Method for the generation of genetically modified cells

Abstract

The invention provides a method for the improved generation of genetically modified cells in vitro, in order to obtain a population of effector cells with immunotherapeutic activity and methods of using such cells in protocols for adoptive cell therapy. The invention further provides non-viral genetically modified cells, cell populations and cell cultures and the use thereof in the treatment or prevention of diseases and disorders.

1 .- 15 . (canceled) 
     
     
         16 . A method of generating genetically modified cells, wherein the genetically modified cells are cytokine induced killer cells expressing one or more T cell receptors (CIK-TCR) or chimeric antigen receptors (CIK-CAR), the method comprising:
 (a) non-viral transfer of one or more nucleic acids or exogenous nucleic acids encoding antigen receptors, T cell receptors, chimeric antigen receptors and optionally encoding suicide genes or genes encoding an inducible Caspase 9 system, or combinations thereof into a population of mononuclear cells in a cell culture optionally by electroporation or nucleofection and optionally in an amount of nucleic acids from about 0.1 to about 100 μg;   (b) addition of one or more differentiating agents to the cell culture before, during or after the transfer of nucleic acids, wherein the differentiating agents differentiate the mononuclear cells in the cell culture into cytokine induced killer cells, optionally the differentiating agents are added in an amount of from about 10 U/ml to about 10,000 U/ml or in an amount of about 1,000 U/ml;   (c) addition of antigen presenting cells to the cell culture before, during or within about 10 days after the transfer of nucleic acids or addition of differentiating agents and optionally the antigen presenting cells are added to the cell culture once before, during or within about 10 days after the transfer of nucleic acids;   (d) addition of one or more stimulating agents to the cell culture before, during or after the transfer of nucleic acids, the addition of differentiating agents or the addition of antigen presenting cells, optionally the stimulating agents are added in an amount of from about 5 ng/ml to about 100 μg/ml or in an amount of about 50 ng/ml and optionally the stimulating agents are added to the cell culture once before, during or after the transfer of nucleic acids or the addition of antigen presenting cells;   optionally step (e), addition of one or more stimulating and expanding agents to the cell culture before, during or after the transfer of nucleic acids, the addition of antigen presenting cells or the addition of stimulating agents and optionally the stimulating and expanding agents are added to the cell culture at least once before, during or after the transfer of nucleic acids, the addition antigen presenting cells or the addition of stimulating agents; and optionally step (f) isolating the cells from the cell culture to obtain a cell population comprising the modified cells.   
     
     
         17 . The method of  claim 16  wherein the population of mononuclear cells in the cell culture comprises: peripheral blood mononuclear cells, bone marrow derived mononuclear cells, umbilical cord blood 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 combinations thereof. 
     
     
         18 . The method of  claim 16  wherein the non-viral transfer of nucleic acids comprises: transposons, Zn-finger nucleases, integrases, transcription activator-like effectors, clustered regularly interspaced short palindromic repeats, sequence-specific recombinase systems able to integrate nucleic acids by recombination between attachment sites, Sleeping Beauty, PiggyBac, TALEs, phiC31 or CRISPR/Cas or combinations thereof and optionally wherein the nucleic acids encode for T cell receptors, chimeric antigen receptors or chimeric antigen receptors for one or more antigens comprising CD19, CD123, CD20, CD23, CRLF2, CD44v6, CD33, CS1, CD38, Her2, EGFr, and CA125. 
     
     
         19 . The method of  claim 16  wherein the antigen presenting cells comprise: irradiated mononuclear cells, irradiated peripheral blood mononuclear cells, Mitomycin C-treated mononuclear cells, Mitomycin C-treated peripheral blood mononuclear cells or combinations thereof, or lymphocytes, monocytes, dendritic cells, macrophages, artificial antigen presenting cells optionally irradiated or treated with Mitomycin C or combinations thereof and optionally wherein the antigen presenting cells are added to the cell culture within about 5 days or between 0 and about 5 days, within about 24. 
     
     
         20 . The method of  claim 16  wherein said stimulating agent is selected from: agents that stimulate antigens; agents that stimulate CD3+ cells; TCR stimulating agents; anti-CD3 antibodies; OKT3; CD3+ cells; anti-CD28 antibodies; anti-TCR antibodies; beads; polyclonal non-TCR restricted stimulation agents; anti-CD3-loaded artificial antigen presenting cells, optionally irradiated or treated with Mitomycin-C; and optionally wherein the stimulating agents are added to the cell culture after the transfer of nucleic acids, within about 10 days or between 0 and about 10 days, within about 5 days or between 0 and about 5 days, or within about 1 day or between 0 and about 1 day after the transfer of nucleic acids. 
     
     
         21 . The method of  claim 16  wherein the stimulating and expanding agents comprise cytokines, cytokines bind the common y chain (CD132), IL-2, IL-7, IL15, IL-21 or combinations thereof and optionally wherein the stimulating and expanding agents are added to the cell culture within about 10 days or between 0 and 10 days, or within about 1 day or between 0 and about 1 day after the transfer of the nucleic acids. 
     
