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Development of a cGMP-compliant process to manufacture donor-derived, CD45RA-depleted memory CD19-CAR T cells

Gene Therapy volume 30, pages 222–231 (2023)Cite this article

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Abstract

Autologous chimeric antigen receptor (CAR) T cells targeting the CD19 antigen have demonstrated a high complete response rate in relapsed/refractory B-cell malignancies. However, autologous CAR T cell therapy is not an option for all patients. Here we optimized conditions for clinical-grade manufacturing of allogeneic CD19-CAR T cells using CD45RA-depleted donor memory T cells (Tm) for a planned clinical trial. Tm were activated using the MACS GMP T Cell TransAct reagent and transduced in the presence of LentiBOOST with a clinical-grade lentiviral vector that encodes a 2nd generation CD19-CAR with a 41BB.zeta endodomain. Transduced T cells were transferred to a G-Rex cell culture device for expansion and harvested on day 7 or 8 for cryopreservation. The resulting CD19-CAR(Mem) T cells expanded on average 34.2-fold, and mean CAR expression was 45.5%. The majority of T cells were CD4+ and had a central memory or effector memory phenotype, and retained viral specificity. CD19-CAR(Mem) T cells recognized and killed CD19-positive target cells in vitro and had potent antitumor activity in an ALL xenograft model. Thus we have successfully developed a current good manufacturing practice-compliant process to manufacture donor-derived CD19-CAR(Mem) T cells. Our manufacturing process could be readily adapted for CAR(Mem) T cells targeting other antigens.

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Fig. 1: Efficient depletion of CD45RA+ and CD14+ cells from cryopreserved apheresis MNCs.
Fig. 2: Optimization of transduction of memory T cells with clinical-grade lentiviral CD19-CAR vector.
Fig. 3: Optimizing cell seeding number in G-Rex device for cell expansion.
Fig. 4: CD19-CAR(Mem) T cells maintain virus specificity post-expansion.
Fig. 5: In vitro functional characterization of CD19-CAR(Mem) T cells.
Fig. 6: CD19-CAR(Mem) and standard CD19-CAR T cells have comparable antitumor activity in vivo.

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Acknowledgements

We thank Suzette Whittaker, Rebecca Banks-Spivey, Jeeba Bellot, Amanda Burton, and Madhuri Kalathur for assistance with engineering runs. We thank Sarah Schell and MaCal Tuggle-Brown for performing Elispot assays. The work was supported by Cookies for Kids’ Cancer and the American Lebanese Syrian Associated Charites. AT received additional support from the American Society of Transplantation and Cellular Therapy (ASTCT) New Investigator Award. Animal imaging was performed by the Center for In Vivo Imaging and Therapeutics, which is supported in part by NIH grants P01CA096832 and R50CA211481. Cellular images were acquired at St. Jude Cell & Tissue Imaging Center, which is supported by St. Jude and NCI P30 CA021765.

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Authors and Affiliations

  1. Experimental Cellular Therapeutics Laboratory, St. Jude Children’s Research Hospital, Memphis, TN, 38105, USA

    Young-In Kim-Hoehamer, Fei Zheng, Jeoungeun J. Park, Na Shang & Sheng Zhou

  2. Department of Bone Marrow Transplantation and Cellular Therapy, St. Jude Children’s Research Hospital, Memphis, TN, 38105, USA

    Janice M. Riberdy, Jean-Yves Métais, Jennifer Moore, M. Paulina Velasquez, Brandon M. Triplett, Aimee C. Talleur & Stephen Gottschalk

  3. Therapeutics Production and Quality, St. Jude Children’s Research Hospital, Memphis, TN, 38105, USA

    Timothy Lockey & Michael M. Meagher

  4. Children’s GMP, LLC, Memphis, TN, 38105, USA

    Catherine Willis

  5. Human Applications Laboratory, St. Jude Children’s Research Hospital, Memphis, TN, 38105, USA

    Salem Akel

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Contributions

MMM, MPV, BMT, ACT, SG and SZ conceived the study, interpreted the data, and reviewed the manuscript. YK, JMR, FZ, JJP, NS, JYM, TL, CW, SA and J.M. conducted the research, analyzed, and summarized the data. YK, JMR, JYM, SG, and SZ wrote the manuscript.

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Correspondence to Stephen Gottschalk or Sheng Zhou.

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Competing interests

SG has a research collaboration with TESSA Therapeutics, is a DSMB member of Immatics, and is on the scientific advisory board of Tidal. BMT had travel support from Miltenyi Biotec.

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Kim-Hoehamer, YI., Riberdy, J.M., Zheng, F. et al. Development of a cGMP-compliant process to manufacture donor-derived, CD45RA-depleted memory CD19-CAR T cells. Gene Ther 30, 222–231 (2023). https://doi.org/10.1038/s41434-021-00307-0

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