Applying the Allogeneic Process to Overcome Challenges in Cell Therapy

Evan Zynda, staff scientist, Thermo Fisher Scientific

Presented by: Evan Zynda, staff scientist, Thermo Fisher Scientific

Cell therapy companies increasingly are pursuing allogeneic therapies to negotiate manufacturing challenges associated with autologous approaches. Products based on high-quality cells from healthy donors could improve therapeutic efficacy. Allogeneic approaches also could facilitate large-scale therapy production, which in turn could reduce time-to-treatment and increase therapy affordability and accessibility. Zynda showed how increased understanding of T-cell biology could help improve allogeneic workflows.

Zynda emphasized the utility of studying signaling pathways associated with T-cell differentiation. Young central memory T cells are highly capable of proliferation, engraftment, persistence, and immunostimulatory cytokine production, all of which correlate strongly with therapeutic efficacy. These cells also take up antitumor programming readily. But continuous signaling transforms such cells into effector memory cells, which become functionally “exhausted” over time and thus lose their therapeutic potential. These insights have clear implications for allogeneic workflows, Zynda stated. Therapy developers and bioprocess suppliers must devise solutions that can delay T-cell differentiation to deliver young, heathy, long-lasting cells to patients.

Thermo Fisher Scientific began development work in that area by optimizing its CTS OpTmizer cell-expansion media. Culture medium was a logical entry point, Zynda noted, because it contacts T cells throughout expansion and because media components can be modified easily to influence cell signaling. The OpTmizer product also presented several manufacturing advantages because it is serum- and phenolred– free and has a strong track record for production of marketed cell therapies. To drive memory-cell maintenance and delay differentiation into effector cells, Thermo Fisher developed its novel CTS OpTmizer Pro expansion medium.

The company evaluated the medium’s performance by using it to culture primary T cells from healthy donors for 17 days. After five, 10, and 17 days, the team tallied the number of central memory cells present and evaluated the cells’ expression of CD62L, CCR7, and CD27 surface markers. The analysts also assessed the cells’ proliferative capacity, survival rate, and production of interferon gamma (IFN-_). That cytokine activates membrane-attack complexes (MACs), natural killer (NK) cells, and other critical immune system responses. All findings were compared with results from similar experiments that used the parent media formulation.

Cultures using OpTmizer Pro media showed robust growth and cell viability rates >90% across all conditions and donors. After 10 and 17 days, the formulation achieved respectively 18% and 93% more growth among central memory cells than did the parent medium. Central memory cells that proliferated in OpTmizer Pro media also showed signs of early enrichment. After five and 10 days of culture, expression of markers of interest increased by 10–20%. Together, those findings suggest that the optimized media indeed delayed the T cells’ differentiation. That ultimately increased their proliferative capacity early, an effect that became compounded between days 10 and 17. Compared with the parent medium, the new formulation also averaged 55% higher production of IFN-_. That could indicate significant increases in therapeutic efficacy.

Those small-scale studies, Zynda explained, were conducted to assess the new medium’s feasibility. Thermo Fisher conducted additional experiments to understand how the medium might perform at larger scales. Successful processing in rocking-motion reactors, for instance, would be useful because such systems enable modest scalability, good control of liquid and gas flow, and in some cases, automation and digitalization of process steps. Using such reactors, both media formulations were used to cultivate T cells for 17 days. The OpTmizer Pro medium showed similar advantages to those observed during small-scale studies.

Thermo Fisher is performing expansion studies in stirredtank bioreactors. Such systems would be ideal for T-cell expansion because they enable high scalability (e.g., to 2,000 L) and can perform sophisticated control functions, including complex feeding strategies, perfusion capabilities, active and passive gassing, and cell bleeding. Thus far, Thermo Fisher scientists have achieved similarly high numbers of central memory cells in rocking-motion and stirred-tank vessels using the OpTmizer Pro formulation. Zynda noted that those results are encouraging, and his company will continue to develop, optimize, and validate allogeneic workflows for such bioreactors and media formulations.