311 development of a high cell density process for cGMP production of VRX496 modified HIV infected CD4+ t lymphocytes

311 Development of a High Cell Density Process for cGMP Production of VRX496 Modified HIV Infected CD4+ T Lymphocytes harvest All 5 GP+envAMI2 cell lines were optimal at 32 °C, but optimal harvest tim[.]

harvest All GP+envAMI2 cell lines were optimal at 32 °C, but optimal harvest time varied The data suggests harvest conditions should be optimized for each MCB generated Also, those MCBs with an optimal titer at 24 hours often have a 12 hour titer that exceeds 50% of the 24 hour titer Therefore, more frequent harvests at shorter intervals can increase the total yield of vector particles and may be preferred when the material can be concentrated Finally, the stability of vector stored was measured over a year period Aliquots of the LNL6 vector (expressing neo) and the Ampho/GFP and GALVIGFP vectors (expressing enhance green f1 uorescent protein, MGF-eGFP) were maintained between -70 and -80 °C and the number oftransducing units was periodically measured using G418 selection and flow cytometry, respectively In all three vectors the titer appeared to be stable during the observation period suggesting an expiration date of5 years would be appropriate for unconcentrated vectors The information obtained serves as a baseline for assessing new packaging cell lines, including those designed to generate novel integrating vector systems such as HIV and foamy viruses 309 Plasmid DNA Production in a Wave Bioreactor under cGMP Conditions Kenneth A Laderman ,' Jonathan M Anderson ,' Ivy V Derecho ,' Nina T Dunphy,' Ross J Mclvlahon,' Valerie R Quezada,' David Hsu,' Larry A Couture.' 'Center for Biomedicine and Genetics Centerfor Applied Technology Development Beckman Research Institute ofCity of Hope Duarte CA Plasmid DNA production is an essential step in the generation of genetic therapies, either as the therapeutic agent or as reagents for the production of viral vectors A robust process for the generation of plasmid bearing E coli biomass is necessary as it is the rate limiting step for plasmid production and it is most variable as there are construct specific factors that affect the plasmid yield There are a number of factors that make the transition ofthe fermentation process from a traditional stirred tank reactor to a Wave bioreactor desirable Foremost among these are the disposable culture vessel in the form of the wave bag The disposable culture vessel minimizes the possibility of prior product contamination in a multi-product facility and decreased production cost while increasing throughput due to the ease of set up and tear down Two lentivirus helper plasmids, which had previously been produced in the facility using a cGMP stirred tank procedure, were used to develop the wave fermentation process Fermentations were completed using media and fermentation parameters adapted from the stirred tank fermentation process To mimic the aeration and agitation of the stirred tank the wave angle and rate were set to their practical maxima The mean specific yield (mg of plasmid I g wet weight) obtained from the wave bioreactor was found to range from equivalent to fold higher than that obtained from the same bacterial master cell bank in the stirred tank fermentor while the bacterial yield (g wet weight I liter of culture) from the wave bioreactor was approximately equivalent to that obtained from the stirred tank The wave fermentation has proven to be scalable with minor increases in specific yield observed as the culture volume is increased to 25 Iitcrs Limited optimization of temperature and pH conditions have established conditions that increase the specific yield approximately 100% from the default fermemation conditions used for the original feasibility assessment The resulting improvement in specific yield ofthe optimized process is approximately 600% greater than that obtained with the stirred tank system Molecular Therapy Volume15 Supplement I ~ br 2007 Copyright © The American Soci ety o r Gene "1l1f:r:lpy 310 A Rapid, Accurate and Precise Assay System To Analyze the Quality and the Quantity of Clinical Grade HSV Vector Stocks Ali Ozucr,' Steve K Wendell,2 Darren Wolfe; William F Goins; David M Krisky," Mohammad M Ataai,' Joseph C Glorioso.' f Molecular Genetics and Biochemistry University ofPittsburgh Pittsburgh PA; ]Dental Medicine, University ofPittsburgh Pittsburgh, PA; 'Chemical and Petroleum Engineering University of Pittsburgh Pittsburgh, PA; "Department ofPathology; University ofPittsburgh Pittsburgh PA As the number of clinical and research applications of herpesvirus-based vectors increase, it becomes critical to develop rapid, accurate and precise assay systems to analyze the quality and the quantity of clinical grade vector stocks Our assay system contains two independent and complementary DNA and protein quantification methods: a real-time quantitative PCR system and two micro-plate assays using PicoGreen and NanoOrange reagents for the quantification of total DNA and protein respectively This assay system is being optimized to quantify the amount of infectious versus defective HSV particles, and the purity ofHSV vector stocks Our real-time quantitative PCR assay employs two independent primer sets The first set, speeific for