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.
Gonzalez
Sergio
S
City of Hope National Medical Center and Beckman Research Institute, 1500 East Duarte Road, Duarte, CA 91010-3000, USA.
Naranjo
Araceli
A
Serrano
Lisa M
LM
Chang
Wen-Chung
WC
Wright
Christine L
CL
Jensen
Michael C
MC
D017252:CD3 Complex / Q000235:genetics; D015704:CD4 Antigens / Q000235:genetics; D018414:CD8-Positive T-Lymphocytes / Q000378:metabolism; D002462:Cell Membrane / Q000235:genetics / Q000378:metabolism; D016207:Cytokines / Q000378:metabolism; D005818:Genetic Engineering / Q000379:methods*; D006801:Humans; D006825:Hybridomas / Q000276:immunology; D007074:Immunoglobulin G / Q000235:genetics; D007135:Immunoglobulin Variable Region / Q000235:genetics / Q000276:immunology; D016219:Immunotherapy, Adoptive / Q000379:methods*; D009447:Neuroblastoma / Q000628:therapy*; D021381:Protein Transport; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology; D013601:T-Lymphocytes / Q000276:immunology / Q000502:physiology; D013602:T-Lymphocytes, Cytotoxic / Q000502:physiology*; D014407:Tumor Cells, Cultured
D017252:CD3 Complex; D015704:CD4 Antigens; C485468:Cd3e protein, mouse; D016207:Cytokines; D007074:Immunoglobulin G; D007135:Immunoglobulin Variable Region; D011994:Recombinant Proteins