjugated antibodies were used to stain cells for the following markers; for T cells anti-CD3 (antibody clone UCHT1), for NK cells anti-CD56 (antibody clone NCAM1) and anti-CD16 (antibody clone B73.1), for B cells anti-CD19 (antibody clone HIB19), for myeloid cells anti-CD14 (antibody clone M ⁇ P9) and anti-CD15 (antibody clone HI98) either on PBMC (day 0) or on CARCIK-CD19 cells during the culture period (Days 7, 14 and 21).
  • PBMC Day 0 PBMC were isolated from whole blood collections by ficoll gradient centrifugation and the percentages of CD3+ T cell, CD56+/CD16+ NK cells, CD19+ B cells, CD14+ and CD15+ myeloid cells were determined on the viable CD45 positive cell population.
  • CD45 positive cells were detected with anti-CD45 antibody clone HI30 which binds to all human mononuclear cells.
  • Viable cells were identified by the addition of a fixable Aqua dye that labels only permeable dead cells leaving viable cells un-labeled.
  • FIG.12 shows data from a representative development run Development Run ID and the percentage of each population. Cells were harvested on days 0, 7, 14, and 21. This observation was repeated in a number of large scale pre-clinical runs with data summarized in Table 9.
  • Table 9 Summary of quantitation of residual cellular impurities in four large scale development runs* Impurities, % Development Run ID CD3+ cells NK cells B lymphocyte Monocytes and Granulocyte 1 97.9 1.6 0.01 0.4 2 98.7 0.5 0.00 0.1 3 97.9 1.6 0.00 0.2 4 96.6 2.5 0.00 0.4 *CAR-CIK cells were engineered using the SB transposon system using 3 rd generation pT4 CD19CAR plasmid. [0420] These data demonstrate that at the end of manufacturing cycle (day 21) the majority of the cellular population is comprised of CAR-CIK-CD19 cells that are CD3 positive with little to no residual cell subsets.
  • Cytokine induced killer (CIK) cells are activated cells derived from peripheral blood mononuclear cells (PBMCs) by the sequential addition of cytokines, such as IFN- ⁇ and then stimulation through the CD3 receptor with the addition of IL-2. Based on the fact that day 2 and day 7 cells still have high CD28 and CCR7 together with the fact that the cells did not yet upregulate CD56 and NKG2D markers, they cannot be called CIK cells yet. However, these activated day 2 cells no longer require any modification with additional stimuli and are committed to CIK phenotype during their proliferation.
  • PBMCs peripheral blood mononuclear cells
  • Committed CIK Precursor cells As used herein, the term for these cells is "Committed CIK Precursor cells.” Furthermore, by day 2, activation of Committed CIK Precursor cells renders them permissible for delivery of CAR transgene gene products via electroporation. However, the activation state of the cell prior to electroporation can influence efficiency of transgene delivery. Therefore, a study was undertaken to characterize the activation state of day 2 activated cells prior to electroporation by tracking the expression of the following surface markers: CD25, CD69 and CD137, which are upregulated on the cell surface.
  • the CD25 marker is the receptor for IL-2 and responsible for cell proliferation
  • CD69 allows activated cells to interact with antigen presenting cells, transducing growth signals
  • CD137 is a costimulatory receptor responsible for memory cell differentiation.
  • HLA-DR is an MHC class II molecule involved in antigen presentation and upregulated during cell activation. Tracking the cell surface expression of these markers characterized the activation state of the cell prior to electroporation.
  • Activation was accomplished by the sequential addition of the cytokine IFN-g (1000 U/mL) on day 0 followed by stimulation through the CD3 receptor via an anti-CD3 specific antibody at 50 ng/mL (clone OKT3) plus the cytokine IL-2 (300 U/mL) on day 1 as described in Example 1 above.
  • the activated cells were harvested on day 2 for electroporation with DNA plasmid pT4-CD19-CAR encoding the CAR transgene and SB100X transposase RNA (Capped SB100X).
  • PBMCs day 0 cells
  • activated day 2 cells were stained with the indicated antibodies listed in Table 10.
  • Viable cells were identified by the addition of a fixable Aquadye that labels only permeable dead cells leaving viable cells un- labeled. Viable cells labeled with the indicated antibodies were then acquired on a flow cytometer and data was analyzed using FlowJo version 10.8.1 software (TreeStar).
  • FIGs.13A-13M for day 0 cells and FIGs.14A-14M for day 2 cells.
  • Single cells were identified by gating on forward scatter height (FSC-H) and forward scatter height area (FSC-A) (FIG. 13A and FIG. 14A).
  • FSC-H forward scatter height
  • FSC-A forward scatter height area
  • Viable cells were gated on the Aquadye-negative cell population (FIG. 13B and FIG. 14B).
  • the viable cell population was gated on CD3+ cell events for day 0 PBMCs (FIG.13C).
  • the CD4+ cell population (FIG.13D) and CD8+ cell population (FIG.13E) were gated within CD3+ cell population.
  • the activation markers CD25, CD69, CD137, and HLA-DR area were reported for both CD8+ cells (FIG.13F, FIG.13G, FIG.13H, FIG.13I, respectively) and CD4+ cells (FIG. 13J, FIG.13K, FIG.13L, FIG.13M, respectively).
  • CD3 On day 2 of culture, detection of CD3 was prevented by the anti-CD3 (OKT3) antibody used to stimulate the CIK cells blocking the anti-CD3 antibody used to detect CD3 surface expression. Therefore, all events in the viable cell gate were included for the day 2 sample analysis (FIG.14C).
  • the CD4+ cell population (FIG.14D) and CD8+ cell population FIG.
