Manufacturing of gene-modified cytotoxic T lymphocytes for autologous cellular therapy for lymphoma.

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 CD3epsilon (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 ganciclovir-mediated killing and display a non-transformed phenotype. TCR Vbeta usage indicated that all populations tested were polyclonal. Ex vivo cell expansion from cryopreserved cell banks is sufficient to produce doses of between 5 x 10(9) and 1 x 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.