Replication Competent Viruses Testing at the

Indiana University Vector Production Facility

K. Cornetta, M.D.

DisclosuresDirector of the Indiana University Vector

Production which focuses on the production of Retroviral and Lentiviral Vectors (Phase I/II) for academic investigators

Co-Founder of Rimedion

Own Stock in Amgen and Starbucks

Research and contract funding through the NIH and USDA

Subcontract on an SBIR awarded to Maxcyte Inc.

Timing of RCR/RCL Testing

Master Cell Bank Final Product

AndEOP Cells

Patient Ex Vivo Transduced Cells

Long-termFollow-up

Biologic Assays qPCR??

Tes

tin

g

Options for RCL TestingOptions for RCL Testing

System Advantages Disadvantages

Biologic assaysBiologic assays Highly sensitiveHighly sensitive

Less prone to false Less prone to false positivespositives

Take 4-6 weeksTake 4-6 weeks

ExpensiveExpensive

Molecular assays (ex. Molecular assays (ex. PCR)PCR)

Moderately sensitiveModerately sensitive

InexpensiveInexpensive

False positivesFalse positives

Serologic assaysSerologic assays Moderately sensitiveModerately sensitive False positive in vivo False positive in vivo

False negative in vivoFalse negative in vivo

Am

plification P

hase (3 weeks)

Indicator P

hase

VectorVirus

MSVMSVMLV

S+/L- Plaque Assay

General Design of RCR Assay

Cells used for amplification phase Cells used for amplification phase are envelope dependentare envelope dependent

Envelopes Receptors Amplification

EcotropicEcotropic11 Murine not Murine not humanhuman

NIH 3T3NIH 3T3

AmphotropicAmphotropic22 Murine andMurine and

humanhumanMus dunniMus dunni

Xenotropic, Xenotropic, GALVGALV33, RD114, RD11444

Human not Human not murinemurine

293 cells293 cells

11Reeves et al. Human Gene Therapy 13:1783-1790, 2002.Reeves et al. Human Gene Therapy 13:1783-1790, 2002.22Cornetta et al. Human Gene Therapy 4, 579-588, 1993Cornetta et al. Human Gene Therapy 4, 579-588, 199333Chen et al. Human Gene Therapy 12:61-70, 2001.Chen et al. Human Gene Therapy 12:61-70, 2001.44Duffy et al. Preclinica May/June:53-59, 2003.Duffy et al. Preclinica May/June:53-59, 2003.

Retroviral Production Methods

Packaging Cell LinePackaging Cell Line Well characterized line Well characterized line

that allows sequential that allows sequential harvestsharvests

Retroviral vectors Retroviral vectors generally not generally not concentratedconcentrated

Most commonly use Most commonly use retroviral envelopesretroviral envelopes

Derived from single cell Derived from single cell clones to 100 vial MCB clones to 100 vial MCB then expansion for then expansion for productionproduction

Expansion allows time Expansion allows time for recombination and for recombination and RCR developmentRCR development

Vector

env

gag/pol

RCR Experience at the IU VPFRCR Experience at the IU VPF

5 Master Cell Banks Failed Sterility / 5 Master Cell Banks Failed Sterility / MycoplasmaMycoplasma

None were generated in IU VPFNone were generated in IU VPF

4 MCB or Final Products Failed due to 4 MCB or Final Products Failed due to RCRRCR

2/2 + PA3172/2 + PA3172/7 + GPE+Am122/7 + GPE+Am12> 17 PG13 have passed> 17 PG13 have passed

In the past 5 years failures due to:In the past 5 years failures due to:Rearrangement of Vector (2) Rearrangement of Vector (2) Low titer from new producer cell lineLow titer from new producer cell line

National Gene Vector Laboratory Program

Repository of gene therapy reagents

Houses a searchable database of gene therapy Pharm/Tox studies

Archives GLP, GMP or patient samples so investigators can comply with FDA requirements

Performs insertion site analysis by LM/LAM-PCR

Performs RCR or RCL testing by qPCR to comply with post-trial monitoring requirements

Post-trial Monitoring for RCR

Investigator Institution Number Samples

Kiem FHCRC 6

Sadelain MSSK 9

Brenner Baylor College 94

Rosenberg NCI 163

Kang NIH 1

Kohn/Condotti UCLA/NHGRI 20

Malek Cincinnati Childrens 4

Gray St Jude Childrens 2

Ribas UCLA 9

Junghans Robert Williams 20

TOTAL 328

Move to Lentiviral VectorsMove to Lentiviral Vectors

Potential efficiency and safety profile.

Present new challenges for RCL detection• RCL has not been detected with current vectorsRCL has not been detected with current vectors

• RCL structure is not knownRCL structure is not known

• Contribution of HERV sequences?Contribution of HERV sequences?