  • Table 10 Antibody clones for detection of CIK cell surface markers Surface Marker description Fluorochrome Antibody Clone marker conjugate CD3 Cell signaling receptor CD3 APC-efluor 780 UCHT1 CD4 Helper cell marker CD4 FITC SK3 CD8 Cytotoxic cell marker CD8 PE HIT8a CD25 Receptor for IL-2 CD25 Alexa Fluor 700 MEM-181 CD69 C-type lectin, early activation CD69 Pacific Blue FN50 marker CD137 Costimulatory receptor CD137 PE Cy7 4B4-1 HLA-DR MCH class II cell surface HLA-DR APC L243 receptor Results [0425] The PBMC population consists of monocytes, B cells, CD3+/CD4+ T cells, and CD3+/CD8+ T cells.
  • FIGs.15A and 15B show data from one representative lot. Low levels of the activation markers CD25, CD69, CD137 and HLA-DR were detected on both CD8+ cells (FIG.15A) and CD4+ cells (FIG.15B). However, on day 2 post activation all the markers were upregulated on both CD8+ cells and CD4+ cells (FIGs.15A and 15B).
  • Table 11 shows data collected at day 0 on both CD8+ cells and CD4+ cells from eight separate cellular production runs.
  • Table 12 shows data collected at day 2 post activation on both CD8+ cells and CD4+ cells from eight separate cellular production runs, with lot number PD008-15 being split into 2 additional arms A and B for a total of nine samples. HLA-DR was only tested on five out of the nine samples on day 2. [0426] A similar trend was seen for both CD8+ and CD4+ cells, with upregulation of all markers tested (Table 12). CD25 expression was upregulated to 41.8% to 88.5% for both CD8+ cells and CD4+ cells. CD69 was upregulated to greater than 80%, with some lots of cells reaching greater than 90% for both CD8+ cells and CD4+ cells.
  • CD8+ cells expressing CD137 were seen compared to CD4+ cells (range of 10.4% to 60.9%).
  • HLA-DR was upregulated on both CD8+ cells and CD4+ cells to ranges of 50% to 80.2% and 30.1% to 78.9% respectively.
  • Table 13 shows the min, max and median values derived at day 0 and day 2 for all the markers tested. To set an acceptable threshold of upregulation for each marker from day 0 to day 2 post activation, the change in the median values from day 0 to day 2 was reported as a percent increase in surface marker expression.
  • values greater than 50% expression for CD25, CD69, CD137, and HLA-DR on CD8+ cells indicated upregulation of these markers post activation.
  • Values greater than 60% expression for CD25 and CD69 on CD4+ cells indicated upregulation of CD25 and CD69 post activation.
  • values greater than 30% would be considered an acceptable upregulation of CD137 and HLA-DR on CD4+ cells.
  • day 2 Committed CIK Precursor cells exhibit an activated phenotype that would render them applicable for electroporation.
  • Table 12 Detection of activation markers on day 2 cells* Lot number PD008- PD008- PD008- PD008- PD008- PD008- PD008- PD008- PD008- PD008- PD008- M arker 09 10 11 12 13 14 15a 15b 16 CD8+ CD25 52.4 41.8 44.3 50.0 73.5 82.5 56.0 66.4 7.8 cells CD69 94.7 94.4 98.3 94.0 92.7 98.6 98.3 98.7 89.2 of the CD137 73.5 46.4 53.6 51.6 78.3 88.8 79.7 77.1 58.3 viable CD3+ HLA- cells DR 61.5 50.0 53.0 58.3 80.2 nt nt nt nt CD4+ CD25 57.0 40.3 64.3 66.3 82.0 88.5 75.8 79.7 38.2 cells CD69 89.6 81.6 96.0 88.7 95.0 96.4 96.1
  • nt not tested.
  • CAR-CIK cells were engineered using the SB transposon system using 3 rd generation pT4 CD19CAR-IL18 bicistronic plasmid (see (FIG.24, pT4-CD19CAR-IL18 plasmid A).
  • Table 13 Min, max and median values for surface marker expression* C D8+ cells Day 0 CD8+ cells Day 2 Increase in median M arker Min (%) Max (%) Median Min Max Median value Day ( %) Marker (%) (%) (%) 0 to Day 2 (% of cells) CD25 0.2 3.5 1 CD25 7.8 82.5 52.4 51.4 CD69 3.3 18.3 11.6 CD69 89.2 98.7 94.7 83.1 CD137 1.3 16.6 3.0 CD137 46.4 88.8 73.5 70.5 HLA-DR 0.2 12.8 3.3 HLA- D R 50 80.2 58.3 55.0 CD4+ cells Day 0 CD4+ cells Day 2 CD25 1.4 12.4 3.7 CD25 38.2 88.5 66.3 62.6 CD69 1.9 15.5 5.1 CD69 81.6 97.2 95 89.9 CD137 0.6 16.7 1.7 CD137 10.4 60.9 34.8 33.1 HLA-DR 0.8 47.6 3.2 HLA- D R 30.1 78.9 44.3 41.1 The minimum and maximum values are reported
  • Example 5 Use of Feeder Free Cells for Electroporation Delivery of CAR Transgene Products
  • Committed CIK Precursor cells are activated cells derived from peripheral blood mononuclear cells (PBMCs) by the sequential addition of cytokines, such as IFN- ⁇ and then stimulation through the CD3 receptor with the addition of IL-2. By day 2, activation of these cells renders them permissible for delivery of CAR transgene gene products and further expansion in culture without the addition of feeder cells.
  • SB100X sleeping beauty transposase
  • Isolated PBMCs were frozen in FBS with 10% DMSO and stored under liquid nitrogen. PBMCs were thawed, washed with advanced RPMI containing 10% FBS media, resuspended at 3x10 ⁇ 6 cell/mL in advanced RPMI/10% FBS media and stimulated by the sequential addition of the cytokine IFN- ⁇ (1000 U/mL) on day 0 followed by stimulation through the CD3 receptor via an anti-CD3 specific antibody at 50 ng/mL (clone OKT3) plus the cytokine IL-2 (300 U/mL) on day 1.
  • the activated cells were harvested on day 2 for electroporation with DNA plasmid pT4- CD19-CAR encoding the CAR transgene and SB100X transposase.
