1. Findings and Challenges in Product Testing and Patient Monitoring for RCR/L in Gene Therapy Clinical Trials
Gwendolyn Binder-Scholl, Ph.D.
University of Pennsylvania
Translational Research Program

2. Overview Challenges of Biologic RCR/L Testing on Cell Products
Summary of Retroviral Gene Therapy Trials in the TRP
Detailed Testing Summaries for each Trial
Discussion Points

3. Challenges biologic RCR/L testing on cell products
Timing
Increases enrollment-infusion time ~4-6 weeks
(an alternative rapid method such as PCR may be used to provide an initial analysis when transduced patient cells must be administered immediately. But a culture based RCR/L assay should be initiated for retrospective confirmation).
Cost
cell manufacture ~$15K-$20K
vector costs ~$15-30K per patient
biological RCL testing is ~$22K
Risk
Theoretical and never observed
Risk is theoretical and never observed with manufacturing methods used for clinical trials to date

4. RCR/L Testing Summary
No RCR/L detected: 12 vector and 34 cell lots; 31 patients
3 different vector types and production systems: MLV, crHIV, sinHIV
Up to 9 years follow-up in vivo
IND
Year open
Vector type
Transgene
Production Method
RCR / RCL Vector
RCR / RCL in Cells
RCR / RCL in vivo
Years follow-up
8568
1999
MLV
CD4-ζ CIR
Producer cells1
0/3
0/11
0/10
5-9yr
12799
2006
cr-HIV
HIV env antisense
Transient transfection2
0/7
0/20
0/18
1-4 yr
13960
2009
SIN-HIV
CD19-CIR
Transient transfection3
0/2
<1
14122
High affinity gag-TCR
0/1
pending
For 8568 there were parallel trials Sponsored by Cell Genesys 44 patients up to 10 year follow-up
For there were additional trials sponsored by VIRxSYS as summarized by Dr. Humeau, including the first 5 lv vector treated patients which were treated at Penn and are up to 7 years follow-up
1 PA317 cell line unpublished
2Lu et al, J. Gene Med., 2004
3Zufferey et al, Nat. Biotech., 1997

5. RCR Testing in IND 8568 (MLV)
Clinical indication: HIV; CD4-zeta CIR
Target cells: CD4 and CD8 T cells
Vector and Cell product: Supernatant and cells cocultured with M. dunni cells
-approximately 1% of total cell product/EOP and 300ml supernatant cocultured with M. dunni cells
-+ control ( ffu amphotrophic MLV)
-5 passages cell product (~3 weeks); 2 passages sup (~1-1.5 week)
- test in feline PG-4 S+L- focus forming assay
Patient Monitoring:
-PCR amplification of a 70 bp region of the amphotrophic envelope in PBMCs
-baseline, ~ quarterly to 1 year, and annually thereafter (protocol later modified to reflect current guidance on monitoring for delayed adverse events)
Now am going to talk about specific RCR/RCL testing under specific TRP sponsored INDs

6. RCL Testing in IND 12799 (crHIV)
Clinical indication: HIV; env antisense
Target Cells: CD4 T cells
Vector product: 5% final vector fill and 1% EOP cells at VIRxSYS Corporation in C cells
-attenuated HIV positive control (Schonely et al, Bioprocessing J, 2003)
Cell product: (no biologic RCL test)
-PCR based assay for VSV-G envelope
-PCR based assay monitoring HIV gag pre and post expansion
Patient Monitoring:
-PCR amplification for VSV-G envelope in PBMCs at a minimum quarterly and then annually (if negative at year 1, then archived for look back)
IND cross referenced VIRxSYS IND-presented by Dr. Humeau, for RCL testing.
Final fill was tested instead of vector supernatant because crude supernatants were found to be inhibitory to RCL detection-as published
Strategy for PCR assay was developed in close proprietary consultation with the FDA. Generally because VIRxSYS submitted data showing that testing of the cell product was not sufficiently sensitive to detect an RCL.

7. RCL Testing in IND 14122 (SIN-HIV)
Clinical indication: HIV; HIV gag CTLs
Target cells: CD8 T cells
Vector product: 300ml sup and 1% EOP cells at Indiana University using the C cell line
-R8.71 VSV-G pseudotyped attenuated HIV positive control
Cell product: (no biologic RCL)
-PCR based assay for VSV-G envelope
Patient Monitoring:
-PCR amplification for VSV-G envelope in PBMCs at a minimum quarterly and then annually (if negative at year 1, then archived for look back)
In close consultation with the FDA, we developed a plan for RCL release testing on the cell product that involved only molecular based assays.