Lentiviral Production MethodsLentiviral Production Methods

TransientTransient No clone selection No clone selection

saving months in saving months in production timeproduction time

Concern of Concern of reproducibility reproducibility

Large plasmid Large plasmid requirementsrequirements

Product generally Product generally concentratedconcentrated

Less cell expansion Less cell expansion which may decrease which may decrease recombination recombination frequencyfrequency

Vector

Gag/pol

env

Rev

HEK293T cells

The challenge of VSV-G envelope

• VSV-G env causes cell fusion

• Limits the number of end-of-production cells

• Are the EOP cells relevant?

Amplify with C8166 cellsHighly

infectableAmplify to

high titer

Am

plification P

hase (3 weeks)

Indicator P

hase

VectorRCL

7 days

Assay by PCR and ELISA

RCL Assay Design

Rational for Indicator Phase

The kinetics of a RCL is currently unknown

Rational for Indicator Phase

Potential to transfer sequences without true RCL

Sastry et al. Mol Therapy 8: 830-839, 2003

Cell to Vector Ratio based in part on vector toxicity

For RCL assay we dilute vector to a concentration of 1000 ng/mL

Ratio of 5 x 106 C8166 cells per mL of test article

Purification may improve toxicity profile

Currently challenging when testing vectors > 20 liter scale

RCL Testing of Anti-HIV VectorRCL Testing of Anti-HIV Vector

rHIV7-shI-TAR-CCR5RZ vector DiGiusto, D.L. et al. Sci. Transl. Med. 2, 36-43 (2010).

RCL Method and Performance

Performed 13 assays under GMP Amplification Phase virus detection

Negative controls - 0/39 by p24, 0/30 psi-gagPositive control – 33/39 by p24, 30/30 by psi-gag

Indicator Phase virus detectionNegative controls – 0/36 by p24 and 1/33 by psi-gagPositive controls – 46/60 by p24 and 39/50 by psi-gag

Acceptance Criteria

p24 PCRMedia

Media

0.5 IU

0.5 IU

0/3 +

1/3 +

0/3 +

1/5 +

Init

ial

Ass

ay

0/3 +

1/3 +

0/3 +

1/5 +

IP NegativeControl

PositiveControl

IP PositiveControl

NegativeControl

IndicatorPhase

AmplificationPhase

RCL Method and Performance

Media

5 IU

50 IU + Vector

0/3 +

1/3 +

2/3 +

AmplificationPhase

IndicatorPhaseM

od

ifie

d A

ssay

0/3 +

1/3 +

2/3 +

InhibitionControl

NegativeControl

PositiveControl

Performed 17 assays under GMPAmplification Phase virus detection

Negative controls – 0/54 by p24, 0/51 psi-gagPositive control – 54/54 by p24, 51/51 by psi-gag

Inhibition controlsAll met acceptance criteria

Acceptance Criteria

p24 PCR

RCL SummaryMaterial generated in 6 different GMP

facilities (20% generated at IU)16 Vector Products17 End-of-Production Cells7 cell lines

Analyzed 1.12 x 107 ng of p24 (1.3 x 1014 virions)1.8 x 109 cells

No evidence of RCL

RCL Assay Moving ForwardRe-evaluate the toxicity as product is

purified

Can we decrease the cell to vector ratio and maintain sensitivity?

Should the procedure be different for anti-HIV-1 vectors?

Validating alternative envelopes.

Is p24 sufficient / is psi-gag needed?

Transgene effects?

Still at the point of qualifying RCL assay on a case by case basis

Detecting RCL in infused product

PCR is likely to give false positive

Biologic assay take 6 weeks and is expensive

Amplification kinetics of primary cells unknown

How much is gained?Consider about 90% of vector available after

testingCurrently testing 5% of final product for RCL If you used the entire lot in a single patient and

tested 1% of transduced cells you are adding 0.9% of final product analyzed (testing 5.9%)

CollaboratorsCollaboratorsLarry Couture and David Larry Couture and David

Hsu, City of HopeHsu, City of HopePhil ZoltickPhil ZoltickRichard Morgan, Steve Richard Morgan, Steve

Feldman, and Steve Feldman, and Steve Rosenberg, NIH, NCIRosenberg, NIH, NCI

IU- VPFIU- VPFLisa DuffyLisa DuffyDaniela Bischof, PhDDaniela Bischof, PhDTroy Hawkins, PhDTroy Hawkins, PhDClara HazelgroveClara HazelgroveSue KoopSue KoopJing YaoJing YaoLina SegoLina SegoMikhaila DouglasMikhaila DouglasAlisha AuberryAlisha AuberryAaron ErnstbergerAaron ErnstbergerAparna JastiAparna Jasti

Lorraine MathesonLorraine MathesonLilith ReevesLilith ReevesErol CetinokErol Cetinok

Support byNHLBI, HHSN26820074820 and PO1 HL53586 (Dinauer) NCRR P40 RR024928NCI N02-RC-67002Lilly Endowment: Indiana Genomics Initiative

Department ofMedical and

Molecular Genetics