Antigen-independent and antigen-dependent methods to numerically expand CD19-specific CD8+ T cells.

OBJECTIVE: Preclinical and clinical trials are investigating the potential of T cells genetically modified to express a first-generation CD19-specific chimeric antigen receptor (CAR), designated CD19R, for adoptive immunotherapy of B-lineage leukemias and lymphomas. Currently, our genetically modified CD19-specific CD8+ (CD19R+CD8+) T cells are expanded ex vivo using a rapid expansion protocol (REP) to clinically meaningful numbers after antigen-independent activation with anti-CD3epsilon and recombinant human interleukin-2 on a double-cell feeder-layer of gamma-irradiated allogeneic peripheral blood mononuclear cells and a lymphoblastoid cell line. We now compare the ability of the REP with CD19-dependent numerical expansion using CD19+ artificial antigen-presenting cells to propagate CD19R+CD8+ T cells. MATERIALS AND METHODS: We evaluated long-term (28 days) propagation, CD19R CAR expression, and cytolytic activity of CD19R+CD8+ T cells expanded by either a REP or an antigen expansion protocol (AEP) using K562-derived artificial antigen-presenting cells coexpressing CD19 antigen and two T-cell costimulatory molecules (4-1BB ligand and major histocompatibility class I-related chains A) in the presence of exogenous recombinant human interleukin-2 and recombinant human interleukin-15. RESULTS: Populations of CD19R+CD8+ T cells could be numerically expanded on AEP to meet anticipated clinical need. The AEP was superior to REP, as this method selected for an outgrowth of T cells with increased CD19R CAR expression and improved redirected cytolytic activity. CONCLUSION: Robust propagation of CD19R+CD8+ T cells achieved by AEP supports qualifying this cell line for use in current good manufacturing practices for CAR+ T cells as an alternative to REP for adoptive immunotherapy clinical trials.

Genetic engineering of cytolytic T lymphocytes for adoptive T-cell therapy of neuroblastoma.

BACKGROUND: Disease relapse is the leading cause of mortality for children diagnosed with disseminated neuroblastoma. The adoptive transfer of tumor-specific T cells is an attractive approach to target minimal residual disease following conventional therapies. We describe here the genetic engineering of human cytotoxic T lymphocytes (CTL) to express a chimeric immunoreceptor for re-directed HLA-independent recognition of neuroblastoma. METHODS: The CE7R chimeric immunoreceptor was constructed by PCR splice overlap extension and is composed of a single-chain antibody extracellular domain (scFv) derived from the L1-CAM-specific murine CE7 hybridoma fused to human IgG1 hinge-Fc, the transmembrane portion of human CD4, and the cytoplasmic tail of huCD3-zeta chain (scFvFc:zeta). Primary human T cells were genetically modified by naked DNA electrotransfer of plasmid expression vector CE7R-pMG then analyzed by Western blotting, flow cytometry for CE7R expression and cell surface trafficking, 4-h chromium release assay for re-directed neuroblastoma lysis, and ELISA for tumor-specific activation of cytokine production. RESULTS: CE7R is expressed as an intact chimeric protein that trafficks to the cell surface as a type I transmembrane protein. Primary human CE7R-expressing CD8(+) CTL clones specifically recognize human neuroblastoma tumor cells and are activated for tumor cell lysis and T(c)1 cytokine production. CONCLUSIONS: These data demonstrate the utility of CE7R for re-directing the effector function of CTL to neuroblastoma and have provided the rationale to initiate a FDA-authorized (BB-IND#9149) pilot clinical trial to establish the feasibility and safety of adoptive transfer of autologous CE7R(+)CD8(+) CTL clones to children with recurrent/refractory neuroblastoma.