  • the SB100X transposase is encoded by either plasmid DNA (pCMV-SB100X) or RNA (Capped SB100X).
  • Day 2 activated cells were resuspended in different buffers for testing during electroporation with a BioRad Gene Pulser Xcell.
  • Cells were resuspended at 1x10 ⁇ 8cell/mL in either BioRad, CoStorSol, Lonza or OptiMEM buffers and 1x10 ⁇ 7cells in 100 ml buffer were transferred to 2 mm gap cuvettes for electroporation.
  • the transposase, plasmid, and cell concentration along with the electroporation voltages and buffers are listed in Table 14.
  • the mock cells were electroporated in CoStorSol buffer in the absence of nucleic acids. Post electroporation, each cuvette of cells was added to a single well of a 6 well plate in 5 mL of complete media (Advanced RPMI/20% FBS).
  • Table 14 Electroporation Parameters* pT4-CD19-CAR SB100X DNA Sample# Voltage Electroporation buffer plasmid D-NA pCMV-SB100X mg/1x10 ⁇ 7 cells mg/1x10 ⁇ 7 cells 250 Mock 0 0 1 250 CoStorSol 15 0.5 2 250 OptiMEM 15 0.5 3 250 Lonza 15 0.5 4 250 BioRad 15 0.5 pT4-CD19-CAR Capped SB100X Sample# Voltage Electroporation buffer plasmid DNA RNA mg/1x10 ⁇ 7 mg/1x10 ⁇ 7 cells cells 5 350 CoStorSol 15 0.5 6 350 OptiMEM 15 0.5 7 350 Lonza 15 0.5 8 350 BioRad 15 0.5 Day 2 activated cells are harvested and washed and resuspended in the indicated electroporation buffers listed above.
  • FIGs.17A-17J show the gating of CAR positive cells in the CD3+ cell gate at day 14 and 21.
  • Activated cells electroporated in the Lonza buffer resulted in the highest expression of CAR positivity (7.28%) at day 14 (FIG.17D).
  • FIG.17F By day 21 the expression of CAR positivity diminishes but was still detectable above mock cells (FIG.17F) for cells electroporated in CoStorSol (FIG. 17H), Lonza (FIG. 17I), or OptiMEM (FIG. 17J) buffers.
  • Day 2 activated cells were also electroporated with the combination of pT4-CD19- CAR plasmid DNA and SB100X RNA.
  • the transposase was encoded by RNA instead of DNA.
  • Electroporation of day 2 activated cells with the combination of pT4-CD19-CAR plasmid DNA and SB100X RNA resulted in the expression of the CAR transgene by day 7, with three out of four buffers, CoStorsol, Lonza and OptiMEM resulting in the sustained presence of CAR positive cells at days 14 and 21 of the culture period (FIG.18).
  • FIGs.19A-19J show the gating of CAR positive cells in the CD3+ cell gate at days 14 and 21. Activated cells electroporated in the Lonza buffer resulted in the highest expression of CAR positivity (8.3%) on day 14 (FIG.
  • CAR-CIK cells were prepared from cord blood cells according to the following protocol. On day 0, cord blood cells obtained from frozen cord blood units were thawed. Cells were centrifuged and seeded in in 6-well plates at 3x10 ⁇ 6/ml in 5 ml complete medium. 1000 U/ml of the cytokine IFN- ⁇ was added.
  • IL-2 300U/ml
  • OKT3 50 ng/ml
  • cells were harvested, washed, and resuspend in electroporation buffer.
  • Cells 10x10 ⁇ 6/cuvette were mixed with DNA/RNA and electroporated.
  • Post electroporation one electroporated reaction was plated per well of 6-well plate in 4 ml of complete medium.
  • a half of the medium was replaced with complete medium + 300 U/ml IL-2.
  • cells were passaged into flasks at 1x10 ⁇ 6/ml in complete medium + 300 U/ml IL-2.
  • PBMCs from healthy donors were isolated from leukapheresis products and processed either fresh or after cryopreservation. Briefly, cells were cultured with IFN- ⁇ (day 0), stimulated with OKT3 and IL-2 (day 1), electroporated with SB100X RNA and pT4-1928YSNVz-IL18 plasmid (see FIG.24, plasmid D) (day 2), and expanded in culture for 15 days, as described in Example 1 above.
  • T cell enrichment and T cell activation were compared before electroporation in lots initiated from cryopreserved versus fresh PBMCs.
  • cells were analyzed using flow cytometry to determine the % of T cells and the expression of the activation marker CD25 in those T cells.
  • PBMC cryopreservation resulted in a significantly higher T cell enrichment (% of T cells in the culture) (FIG.20A) and higher T cells activation (% CD25+ T cells) (FIG.20B and FIG.23).
  • CAR expression was measured using flow cytometry to determine transfection efficiency.
  • the memory phenotype of the final product CARCIK-1918 cells was evaluated by assessing the expression of CD62L, CD45RO markers on CD3+CAR+ cells. Central memory cells are identified as CD62L+CD45RO+, while effector memory cells are identified as CD62L-CD45RO+.
  • FIG. 21B shows that cryopreservation of PBMCs does not impact memory phenotype of CARCIK-1918 cells.
  • FIG. 21B shows that cryopreservation of PBMCs does not impact memory phenotype of CARCIK-1918 cells.
  • the effect of PBMC cryopreservation in the function of the final product CARCIK-1918 cells was assessed.
  • CARCIK-1918 cells manufactured from either fresh or cryopreserved (frozen) PBMCs were characterized by in vitro stimulation with CD19+ REH tumor cells to assess tumor killing activity and IL-18 secretion.
  • CARCIK-1918 cells demonstrated potent in vitro cytotoxicity and IL-18 secretion towards the CD19+ REH target cell line. No significant differences were detected in products manufactured from fresh or frozen PBMCs (% cytotoxicity (FIG.22A) and IL- 18 secretion (FIG.22B)).
