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 5 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 hoursoften have a 12 hour titer that

ex-ceeds 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 5 year period

Aliquots of the LNL6 vector (expressing neo) and the Ampho/GFP

and GALVIGFPvectors (expressing enhance greenf1uorescent

pro-tein,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,' RossJ.Mclvlahon,' Valerie R Quezada,' David

Hsu,' Larry A.Couture.'

'Center for Biomedicine and G enetics 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

genera-tion of plasmid bearingE colibiomass 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

through-put due to the ease of set up and tear down.Twolentivirus helper

plasmids,which had previously been produced in the facility using

a cGMP stirred tank procedure, were used to develop the wave

fer-mentation process Ferfer-mentations 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 practicalmaxima The mean

specific yield (mg of plasmid Ig wet weight) obtained from the

wave bioreactor was found to range from equivalent to 3 fold higher

than that obtained from the same bacterial master cell bank in the

stirred tank fermentor while the bacterial yield (g wet weightIliter

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

assess-ment The resulting improvement in specific yield ofthe optimized

process is approximately 600% greater than that obtained with the

stirred tank system

Molecular Therapy Volume15 SupplementI ~ br 2 007

Copyright © The American So ci etyo 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; WilliamF.Goins; David M Krisky,"Mohammad M.Ataai,'Joseph C Glorioso.'

f Molecular Genetics and Biochemistry University ofPittsburgh Pittsburgh PA;]Dental Medicine, University ofPittsburgh Pitts-burgh, PA; 'Chemical and Petroleum Engineering Universityof Pittsburgh Pittsburgh, PA; "Department ofPathology ; University ofPittsburgh Pittsburgh PA.

As the number of clinical and research applications of herpes-virus-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 quanti-fication 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-timequantitative 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 virologi-cal 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 4 to 4x 103HSV 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 micro-plate 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 0.5ng 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,'TonyEncinas.'Yclena Skripchcnko.! James Rogers,'ReubenCohen,'VladimirSlepushkin,' Laurent Hu-meau.'

f Researchand Development VIRxSYS Corporation Gaithers-burg AID; l Operations 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 lenti-viral vector, VRX496, carrying a 937-base antisense targeting the HIV envelope.Aftera 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 optimaltherapeutic dose A large scale cGMP cell process was developed and has been presented (Humeau et aI.,ASGT 2006) Briefly, the positively

se-SI17

lected CD4+T cells from 35 HIV infected patients were genetically

modified with VRX496 and expanded in a Wave™ bioreactor toa

final cell number averaging 84 billion, with expansion up to 300

fold in some cases However,in order toreachsuch cellnumbers,

expansionshave been performedin a 50L perfusionbag (25Lculture

volume) with a cell concentration averaging4.3million per ml and

yielding excellent viabilities in the range of 92% In preparation

of our phaseIIIclinical trial, we sought to reduce culture volumes

from25Lto IOL while retainingrobustexpansionsand excellentcell

viabilities Cell processingdevelopment runs conductedin 20L

Wa-vc" perfusion bags (IOL culture volume) lead to final cellnumber

averaging 134billion.More importantly,the averagemaximumcell

concentration increased up to 23 million cells per ml with excellent

viabilitiesin the 95% range.Thus we have beenableto 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 tostreamliningand optimizing the cell process for the mass

production of our VRX496 therapy

in a GMP Facilty

Lara J Ausubcl,IKen A Laderman,IMisty ShakeIcy,IAnupriya

Sharma,ISylvana Couture,IPatricia Lopez,IChristine

Knob-lauch,' Jonathan Anderson,' IvyDerecho,' Ross McMahon,1

David Hsu,ILarry Couture.'

'Center for Biomedicine and Gen etics, Center for Applied

Lentiviral Vectorsare emerging as important tools in the field of

gene therapy.They have many advantagesover 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 transienttransfectionof293T cells basedon its ability to support

high titer vector productionand itsconsistency in vector production

over time in culture A Lentiviral vector packaging system using4

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 addressingsafety concerns by minimizing the

probabilityofthe generationofreplicationcompetent virus In order

to produce largequantitiesofhigh titer virus under GMPconditions,

we have optimizedseveral parametersofthe manufacturingprocess

These include conditions for cell growth and adherence,

transfec-tion,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

Benzo-nase is added to the clarified supernatant to digest residual plasmid

and host cell DNA.A tangentialflow 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 109TUlmL, when titered on HTI 080 cells

Our multi-sub-batch system provides for virtually unlimitedscale

up capacity

SII8

Proteins That Inhibit Viral Propagation Gina Nichols,' Tony Stepan" Kristin Parker" Eric J Pastor" Richard W.Peluso" BarbaraA Thorne.'

F Process Development, Targeted Genetics, Seattle, W:4.

For adeno-associated viral vectors to fulfill their promise as gene therapy delivery vehicles,scaleableproduction methods are required The componentsrequired for rAAV production include cells, the AAVrep andcapgenes,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 contain-ing the vector genome, are used to co-infect a packagcontain-ing 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 arerequiredas critical raw materials,including a packagingcell line bank,Ad5,

Ad!AAV-hybrid stocksand cells to producethem.This study focuses

on the generationofthe Ad!AAV-hybrid stocks.The necessarysteps 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 expressingthe 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, regulationof 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·lumanGene 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

Vitro Generation of Human Tumor-Reactive

T Lymphocytes for Adoptive T Cell Transfer Therapy

Seung Shin YU,IErniChatani,I Kazuyuki Dan,IIkueiNukaya,I Hideto Chono,' Junichi Mineno,'IkunosinKato.'

'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 ofcancer.Previously

we reported that peripheral blood T cells stimulated and expanded

monoclonal antibody(OKT3) and IL-2, contain approximately two fold higher numbers of Naive T cells over Tcells expanded

Molecul ar Therapy Volume 15 S upplement I•.\by:!007

C opyright © The Am eric m Society {I t Gene Therapy