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The production of clinical-grade T cells for adoptive immunotherapy has evolved from the ex vivo numerical expansion of tumor-infiltrating lymphocytes to sophisticated bioengineering processes often requiring cell selection, genetic modification and other extensive tissue culture manipulations, to produce desired cells with improved therapeutic potential. Advancements in understanding the biology of lymphocyte signaling, activation, homing and sustained in vivo proliferative potential have redefined the strategies used to produce T cells suitable for clinical investigation. When combined with new technical methods in cell processing and culturing, the therapeutic potential of T cells manufactured in academic centers has improved dramatically. Paralleling these technical achievements in cell manufacturing is the development of broadly applied regulatory standards that define the requirements for the clinical implementation of cell products with ever-increasing complexity. In concert with academic facilities operating in compliance with current good manufacturing practice, the prescribing physician can now infuse T cells with a highly selected or endowed phenotype that has been uniformly manufactured according to standard operating procedures and that meets federal guidelines for quality of investigational cell products. In this review we address salient issues related to the technical, immunologic, practical and regulatory aspects of manufacturing these advanced T-cell products for clinical use. [ABSTRACT FROM AUTHOR]

Background The production of therapeutic T-cell populations for adoptive immunotherapy of cancer requires extensive ex vivo cell processing, including the isolation or creation of Ag-specific T cells and their subsequent propagation to clinically relevant numbers. These procedures must be performed according to the principles of current good manufacturing practices (cGMP) for phase I clinical trials to ensure the identity, purity potency and safety of the cellular product. In this report we describe our approach to manufacturing and characterizing bulk populations of gene-modified autologous T cells for use in treating follicular lymphoma. Methods PBMC from healthy donors, obtained after informed consent, were stimulated in vitro with Ab to CD3ε (OKT3) and recombinant human IL-2 and then electroporated with plasmid DNA containing a human CD19-specific chimeric Ag receptor (CAR) gene and HSV-1 thymidine kinase (TK) gene. Stably transfected cells were selected in cytocidal concentrations of hygromycin B over multiple 14-day stimulation culture cycles and then cryopreserved. Vials of cryopreserved/selected T cells were used to initiate T-cell expansion cultures to produce cell products for clinical infusion. These cultures were characterized for phenotype, function and suitability for use in adoptive immunotherapy studies. Results Our results demonstrate that bulk populations of gene-modified T cells derived from peripheral blood of healthy donors express CD19 + chimeric Ag receptor at low levels and can specifically lyse CD19 + target cells in vitro . These cells display a differentiated T-effector phenotype, are sensitive to gancilovir-mediated killing and display a non-transformed phenotype. TCR Vβ usage indicated that all populations tested were polyclonal. Ex vivo cell expansion from cryopreserved cell banks is sufficient to produce doses of between 5×10 9 and 1×10 10 cells/run. One of three transductions resulted in a population of cells that was not suitable for infusion but was identified during release testing. No populations displayed any evidence of bacterial, fungal or mycoplasma contamination. Discussion We have established a manufacturing strategy that is being used to produce T cells for a phase I clinical trial for follicular lymphoma. Genetically modified T cells have been characterized by cell-surface marker phenotype, functional activity against CD19 + targets and requisite safety testing. These pre-clinical data confirm the feasibility of this approach to manufacturing T-cell products. [ABSTRACT FROM AUTHOR]

Background: Immunotherapy for B-cell lymphomas has evolved significantly with the advent of CD20-targeted Ab-based therapeutics. Strategies to invoke or augment cellular anti-lymphoma immune responses may also have considerable therapeutic potential and serve to further augment the clinical efficacy of MAbs., Methods: We report here the aquisition by priming human cytotoxic T lymphocyte (CTL) effectors of re-directed CD20 specificity by their genetic modification to express a chimeric immunoreceptor consisting of an anti-CD20 single chain Ab extracellular domain molecularly fused to the T-cell receptor complex CD3-zeta cytoplasmic tail (scFvFczeta). Peripheral blood-derived human T-cells were transduced with naked DNA plasmid vector by electoporation then selected for G418 resistance., Results: Following cloning in limiting dilution and ex vivo expansion to large numbers scFvFczeta+ TCRalpha/beta+ CD4- CD8+ CTL display re-directed HLA-unresricted CD20-specific lymphoma cell cytolysis proportional to the cell-surface density of the chimeric immunoreceptor. Engineered CTL clones are also activated through the chimeric immunoreceptor to produce Tc1 cytokines (IFN-gamma) upon co-culture with CD20+ lymphoma stimulator cells. Additionally, CD20-specific CTL proliferate in the presence of lymphoma stimulators and IL-2 (5 U/mL)., Discussion: These studies provide the rationale for exploring the clinical utility of adoptive therapy with CD20-specific CTL as a component of immunotherapeutic targeting of CD20+ malignancy.