573 High Throughput Screening Identifies Effective Enhancers of Lentiviral Transduction Molecular Therapy Volume 20, Supplement 1, May 2012 Copyright © The American Society of Gene & Cell Therapy S222[.]
CELL PROCESSING AND VECTOR MANUFACTURE Cell Processing and Vector Manufacture 571 Manufacture of Clinical-Grade Lentiviral Vectors for Ex Vivo Use Anne Galy,1 Emmanuel Galene,1 Than-Hoa Le,1 Otto Merten,1 Frederic Barnay-Toutain,1 Sabine Charrier,1 Mehdi Gasmi.1 Genethon, Evry, France Hematopoietic gene therapy has now been tested for over 10 years in pilot phase I/II trials in several inherited diseases such as primary immune deficiencies (PID) As vector technology is evolving, new bioprocesses are needed to manufacture clinical-grade products Lentiviral vectors (LV) present significant advantages for gene transfer into hematopoietic stem/progenitor cells, in terms of biological efficacy, biosafety and medicinal product characterization We have developed an advanced generation LV derived from HIV1 and pseudotyped with VSVg to treat Wiskott Aldrich syndrome (WAS), a rare PID characterized by a combined platelet and immune defect This LV leads to the physiological expression of the WAS gene in hematopoietic cells To produce the clinical lots we have developed a scaleable lentiviral manufacturing process that has been approved for phase I/II clinical trials in the EU (UK, France) and in the US The LV upstream process involves a 4-plasmid transfection system in HEK293T cells in Cell Factory stacks Downstream process involves ion exchange chromatography coupled to ultrafiltration and gel filtration steps to purify, concentrate and formulate the bulk vector, which in turn is filter-sterilized to generate the product filled for clinical use At present six clinical grade preparations of the WAS LV have been manufactured and released from our facility Although the process has not yet been validated, to a large extent analytical data generated show good reproducibility and robustness The vector is stable for at least 36 months The process is now adapted to manufacture another VSVg-pseudotyped LV vector for a different PID, X-linked chronic granulomatous disease In an effort to improve residual contaminant profiles of the vector, we have introduced a modification of the DNA removal step in the process The first GMP lot of the X-CGD vector has been manufactured Process yield and analytical data will be discussed in the perspective of regulatory requirements 572 Efficient High Titer Production of Recombinant Lentiviral Vectors for Cancer Immunotherapy Bernd Hauck,1 Guang Qu,1 Tiffany Kincaid,1 William Righter,1 John Scholler,2 Olga Zelenaia,1 Anne Chew,2 Junwei Sun,1 Carl June,2 Katherine A High,1,3 J Fraser Wright.1,2 Center for Cellular and Molecular Therapeutics, Children’s Hospital of Philadelphia, Philadelphia, PA; 2Pathology and Laboratory Medicine, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA; 3Howard Hughes Medical Research Institute, Children’s Hospital of Philadelphia, Philadelphia, PA Recombinant lentiviral vectors (rLenti) expressing a chimeric antigen receptor with specificity for the B-cell antigen CD19 used to modify autologous T lymphocytes have recently shown great promise for effective immunotherapy in human clinical trials for chronic lymphocytic leukemia (Porter et al, NEJM 365:725) One challenge facing development and large scale implementation of this therapeutic approach is the ability to manufacture a sufficient quantity of clinical grade rLenti We have developed and optimized a clinical Phase I/II GMP manufacturing process that has provided consistent high level production and efficient purification Vector generation is performed by four plasmid transient transfection of HEK293T cells in CF5 cell culture units using an optimized scalable methodology to achieve efficient DNA transfection A two harvest strategy was developed following matrix optimization experiments to maximize S222 rLenti TU recovery from the transfected cells using a strategy that balances vector generation during culture and loss of infectivity of the labile rLenti particles that occurs in a time dependent manner after budding from production cells The harvest strategy involved 90%) and direct them to a target cell Using this approach, vector particles could be directed to target cells expressing the same surface molecule An enhanced transduction was observed (3-fold) when both magnetically labeled vector and cells were combined in MACS columns as described above (T cells, MOI=0.5, 30% GFP+; CD34+ cells, MOI=25, 69% GFP+) These novel transduction reagents and protocols enable a fast, flexible and reproducible transduction of target cells to be performed that is independent of vector titer As current cell therapy and gene therapy approaches require many manual handling steps between collecting