Development and Validation of a Good Manufacturing Process for IL-4-Driven Expansion of Chimeric Cytokine Receptor-Expressing CAR T-Cells.

May C I Van Schalkwyk,Leticia Bosshard-Carter,Farzin Farzaneh,Sophie Papa,Helen Graves,Andrew Hope,Sjoukje J C Van Der Stegen,Christopher D Fisher,Ana C Parente-Pereira,Antonella Adami,John Maher

doi:10.3390/cells10071797

Abstract

Adoptive cancer immunotherapy using chimeric antigen receptor (CAR) engineered T-cells holds great promise, although several obstacles hinder the efficient generation of cell products under good manufacturing practice (GMP). Patients are often immune compromised, rendering it challenging to produce sufficient numbers of gene-modified cells. Manufacturing protocols are labour intensive and frequently involve one or more open processing steps, leading to increased risk of contamination. We set out to develop a simplified process to generate autologous gamma retrovirus-transduced T-cells for clinical evaluation in patients with head and neck cancer. T-cells were engineered to co-express a panErbB-specific CAR (T1E28z) and a chimeric cytokine receptor (4αβ) that permits their selective expansion in response to interleukin (IL)-4. Using peripheral blood as starting material, sterile culture procedures were conducted in gas-permeable bags under static conditions. Pre-aliquoted medium and cytokines, bespoke connector devices and sterile welding/sealing were used to maximise the use of closed manufacturing steps. Reproducible IL-4-dependent expansion and enrichment of CAR-engineered T-cells under GMP was achieved, both from patients and healthy donors. We also describe the development and approach taken to validate a panel of monitoring and critical release assays, which provide objective data on cell product quality.

Highlights

Adoptive immunotherapy using chimeric antigen receptor (CAR) engrafted T-cells holds great promise as a novel and effective approach to the treatment of cancer
Human AB Serum Is Required for Manufacture of T4 Immunotherapy
We confirmed that several good manufacturing practice (GMP)-grade media were suitable for this purpose, generating comparable cell yields to research grade media

Summary

Introduction

Adoptive immunotherapy using chimeric antigen receptor (CAR) engrafted T-cells holds great promise as a novel and effective approach to the treatment of cancer. One key hurdle relates to the technical challenges associated with manufacture of cell products, leading to poor standardization and variability in quality [1,2]. The starting material is obtained using a leukapheresis procedure, which is widely considered to be an essential step in the manufacturing process [3]. Leukapheresis scheduling can prove challenging since it is used for several additional applications, including the removal of malignant leukocytes, harvesting of stem cells for transplantation and treatment of selected non-malignant indications such as inflammatory bowel disease. Thereafter, CAR T-cells are expanded using complex and costly cell processing methodologies in highly specialized good manufacturing practice (GMP) facilities [4,5,6]. There has been a move towards the use of automated systems with a view to upscaling manufacture to industrial levels [7]

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