  • the use of frozen PBMCs results in high electroporation rates and the cell phenotype, number, and potency by the end of the production process is comparable to the products initiated from fresh PBMCs.
  • Example 8 In vivo evaluation of CARCIK cells in CD19+ human Burkitt’s lymphoma mouse model (NSG/Raji) [0443] The anti-tumor efficacy of CARCIK-1918 cells was evaluated in the CD19+ human Burkitt’s lymphoma model generated by xeno-transplantation of the human Raji cell line into NOD scid gamma (NSG) immunocompromised mice. [0444] CARCIK-CD19 cells with three modifications to the CD19.CAR DNA plasmid construct were generated to enhance in vivo anti-tumor activity and cell persistence.
  • the first modification incorporated the gene for IL-18 was linked to the CAR transgene in a bicistronic DNA plasmid used to generate CARCIK-CD19 cells.
  • the second modification for the CAR construct design was to test two versions of the CAR transgene.
  • a 3 rd generation CAR which uses the CD19 antigen recognition domain derived from the FMC63 monoclonal antibody (mAb) (Jackson ImmunoResearch laboratories, catalog # 109-136-098), joined to the CD28 transmembrane domain with an intracellular CD28-OX40-CD3 ⁇ signaling domain. (See e.g., Magnani et al. J Clin Invest. 130(11): 6021-6033 (2020)).
  • the other CAR transgene evaluated is a 2 nd generation CAR which uses a CD19 antigen recognition domain derived from the SJ25C1 mAb (Memorial Sloan Kettering Cancer Center (MSKCC) Antibody Core Facility) joined to a CD28 transmembrane domain with an intracellular CD28-CD3z signaling domain.
  • SJ25C1 mAb Memorial Sloan Kettering Cancer Center (MSKCC) Antibody Core Facility
  • CD28 transmembrane domain with an intracellular CD28-CD3z signaling domain.
  • the third modification incorporated an amino acid change to the CD28 cytoplasmic signaling domain.
  • the YMNM motif of the CD28 signaling domain was substituted to YSNV (See e.g., International Publ. No. WO2021158850A1) in order to attenuate CD28 signaling. Therefore, both 2 nd and 3 rd generation CAR.CD19 IL-18 bicistronic constructs were generated with the YSNV CD28 signaling motif.
  • Four DNA plasmids were designed incorporating either 2 nd or 3 rd generation CAR transgenes all with the IL-18 gene as a bicistronic construct (FIG. 24).
  • the first two contained the 3 rd generation CAR transgene with either the parental CD28 signaling domain (FIG.24, pT4-CD19CAR-IL18 plasmid A) or the signaling domain with the amino acid substitution CD28-YSNV (FIG. 24, pT4-CD19CARYSNV-IL18 plasmid B).
  • the constructs with the 2 nd generation CAR transgene contained either the parental CD28 signaling domain (FIG.24, pT4-1928z-IL18 plasmid C) or the signaling domain with the amino acid substitution CD28-YSNV (FIG. 24, pT4-1928YSNVz-18 plasmid D).
  • Donor-derived CIK cells were engineered using the SB transposon system to express the CD19.CAR-IL18 transgenes and are referred to as “CARCIK-1918” cells.
  • CARCIK- CD1918 cells were produced from 2 independent donors and tested in tumor bearing NOD scid gamma (NSG) mice (Roswell Park COSR facility, Buffalo. NY). Selection of the optimal CAR.CD19 transgene was based on the ability of CARCIK-CD1918 cells to control tumor growth, enhance animal survival, and cell persistence in vivo. Table 15 outlines the four sets of CARCIK-1918 cells tested each generated with the indicated CAR transgene plasmid DNA.
  • Table 15 List of the Four Arms of CARCIK-1918 cells containing each plasmid A rm CAR generation Plasmid scFv CD28 signaling rd domain Arm A 3 gen pT4-CD19CAR-IL18 FMC63 YMNM Arm B 3 rd gen pT4-CD19CARYSNV-IL18 FMC63 YMNM Arm C 2 nd gen pT4-1928z-IL18 SJ25C1 YSNV Arm D 2 nd gen pT4-1928YSNVz-18 SJ25C1 YSNV [0449] FIG.
  • FIG. 25 shows a schematic representation of study design in NSG/Raji survival model.
  • Raji Tumor Cell and Test System Preparation [0450] GFP/FireFlyLuciferase (GFP/FFLuc) (construct described in Santos, E.B., et al. Nat. Med.15(3):338-344 (2009)) positive Raji cells were thawed at least 1 week prior to the start of the in vivo experiment and seeded at a concentration of 0.5 x 10 6 cells/mL in 5 mL RPMI supplemented with 10% Fetal Bovine Serum (FBS), 2 mM L-glutamine, 1% Penicillin/Streptomycin, 1% NEAA, 1 mM sodium pyruvate (Complete Medium).
  • FBS Fetal Bovine Serum
  • Penicillin/Streptomycin 1%
  • NEAA 1 mM sodium pyruvate
  • the cells were maintained twice weekly by splitting 2.5 x 10 6 cells in 5 mL of fresh complete media (0.5 X 10 6 cells/mL). When required, cells were expanded by determining cell count and increasing the culture volume to achieve 0.5 x 10 6 cells/mL. The total number of cells seeded was the number of cells required to inject 0.5 x 10 6 cells/animal on Day 0.
  • the expanded cells were qualified prior to use for the presence of human CD19 and GFP by flow cytometry analysis using antibody specific for human CD19 and fluorescent emission consistent with GFP.
  • the cells were qualified for luciferase by treating the cells with luciferin and measuring the bioluminescent output via a luminescence plate reader (Tecan).
  • Lot A of CARCIK-1918 cells generated four sets of CARCIK-1918 cells Arms A, B, C and D with the indicated CAR transgene IL-18 bicistronic plasmid DNA (FIG.24, Table 15).
