Phenotypic and functional attributes of lentivirus-modified CD19-specific human CD8+ central memory T cells manufactured at clinical scale

J Immunother. 2012 Nov-Dec;35(9):689-701. doi: 10.1097/CJI.0b013e318270dec7.


A key determinant of the therapeutic potency of adoptive T-cell transfer is the extent to which infused cells can persist and expand in vivo. Ex vivo propagated virus-specific and chimeric antigen receptor (CAR)-redirected antitumor CD8 effector T cells derived from CD45RA(-) CD62L(+) central memory (TCM) precursors engraft long-term and reconstitute functional memory after adoptive transfer. Here, we describe a clinical scale, closed system, immunomagnetic selection method to isolate CD8(+) T(CM) from peripheral blood mononuclear cells (PBMC). This method uses the CliniMACS device to first deplete CD14(+), CD45RA(+), and CD4(+) cells from PBMC, and then to positively select CD62L(+) cells. The average purity and yield of CD8(+) CD45RA(-) CD62L TCM obtained in full-scale qualification runs were 70% and 0.4% (of input PBMC), respectively. These CD8(+) T(CM) are responsive to anti-CD3/CD28 bead stimulation, and can be efficiently transduced with CAR encoding lentiviral vectors, and undergo sustained expansion in interleukin (IL)-2/IL-15 over 3-6 weeks. The resulting CD8(+) T(CM)-derived effectors are polyclonal, retain expression of CD62L and CD28, exhibit CAR-redirected antitumor effector function, and are capable of huIL-15-dependent in vivo homeostatic engraftment after transfer to immunodeficient NOD/Scid IL-2RgCnull mice. Adoptive therapy using purified T(CM) cells is now the subject of a Food and Drug Administration-authorized clinical trial for the treatment of CD19(+) B-cell malignancies, and 3 clinical cell products expressing a CD19-specific CAR for IND #14645 have already been successfully generated from lymphoma patients using this manufacturing platform.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adoptive Transfer
  • Animals
  • Antigens, CD19 / metabolism*
  • CD28 Antigens / metabolism
  • CD3 Complex / metabolism
  • CD8-Positive T-Lymphocytes / cytology
  • CD8-Positive T-Lymphocytes / immunology*
  • CD8-Positive T-Lymphocytes / metabolism*
  • Cell Line, Tumor
  • Cryopreservation
  • Cytokines / biosynthesis
  • Cytotoxicity, Immunologic
  • Genetic Vectors / genetics
  • Humans
  • Immunologic Memory*
  • Immunomagnetic Separation / methods
  • Immunophenotyping
  • L-Selectin / metabolism
  • Lentivirus / genetics
  • Lymphocyte Activation / immunology
  • Lymphoma / immunology
  • Lymphoma / therapy
  • Mice
  • Mice, Inbred NOD
  • Mice, SCID
  • Phenotype*
  • Receptors, Antigen, T-Cell / metabolism
  • Transduction, Genetic
  • Tumor Necrosis Factor Receptor Superfamily, Member 7 / metabolism
  • Xenograft Model Antitumor Assays


  • Antigens, CD19
  • CD28 Antigens
  • CD3 Complex
  • Cytokines
  • Receptors, Antigen, T-Cell
  • Tumor Necrosis Factor Receptor Superfamily, Member 7
  • L-Selectin