the patient or donor cell sample and returning the modified cell product to the patient, these protocols are currently being assessed for their suitability for incorporation into a functionally closed and fully automated cell processing device for the manufacture of gene therapeutic cellular products Molecular Therapy Volume 20, Supplement 1, May 2012 Copyright © The American Society of Gene & Cell Therapy 575 Optimizing the Manufacture of CAR-T Cells for Clinical Applications Roopa Mucharla,1 Usanarat Anurathapan,2 Natalia Lapteva,3 Ann M Leen,4 Cliona Rooney,5 Juan F Vera.6 Center for Cell and Gene Therapy, Baylor College of Medicine, Houston, TX Chimeric antigen receptors (CARs) are artificial molecules which can be use to redirect T cell immune response against antigens expressed on the surface of tumor cells Recent encouraging clinical data from our group and others has shown that T cells engineered with these molecules can produce complete clinical response Although promising, most current protocols expand engineered T cells nonspecifically (using IL2 and OKT3), which often results in a decrease in transgenic populations over time Additionally, cell expansion using conventional cultureware is complicated and labor intensive, limiting the broader application of this therapy With the purpose of optimizing and streamlining CAR-T cell manufacture we assessed whether cell expansion could be improved by; (i) substituting non-specific stimuli with an artificial antigen presenting cell (a-APC) expressing cognate antigen, and (ii) culturing cells in a simple and scalable gas permeable culture device (G-Rex) To expand T cells engineered with a CAR targeting the prostate cancer antigen PSCA we first generated an a-APC cell line using K562 cells engineered to express PSCA antigen and different co-stimulatory molecules including CD80, CD86 and 41BB When co-cultured with CAR-PSCA T cells in vitro we found that a-APCs co-expressing PSCA, CD80 and 41BB in combination were most effective in inducing T cell expansion, with a 1.9 fold increase in cell numbers when compared with CAR T cells cultured in the presence of IL2 alone In addition this culture condition enriched for engineered T cells as illustrated by a 2.4±1.2 fold increase in the frequency of transgenic cells after only days of culture To next assess whether we could simply CAR T cell manufacture we transferred our engineered a-APCs and transgenic T cells to a static G-Rex with a surface area of 600cm2, developed by Wilson Wolf Manufacturing as a closed and GMP-compliant culture system In this flask O2 and CO2 are exchanged across a silicone membrane at the base, allowing an increased depth of medium above the cells, which provides more nutrients while waste products are diluted These culture conditions resulted in an increase in the cell output when compared with conventional commercial products such as bags, flasks, and 24-well tissue culture plates, without increasing the number of cell doublings From an initial seeding density of 1.5E+08 T cells (0.25E+06 cells per cm2) we achieved on average a 110 fold cell expansion in days of culture resulting in the production of 1.51.8E+10 CAR-T cells (25-30E+06 T cells per cm2) using only 6L of culture media (without media change) We found that 10ml media/cm2 (volume:surface area) supported maximal cell output and cell viability was maintained at >95% for days However, while cell output could not be improved by adding additional media the use of an additional 5ml/cm2 sustained cell viability at >95% for days This optimized bioprocess is GMP compatible and could be adapted for clinical manufacture of CAR-T cells, decreasing the cost and complexity of this technology and making this therapy more accessible 576 Exploiting the Mechanism of IntronSplicing in Insect Cells To Produce Viral Vectors Harboring Toxin Genes for Cancer Gene Therapy Haifeng Chen.1 VIROVEK Incorporation, Hayward, CA Suicide gene therapy has been widely investigated for the treatment of human immunodeficiency virus (HIV) infection, for controlling graft-versus-host disease, and also for the treatment of cancer While the production of viral vectors carrying suicide genes such as the herpes simplex virus thymidine kinase (HSV-TK) or S223 ... absence of PMA to greater than 20% in the presence of 1nM PMA, and cell division was hindered The lack of enhanced viral transduction with sca-1+ cells may be due to the different PKC isoform found... GFP-encoding adenoviral (AdV) and lentiviral (LV) vector particles effectively After AdV transduction (pMOI, physical particles per cell=200-500) of human cells of low permissivity (K562, M-07e,... Department of Gene Therapy, University of Ulm, Ulm, Germany A major factor limiting viral transduction of target cells in cell culture is the diffusion of virus particles to the cell surface