  • Table 16 Characteristics of Lot A for each Arm of CARCIK-1918 cells Lot ID A A ttribute Release C riteria Arm A Arm B Arm C Arm D Viability (%) ⁇ 70 93 91 91 89 CD3 + of total viable (%) ⁇ 90 99 99 99 99 99 CD56 + of CD3 (%) Report 40.6 39.1 36.7 35.9 CAR + of CD3 (%) ⁇ 15 42.0 57.1 31.5 34.3 IL-18 pg/mL Report 616 511 368 411 Cytotoxicity (%) ⁇ 13 28 30 33 53 VCN 1 (cp/mL) Report 1.61 2.40 9.80 9.09 Sterility No Growth No Growth No Growth No Growth Endotoxin (EU/mL) ⁇ 6.25 ⁇ 1.25 ⁇
  • Tumor burden was assessed by bioluminescence imaging (BLI) of mice treated with CARCIK-1918 cells. (FIG.26). Mice were either left untreated or treated with Arm A, B, C, and D CARCIK-1918 cells. BLI measurements were taken at weekly intervals, starting at week 1 and at weeks 2, 3, 4, and 5. At week 4 one animal in Arm D (#5 left to right) did not show tumor burden by BLI, but did not survive the anesthesia treatment. Therefore, 4 animals remained out to week 5 for analysis. [0455] Region of interest (ROI) was recorded (FIG.27) for each animal at weekly intervals starting at week one for five weeks.
  • ROI Region of interest
  • mice in the untreated group and Arms A and B developed progressive highly disseminated tumor and succumbed to the disease by 3 weeks (days 21-23).
  • Mice treated with Arm C and D CARCIK-1918 cells controlled tumor burden out to 5 weeks as demonstrated by decreased BLI signaling following CARCIK-1918 treatment. This is consistent with CARCIK-1918 derived anti-leukemic activity.
  • the mean body weight for each experimental group was plotted versus sampling point for the duration of the study (FIG.29).
  • untreated animals and animals treated with CARCIK-1918 cells expressing a 3 rd generation CAR transgene substantial weight loss, corresponding with animals succumbing to disease, was observed between Days 17 and 22, prior to animals reaching the humane endpoint.
  • animals treated with CARCIK cells expressing a 2 nd generation CAR transgene demonstrated steady body weight out to Day 42.
  • Baseline levels of 750 pg/mL of GM-CSF were measured in a single untreated mouse.
  • Table 17 Characteristics of Lot B for each arm of CARCIK-1918 cells Lot ID B A ttribute Release C riteria Arm A Arm B Arm C Arm D Viability (%) ⁇ 70 91 91 92 91 CD3 + of total viable (%) ⁇ 90 99 99 99 99 CD56 + of CD3 (%) Report 33 38 30 34 CAR + of CD3 (%) ⁇ 15 50 69 32 18 IL-18 pg/mL Report 516 683 260 107 Cytotoxicity (%) ⁇ 13 52 65 60 47 VCN 1 (cp/mL) Report 2.7 3.8 5.9 2.7 Sterility No Growth No growth No growth No growth No growth No growth No growth No growth Endotoxin (EU/mL) ⁇ 6.25 ⁇ 1.00 ⁇ 1.00 ⁇ 1.00 ⁇ 1.00 Mycoplasma Absent Absent Absent Absent absent 1VCN was tested on D17 cells prior to formulation and freezing.
  • Tumor burden was assessed by BLI (FIG. 32), and region of interest (ROI) was recorded for each animal at weekly intervals starting at week two for five weeks (FIG.33). Animals in the untreated group and Arms A and B developed progressive highly disseminated tumor and succumbed to disease by day 18 (FIG.35). To assess early tumor burden control in the absence of a reading on day 7 (week 1) individual ROI was plotted for each animal in groups, untreated, Arms A, B, C and D at day 14 (FIG.34).
  • mice treated with CARCIK-1918 cells containing the 2 nd generation CAR transgene either Arms C and D
  • mice treated with CARCIK-1918 cells containing the 3 rd generation CAR transgene were compared to the untreated mice and mice treated with CARCIK-1918 cells containing the 3 rd generation CAR transgene (Arms A and B).
  • This decrease observed on day 14 was statistically significant (p ⁇ 0.01).
  • Mice treated with Arms C and D CARCIK-1918 cells were capable of controlling tumor burden out to 5 weeks as demonstrated by decreased BLI signaling following CARCIK-1918 treatment, which is consistent with CARCIK-1918 derived anti- leukemic activity (FIG.32).
  • RO retro-orbital
  • the presence of circulating tumor cells was measured by flow cytometry by staining viable cells for human CD19 expressed on circulating Raji tumor cells (hCD19+ cells).
  • Lower numbers of circulating tumor cells were detected in all treated groups compared to the untreated animals, except for a single animal in Arm B having high tumor burden (FIG. 37A).
  • a similar pattern of reduction in the frequency of circulating tumor cells was observed for all three mice treated with CARCIK-1918 cells on Arm D (FIG.37B).
  • CARCIK-1918 cell persistence in peripheral blood of treated animals Peripheral blood from untreated mice or mice treated with each Arm of CARCIK- 1918 cells was collected by RO bleeding on day 8. The persistence of CARCIK-1918 cells in the peripheral blood was measured with flow cytometry by staining viable cells for the human CD3 marker expressed on the infused CARCIK-1918 cells (hCD3+ cells). Greater numbers of CARCIK-1918 cells were seen in mice treated with Arms C and D CARCIK- 1918 cells compared to untreated animals or animals treated with Arms A and B CARCIK- 1918 cells (FIG. 38A). Only one of three mice on Arm B did exhibit high numbers of circulating CARCIK-1918 cells (FIG.38A).
  • mice treated with Arm C or Arm D CARCIK- 1918 cells showed the highest frequency of CARCIK-1918 cells (FIG.38B), with 2 of 3 mice treated with CARCIK-1918 cells with the attenuated CD28 signaling domain having the highest frequency of CARCIK-1918 cells.
