JPWO2019104269A5 - - Google Patents

Claims (26)

Steps to isolate γδ T cells from blood samples of human subjects,
The step of activating the isolated γδ T cells in the presence of aminobisphosphonates, human recombinant interleukin 2 (IL-2) and human recombinant interleukin 15 (IL-15).
The step of transducing the activated γδ T cells with a vector containing a nucleic acid encoding a T cell receptor (TCR), and
Including the step of growing the transduced γδ T cells in the absence of aminobisphosphonates and in the presence of human recombinant interleukin 2 (IL-2) and human recombinant interleukin 15 (IL-15). An in vitro method that activates and proliferates γδ T cells. The method of claim 1, wherein the γδ T cells are isolated from a leukocyte apheresis human sample. The method according to claim 1 or 2, wherein the aminobisphosphonate comprises pamidronic acid, alendronic acid, zoledronic acid, risedronic acid, ibandronic acid, incadronic acid, a salt thereof and / or a hydrate thereof. The method according to any one of claims 1 to 3, wherein the aminobisphosphonate is zoledronic acid. The method according to any one of claims 1 to 4, wherein the activation step is in the presence of zoledronic acid and a cytokine composition consisting of IL - 2 and IL - 15. The method of any one of claims 1-5, wherein the activation step is in the presence of a Toll-like receptor 2 (TLR2) ligand. The method of claim 6, wherein the TLR2 ligand is selected from the group consisting of amphotericin B, L-theanine, tannins, and polyphenols. The method of any one of claims 1-7, wherein the activation step is in the presence of N-acetylcysteine (NAC). The method according to any one of claims 1 to 8, wherein the activation step is in the presence of a COX-2 inhibitor. The method of claim 9, wherein the COX-2 inhibitor is ibuprofen. The method of any one of claims 1-10, wherein the activation step is in the presence of zoledronic acid at a concentration of about 1 μM to about 10 μM. The method of any one of claims 1-11, wherein the activation step is performed in the presence of IL-2 at a concentration of about 10 IU / ml to about 100 IU / ml. The activation step is performed in the presence of zoledronic acid at a concentration of about 1 μM to about 100 μM, IL-2 at a concentration of about 10 IU / ml to about 200 IU / ml, and IL-15 at a concentration of about 10 to 500 ng / ml. The method according to any one of claims 1 to 10. Any of claims 1-10 and 13, wherein the growing step is in the presence of IL-2 at a concentration of about 10 IU / ml to about 100 IU / ml and / or IL-15 at a concentration of about 50-200 ng / ml. The method described in item 1. The proliferated γδT cells have a density of at least about 1 × 10 ⁵ cells / ml, at least about 1 × 10 ⁶ cells / ml, at least about 1 × ¹⁰⁷ cells / ml, at least about 1 × 10 ⁸ cells / ml, or A population of proliferated γδT cells prepared by the method according to any one of claims 1-14 , which is at least about 1 × 10 ⁹ cells / ml. It comprises the step of isolating γδ T cells from peripheral blood mononuclear cells (PBMC) of a human subject.
The isolation step
The step of contacting the PBMC with anti-α and anti-βT cell receptor (TCR) antibodies,
Including the step of depleting α- and / or β-TCR positive cells from the PBMC.
The step of activating the isolated γδ T cells in the presence of aminobisphosphonates, human recombinant interleukin 2 (IL-2) and human recombinant interleukin 15 (IL-15).
The step of transducing the activated γδ T cells with a vector containing a nucleic acid encoding a T cell receptor (TCR), and
Including the step of growing the transduced γδ T cells in the absence of aminobisphosphonates and in the presence of human recombinant interleukin 2 (IL-2) and human recombinant interleukin 15 (IL-15). An in vitro method that activates and proliferates γδ T cells. The method according to any one of claims 1 to 19, wherein the vector is a recombinant viral vector. 17. The method of claim 17 , wherein the viral vector is a retroviral vector, a lentiviral vector, an adeno-associated virus (AAV), or a transposon. 17. The method of claim 17 or 18 , wherein the viral vector is a lentiviral vector. The method of any one of claims 17-19 , wherein the viral vector comprises the RD114TR gene having at least 95% identity with SEQ ID NO: 1 encoding the enveloped protein. The method of any one of claims 1-14 and 16-20 , wherein the viral vector further comprises a nucleic acid encoding CD8. The method of any one of claims 1-14 and 16-21 , wherein the transduction step is in the presence of a transduction enhancer. 22. The method of claim 22 , wherein the transduction enhancer is RetroNectin® or Vectorofsin-1®. Manipulated γδ T cells prepared by the method according to any one of claims 16 to 23 . A population of proliferated and activated γδ T cells prepared by the method according to any one of claims 1-14 and 16-23 . (E) -4-Hydroxy-3-methyl-but-2-enylpyrophosphate (HMBPP), isoprenoid pyrophosphate (farnesyl pyrophosphate (FPP), geranylgeranylpyrophosphate (GGPP), isopentenyl pyrophosphate (IPP), and The method according to any one of claims 1 to 14 and 16 to 23 , wherein activation is carried out in the presence of a phosphoantigen selected from the group consisting of dimethylallyl diphosphate (DMAPP).