Enabling Allogeneic T Cell-Based Therapies: Scalable Stirred-Tank Bioreactor Mediated Manufacturing

Abstract

Allogeneic T cells are key immune therapeutic cells to fight cancer and other clinical indications. High T cell dose per patient and increasing patient numbers result in clinical demand for a large number of allogeneic T cells. This necessitates a manufacturing platform that can be scaled up while retaining cell quality. Here we present a closed and scalable platform for T cell manufacturing to meet clinical demand. Upstream manufacturing steps of T cell activation and expansion are done in-vessel, in a stirred-tank bioreactor. T cell selection, which is necessary for CAR-T-based therapy, is done in the bioreactor itself, thus maintaining optimal culture conditions through the selection step. Platform's attributes of automation and performing the steps of T cell activation, expansion, and selection in-vessel, greatly contribute to enhancing process control, cell quality, and to the reduction of manual labor and contamination risk. In addition, the viability of integrating a closed, automated, downstream process of cell concentration, is demonstrated. The presented T cell manufacturing platform has scale-up capabilities while preserving key factors of cell quality and process control.

Keywords: T cell manufacturing; allogeneic T cells; automation; bioreactor; closed process.

Figure 1

T cells expand better in agitation, compared to 2D flasks: (A) Viable cell density of T cells for 11 days expansion in 125-mL spinner flasks and T25 (2D) flask. The agitation was increased from 50 to 100 RPM on day 5; (B) Total number of T cells during expansion in 125 mL spinner flasks and T25 (2D) flask, as evaluated on specified days till day 11; (C) VCD of T cells expanded in 1-L STR at different agitation rates. The values represent mean ± S.E. of three experiments.

Figure 2

Alternating tangential flow mediated continuous perfusion in stirred tank bioreactor enables T cell expansion: (A) Prominent subsets of T cells before and after isolation from PBMCs. The values represent an average of 50,000 single cells per condition; (B) Viable cell density of T cells during 14 days expansion. Cell count in STR was performed at inoculation (day 0), before and after media addition (Day 3) and then each day till day 14. Cell count in G-Rex® was performed on day 0 at inoculation, on days 10 and 14. The values represent mean ± S.E. of three technical replicates; (C) Lactate levels during 14 days of T cell cultivation in the stirred tank bioreactor in comparison to viable cell density; (D) Glucose levels during 14 days of T cell cultivation in the stirred tank bioreactor in comparison to viable cell density; (E) Cell viability of T cells during 14 days of T cell cultivation in the stirred tank bioreactor. The values represent mean ± S.E. of three technical replicates.

Figure 3

The T cell activation in STR with ATF mediated continuous perfusion results in T cells with stemness over terminal differentiation or exhaustion: (A) Prominent subsets of T cells before, during and after expansion; (B) Differentiation status of T cells before, during and after expansion; and (C) Senescence and exhaustion of T cells before, during and after expansion. The values represent average of 50,000 single cells per condition.

Figure 4

The T cell activation and expansion in stirred-tank bioreactor maintains the polyfunctionality of the T cells: (A) T cell subsets on Day 0 and Day 14 before and after isolation. The values represent average of 50,000 single cells per condition; (B) Polyfunctionality of T cells on day 0 and 14 after stimulation. The values represent average of 50,000 single cells per condition; (C) Heat map of polyfunctionality of T cells shown based on the number of cytokines produced; (D) Polyfunctional strength index of T cells on day 0 and 14 after stimulation. The values represent the average of 3,000 single cells per condition.

Figure 5

In-vessel cell depletion results in CD4⁺ T cell and bead depletion effectively: (A) Dot plots from flow cytometry analysis of CD8⁺ T cells vs. CD4⁺ T cells pre depletion and 30 min after depletion. The values represent average of 50,000 single cells per condition; (B) Images of beads in cell culture at 20X magnification before bead depletion and after incubation with magnets for specified time. The values represent reading from one experiment, unless mentioned otherwise.

Figure 6

Representation of closed, GMP compatible end-to-end platform for activation, expansion, selection and downstream processing of T cells. T cell activation is performed in the STR. Cell retention and continuous media perfusion is enabled by ATF. Depletion of undesired cells is performed in the STR with proprietary magnetic technology. Cells are concentrated and formulated using automated closed systems.