  • Further breakout of hCD3+ cells by either CD4+ or CD8 positivity revealed the hCD3+ cell population to be predominantly composed of CD8+ cells (FIG. 39A) for animals treated on Arms C and D.
  • Example 9 In vivo evaluation of CARCIK cells in CD19+ human Burkitt’s lymphoma mouse model (NSG/Daudi) [0467] The anti-tumor efficacy of CARCIK-1918 cells was evaluated in Daudi cell tumor bearing NOD scid gamma (NSG) immunocompromised mice.
  • FIG.41 shows a schematic representation of study design in NSG/Daudi survival model.
  • Daudi Tumor Cell and Test System Preparation Daudi cells were thawed 10 days prior to the start of the in vivo experiment and seeded at a concentration of 1 x 10 6 cells/mL in RPMI 1640 medium supplemented with 10% Fetal Bovine Serum (FBS), 1% L-glutamine and Penicillin/Streptomycin (Complete Medium). The cells were expanded by passage twice weekly to achieve at least 0.2 x 10 5 cells/animal on Day 0. The expanded cells were qualified prior to use for the presence of human CD19 by flow cytometry analysis using antibody specific for human CD19.
  • FBS Fetal Bovine Serum
  • Complete Medium Penicillin/Streptomycin
  • mice (Charles River Laboratories, Calco, Italy) were injected with Daudi cells on Day 0 of the study to establish the test system.
  • Each mouse was injected intravenously (tail vein) with 200 ⁇ L of formulated Daudi cells (0.2 X 10 5 cells/mouse) in a single administration.
  • a single lot of test article was thawed, formulated, and injected at two dose levels into 6 mice per level (5 x 10 ⁇ 6 and 10 x 10 ⁇ 6 CARCIK-1918 cells/mouse). The remaining 5 mice were left untreated.
  • peripheral blood was collected from the mice in each experimental group for flow cytometric analysis.
  • CARCIK-1918 cells were generated with the pT4-1928YSNVz-IL18 (FIG. 24, plasmid D) donor transposon and evaluated in Daudi cell tumor bearing NSG mice. [0471] The key parameters for CARCIK-1918 cells generated from Lot C are provided in Table 18.
  • Table 18 Characteristics of Lot C CARCIK-1918 cells Lot ID C Attribute Release Criteria Result Viability (%) ⁇ 70 87 CD3 of total viable (%) ⁇ 90 96 CD56 of CD3 (%) Report 86 CAR of CD3 (%) ⁇ 15 48 Cytotoxicity (%) ⁇ 13 71 IL-18 (pg/mL) Report 213.42 VCN (cp/cell) Report 10.0 1 Sterility No Growth No Growth Endotoxin (EU/mL) ⁇ 6.25 ⁇ 1.00 Mycoplasma Absent Absent 1VCN was tested on D17 cells prior to formulation and freezing [0473] The mean concentration of hCD45+/hCD19+ cells/mL of blood for each experimental group (DAUDI only, CARCIK-19185x10 ⁇ 6, CARCIK-191810x10 ⁇ 6) was plotted versus sampling point to monitor cell expansion over the course of the study (FIG.
  • the leukemic cells expanded in the untreated group to a peak around Day 40 after which the animals succumbed to disease or were sacrificed. Control of disease was observed in the treated groups as indicated by the low mean concentration of circulating Daudi cells over the course of the study, particularly at the 10 x 10 6 dose level.
  • the mean concentration of hCD45+/hCD3+ cells for each experimental group (CARCIK-19185x10 ⁇ 6, CARCIK-191810x10 ⁇ 6) was plotted versus sampling point to monitor cell expansion over the course of the study (FIG.43). CARCIK-1918 cells were detected in the peripheral blood of mice treated with both dose levels over the entire course of the study.
  • the mean number of CARCIK-1918 cells/mL in the blood in the lower dose treatment group began to increase after Day 40, reaching peak detection at Day 60.
  • Individual mouse body weights were measured and recorded at least twice weekly until the animals succumbed to disease or reached the humane endpoint. The mean body weight for each experimental group was plotted versus sampling point for the duration of the study (FIG.44). In the untreated animals, substantial weight loss, corresponding with the peak detection of circulating tumor cells, was observed between Days 30 and 40 just prior to the humane endpoint for this experimental group.
  • the animals treated with 5 x 10 6 CARCIK-1918 cells demonstrated a steady increase in body weight out to Day 50 of the study after which a substantial decline in weight between Days 58 and 62 was observed, corresponding to the peak detection of circulating hCD45+/hCD19+ leukemic cells (FIG.42). Except for a noticeable and transient weight decline over the first 5 days of the study, which fully recovered by Day 10, the animals treated with 10 x 10 6 CARCIK- 1918 cells exhibited a steady increase in weight over the entire study consistent with control of disease as corroborated by the low level of detected circulating tumor cells (FIG.42).
  • mice All the untreated mice were dead or at the humane endpoint by Day 44 of the study and had exhibited signs of disease burden including ruffled fur, closed eyes, hunched back and paralysis in both limbs (Table 19). Of the 6 mice treated with 5 x 10 6 CARCIK-1918 cells, 5 were still alive at Day 90 and only 1 of the surviving 5 was exhibiting clinical signs of disease burden. All 6 of the mice treated with 10 x 10 6 CARCIK-1918 cells were still alive on Day 90 and none of these 6 were displaying signs of suffering or paralysis.
  • CARCIK-1918 cells at both dose levels demonstrated an inhibitory effect on disease progression.
  • Five of the 6 mice treated at the 5 x 10 6 CARCIK-1918 dose level and all 6 of the 6 mice treated at the 10 x10 ⁇ 6 level were still alive at the study termination on day 90 (FIG. 45).