     
         22 . The method of  claim 16  wherein the differentiating agents comprise molecules that promote differentiation, cytokines, IFN-y, IL-4, IFN-cx, IL-10, IL-12, IL-6, IL-21, IL-23, IL-1p, TGF-P or combinations thereof and optionally wherein the differentiating agents are added within about 10 days or between 0 and about 10 days, within about 5 days or between 0 and about 5 days, within about 1 day or between 0 and about 1 day, or within about 2 hours or between 0 and about 2 hours after the transfer of nucleic acids. 
     
     
         23 . Non-viral genetically modified cells, cell populations or cell cultures comprising CIK-TCR, CIK-CAR, CIK-CAR19, CIK-CAR123 cells, CIK-CAR cells comprising nucleic acids encoding for one or more antigens comprising CD19, CD123, CD20, CD23, CRLF2, CD44v6, CD33, CS1 CD38, Her2, EGFr and CA125 antigens, or combinations thereof, wherein the cell populations and cell cultures further comprise: a) expression levels of TCR, CAR or combinations thereof, of at least about 10-60%, preferably at least about 20-30% and more preferably at least about 50-60%; b) optionally at least about 10%, preferably at least about 25% of CIK-TCR, CIK-CAR, CIK-CAR19, CIK-CAR123 cells, CIK-CAR cells comprising nucleic acids encoding for one or more antigens comprising CD19, CD123, CD20, CD23, CRLF2, CD44v6, CD33, CS1 CD38, Her2, EGFr and CA125 antigens, or combinations thereof; c) optionally a fold increase in expansion of the cell population greater than about 10 at about 21-28 days of culture; and d) optionally at least about 10-90%, preferably about 60-90% and more preferably about 80-90% of viable CIK-TCR, CIK-CAR, CIK-CAR19, CIK-CAR123 cells, CIK-CAR cells comprising nucleic acids encoding for one or more antigens comprising CD19, CD123, CD20, CD23, CRLF2, CD44v6, CD33, CS1 CD38, Her2, EGFr and CA125 antigens, or combinations thereof. 
     
     
         24 . Genetically modified cells, cell populations, cell populations comprising T cell receptor cells, chimeric antigen receptor cells or combinations thereof obtained by the methods of  claim 16 , optionally comprising expression levels of transgenes of at least about 10-60%, at least about 20-30% or at least about 50-60%, optionally at least about 10% or at least about 25% CIK-TCR, CIK-CAR, CIK-CAR19, CIK-CAR123 cells or combinations thereof, optionally a fold increase in expansion of the cell population greater than about 10 at about 21-28 days of culture and optionally at least about 10-90%, about 60-90% or about 80-90% of viable T cell receptor cells, chimeric antigen receptor cells or combinations thereof. 
     
     
         25 . Genetically modified cells, cell populations, cell populations comprising CIK-TCR, CIK-CAR, CIK-CAR19, CIK-CAR123 cells, CIK-CAR cells comprising nucleic acids encoding for one or more antigens comprising CD19, CD123, CD20, CD23, CRLF2, CD44v6, CD33, CS1 CD38, Her2, EGFr and CA125 antigens, or combinations thereof made by the method of  claim 16 , optionally comprising expression levels of transgenes of at least about 10-60%, at least about 20-30% or at least about 50-60%, optionally at least about 10% or at least about 25% CIK-TCR, CIK-CAR, CIK-CAR19, CIK-CAR123 cells, CIK-CAR cells comprising nucleic acids encoding for one or more antigens comprising CD19, CD123, CD20, CD23, CRLF2, CD44v6, CD33, CS1 CD38, Her2, EGFr and CA125 antigens, or combinations thereof, optionally a fold increase in expansion of the cell population greater than about 10 at about 21-28 days of culture and optionally at least about 10-90%, about 60-90% or about 80-90% of viable CIK-TCR, CIK-CAR, CIK-CAR19, CIK-CAR123 cells, CIK-CAR cells comprising nucleic acids encoding for one or more antigens comprising CD19, CD123, CD20, CD23, CRLF2, CD44v6, CD33, CS1 CD38, Her2, EGFr and CA125 antigens, or combinations thereof. 
     
     
         26 . A formulation comprising the genetically modified cells or cell populations of  claim 23 . 
     
     
         27 . A method of treating or preventing a disease or disorder in a mammal in need thereof comprising administering to the mammal an effective amount of the genetically modified cells or cell populations of  claim 23 . 
     
     
         28 . The method of  claim 27  wherein the disease or disorder is selected from a cancer, a tumor, a hematologic disorder, a leukemia, a lymphoma, a solid tumor, a viral infection, an inflammatory disease or disorder, or an autoimmune disease or disorder. 
     
     
         29 . The method of  claim 27  wherein the disease or disorder is selected from a cancer, a tumor, a hematologic disorder, a leukemia, a lymphoma or a solid tumor.