8. Molecular RCL Testing for Cell Product Release 14122 case study
+vector
+ARV
High affinity HIV gag TCR targeting SL9 epitope expressed by a SIN–HIV vector
CD8
20% CD4
Harvest
Day: 3
9-12
RCL testing
VSV-G DNA
In consulation with FDA, determined no biologic RCL necessary:
ARV in media, no RCL amplification
CD4 T cells added after vector washout
Retroviruses are not stable at 37°C, losing 90% of titer each 48 hours (Higashikawa and Chang, Virology, 2001). Without amplification, residual RCL from the vector reduced 10,000 fold in 8 day culture. Fewer than 250 IU RCL are added to culture based on the LOD of the vector RCL assay.
20% CD4 T cells and 20% v/v conditioned media
Post gag is not statistically great than pre (unique situation since final product is typically <5% CD4 T cells)
CD8 T cells are enriched by elutriation and negative selection
Concentration of ARV is 1ug/ml of AZT and 500 nM ritonavir

9. Molecular RCL Testing on Cell Products 14122 case study-PCR sensitivity
Testing 1% of cell product not feasible.
The # of cells required to have 95% confidence that the sample is negative =
10,000 cells / 0.06 µg DNA
Established test evaluates 450,000 cells / 3 µg DNA
Adapt p=1-exp(-cVt) : the probability of not detecting at least 1 RCR
(FDA 2006 Supplemental Guidance on RCR testing)
We see a 1/2 log reduction in sensitivity of spike recovery if spike is performed pre isolation. May be different if td cell line used. Given the number of cells in the final cell product, we know there are fewer than 6666 copies of VSV-G in the cell culture

10. Molecular RCL Testing on Cell Products 14122 case study-PCR sensitivity
Vt=-(1/c)ln(1-p)
Current Guidance
-Vt is the volume
-c is the 1 IU/100ml
-p is 95% confidence
Adapted for DNA testing
-Vt is the ug of DNA (# cells)
-c is 50 copies / ug genomic DNA
(1:3000 cells)
-p is 95% confidence
Assay can detect ≤ 50 VSV-G copies / µg genomic DNA: Vt=-(1/50)ln(1-0.95)
-FDA Guidance
-Feasible LOD for a PCR assay
-Empirically determined through spike and recovery studies
This approach was discussed with and approved by the FDA. A potential discussion point for the committee may be the biological relevance of this method and it’s usefulness for a broader application across retroviral vector based cell therapies.

11. RCL Testing in IND 13960 (SIN-HIV)
Clinical indication: CD19+ leukemia and lymphomas; CD19-CAR
Target cells: CD4 and CD8 T cells
Vector product: 300ml sup and 1% EOP cells at Indiana University using the C cell line
Cell product:
-p24 in T cell culture
-PCR based assay for VSV-G envelope and HIV gag
-Coculture of final product (1% or 1e8 cells) with C cells in method used in Escarpe et al, Mol Ther., 2006)
-R8.71 positive control ~100 IU
Patient Monitoring:
-PCR amplification for HIV-gag in PBMCs at a minimum quarterly and then annually (if negative at year 1, then archived for look back)

12. Molecular RCL Testing on Cell Products IND 13960 Case Study
+vector
CD19-41BB-ζ CAR, expressed by a SIN–HIV vector
CD4/8
Harvest
Day: 3
9-12
RCL testing
HIV gag DNA
P24
VSV-G DNA
Co-culture with C8166 cells
Biological RCL assay because:
Antiretrovirals are not included in the culture media,
Spiking studies with R8.71 showed that 100 IU RCL could be carried through to the end of culture
-not detectable by end of culture p24
-not detectable by amplification of end of culture supernatant with C8166
-detectable by co-culture with C8166 cells
The amount of vector used could theoretically contain >500 IU RCL given a LOD of 5 IU/ml from the vector RCL assay
Cells expanded post vector wash out in a static system or in a perfusion bioreactor which allows for greater exchange of media to remove residual vector proteins and DNA.

13. Molecular RCL Testing on Cell Products IND 13960 Case Study-Residual vector protein/DNA
Patient 1
Patient 2
Patient 3
Cell population
doubling
Negative
HIVgag copies/µg
Media exchange is proportional to the amount of cell expansion. Patients 1 and 3 underwent expansion in a perfusion bioreactor, but patient 2 did not. Mention here the residual testing, and cite that the LV vector titer was low and so a large volume (~110 ml) had to be added. Old vector process only used TFF-new process now available that uses anionic exchange followed by TFF and size exclusion chromatography.
p24 pg/ml
Culture day

14. Discussion Points
Not all cell manufacturing processes are equal. When might biologic RCL testing for cell products be informative?:
-when using vectors with high levels of residual packaging DNA/protein
-addition of high amounts of vector (low titer)
Biological RCL testing on cell products is a major burden. Potential solutions are:
a. Use molecular approaches for RCR/L testing on cell products-in which cases would this be acceptable?
b. Develop a funding initiative to defray the costs associated with amplification based RCR/L testing, or,
c. Establish discounted services to perform amplification based RCL testing (such as the NGVL initiative)
FDA Guidance for RCR/L annual archive could follow current guidance for monitoring for delayed adverse events. If gene modified cells no longer detected at 5 year, no RCR/L sample collection for archive should be required.
In what cases would molecular testing be acceptable?

15. Acknowledgements
University of Pennsylvania City of Hope, Center for Biomedicine & Genetics
Carl June Larry Couture
Bruce Levine David Hsu
Zoe Zheng
Cell Genesys NIH, NIAID
Kristen Hege Sarah Read
Mitch Finer Frosso Voulgaropoulou
Sandra Bridges
VIRxSYS U19 AI066290
Gerard McGarrity U19 AI082628
Laurent Humeau
Alliance for Cancer Gene Therapy
Lentigen
Boro Dropulic