  • a statistically significant improvement in survival relative to the untreated control group was observed only at the 10 x 10 6 CARCIK-1918 dose level (p-value 0,0011, Log-rank Mantel Cox test). Lack of statistical significance in the survival difference between the untreated (Daudi only) group and the group treated with 5 x 10 6 CARCIK-1918 cells is likely due to the small number of animals evaluated.
  • mice treated at the low dose level Nevertheless, a substantial qualitative improvement in survival for mice treated at the low dose level was readily observed.
  • the post-mortem analysis of peripheral blood and tissues demonstrated that CARCIK-1918 cells from both dose levels persisted over time and limited the level of leukemic cell dissemination in the animals.
  • the 10 x 10 6 regimen allowed an almost total control of the disease progression in all tissues examined (in the bone marrow (BM) (FIG.46A), peripheral blood (PB) (FIG.46B), spleen (FIG.46C), and kidney (FIG. 46D)).

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Abstract

La présente invention propose des procédés de génération de cellules tueuses induites par des cytokines (CIK) génétiquement modifiées, comprenant les étapes successives suivantes : (a) culture d'une population de cellules mononucléaires dans un milieu de culture comprenant au moins un agent de différenciation pour induire la différenciation des cellules mononucléaires dans une culture cellulaire en cellules précurseurs de CIK engagées ; (b) ajout d'au moins un agent stimulant et d'au moins un agent multiplicateur à la culture cellulaire ; et (c) multiplier les cellules de la culture cellulaire pour obtenir une population cellulaire comprenant des cellules précurseurs de CIK engagées, transfecter la population cellulaire comprenant les cellules précurseurs de CIK engagées avec un ou plusieurs acides nucléiques pour produire des cellules précurseurs de CIK engagées génétiquement modifiées, et multiplier les cellules précurseurs de CIK engagées génétiquement modifiées dans un milieu de culture pour produire les cellules CIK génétiquement modifiées ; ou (c) multiplier les cellules de la culture cellulaire pour obtenir une population cellulaire comprenant des cellules précurseurs de CIK engagées et transfecter la population cellulaire comprenant les cellules précurseurs de CIK engagées avec un ou plusieurs acides nucléiques ; (d) formulation des cellules transfectées comprenant les cellules précurseurs de CIK engagées en vue de leur administration à un sujet sur le point d'en avoir besoin ; et (e) administrer les cellules transfectées au sujet, les cellules précurseurs CIK transfectées étant multipliées pour produire les cellules CIK génétiquement modifiées chez le sujet ; les étapes (a), (b) et (c) étant réalisées en l'absence de cellules nourricières non irradiées ou irradiées.
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Citations (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3773919A (en) 1969-10-23 1973-11-20 Du Pont Polylactide-drug mixtures
EP0036676A1 (fr) 1978-03-24 1981-09-30 The Regents Of The University Of California Procédé de préparation de liposomes de taille identique et les liposomes ainsi obtenus
EP0058481A1 (fr) 1981-02-16 1982-08-25 Zeneca Limited Compositions pharmaceutiques pour la libération continue de la substance active
EP0088046A2 (fr) 1982-02-17 1983-09-07 Ciba-Geigy Ag Lipides en phase aqueuse
EP0133988A2 (fr) 1983-08-02 1985-03-13 Hoechst Aktiengesellschaft Préparations pharmaceutiques contenant des peptides régulateurs à libération retardée et procédé pour leur préparation
EP0143949A1 (fr) 1983-11-01 1985-06-12 TERUMO KABUSHIKI KAISHA trading as TERUMO CORPORATION Composition pharmaceutique contenant de l'urokinase
US4683195A (en) 1986-01-30 1987-07-28 Cetus Corporation Process for amplifying, detecting, and/or-cloning nucleic acid sequences
US20040014194A1 (en) 2002-03-27 2004-01-22 Schering Corporation Beta-secretase crystals and methods for preparing and using the same
US9300829B2 (en) 2014-04-04 2016-03-29 Canon Kabushiki Kaisha Image reading apparatus and correction method thereof
WO2016071513A1 (fr) * 2014-11-07 2016-05-12 Fondazione Matilde Tettamanti E Menotti De Marchi Onlus Procédé amélioré de production de cellules génétiquement modifiées
WO2021158850A1 (fr) 2020-02-05 2021-08-12 Memorial Sloan-Kettering Cancer Center Récepteurs antigéniques chimériques présentant des mutations en cd28 et leur utilisation

Patent Citations (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3773919A (en) 1969-10-23 1973-11-20 Du Pont Polylactide-drug mixtures
EP0036676A1 (fr) 1978-03-24 1981-09-30 The Regents Of The University Of California Procédé de préparation de liposomes de taille identique et les liposomes ainsi obtenus
EP0058481A1 (fr) 1981-02-16 1982-08-25 Zeneca Limited Compositions pharmaceutiques pour la libération continue de la substance active
EP0088046A2 (fr) 1982-02-17 1983-09-07 Ciba-Geigy Ag Lipides en phase aqueuse
