Closed System Automated Manufacturing – Benefits and Challenges

Cheryl is the Laboratory Director of the Cellular Therapy Laboratory, Cancer Care Services at the Royal Brisbane & Women’s Hospital. The laboratory is a large processing facility for the haemopoietic progenitor cell transplant and CAR T-cell programs within the hospital. As part of the CAR T- cell program, the laboratory has developed point-of-care production of CAR T-cells using the Miltenyi Biotec CliniMACs Prodigy® to provide treatment options for patients who are ineligible for government funded commercial CAR T-cell therapies.

Hutchins CJ ^1, Henderson AJ ^1, Henden AS ^1,2, Abaca-Cleopas ME ^1, Acworth M ^1, Lynam E ^1, Barnes EC ^1, McEnroe B ^2, McLean A ^1, Mudie K ^1, Kennedy GA¹ and Tey SK^1,2
Cellular Therapy Laboratory, Cancer Care Services, Royal Brisbane & Women’s Hospital¹
QIMR Berghofer Medical Research Institute²

The Cellular Therapy Program at the Royal Brisbane and Women’s Hospital, in collaboration with QIMR Berghofer, has developed Point-of-Care manufacturing of CAR T-cells using the Miltenyi Biotec CliniMACS Prodigy® and a lentiviral vector (Lentigen, Miltenyi Biotec) to expand the availability of CAR T- cells to patients who are ineligible for government funded commercial CAR T-cells.
Three validation procedures were performed for the in-house production of CD19 CAR T-cells using the T-cell Transduction (TCT) program on the Prodigy®. Mononuclear cells (MNC(A)) were collected from healthy donors by apheresis and a maximum of 20 x 10^9 WCC or 3 x 10^9 CD3+ T-cells were loaded onto the device for immunomagenetic selection of CD4+ and CD8+ T cells. Following selection, 1 x 10^8 CD3+ T-cells were activated using TransAct®. Transduction with lentiviral vector was scheduled 24h post T cell activation, followed by T cell expansion in TexMACS GMP medium supplemented with IL7 and IL15 for 11 days. Quality assurance testing was performed on starting material MNC(A), CD4+/CD8+ selected MNC(A), day 5, day 9 and day 12 (formulated CD19 CAR T-cell product).
The three POC validation procedures resulted in CD19 CAR T-cell products with adequate viable CAR T-cell doses. All CAR19 T-cell products met specification for viability, transduction efficiency, absence of microbial contamination during in-process sampling, and in the final formulated product, mycoplasma and endotoxin, and qPCR testing for replication competent lentivirus (RCL) and lentiviral copy number. Preliminary stability studies were performed on the formulated, and cryopreserved CD19 CAR T-cell products.
POC manufacture of CAR T-cells enables the infusion of fresh rather than cryopreserved CAR T-cells with reduced turnaround time for production (12 days) and decreased cost (anticipated 40 – 80% reduction in cost per product compared to current commercial products). It also provides a platform for the manufacture of novel CAR T-cell products for use in clinical trials.
Following successful completion of the validation studies, a Phase 1 clinical trial of POC manufactured CD19 CAR T-cells is now open to patients with relapsed or refractory CD19+ haematological malignancy who are ineligible for government-funded commercial CAR T-cells.

An overview of the Malaghan Institute of Medical Research’s experience of launching a phase 1 trial of a autologous third generation CAR T-cell therapy incorporating a novel TLR2 costimulatory domain, and our early experience of an automated CAR T-cell manufacturing protocol using the Lonza Cocoon^®.

The toll-like receptor (TLR) and MyD88 signalling pathway is emerging as a promising new avenue for chimeric antigen receptor (CAR) co-stimulation¹. Developed in partnership with Guangzhou Institutes of Biomedicine & Health, our 3^rd generation 1928T2z CAR construct consists of a murine anti-CD19 scFv (FMC63) with CD28 and CD3z signaling domains, alongside a novel TLR2-derived costimulatory domain. Our translational research programme aims to deepen understanding of the mechanism of the TLR2 domain within CARs. In parallel, we have established local Good Manufacturing Practice (GMP) lentiviral vector and CAR T-cell manufacture, and are recruiting to a phase I dose-escalation trial of autologous 1928T2z CAR T-cells for relapsed and refractory B-cell lymphomas (ENABLE; ClinicalTrials.gov NCT04049513)². We are now adapting our GMP protocols for automated, closed-system manufacture using the Lonza Cocoon^®, to facilitate scale-up for future clinical trials of 1928T2z, and other CAR T-cell products.


1. Nouri Y, et al. J Immunother Cancer 2021;0:e003065. doi:10.1136/jitc- 2021-003065
2. George P, et al. Third-generation anti-CD19 chimeric antigen receptor Tcells incorporating a TLR2 domain for relapsed or refractory B-cell lymphoma: a phase I clinical trial protocol (ENABLE). BMJ Open. 2020 Feb 9;10(2):e034629. doi: 10.1136/bmjopen-2019-034629.