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

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.

Intratumoral pan-ErbB targeted CAR-T for head and neck squamous cell carcinoma: interim analysis of the T4 immunotherapy study

“…Manufacture using a bespoke semiclosed fresh product platform was successful for all subjects from whole blood as starting material. 14 Vein to treatment time was only 14 days owing to the delivery of a fresh product in which final sterility testing data was only available post-treatment. The CAR T-cell drug product demonstrated considerable donor to donor variability in immunophenotype, which is a recognized limitation of autologous CAR T-cell immunotherapy.…”

“…Interleukin-4 was the sole cytokine support provided after gene transfer. 14 The drug substance was subject to release testing as summarized in online supplemental table S1 . 15 Drug product was suspended in X-VIVO medium (Lonza, Basel Switzerland) + 1X GlutaMAX (ThermoFisher Scientific, Horsham, UK) + 10% human AB serum (BioIVT, West Sussex, UK).…”

“…The bag was carefully mixed to resuspend aggregates before the appropriate therapeutic volume (1–4 mL: online supplemental table S2 ) was drawn up into a Luer syringe. 14 Following attachment of a 21 gauge needle, T4 immunotherapy was immediately delivered by intratumoral (single or multisite) injection to one or more predefined HNSCC tumor(s). 15 In one case, ultrasound guidance was used to facilitate accuracy of injection.…”

Generation of human parallel chimeric antigen receptor (pCAR) T cells to achieve synergistic T cell co-stimulation

“… Note: Numbers of PBMC between 3 – 100 million can be routinely activated in this manner. However, for scalable manufacture with a view to clinical application, alternative culture vessels and activation stimuli should be used ( van Schalkwyk et al., 2021 ). Incubate at 37°C & 5% CO 2 for 48 h. Do not add or change medium over this period since some T cell loss typically occurs and medium is conditioned with cytokines derived from activated T cells.…”

“…Note: Numbers of PBMC between 3 – 100 million can be routinely activated in this manner. However, for scalable manufacture with a view to clinical application, alternative culture vessels and activation stimuli should be used ( van Schalkwyk et al., 2021 ).…”