EP0133988A2 (fr) 1983-08-02 1985-03-13 Hoechst Aktiengesellschaft Préparations pharmaceutiques contenant des peptides régulateurs à libération retardée et procédé pour leur préparation
EP0143949A1 (fr) 1983-11-01 1985-06-12 TERUMO KABUSHIKI KAISHA trading as TERUMO CORPORATION Composition pharmaceutique contenant de l'urokinase
US4683195A (en) 1986-01-30 1987-07-28 Cetus Corporation Process for amplifying, detecting, and/or-cloning nucleic acid sequences
US4683195B1 (fr) 1986-01-30 1990-11-27 Cetus Corp
US20040014194A1 (en) 2002-03-27 2004-01-22 Schering Corporation Beta-secretase crystals and methods for preparing and using the same
US9300829B2 (en) 2014-04-04 2016-03-29 Canon Kabushiki Kaisha Image reading apparatus and correction method thereof
WO2016071513A1 (fr) * 2014-11-07 2016-05-12 Fondazione Matilde Tettamanti E Menotti De Marchi Onlus Procédé amélioré de production de cellules génétiquement modifiées
WO2021158850A1 (fr) 2020-02-05 2021-08-12 Memorial Sloan-Kettering Cancer Center Récepteurs antigéniques chimériques présentant des mutations en cd28 et leur utilisation

Non-Patent Citations (39)

* Cited by examiner, † Cited by third party
Title
"Concise Dictionary of Biomedicine and Molecular Biology", 2002, CRC PRESS
"DNA Cloning", vol. 1,2, 1985
"Epitope Mapping Protocols in Methods in Molecular Biology", vol. 66, 1996
"Handbook Of Experimental Immunology", vol. 1-4, 1986, COLD SPRING HARBOR LABORATORY PRESS
"Immunochemical Methods In Cell And Molecular Biology", 1987, COLD SPRING HARBOR LABORATORY
"Molecular Cloning: A Laboratory Manual", 1992, COLD SPRINGS HARBOR LABORATORY
"Remington's Pharmaceutical Sciences", 1995, MACK PUBLISHING COMPANY
"The Dictionary of Cell and Molecular Biology", 1999, ACADEMIC PRESS
ARAFAR, A., BIOMEDICAL RESEARCH AND THERAPY, vol. 1, no. 2, 2014, pages 71 - 77
AUSUBEL ET AL.: "Current Protocols in Molecular Biology", 1989, COLD SPRING HARBOR LABORATORY PRESS
BRENTJENS ET AL., BLOOD, vol. 118, no. 18, 2011, pages 4817 - 4828
BRICOGNE G, ACTA CRYSTALLOGR D BIOL CRYSTALLOGR, vol. 49, no. 1, 1993, pages 37 - 60
BRICOGNE G, METH ENZYMOL, vol. 276A, 1997, pages 361 - 423
CHAMPE M ET AL., J BIOL CHEM, vol. 270, 1995, pages 1388 - 1394
CHAYEN NE, STRUCTURE, vol. 5, 1997, pages 1269 - 1274
CROOKS: "Principles, strategies and applications", vol. 154,155, 2007, CRC PRESS
CUNNINGHAM BCWELLS JA, SCIENCE, vol. 244, 1989, pages 1081 - 1085
EPPSTEIN ET AL., PROC. NATL. ACAD. SCI. USA, vol. 114,115, 1985, pages 3688 - 3692
GIEGE R ET AL., ACTA CRYSTALLOGR D BIOL CRYSTALLOGR, vol. 50, no. 4, 1994, pages 339 - 350
HUDECEK ET AL., CRIT. REV. IN BIOCHEM AND MOL BIO, vol. 52, no. 4, 2017, pages 355 - 380
LANGER ET AL., J. BIOMED. MATER. RES., vol. 15, 1981, pages 167 - 277
LANGER, CHEM. TECH., vol. 12, 1982, pages 98 - 105
LEUCI VALERIA ET AL: "CSPG4-Specific CAR.CIK Lymphocytes as a Novel Therapy for the Treatment of Multiple Soft-Tissue Sarcoma Histotypes", CLINICAL CANCER RESEARCH, AMERICAN ASSOCIATION FOR CANCER RESEARCH INC, US, vol. 26, no. 23, 1 December 2020 (2020-12-01), pages 6321 - 6334, XP009530021, ISSN: 1557-3265, DOI: 10.1158/1078-0432.CCR-20-0357 *
LINN, Y.C.HUI, K.M., J BIOMEDBIOTECHNOL, 2010, pages 435745
LODISH, H. ET AL.: "Molecular Cell Biology", 2000, W.H. FREEMAN AND COMPANY
MAGNANI CHIARA F ET AL: "Immunotherapy of acute leukemia by chimeric antigen receptormodified lymphocytes using an improved Sleeping Beauty transposon platform", 13 June 2016 (2016-06-13), pages 51581 - 51597, XP093138277, Retrieved from the Internet <URL:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5239498/pdf/oncotarget-07-51581.pdf> [retrieved on 20240306] *
MAGNANI ET AL., J CLIN INVEST., vol. 130, no. 11, 2020, pages 6021 - 6033
MARTIN: "REMINGTON'S PHARM. SCI.", 1990, MACK PUBL. CO.
MCPHERSON A, EUR JBIOCHEM, vol. 189, 1990, pages 1 - 23
MCPHERSON A, J BIOL CHEM, vol. 251, 1976, pages 6300 - 6303
MERKER MICHAEL ET AL: "ERBB2-CAR-Engineered Cytokine-Induced Killer Cells Exhibit Both CAR-Mediated and Innate Immunity Against High-Risk Rhabdomyosarcoma", FRONTIERS IN IMMUNOLOGY, vol. 11, 19 October 2020 (2020-10-19), Lausanne, CH, XP093138625, ISSN: 1664-3224, DOI: 10.3389/fimmu.2020.581468 *
PARK ET AL., N ENGL J MED., vol. 378, no. 5, 2018, pages 449 - 459
PERBAL: "Transcription And Translation; Freshney", 1984, ACADEMIC PRESS, INC., article "A Practical Guide To Molecular Cloning; the treatise"
ROVERSI P ET AL., ACTA CRYSTALLOGR D BIOL CRYSTALLOGR, vol. 56, no. 10, 2000, pages 1316 - 1323
SANTOS, E.B. ET AL., NAT. MED., vol. 15, no. 3, 2009, pages 338 - 344
SIDMAN ET AL., BIOPOLYMERS, vol. 22, 1983, pages 547 - 556
TILLY, G ET AL.: "TEMRA CD8 T Cells Are Highly Cytopathic Cells That Escape From Costimulatory Based-Therapy.: Abstract# B1159", TRANSPLANTATION, vol. 98, 2014, pages 318 - 319
WANG ET AL., ONCOLYTICS, vol. 3, 2016, pages 16015
XU ET AL., CELL DISCOVERY, vol. 4, 2018, pages 62

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