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320 CAR-T cell manufacturing from fresh whole blood facilitates point-of-care therapeutic cell production
  1. Ying Xiong,
  2. Yanping Xie,
  3. Yang Liu,
  4. Matthew Addington-Hall,
  5. Beatrix Ferencz,
  6. Tony Luo,
  7. Maggie Yates,
  8. Oxana Slessareva,
  9. Ibeawuchi Oparaocha,
  10. Zhongyu Zhu,
  11. Boro Dropulić and
  12. Rimas J Orentas
  1. Caring Cross, Gaithersburg, MD, USA
  • Journal for ImmunoTherapy of Cancer (JITC) preprint. The copyright holder for this preprint are the authors/funders, who have granted JITC permission to display the preprint. All rights reserved. No reuse allowed without permission.


Background Chimeric antigen receptor modified (CAR-T) cell therapy has shown tremendous clinical efficacy for hematological malignancies,1 2 and limited efficacy in solid tumor therapy.3 4 However, bottlenecks in the ability to manufacture and deliver these engineered cell products, combined with their high cost, have limited patient accessibility of CAR T therapy, especially those in underserved areas or who are uninsured. Here, we describe the development of a flexible, cost-effective CAR T manufacturing process starting with fresh whole blood that generates sufficient CAR T cells numbers with excellent functionality.

Methods and Results For T cell Enrichment, a Sepax C-Pro (Cytiva, Inc.) was used to reduce 450ml fresh whole blood volume to 100ml. Concentrated cells were labeled for negative T cell enrichment (RosetteSep, Stemcell Research, Inc.), then fractionated on the Sepax C-Pro by density gradient centrifugation. Purified T cells were harvested, washed, and analyzed by flow cytometry. A cell product with >85% T cell purity, and 200x106 total T cells was routinely recovered. For large-scale T cell transduction with CAR LV expression vector, 1x 108 T cells were suspended in 100ml TexMACSTM medium (Miltenyi Biotec) with 200 IU/ml IL-2 and transferred to a G-Rex cell culture device (Wilson-Wolf), followed by addition of T cell TransActTM reagent and HIV gp120 DuoCAR lenti-vector5 (MOI 40). On Day 2, a complete medium exchange was carried out, cells were re-suspended in 400ml of fresh medium with 200 IU/ml IL-2, and returned to the same G-Rex. The cells were cultured in a tissue culture incubator to Day 8, when the final cell product was harvested. On days 3, 6 and 8, cultures were sampled to monitor T cell growth, phenotype, CAR expression, and function. Evaluating 4 healthy donors, at least 5x108 CAR-T cells could be harvested on Day 6, with ~90% cell viability. Day 6 CAR-T showed similar transduction efficiency and increased Tscm% and Tcm% compared to the standard Day 8 manufacturing time frame.5 6 Comparable target leukemia cell cytolysis was also observed.

Conclusions This large-scale CAR T production process can: 1) replace the complicated and expensive leukapheresis process using fresh whole blood for starting material; 2) provide the flexibility of earlier culture harvest. We thereby approach our goal to generate an economical process for CAR T production that can be administered to all who would benefit. Future work will include evaluation of this process with patient-derived material.


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  2. Neelapu SS, Locke FL, et al. Axicabtagene Ciloleucel CAR T-Cell Therapy in Refractory Large B-Cell Lymphoma. N Engl J Med 2017 Dec;377(26):2531–2544.

  3. Vitanza NA, Wilson AL, et al. Intraventricular B7-H3 CAR T Cells for Diffuse Intrinsic Pontine Glioma: Preliminary First-in-Human Bioactivity and Safety. Cancer Discov 2023 Jan;13(1):114–131.

  4. Del Bufalo F, De Angelis B, et al. GD2-CART01 for Relapsed or Refractory High-Rish Neuroblastoma. N Engl J Med 2023 Apr;388(14):1284–1295.

  5. Anthony-Gonda K, Barhi A, et al. Multispecific anti-HIV duoCAR-T cells display broad in vitro antiviral activity and potent in vivo elimination of HIV-infected cells in a humanized mouse model. Sci Transl Med. 2019 Aug;11(504):eaav5685.

  6. Jackson Z, Roe A, et al. Automated Manufacture of Autologous CD19 CAR-T Cells for Treatment of Non-Hodgkin Lymphoma. Front Immunol. 2020 Aug;11:1941.

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