NCI experience using engineered T cells for the treatment of cancer (Implication for RCR testing)

1. NCI experience using engineered T cells for the treatment of cancer(Implication for RCR testing) Richard A. Morgan, Ph.D. Surgery Branch National Cancer Institute

2. 20 Years of Clinical Gene Therapy at NCI Since this initial report, >200 patients have been treated at NCI with transduced lymphocytes

3. Development of gene therapy as an alternative to TIL therapy

4. SB Clinical Gammaretroviral Product Summary 14 products total.

5. Surgery Branch Vector Production Facility (SBVPF) Product Summary

6. Production Process - MCB MSGV1 backbone: Combination of MSCV+SFG Hughes et al., Hum Gene Ther 2005; 16:457--‐472. Clone Selection (RNA Dot Blot) Transduction Transient Transfection PG13 Phoenix-Eco • Select 6 best clones • Functional evaluation • Select final clone • Produce master cell bank

7. Production Process - Retroviral Vector SBVPF Preclinical Vector Development 25 x 1700cm2 Roller bottles Generate PG13 MCB Cell Thaw 120/240 ml Upstream Cell Expansion Seed Roller Bottles Medium Exchange/Harvest Clarification Downstream Fill Modified from: Reeves and Cornetta, Gene Ther. 23:1993-8, 2000

8. SBVPF Product Biosafety Testing: Product RCR testing:

9. Active Surgery Branch Gene Therapy Protocols: * * * * New protocols

10. Procedure for TCR Gene Therapy Protocols Carry out patient pheresis to obtain PBMC Activate T cells using anti-CD3 antibody for two days Transduce with gammaretroviral vectors using RetroNectin-coated plates Pre-condition patients with cyclophosphamide and fludarabine Administered T cells with IL-2 to tolerance +/- Vaccine Post-treatment----Test/archive samples for further RCR testing RCR-PCR

11. NY-ESO-1 TCR gene therapy: Tumor regression in melanoma and synovial cell sarcoma patients * No on or off target toxicities have been observed that could be attributed to TCR engineered cells. Paul F. Robbins, et al., In press, Journal of Clinical Oncology

12. Summary: clinical findings: • Both TCR and CAR gene therapy targeting multiple tumor antigens can lead to cancer regression in humans (melanoma, colorectal cancer, synovial cell sarcoma, and lymphoma). • Targeting self-antigens also leads to on-target recognition of antigen expressing normal cells and mild to severe toxicity.

13. RCR testing implications(I) • A total of 144 patients have been treated on active INDs. The total number of all current and former patient treatments is 231. • All 231 patient treatments have been tested by PCR-based lot release assays for RCR and all 231 patient treatment products were negative. 22 patient treatments were tested by cell-based RCR assays and all were negative. All remaining patient samples have been archived for the purposes of future testing if necessary

14. RCR testing implications(II) • Issues with cell-based RCR testing: • It is difficult and unwise to perform a cell culture based RCR “in house” because it requires an infectious retrovirus control, which could lead to contamination.  Therefore, the assay must be contracted to an outside vendor.  • The test is extremely expensive (>$3000/sample), and contractors capable of performing the assay are scarce.    The government procurement procedures for outside contractors are complex, involved, and protracted. • Achieving samples is also an onerous prospect in particular when long-term storage is required. 

15. RCR testing implications(III) • Why RCR testing at all? • Current gamma-retroviral vector backbones in combination with split-gene packaging cell lines do not generate RCR. Furthermore, each and every retroviral product and master cell bank is tested for RCR using cell based assays before it can be released for patient use. It is highly unlikely that RCR (which is a theoretical risk to begin with) would escape detection in this extremely thorough pre-transduction testing. So why test for something that is not there to begin with????

16. RCR testing implications(IV) • Recommendations: • The simplest solution is to eliminate patient-specific cell-based RCR testing.  The previously certified vectors and processes are theoretically extremely unlikely to generate RCR (or RCL) and other patient safety issues vastly supersede the effort and expenses spent on this issue.  •  An alternative would be to archive all samples, and test later only if a problem were suspected for clinical or other reasons.  The EU model currently does not require cell-based RCR testing of transduced patient.

17. Acknowledgments: Morgan Lab Steven A. Rosenberg, M.D., Ph.D. Chief, Surgery Branch NCI Surgery Branch Colleagues: Mark Dudley Steve Feldman Carolyn Laurencot John Wunderlich Paul Robbins James Yang Nick Restifo Jim Kochenderfer Maria Parkhurst TIL LAB FACS LAB Clinical Staff Vector production: Kenneth Cornetta Indiana University Takara Bio for supplying clinical grade RetroNectin