GMP manufacture of Regulatory T cells (Tregs) to facilitated reduced reliance on immunosuppressive drugs following renal transplantation

Abstract

Seetha Abdul Wahab, Matthew Brook, Giovanna Lombardi, Joanna Hester, Paul Harden, Fadi Issa

Due to significant side effects, it is desirable to be able to reduce patient’s dependency on immunosuppressive drugs after organ transplantation. Personalised Treg therapy offers a promising route to facilitating a reduction in immunosuppression.  The TWO Study employs a GMP-compliant manufacturing process to produce autologous Tregs for therapy. GMP is a quality management system that entails production and quality control of advanced therapy medicinal products (ATMPs) ensuring that ATMPs are consistently produced per clinical trial authorisation or product specification.  This work is undertaken at a purpose-built GMP facility at Guy’s Hospital, London. Patient samples of peripheral blood are received from Oxford and then volume reduced aseptically to 40 mls using Sepax. CD8⁺cells are eliminated using CliniMACS CD8 reagent. CliniMACS CD25 reagent is then added to isolate CD25⁺ cells. Isolated cells are expanded using Expact Treg bead suspension kit and Rapamycin. Cells are fed every two days and subsequently re-stimulated twice at five feeding cycle intervals or until the required cell number is reached.  The cells are then pooled and centrifuged at 1000xg. The resulting pellet is re-suspended and the Expact expansion beads removed using CliniMACS tubing set LS.  Finally, the cell product is formulated in CryoStor CS-10 to a maximum dose concentration of 10x10⁶ cells/kg patient body weight. At the same time, a back-up dose is prepared as well as reference and QC samples. These are all cooled to -80^oC at c.1^oC/min and stored in liquid nitrogen vapour phase until dispatch.  As of end 2019, three patient samples have been processed. Tregs were purified and propagated to 194.4, 282.1, and 183.4-fold expansion yielding 3.5x10⁹, 2.2x10⁹, and 3.1x10⁹ cells respectively. Samples 1 and 2 have been released for treatment after phenotyping and testing for suppression activity, sterility, and contamination with aerobic/anaerobic pathogens and mycoplasma. The third product is scheduled to be released for treatment in May 2020.