ICP47 sequences present in all HSV genomes, allows for the quantification of total viral particles and for defective DNA containing particles when compared with the number of plaque forming units generated by standard virological plaque assay A second primer set, specific for the human 18S gene, enables the estimation of purity ofgene therapy vector stocks in combination with NanoOrange protein assay system Our real time PCR assays are linear from to 4x 103 HSV genome copies per reaction, and 0.005 to 50 ng host cell genomic DNA per reaction In contrast to our PCR method that quantifies the viral and host cellular DNA concentration, we developed an independent microplate assay measuring the total DNA and protein concentrations of vector stocks Our micro-plate assays are fast and accurate, with detection limit as low as 0.5 ng of DNA and 0.4 mg/mL protein The resultant combination of real-time PCR and micro-plate DNA and protein quantitation assays represents a standard in the field of HSV vector quality assessment 311 Development of a High Cell Density Process for cGMP Production of VRX496 Modified HIV Infected CD4+ T Lymphocytes Andrew Worden,' Tony Encinas.' Yclena Skripchcnko.! James Rogers,' Reuben Cohen,' Vladimir Slepushkin,' Laurent Hu- meau.' f Research and Development VIRxSYS Corporation Gaithersburg AID; lOperations VIRxSYS Corporation Gaithersburg, MD VIIb:SYS Corporation has developed an autologous CD4+ T cell therapy for the treatment ofI-IlV infection, which uses a patient's own CD4+ T cells that have been genetically modified by a lentiviral vector, VRX496, carrying a 937-base antisense targeting the HIV envelope After a patient's white blood cells are harvested, the cells are then purified to obtain the CD4+ T cells, transduced with VRX496 , expanded, and then rcinfused into the patient We reported the successful completion of our Phase I clinical trial where we demonstrated the safety and tolerability ofa single dose consisting ofapproximately 10 billion autologous I-IIV infected CD4+ T cells transduced with VRX496, (Levine and Humeau et al., PNAS 2006) We immediately initiated a Phase II clinical trial testing the safety and tolerability ofsingle and repeated infusions ofVRX496-modifed CD4+ T cells with the aim to determine the optimal therapeutic dose A large scale cGMP cell process was developed and has been presented (Humeau et aI., ASGT 2006) Briefly, the positively seSI17 lected CD4+ T cells from 35 HIV infected patients were genetically modified with VRX496 and expanded in a Wave™ bioreactor to a final cell number averaging 84 billion, with expansion up to 300 fold in some cases However, in order to reach such cell numbers, expansionshave been performedin a 50L perfusionbag (25Lculture volume) with a cell concentration averaging 4.3 million per ml and yielding excellent viabilities in the range of 92% In preparation of our phase III clinical trial, we sought to reduce culture volumes from25L to IOL while retainingrobustexpansionsand excellentcell viabilities Cell processingdevelopment runs conducted in 20L Wavc" perfusion bags (IOL culture volume) lead to final cell number averaging 134billion More importantly,the average maximum cell concentration increased up to 23 million cells per ml with excellent viabilitiesin the 95% range.Thus we have been able to obtainsimilar or superior levels of expansion and viability in 20L perfusion bags and saved approximately 15L of medium per run while producing an average 01'37% more cells at time of harvest.These findings are critical to streamlining and optimizing the cell process for the mass production of our VRX496 therapy 312 Large Scale Lentiviral Vector Production in a GMP Facilty Lara J Ausubcl, I Ken A Laderman, I Misty ShakeIcy,I Anupriya Sharma,I Sylvana Couture, I Patricia Lopez, I Christine Knoblauch,' Jonathan Anderson,' Ivy Derecho,' Ross McMahon,1 David Hsu,I Larry Couture.' 'Center for Biomedicine and Genetics, Center for Applied Technology Development, Beckman Research Institute ofCity of Hope , Duarte, CA Lentiviral Vectorsare emerging as important tools in the field of gene therapy They have many advantages over other techniques that have been used for gene transfer in that the vectors are able to infect non-dividing cells Wehave compared both the 293 and 293T cells for the lentivirus production and have chosen to produce virus by transient transfection of293T cells based on its ability to support high titer vector production and its consistency in vector production over time in culture A Lentiviral vector packaging system using separate plasmids developed at City of Hope is used to produce the vectors This system provides maximum flexibility in the type of virus produced while addressing safety concerns by minimizing the probabilityofthe generation ofreplicationcompetent virus In order to produce largequantitiesofhigh titer virus under GMP conditions, we have optimizedseveral parametersofthe manufacturingprocess These include conditions for cell growth and adherence, transfection, viral production as well as the conditions for downstream purification of the virus The optimized conditions were then used to produce virus in multiple sub-batches of 10 cell factories, each factory having 10 layers Using these methods, we have been able to achieve large scale vector production (100 liter quantities) with crude titers on the order of 106 • The crude supernatant is harvested following transfection from each sub-batch and is clarified Benzonase is added to the clarified supernatant to digest residual plasmid and host cell DNA A tangential flow ultrafiltration system is used to concentrate the c1arificd supernatant 10-20fold and to formulate the virus The concentrated!diafiltered material is centrifuged to pellet the virus which is subsequently resuspended in a volume of the final formulation representing a 100-200 fold concentration of the crude supernatant These methods have been used under GMP conditions to produce a number of lots with an infectious titer of approximately 1-3 x 109 TUlmL, when titered on HTI 080 cells Our multi-sub-batch system provides for virtually unlimited scale up capacity SII8 313 Production of Ad/AAV-Hybrids Encoding Proteins That Inhibit Viral Propagation Gina Nichols,' Tony Stepan" Kristin Parker" Eric J Pastor" Richard W Peluso" Barbara A Thorne.' Process Development, Targeted Genetics, Seattle, W:4 F For adeno-associated viral vectors to fulfill their promise as gene therapy delivery vehicles, scaleable production methods are required The components required for rAAV production include cells, the AAV rep and cap genes, viral helper functions, and the vector genome Onc of the more flexible and scaleable approaches to bring these components together for rAAV production is to use an Ad/AAV-hybrid process, where wild type adenovirus (Ad5) and an Ad/AAV-hybrid, a non-replicating EI-deleted adenovirus containing the vector genome, are used to co-infect a packaging cell line containingthe rep and cap genes (I) Thus, in order to producerAAV at large scale in a cGMP environment using the Ad!AAV.hybrid system, several cGMP cell banks and viral stocks are required as critical raw materials, including a packaging cell line bank, Ad5, Ad!AAV-hybrid stocksand cells to producethem.This study focuses on the generation ofthe Ad!AAV-hybrid stocks The necessary steps for producing Ad!AAV-hybrid as a product-specific raw material include virus construction, generation of seed stocks and cell banks for viral production, and development of a scaleable production process However, the virus generation and subsequent production can present challenges with some transgenes whose expression can inhibit adenovirus production (2) A tetracycline regulated protein expression system (3) was evaluated with an Ad!AAV-hybrid that exhibited such a phenotype in unmodified EI-complementing 293 cells This system has two components, an El-complementing cell line expressing the Tet repressor protein and a construct containing Tetoperatorclementsupstreamofthe gene ofinterest In the absence of tetracycline, repressor proteins in the cell bind to the operator region of the construct, inhibiting transcription and subsequent protein expression Using the tetracycline regulated system, rescue and production of the problematicAd!AAV-hybrid was successful Adapting the system for large scale Ad/AAV-hybrid raw material supply will be discussed Issues addressed include adaptation to serum-free suspension growth, regulation of transgene expression, and serum-free production of the Ad/AAV hybrid for use in GMP rAAV manufacturing References: (1) Gao et al (1998) Human Gene Therapy 9: 2353 (2) D.A Matthews et AI (1999) Journal of General Virology 80:345-353 (3) Yao et al (1998) 1·luman Gene Therapy 9:1939-1950 Federal funding is being provided for this project by the National Institute ofAllergy and Infectious Diseases, National Institutes of Health, Department of Health and Human Services, under Contract No HHSN266200500008C 314 Retronectin Stimulation for the In Vitro Generation of Human Tumor-Reactive T Lymphocytes for Adoptive T Cell Transfer Therapy Seung Shin YU,I Erni Chatani, I Kazuyuki Dan, I Ikuei Nukaya, I Hideto Chono,' Junichi Mineno,' Ikunosin Kato.' 'Center for Cell and Gene Therapy; 'Biotechnology Research Laboratory, Takara Bio, Sela3-4-1, OISIl, Shiga, Japan Retronectin, chimeric peptide of human fibronectin has been widely used for Retroviral Gene Therapy protocols based on the observation that it significantly enhances retrovirus-mediated gene transfer to mammalian cells In this study,we report its potential use for Adoptive T cell Therapy for the treatment of cancer Previously we reported that peripheral blood T cells stimulated and expanded in vitro on immobilized Retronectin in the presence of anti-CD3 monoclonal antibody(OKT3) and IL-2, contain approximately two fold higher numbers of Naive T cells over T cells expanded Molecular Therapy Volume 15.Supplement Iã.\by:!007 Copyright â The Americ m Society {I t Gene Therapy ... supernatant to digest residual plasmid and host cell DNA A tangential flow ultrafiltration system is used to concentrate the c1arificd supernatant 10-20fold and to formulate the virus The concentrated!diafiltered... Development, Beckman Research Institute ofCity of Hope , Duarte, CA Lentiviral Vectorsare emerging as important tools in the field of gene therapy They have many advantages over other techniques that... Retroviral Gene Therapy protocols based on the observation that it significantly enhances retrovirus-mediated gene transfer to mammalian cells In this study,we report its potential use for Adoptive T