Replication Competent Viruses Testing at the Indiana University Vector Production Facility K. Cornetta, M.D.

Disclosures Director 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 Long-term Follow-up Master Cell Bank Patient Ex Vivo Transduced Cells Final Product And EOP Cells Biologic Assays ?? qPCR

Options for RCL Testing System Advantages Disadvantages Biologic assays Highly sensitive Less prone to false positives Take 4-6 weeks Expensive Molecular assays (ex. PCR) Moderately sensitive Inexpensive False positives Serologic assays False positive in vivo False negative in vivo

General Design of RCR Assay Amplification Phase (3 weeks) Indicator Phase Vector Virus MSV MLV S+/L- Plaque Assay

Cells used for amplification phase are envelope dependent Envelopes Receptors Amplification Ecotropic1 Murine not human NIH 3T3 Amphotropic2 Murine and human Mus dunni Xenotropic, GALV3, RD1144 Human not murine 293 cells 1Reeves et al. Human Gene Therapy 13:1783-1790, 2002. 2Cornetta et al. Human Gene Therapy 4, 579-588, 1993 3Chen et al. Human Gene Therapy 12:61-70, 2001. 4Duffy et al. Preclinica May/June:53-59, 2003.

Retroviral Production Methods Packaging Cell Line Well characterized line that allows sequential harvests Retroviral vectors generally not concentrated Most commonly use retroviral envelopes Derived from single cell clones to 100 vial MCB then expansion for production Expansion allows time for recombination and RCR development env gag/pol Vector

RCR Experience at the IU VPF 5 Master Cell Banks Failed Sterility / Mycoplasma None were generated in IU VPF 4 MCB or Final Products Failed due to RCR 2/2 + PA317 2/7 + GPE+Am12 > 17 PG13 have passed In the past 5 years failures due to: Rearrangement of Vector (2) Low 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 Junghans Robert Williams TOTAL 328

Move to Lentiviral Vectors Potential efficiency and safety profile. Present new challenges for RCL detection RCL has not been detected with current vectors RCL structure is not known Contribution of HERV sequences?

Lentiviral Production Methods Vector Gag/pol env Rev Transient No clone selection saving months in production time Concern of reproducibility Large plasmid requirements Product generally concentrated Less cell expansion which may decrease recombination frequency 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?

RCL Assay Design Amplify with C8166 cells Amplification Phase (3 weeks) Indicator Phase Vector RCL 7 days Assay by PCR and ELISA Amplify with C8166 cells Highly infectable Amplify to high titer

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

RCL Method and Performance Media 0.5 IU 0/3 + 1/3 + 1/5 + Initial Assay IP Negative Control Positive IP Positive Negative Indicator Phase Amplification Acceptance Criteria p24 PCR Performed 13 assays under GMP Amplification Phase virus detection Negative controls - 0/39 by p24, 0/30 psi-gag Positive control – 33/39 by p24, 30/30 by psi-gag Indicator Phase virus detection Negative controls – 0/36 by p24 and 1/33 by psi-gag Positive controls – 46/60 by p24 and 39/50 by psi-gag

RCL Method and Performance Media 5 IU 50 IU + Vector 0/3 + 1/3 + 2/3 + Amplification Phase Indicator Modified Assay Inhibition Control Negative Positive Acceptance Criteria p24 PCR Performed 17 assays under GMP Amplification Phase virus detection Negative controls – 0/54 by p24, 0/51 psi-gag Positive control – 54/54 by p24, 51/51 by psi-gag Inhibition controls All met acceptance criteria

RCL Summary Material generated in 6 different GMP facilities (20% generated at IU) 16 Vector Products 17 End-of-Production Cells 7 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 Forward Re-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 testing Currently 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%)

Larry Couture and David Hsu, City of Hope Phil Zoltick IU- VPF Lisa Duffy Daniela Bischof, PhD Troy Hawkins, PhD Clara Hazelgrove Sue Koop Jing Yao Lina Sego Mikhaila Douglas Alisha Auberry Aaron Ernstberger Aparna Jasti Lorraine Matheson Lilith Reeves Erol Cetinok Department of Medical and Molecular Genetics Collaborators Larry Couture and David Hsu, City of Hope Phil Zoltick Richard Morgan, Steve Feldman, and Steve Rosenberg, NIH, NCI Support by NHLBI, HHSN26820074820 and PO1 HL53586 (Dinauer) NCRR P40 RR024928 NCI N02-RC-67002 Lilly Endowment: Indiana Genomics Initiative