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Gianpietro Dotti G Center for Cell and Gene Therapy Baylor College of Medicine, Houston TX

Clinical Experience at Baylor College of Medicine with T Lymphocytes Genetically Modified Using Retroviral Vectors. Gianpietro Dotti G Center for Cell and Gene Therapy Baylor College of Medicine, Houston TX . Type of vector and packaging cell lines. Moloney-derived retroviral vector

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Gianpietro Dotti G Center for Cell and Gene Therapy Baylor College of Medicine, Houston TX

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  1. Clinical Experience at Baylor College of Medicine with T Lymphocytes Genetically Modified Using Retroviral Vectors Gianpietro Dotti G Center for Cell and Gene Therapy Baylor College of Medicine, Houston TX

  2. Type of vector and packaging cell lines • Moloney-derived retroviral vector • (SFG from Mulligan R.) • Packaging cell line Phoenix-Eco • original cells from ATCC • generation of a master cell bank under GMP conditions • Packaging cell line PG13 • original cells from ATCC • generation of a master cell bank under GMP conditions

  3. Generation of stable packaging cell line • Multiple infection of PG13 cells using Eco-pseudotyped retroviral particles (transient supernatant) • Generation of single cell clone from infected PG13 cells • Expansion of the single cell clone to make a master cell bank (MCB) • Production and collection of supernatant from the MCB

  4. Manufacturing • Manufacturing of the single cell clone under GLP conditions • Expansion of the master cell bank (MCB) and production of the retroviral supernatant under GMP conditions

  5. Packaging cell lines • 9 Packaging cell lines generated and tested • RCR detection negative in all cases for both Ecotropic and Galv envelopes (co-culture assay) • 18 lots of supernatant produced and tested • RCR detection negative in all cases for GalV envelope (co-culture assay)

  6. Clinical protocols IND Transgene Cell product Disease #11217 CAR.GD2z Activated T cells vs Neuroblastoma EBV-CTLs 19 patients treated. 38 T-cell/CTL products infused all negative by co-culture assay.

  7. Clinical protocols IND Transgene Cell product Disease #13296 CAR.CD19.CD28z vsActivated T cells CD19+ Lymphoma CAR.CD19z #13902 CAR.CD19.CD28z vs Activated T cells vs CD19+ Lymphoma CAR.CD19z EBV-CTLs #13958 CAR.CD19.28z Virus-specific CTLs CD19+ Lymphoma 8 patients treated (6 in # and 2 in #). 16 T-cell/CTL products infused. 6 negative by co-culture assay. 8 pending and 2 not needed (frozen within 4 days of culture).

  8. Clinical protocols IND Transgene Cell product Disease #14050 CAR.HER2.CD28z Activated T cells Osteosarcoma #14036 CAR.HER2.CD28z and EBV-CTLs Lung cancer TGFbRIIdcyt (DNRII) #14050 CAR.HER2.CD28.z CMV-specific CTLs Glioblastoma 4 patients treated (2 in #14050 and 2 in #14036). 4 T-cell/CTL products infused. 1 negative by co-culture assay. 3 pending.

  9. Clinical protocols IND Transgene Cell product Disease #12830 TGFbRIIdcyt (DNRII) LMP-2a-CTLs EBV+ Lymphoma 4 patients treated. 4 CTL products infused. All negative by co-culture assay.

  10. Clinical protocols IND Transgene Cell product Disease # 13813 icaapase9.2A.DCD19 allodepleted T cells hematological malignancies/haplo HSC transplant 4 patients treated. 4 T-cell products infused. All negative by co-culture assay.

  11. T/CTL line infused summary 92 lines infused to 73 patients 2 did not require assay 8 results pending 82 lines negative RCR co-culture assay for GalV envelope

  12. Follow up patient samples - 73 patients infused (follow up from 1 month to 15 years) - 371 samples analyzed - All RCR negative by Q-PCR (GalV envelope)

  13. Previous experience at St. Jude Children’s Research Hospital (Gene marking) • 34 patients infused with gene marked EBV-specific CTLs • All 34 CTL lines negative by co-culture assay • - 271 follow up samples all negative by PCR assay (samples continue to be archived from 2002 but no longer tested)

  14. Conclusions and Suggestions • T cell/CTLs modified with retroviral vectors are consistently RCR negative • RCR assay based on co-culture is time consuming and extremely expensive limiting the possibility to extend the use of promising immunotherapy approaches • Based on the current experience samples can be stored and analyzed for RCR contamination in case of severe adverse events

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