Manufacture of tumor- and virus-specific T lymphocytes for adoptive cell therapies

Abstract

Adoptive transfer of tumor-infiltrating lymphocytes (TILs) and genetically engineered T lymphocytes expressing chimeric antigen receptors (CARs) or conventional alpha/beta T-cell receptors (TCRs), collectively termed adoptive cell therapy (ACT), is an emerging novel strategy to treat cancer patients. Application of ACT has been constrained by the ability to isolate and expand functional tumor-reactive T cells. The transition of ACT from a promising experimental regimen to an established standard of care treatment relies largely on the establishment of safe, efficient, robust and cost-effective cell manufacturing protocols. The manufacture of cellular products under current good manufacturing practices (cGMPs) has a critical role in the process. Herein, we review current manufacturing methods for the large-scale production of clinical-grade TILs, virus-specific and genetically modified CAR or TCR transduced T cells in the context of phase I/II clinical trials as well as the regulatory pathway to get these complex personalized cellular products to the clinic.

Figure 1

Schematic illustration of representative TIL, TCR/CAR-T, and EBV-CTL manufacturing platforms. (a) Traditional and young TIL manufacturing scheme. Single-cell digests of resected tumor are plated in 24-well plates as microcultures. For traditional TILs, the microcultures are passaged as independent cultures. Screened and selected TIL cultures are further expanded with a 2-week rapid expansion procedure using OKT-3 antibody. For young TILs, microcultures are pooled without screening. The pooled cells undergo the same 2-week rapid expansion procedure to reach the target dose. During the whole process, cells are maintained in culture with 6000 IU mL⁻¹ of IL-2. (b) TCR/CAR-T manufacturing process. T cells are selected from washed apheresis product and activated by using CD3/28 Dynabeads and ClinExVivo magnetic particle concentrator (MPC). Activated T cells are transduced with TCR/CAR retroviral or lentiviral vectors, and transduced cells are expanded with WAVE bioreactor. CD3/28 magnetic Dynabeads are removed from the cells with MPC and end of the process cells are formulated for infusion. (c) CD8⁺ central memory TCR/CAR T-cell manufacturing process. PBMCs are first purified from apheresis product using Ficoll-Plaque gradient centrifugation, followed by CD4⁺, CD14⁺ and CD45RA⁺ cell depletion using anti-CD4, anti-CD14 and anti-CD45RA microbeads and CliniMACS. Collected cells undergo an additional CD62L positive selection procedure using anti-CD62L microbeads and CliniMACS. Selected CD8⁺CD62L⁺ cells are further activated with CD3/28 Dynabeads. Activated memory CD8⁺ cells are transduced with TCR/CAR vectors and expanded in vitro. Dynabeads are removed from the EOP cells using MCP before formulation. (d) Generation of multiviral antigen-specific T cells using G-Rex bioreactor. Donor PBMCs are pulsed with 15mer peptides mix spanning EBV, adenovirus, CMV, BK virus and human herpes virus antigen epitopes and transferred into G-Rex device. Multivirus antigen-specific T cells are expanded to high quantities in ~ 2 weeks of time in the presence of IL-4 and IL-7. CAR, chimeric antigen receptor; CTL, cytotoxic T lymphocytes; EBV, Epstein–Barr virus; IL, interleukin; PBMC, peripheral mononuclear blood cell; TCR, T-cell receptor; TIL, tumor-infiltrating lymphocyte.

Figure 2

Retroviral and lentiviral vectors’ manufacturing platform. (a) Generation of retroviral vector. High titer-producer cells from master cell bank are thawed and expanded in T flasks. Cells are further expanded in either roller bottles, cell factories or bioreactors. Vector stocks are harvested in the optimized harvesting window, filtered to removed contaminants and cryopreserved for biosafety testing before release for clinical use. (b) Manufacturing of lentiviral vector using transient transfection in 10-layer cell factories. 293T cells or derivatives are expanded to large quantity to inoculate multiple 10-layer cell factories. Cells are transiently transfected with packaging, envelope, and SIN-vector plasmids. Crude vector stocks are harvested and filtered. Benzonase is added to the crude harvests to remove plasmid contaminants. Vector stocks need to be further purified and concentrated using diafiltration or chromatography methodologies. Purified and concentrated vector stocks are cryopreserved for biosafety testing to be qualified for clinical use. MCB, master cell bank; SIN, self-inactivating.